Patent Publication Number: US-2013243855-A1

Title: Modified coagulation factor IX polypeptides and use thereof for treatment

Description:
RELATED APPLICATIONS 
     This application is a continuation of now allowed co-pending U.S. application Ser. No. 11/818,985, entitled “MODIFIED COAGULATION FACTOR IX POLYPEPTIDES AND USE THEREOF FOR TREATMENT,” to Jorge Oyhenart, Xavier Gallet, Gilles Borelly, Thierry Guyon, Manuel Vega and Lila Drittanti, filed Jun. 15, 2007, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/815,113, entitled “MODIFIED COAGULATION FACTOR IX POLYPEPTIDES AND USE THEREOF FOR TREATMENT,” to Jorge Oyhenart, Xavier Gallet, Gilles Borelly, Thierry Guyon, Lila Drittanti and Manuel Vega, filed Jun. 19, 2006. 
     This application is related to International Application Serial No. PCT/US2007/14219, entitled “MODIFIED COAGULATION FACTOR IX POLYPEPTIDES AND USE THEREOF FOR TREATMENT,” which also claims priority to U.S. Provisional Application Ser. No. 60/815,113. The subject matter of each of these applications is incorporated by reference in its entirety. 
     This application is also related to U.S. application Ser. No. 11/176,830, to Rene Gantier, Thierry Guyon, Manuel Vega and Lila Drittanti, entitled “RATIONAL EVOLUTION OF CYTOKINES FOR HIGHER STABILITY, THE CYTOKINES AND ENCODING NUCLEIC ACID MOLECULES,” filed Jul. 6, 2005 and published as U.S. Application No. US 2006-0020116, which is a continuation of U.S. application Ser. No. 10/658,834, to Rene Gantier, Thierry Guyon, Manuel Vega and Lila Drittanti entitled “RATIONAL EVOLUTION OF CYTOKINES FOR HIGHER STABILITY, THE CYTOKINES AND ENCODING NUCLEIC ACID MOLECULES,” filed Sep. 8, 2003 and published as U.S. Application No. US-2004-0132977-A1. This application also is related to U.S. application Ser. No. 11/196,067, to Rene Gantier, Thierry Guyon, Hugo Cruz Ramos, Manuel Vega and Lila Drittanti entitled “RATIONAL DIRECTED PROTEIN EVOLUTION USING TWO-DIMENSIONAL RATIONAL MUTAGENESIS SCANNING,” filed Aug. 2, 2005 and published as U.S. Application No. US-2006-0020396-A1, which is a continuation of U.S. application Ser. No. 10/658,355, to Rene Gantier, Thierry Guyon, Hugo Cruz Ramos, Manuel Vega and Lila Drittanti entitled “RATIONAL DIRECTED PROTEIN EVOLUTION USING TWO-DIMENSIONAL RATIONAL MUTAGENESIS SCANNING”, filed Sep. 8, 2003 and published as U.S. Application No. US 2005-0202438. 
     This application also is related to U.S. application Ser. No. 10/658,834, filed Sep. 8, 2003, and to published International PCT Application WO 2004/022593, to Rene Gantier, Thierry Guyon, Manuel Vega and Lila Drittanti entitled, “RATIONAL EVOLUTION OF CYTOKINES FOR HIGHER STABILITY, THE CYTOKINES AND ENCODING NUCLEIC ACID MOLECULES.” This application also is related to U.S. application Ser. No. 10/658,355, filed Sep. 8, 2003, and to International PCT Application WO 2004/022747, to Rene Gantier, Thierry Guyon, Hugo Cruz Ramos, Manuel Vega and Lila Drittanti entitled “RATIONAL DIRECTED PROTEIN EVOLUTION USING TWO-DIMENSIONAL RATIONAL MUTAGENESIS SCANNING.” 
     Where permitted the subject matter of each of the above-referenced applications is incorporated by reference in its entirety. 
    
    
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS 
     An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy #1 and Copy #2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Nov. 13, 2012, is identical, 6,982 kilobytes in size, and titled 4930Bseq.001.txt. 
     FIELD OF INVENTION 
     Modified coagulation factor IX (factor IX; FIX; F9) polypeptides are provided. The FIX polypeptides are modified to exhibit physical properties and activities that differ from unmodified and wild-type FIX polypeptides. Nucleic acid molecules encoding these polypeptides also are provided. Also provided are methods of treatment and diagnosis using the modified FIX polypeptides. 
     BACKGROUND 
     Effective delivery of therapeutic proteins for clinical use is a major challenge to pharmaceutical science. Once in the blood stream, these proteins are constantly eliminated from circulation within a short time by different physiological processes, involving metabolism as well as clearance using normal pathways for protein elimination, such as (glomerular) filtration in the kidneys or proteolysis in blood. Once in the luminal gastrointestinal tract, these proteins are constantly digested by luminal proteases. The latter can be a limiting process affecting the half-life of proteins used as therapeutic agents in per-oral administration and either intravenous or intramuscular injection. The problems associated with these routes of administration of proteins are known and various strategies have been used in attempts to solve them. 
     A protein family that has been the focus of clinical work and effort to improve its administration and bio-assimilation is the blood coagulation factor family, which includes procoagulation factors, such as factor IX (FIX) and factor VIII (FVIII). Deficiencies in the levels of functional coagulation pathway proteins can cause mild to severe bleeding disorders. For example, hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX polypeptides, respectively. The underlying cause of the deficiencies is frequently the result of mutations in FVIII and FIX genes, both of which are located on the X chromosome. Traditional therapy for hemophilias often involves intravenous administration of pooled plasma or semi-purified coagulation factors from normal individuals. These preparations can be contaminated by pathogenic agents or viruses, such as infectious prions, HIV, parvovirus, hepatitis A, and hepatitis C. Hence, there is an urgent need for therapeutic agents that do not require the use of human serum. 
     Recombinantly produced FIX polypeptides have been approved for procoagulation treatment of hemophilia. Also of therapeutic interest are FIX polypeptides that lack peptidase activity and can have anticoagulant activities useful in the treatment of thrombolytic diseases. Hence, FIX along with other coagulation factors are important therapeutic agents for procoagulant and anticoagulation therapies. Since naturally occurring variants can have undesirable side effects as well as the problems of administration, bioavailability, and short half-life, there is a need to improve properties of FIX for its use as a biotherapeutic agent. Therefore, among the objects herein, it is an object to provide modified FIX polypeptides that have improved therapeutic properties. 
     SUMMARY 
     Provided herein are modified Factor IX (FIX) polypeptides that exhibit increased protein stability compared to an unmodified FIX polypeptide. The modified FIX polypeptides provided herein are mutant variants of FIX that display improved protein stability. Modified FIX polypeptides provided herein exhibiting increased protein stability display increased protein half-life in vivo or in vitro compared to an unmodified FIX polypeptide. For example, modified FIX polypeptides provided herein exhibit increased stability in the bloodstream, following oral administration, and/or under storage conditions. Increased protein stability of a modified FIX provided herein can be manifested as increased resistance to digestion by proteases. Such increased stability includes stability as assessed by resistance to blood, intestinal or any other proteases. 
     Provided herein are modified FIX polypeptides containing one or more amino acid modifications corresponding to amino acid positions E36, R37, T39, E40, S61, S68, E70, P74, K80, D85, T87, S102, E119, K122, E125, P126, P129, R134, S136, T144, E147, P151, D166, T169, S171, T172, S174, D177, E224, T225, T241, E242, T244, K247, E294, T371, E372, and L414 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the one or more amino acid positions can include replacements of any of E36Q, E36H, E36N, R37H, R37Q, T39Q, T39H, T39N, E40Q, E40H, E40N, S61Q, S61H, S61N, S68Q, S68H, S68N, E70Q, E70H, E70N, P74A, P74S, K80N, K80Q, D85N, D85Q, T87Q, T87H, T87N, S102Q, S102H, S102N, K122N, K122Q, E125Q, E125H, E125N, P126A, P126S, P129A, P129S, R134H, R134Q, S136Q, S136H, S136N, S138Q, T144Q, T144H, T144N, E147Q, E147H, E147N, P151A, P151S, D166N, D166Q, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, D177N, D177Q, E224Q, E224H, E224N, T225Q, T225H, T225N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, K247N, K247Q, E294Q, E294H, E294N, T371Q, T371H, T371N, E372Q, E372H, E372N, L414I, and L414V of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     Provided herein are modified FIX polypeptides containing one or more amino acid modifications corresponding to any of FIX polypeptides with modifications corresponding to Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9V, E15Q, E15N, R16H, R16Q, E17H, E17N, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24N, F25V, E26H, E26N, E27Q, E27N, R29H, E30Q, E30N, F321, F32V, T35Q, T35H, T35N, T38Q, T38H, T38N, F41I, K43N, K43Q, Y45I, D47Q, G48Q, G48H, G48N, D49N, E52Q, E52H, E52N, S53Q, S53N, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60N, K63N, K63Q, D64Q, D65N, D65Q, Y69I, F751, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, E83Q, E83H, E83N, L84I, L84V, K91N, K91Q, N92Q, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, D104N, D104Q, K106N, K106Q, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113N, Y115I, R116H, R116Q, L117I, L117V, E119Q, E119H, E119N, S123Q, S123N, A127Q, A127H, A127N, P131A, G133Q, G133H, G133N, S138N, T140Q, T140N, S141Q, S141H, S141N, K142N, K142Q, R145Q, A146Q, A146H, A146N, T148Q, T148N, D152N, D152Q, D154N, Y155I, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, E162Q, E162H, E162N, T163Q, T163H, T163N, L165I, L165V, F175I, F175V, F178I, F178V, T179Q, T179H, T179N, E185Q, E185N, D186N, D186Q, K188N, K188Q, P189A, P189S, F192I, P193A, W194I, L198V, N199Q, G200Q, G200H, G200N, D203N, D203Q, F205I, G207Q, G207H, G207N, S209Q, S209H, S209N, E213Q, E213N, K214N, K214Q, T218Q, T218H, T218N, A219Q, A219N, A220Q, A220H, A220N, G226Q, G226H, G226N, K228N, K228Q, T230Q, T230H, T230N, E239Q, E239H, E239N, E240Q, E240N, E245Q, E245H, E245N, R248H, R252H, R252Q, P255A, P255S, Y259I, K265N, K265Q, Y266I, L272I, L272V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, S283Q, S283H, S283N, Y284I, T286Q, T286H, T286N, P287A, P287S, D292N, D292Q, K293N, K293Q, Y295I, T296Q, T296H, T296N, F299I, K301N, K301Q, F302I, F302V, G303Q, G303N, S304Q, S304H, S304N, Y306I, S308Q, S308H, G309Q, G309H, G309N, G311Q, G311N, R312H, R312Q, F314V, K316N, K316Q, R318H, R318Q, L321I, L321V, Y325I, R327H, R327Q, P329A, P329S, D332N, D332Q, T335Q, T335H, T335N, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341Q, F342I, F342V, T343Q, T343H, T343N, Y345I, F349V, G352Q, G352H, G352N, F353V, G363Q, G363H, G363N, D364Q, S365Q, S365H, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, E374Q, E374H, E374N, G375Q, G375N, T376Q, T376H, S377Q, S377H, S377N, F378I, L379V, T380Q, T380H, T380N, S384Q, S384H, G386Q, G386H, G386N, E387Q, E387N, M391V, K392N, K392Q, K394Q, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398I, T399Q, T399H, K400Q, S402Q, S402H, S402N, R403H, Y404I, K409N, K409Q, E410Q, E410N, K411N, K411 Q, T412Q, T412H, T412N, K413N, K413 Q, T415 Q, T415H, and T415N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In a particular example, the modifications in FIX polypeptide are any of T163N, T163H, I164N, I164Q, L165I, I168N, I168H, T169N, T169Q, T169H, S171N, S171Q, S171H, T172Q, T172H, S174N, S174Q, S174H, F175I, T179N, T179Q, T179H, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In another example, the modifications in FIX polypeptide are any of D152Q, D154N, T163H, I164Q, I164H, D166Q, D166N, I168Q, I168H, T169H, S171N, S171Q, T172Q, S174Q, S174H, F175H, D177Q, D177N, T179N, T179Q, T179H, T179N, T179Q, T179H, V181Q, V182N, G184N, D186Q, D186N, A187Q, F192I, V197H, L198I, L198V, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In yet another example, the modifications in FIX polypeptide are any of T163H, I164Q, I168H, I169H, S171N, S171Q, T172Q, S174Q, S174H, T179N, T179Q, T179H, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     Provided herein are modified FIX polypeptides containing two or more amino acid modifications corresponding to modifications at any two or more positions of Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, I66, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, S141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, E294, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, T371, E372, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, L414, and T415 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid amino acid replacements at any two or more of the amino acid positions can include replacements of any of Y1H, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192H, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V19′7N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, I303Q, G303H, I303N, S304Q, S304H, S304N, G305Q, G305H, I305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, I311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, I375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, I381Q, I381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     Provided herein are any of the above modified FIX polypeptides above that are further modified at one or more positions corresponding to amino acid positions Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, K63, D64, D65, I66, Y69, W72, F75, G76, F77, E78, G79, E83, L84, V86, I90, K91, N92, G93, R94, E96, F98, K100, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, S123, A127, V128, P131, G133, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, I168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, E245, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, and T415 of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the one or more further amino acid positions can include replacements of any of Y1H, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L61, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, T38Q, T38H, T38N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y451, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, Y69H, Y691, W72S, W72H, F75I, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, E83Q, E83H, E83N, L84I, L84V, V86Q, V86H, V86N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, P131A, P131S, G133Q, G133H, G133N, V135Q, V135H, V135N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, R145H, R145Q, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, I168Q, I168H, I168N, F175I, F175V, F175H, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192H, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, E245Q, E245H, E245N, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q; E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, T415Q, T415H, and T415N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     Provided herein are modified FIX polypeptides containing an amino acid modification at a position corresponding to amino acid residue F 192 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Provided herein is a modified FIX polypeptide in which the amino acid modification at position F192 is a replacement of phenylalanine (F) by isoleucine (I), valine (V), or histidine (H). In a particular example, the amino acid modification at position F192 is isoleucine (I). Provided herein are modified FIX polypeptides containing an amino acid modification at F 192 that also contains a further amino acid modification at another position. For example, such a modified FIX polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications. In one example, the further amino acid replacement is at positions corresponding to any of amino acid positions Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, K63, D64, D65, I66, Y69, W72, F75, G76, F77, E78, G79, E83, L84, V86, I90, K91, N92, G93, R94, E96, F98, K100, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, S123, A127, V128, P131, G133, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, I168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, E245, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, and T415 of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the further amino acid positions can include replacements of any of Y11, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F91, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y691, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F751, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     In some examples, exemplary amino acid modifications of a FIX polypeptide include amino acid modifications of any of T163H/F192I, I164Q/F192I, T169H/F192I, S171Q/F192I, D152N/D154N/F192I, S174Q/F192I, S174H/F192I, T179Q/F192I, T179H/F192I, T163N/I164N/F192I, D166Q/F192I, D166N/F192I, D186Q/F192I, D186N/F192I, T148A/D152N/D154N/F192I, F192I/D203Q, F192I/D203N, D152N/F192I, D152Q/F192I, T148A/D154N/F192I, D154N/F192I, D154Q/F192I, T163N/I164N, A187N/G190N, V196N/V197N, T140N/T144N, F175I/F178I, F192I/W194I, T148A/D152N, T148A/D152Q, T148A/D154N, T148A/D154Q, D152N/D154N, I168N/T169N/S171N/T172N/S174N, T179N/V181N/V182N/G183N/G184N, D152N/D154N/T148A, T163H/D166Q/F192I, T163H/D166N/F192I, T163H/D186Q/F192I, T163H/D186N/F192I, T163H/F192I/D203Q, T163H/F192I/D203N, T148A/D152N/D154N/T163H/F192I, T148A/D154N/T163H/F192I, I164Q/D166Q/F1921, I164Q/D166N/F1921, I164Q/D186Q/F192I, I164Q/D186N/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D152N/D154N/I164Q/F192I, T148A/D154N/I164Q/F192I, T163N/I164N/D166Q/F192I, T163N/I164N/D166N/F192I, T163N/I164N/D186Q/F192I, T163N/I164N/D186N/F192I, T163N/I164N/F192I/D203Q, T163N/I164N/F192I/D203N, T148A/D152N/D154N/T163N/I164N/F192I, T148A/D154N/T163N/I164N/F192I, D166Q/T169H/F192I, D166N/T169H/F192I, T169H/D186Q/F192I, T169H/D186N/F192I, T169H/F192I/D203Q, T169H/F192I/D203N, T148A/D152N/D154N/T169H/F192I, T148A/D154N/T169H/F192I, D166Q/S171Q/F192I, D166N/S171Q/F192I, S171Q/D186Q/F192I, S171Q/D186N/F192I, S171Q/F192I/D203Q, S171Q/F192I/D203N, T148A/D152N/D154N/S171Q/F192I, T148A/D154N/S171Q/F192I, D166Q/S174Q/F192I, D 166N/S174Q/F192I, S174Q/D186Q/F192I, S174Q/D186N/F192I, S174Q/F192I/D203Q, S174Q/F192I/D203N, T148A/D152N/D154N/S174Q/F192I, T148A/D154N/S174Q/F192I, D166Q/S174H/F192I, D166N/S174H/F192I, S174H/D186Q/F192I, S174H/D186N/F192I, S174H/F192I/D203Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T148A/D154N/I174H/F192I, D166Q/T179Q/F192I, D166N/T179Q/F192I, T179Q/D186Q/F192I, T179Q/D186N/F192I, T179Q/F192I/D203Q, T179Q/F192I/D203N, T148A/D152N/D154N/T179Q/F192I, T148A/D154N/T179Q/F192I, D166Q/T179H/F192I, D166N/T179H/F192I, T179H/D186Q/F192I, T179H/D186N/F192I, T179H/F192I/D203Q, T179H/F192I/D203N, T148A/D152N/D154N/T179H/F192I, and T148A/D154N/T179H/F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, a modified FIX polypeptide provided herein has a sequence of amino acids set forth in any of SEQ ID NOS: 917-1024 or 1927-2034. In particular examples, the modifications in FIX polypeptide are any of D186Q/F192I, T148A/D152N/D154N/F192I, T163H/D166Q/F192I, T163H/F192I/D203Q, T148A/D154N/T163H/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D154N/T163N/I164N/F192I, T169H/F192I/D203N, S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203Q of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In further examples, the modifications in FIX polypeptide are any of S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203Q of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     The modified FIX polypeptides provided herein include precursor forms and mature forms, such as modification of a wild-type human FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 1 (precursor form) or SEQ ID NO: 2 or 1035 (mature forms). Mature forms of FIX polypeptides can include chimeric forms of FIX polypeptides such as set forth in SEQ ID NO: 1035, where homologous regions of coagulation factor family members, such as factor VII or factor X are inserted or replaced in the FIX amino acid sequence. Exemplary modified FIX polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS: 3-891, 917-1034, or 1036-2044. It also is understood that amino acid modification of a FIX polypeptide can be in an allelic, species, or isoform variant of SEQ ID NO: 2 or 1035, where the allelic or species variant has 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide set forth in SEQ ID NO: 2 or 1035, excluding the modified positions. A modified FIX polypeptide can be a human polypeptide or a non-human polypeptide. Modified loci are identified with reference to the amino acid numbering of a known unmodified mature FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2 or 1035. One of skill in the art can readily determine corresponding positions on a particular polypeptide, such as by alignment of unchanged residues. Furthermore, shortened or lengthened variants with insertions or deletions of amino acids, particularly at either terminus that retain an activity readily can be prepared and the loci for corresponding mutations identified. 
     In one example, provided herein is a modified structural homologue of a modified FIX as described herein containing one or more amino acid replacements in the structural homologue at positions corresponding to the 3-dimensional-structurally-similar positions within the 3-D structure of the modified FIX. 
     Modified FIX polypeptides provided herein include FIX polypeptides modified at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions as compared to an unmodified FIX polypeptide. The modified FIX polypeptides provided herein exhibit increased protein stability compared to the unmodified FIX polypeptide. Provided herein are FIX polypeptides in which increased protein stability of the modified FIX polypeptide is the result of modification to the primary sequence of the FIX polypeptide. The increased protein stability exhibited by a FIX polypeptide can be manifested as increased protease resistance. In some cases, increased protein stability of a FIX polypeptide can be due to increased resistance to proteolysis that can occur in serum, blood, saliva, digestive fluids, or in vitro when exposed to one or more proteases. The increased resistance to proteolysis is exhibited by the modified FIX when it is administered intravenously, orally, nasally, pulmonarily, or is present in the digestive tract. Such modified FIX polypeptides exhibit increased resistance to proteolysis by one or more proteases compared to the unmodified FIX. For example, the proteases can by any of pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. 
     In some examples, a modified FIX polypeptide exhibits increased protein stability due to increased protease resistance to elastase. Exemplary of those modified FIX polypeptides with increased protease resistance to elastase are those with modifications corresponding to S3Q, S3H, S3N, G4Q, G4H, G4N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V197Q, V197H, V197N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     A modified FIX polypeptide provided herein that contains one or more amino acid modifications and exhibits increased protein stability manifested as increased protease resistance to elastase also can contain any one or more further amino acid modification at amino acid positions corresponding to positions Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, K63, D64, D65, I66, Y69, W72, F75, G76, F77, E78, G79, E83, L84, V86, I90, K91, N92, G93, R94, E96, F98, K100, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, S123, A127, V128, P131, G133, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, I168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, E245, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, and T415 of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the further amino acid positions can include replacements of any of Y1H, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L14I, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F251, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F321, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, T38Q, T38H, T38N, F411, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L57I, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, K63N, K63Q, D64N, D64Q, D65N, D65Q, I66Q, I66H, I66N, Y69H, Y69I, W72S, W72H, F751, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, E83Q, E83H, E83N, L84I, L84V, V86Q, V86H, V86N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, P131A, P131S, G133Q, G133H, G133N, V135Q, V135H, V135N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, R145H, R145Q, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, I168Q, I168H, I168N, F175I, F175V, F175H, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192H, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, E245Q, E245H, E245N, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, T415Q, T415H, and T415N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     Modified FIX polypeptides provided herein can be naked polypeptides chains or can be post-translationally modified. Exemplary post-translational modifications include glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, or conjugation to albumin or a polyethylene glycol (PEG) moiety. The modified FIX polypeptides provided herein can be further modified at one or more amino acid positions, where the modification contributes to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, PEGylation or protease resistance of the modified FIX polypeptide. Exemplary amino acid modifications include replacement with natural amino acids, non-natural amino acids and combinations of natural and non-natural amino acids. Such modifications can increase the stability of the FIX polypeptide. 
     Provided herein are any of the above modified FIX polypeptides, further containing one or more pseudo-wild type mutations. In one embodiment, the pseudo-wild-type mutations include, but are not limited to, one or more of insertions, deletions or replacements of the amino acid residue(s) of the unmodified FIX polypeptide. 
     Typically, the modified FIX polypeptides provided herein exhibit increased protein stability compared to the unmodified FIX polypeptide and retain one or more activities of the unmodified FIX polypeptide. Provided herein are any of the above modified FIX polypeptides exhibiting increased activity compared to the unmodified FIX. Provided herein are any of the above modified FIX polypeptides exhibiting decreased activity compared to the unmodified FIX. Activity can be assessed, for example, by measuring peptidase activity or blood coagulation in vitro or in vivo. The results of such assays correlate with an in vivo activity and hence a biological activity. In some examples the modified FIX polypeptide promotes coagulation. In other examples, the modified FIX polypeptide inhibits coagulation. 
     In some examples, a modified FIX polypeptide containing a modification corresponding to position F192 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035, and that also contains a further amino acid modification, exhibits increased protein stability and retains one or more activities of the unmodified FIX polypeptide. 
     Provided herein are any of the above modified FIX polypeptides, in which the increased protein stability is manifested as an increased half-life in vivo or in vitro. In one example, the increased stability is manifested as an increased half-life when administered to a subject. In another example, the modified FIX has a half-life increased by at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of unmodified FIX. In yet another example, the modified FIX also has a half-life increased by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times and 1000 times, or more times when compared to the half-life of unmodified FIX. 
     Provided herein are any of the above modified FIX polypeptides that is a precursor polypeptide containing a signal peptide and a propeptide. In one example, the signal sequence is amino acids 1-28 and the propeptide is amino acids 29-46 of the sequence of amino acids set forth in SEQ ID NO: 1. Provided herein are any of the above modified FIX polypeptides that do not have a signal peptide and/or propeptide. Modified FIX polypeptides without a signal peptide and propeptide are mature FIX polypeptides. In some cases, the modified FIX polypeptides provided herein are secreted. Such a secreted polypeptide has a signal sequence and propeptide sequence that is processed prior to secretion. Such secreted FIX polypeptides can contain other post-translational modifications, such as for example, glycosylation, carboxylation, hydroxylation, sulfation, and/or phosphorylation. 
     Provide herein are modified FIX polypeptides that include a heavy chain and a light chain of the FIX polypeptide. In some examples, a modified FIX polypeptide can include a heavy chain and lack a light chain. In some other examples, a modified FIX polypeptide can include a light chain and lack a heavy chain. 
     Provided herein are libraries (collections) of modified FIX polypeptides containing two, three, four, five, 10, 50, 100, 500, 1000, 10 3 , 10 4  or more modified FIX polypeptides as described herein. 
     Provided herein are nucleic acid molecules containing a sequence of nucleotides encoding a modified FIX polypeptide as described herein. Provided herein are libraries (collections) of nucleic acid molecules comprising a plurality of the molecules as described herein. 
     Provided herein are vectors containing the nucleic acid molecules encoding modified FIX polypeptides as described herein. Exemplary vectors include, but are not limited to, a prokaryotic vector, a viral vector, or a eukaryotic vector. Exemplary viral vectors include adenovirus, adeno-associated-virus, retrovirus, herpes virus, lentivirus, poxvirus, and cytomegalovirus vectors. In some examples, the nucleic acid in the vector is operably linked to a promoter, such as a viral promoter or a eukaryotic promoter. Such promoters can be constitutive promoters or inducible promoters. Provided herein are libraries (collections) of such vectors containing two, three, four, five, 10, 50, 100, 500, 1000, 10 3 , 10 4  or more vectors containing the nucleic acid molecules encoding modified FIX polypeptides as described herein. 
     Provided herein are cells containing nucleic acid molecules encoding modified FIX polypeptides as described herein. Provided herein are cells containing vectors that contain the nucleic acid molecules encoding modified FIX polypeptides as described herein. Such cells can be prokaryotic or eukaryotic cells, and can express the modified FIX polypeptides. 
     Provided herein are methods for expressing a modified FIX polypeptide. Such methods can include the steps of: i) introducing a nucleic acid encoding a modified FIX polypeptide or a vector containing a nucleic acid encoding a modified FIX into a cell, and ii) culturing the cell under conditions in which the encoded modified FIX is expressed. In one embodiment, cells are eukaryotic cells or prokaryotic cells. In one embodiment, the modified FIX polypeptide is post-translationally modified. In another method for expression of a modified FIX polypeptide, a nucleic acid molecule encoding a modified FIX or a vector containing a nucleic acid encoding a modified FIX is introduced into a cell-free translation system, whereby the encoded modified FIX polypeptide is expressed. In such expression methods above, the method can further include detection of the modified FIX polypeptide. 
     Provided herein are non-human transgenic animals containing an exogenous nucleic acid encoding a modified FIX polypeptide. Exemplary non-human transgenic animals include pigs, goats, sheep, rabbits, rats, and cows. Provided herein are methods of expressing modified FIX polypeptides in non-human transgenic animals and detecting the expressed modified FIX polypeptides. Modified FIX polypeptides can be isolated from tissues or fluids of the non-human transgenic animals, such as in serum, milk, or eggs. Such expressed modified FIX polypeptides can be post-translationally modified. For example, the modified FIX polypeptides can be glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to a polyethylene glycol (PEG) moiety. 
     Provided herein are pharmaceutical compositions including any of the modified FIX polypeptides described herein. In some examples, the modified FIX polypeptides are any of amino acids set forth in SEQ ID NOS: 3-891, 917-1034, or 1036-2044. 
     In one example, the pharmaceutical compositions provided herein are formulated for local, systemic, or topical administration. For example the pharmaceutical compositions provided herein can be formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parenteral, intravenous, or intramuscular administration. In a particular example, the pharmaceutical compositions are formulated for oral administration. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide, where the FIX polypeptide is modified by replacement of one or more amino acids in its primary structure to be resistant to a protease and the composition is formulated for oral administration. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the modified FIX polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications. Provided herein are pharmaceutical compositions of a FIX polypeptide, in which only the primary sequence of the FIX polypeptide is modified, and the polypeptide exhibits increased protein stability. Such pharmaceutical compositions of a FIX polypeptide can include removal of proteolytic digestion sites. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which increased protein stability is manifested as increased resistance to proteolysis. In some instances, the modified FIX polypeptide in the pharmaceutical formulation exhibits increased protein stability in the gastrointestinal tract under conditions selected from exposure to saliva, exposure to proteases in the gastrointestinal tract and exposure to low pH conditions compared to an unmodified FIX polypeptide. In some instances, the increased resistance to proteolysis occurs in serum, blood, saliva, digestive fluids or in vitro when exposed to one or more proteases. Proteases include, but are not limited to one or more of a pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. In a particular example, that protease is elastase. In some instances, the modified FIX polypeptide in the pharmaceutical formulation exhibits increased protease resistance when it is administered orally or is present in the digestive tract. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide that are a FIX naked polypeptide or a post-translationally modified polypeptide, such as a FIX polypeptide that is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated albuminated, or conjugated to a polyethylene glycol (PEG) moiety. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide that further comprise one or more additional amino acid modifications that contribute to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, PEGylation or protease resistance of the modified FIX polypeptide. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which one or more additional amino acid modifications are selected from natural amino acids, non-natural amino acids or a combination of natural and non-natural amino acids. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which one or more additional amino acid modifications increase stability of the FIX polypeptide. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide that further comprise one or more pseudo-wild type mutations, including insertions, deletions, and replacements of the amino acid residue(s) of the unmodified FIX polypeptide. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide that exhibits increased protein stability compared to the unmodified FIX polypeptide and retains one or more activities of the unmodified FIX polypeptide. Provided herein are pharmaceutical compositions of a modified FIX polypeptide that promote coagulation. Provided herein are pharmaceutical compositions of a modified FIX polypeptide that inhibit coagulation. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide in which increased protein stability of the FIX polypeptide leads to increased protein half-life of the FIX polypeptide in vivo or in vitro. Increased protein stability can lead to increased protein half-life following administration to a subject. Protein half-life can be increased in an amount of at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of the unmodified FIX polypeptide. Alternatively, protein half-life can be increased in an amount of at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times or at least 1000 times or more compared to an unmodified protein. In one example, the modified FIX in the pharmaceutical composition exhibits increased protein half-life or bioavailability in the gastrointestinal tract. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide containing a composition that also contains a pharmaceutically acceptable carrier, diluent, or excipient. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the excipient is a binding agent, a filler, a lubricant, a disintegrant or a wetting agent. In a particular example, the excipient is anhydrous crystalline maltose or magnesium stearate. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the additive is a suspending agent, an emulsifying agent, a non-aqueous vehicle, or a preservative. 
     Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the pharmaceutical composition is formulated for administration in a form selected from among liquid, a pill, a tablet, a lozenge, and a capsule. In such pharmaceutical compositions, the modified FIX polypeptide is delivered to the gastrointestinal tract or the mucosa of the mouth, throat, or gastrointestinal tract. In some pharmaceutical compositions, the pill or tablet is chewable. In another example, the pill or tablet dissolves when exposed to saliva on the tongue or in the mouth. In other pharmaceutical compositions the capsule is in liquid form. Exemplary liquid pharmaceutical compositions include a solution, a syrup, or a suspension. In some pharmaceutical compositions, the tablet or capsule is enterically coated. In some pharmaceutical compositions, the modified FIX polypeptide is formulated for controlled-release. 
     Provided herein are pharmaceutical compositions prepared without the use of protease inhibitors, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat. Provided herein are pharmaceutical compositions formulated without protective compounds. 
     Provided herein are pharmaceutical compositions of nucleic acid molecules encoding any of the modified FIX polypeptides described herein or a vector containing a nucleic acid molecule encoding any of the modified FIX polypeptides described herein and a pharmaceutically acceptable excipient. 
     Provided herein are methods of treating a subject exhibiting symptoms of or having FIX-mediated disease or condition by administering any of the pharmaceutical compositions described herein. For example, such pharmaceutical compositions can contain modified FIX polypeptides having a sequence of amino acids set forth in any one of SEQ ID NOS: 3-891, 917-1034, or 1036-2044. In some examples, the FIX-mediated disease or condition is treated by administration of an active FIX polypeptide. In other examples the FIX-mediated disease or condition is treated by administration of an inactive FIX polypeptide. Typically, treatment with the pharmaceutical composition ameliorates or alleviates the symptoms associated with the disease or condition. Provided herein are methods of monitoring the subject for changes in the symptoms associated with the FIX-mediated disease or condition. 
     In one example, the FIX-mediated disease or condition includes, but is not limited to hemophilia, thrombotic disease, disseminated intravascular coagulation (DIC), or hemorrhagic disease. In such an example where the disease to be treated is hemophilia, the disease or condition can be congenital or acquired. In another example where the disease to be treated is a thrombotic disease or hemorrhagic disease, the disease or condition can be, but is not limited to, ischemia, stroke, atherosclerosis, antithrombin III deficiency, or protein C deficiency. In cases where increased coagulation is desired (i.e. for hemophilia), modified FIX polypeptides that promote blood clotting are administered. In cases where decreased coagulation is desired (i.e. for thrombotic diseases), modified FIX polypeptides that inhibit blood clotting are administered. 
     Provided herein are methods of treating a subject exhibiting symptoms of or having a FIX-mediated disease or condition by administering modified FIX polypeptides with additional pro-coagulation factors, such as factor VIII, factor VII, vitamin K, pro-coagulants, platelet activators, vasoconstriction agents, plasminogen inhibitors, or fibrolytic inhibitors. 
     Provided herein are methods of treating a subject exhibiting symptoms of or having a FIX-mediated disease or condition by administering modified FIX polypeptides with additional anticoagulation factors, such as platelet inhibitors, vasodilators, fibrolytic activators, or other anticoagulants. Exemplary anticoagulants include heparin, coumarin, and hirudin. Exemplary platelet inhibitors include aspirin, naproxen, meclofenamic acid, ibuprofen, indomethacin, phenylbutazare, and ticlopidine. Exemplary fibrolytic activators include streptokinase, urokinase, and tissue plasminogen activator (t-PA). 
     Provided herein are articles of manufacture including, but not limited to, packaging material and a pharmaceutical composition of a modified FIX polypeptide described herein contained within the packaging material. In a particular embodiment, the pharmaceutical composition packaged within the article of manufacture is effective for treatment of a FIX-mediated disease or disorder, and the packaging material includes a label that indicates that the modified FIX is used for treatment of a FIX-mediated disease or disorder. 
     Provided herein are kits including a pharmaceutical composition of a modified FIX polypeptide as described herein, a device for administration of the modified FIX polypeptide and optionally instructions for administration. 
     Provided herein are methods for producing a modified FIX polypeptide, having an evolved predetermined property, wherein the evolved predetermined property is increased protein stability manifested as increased protease resistance. In such examples, the increased protein stability of the FIX polypeptide that is evolved is due to amino acid modifications, such that only the primary sequence of the polypeptide is modified to confer the property. In some examples, the methods include modifications of one or more additional amino acids that contribute to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, or PEGylation of the modified FIX polypeptide. 
     In one example, a method of increasing protein stability involves the step of introducing one or more amino acid modification that leads to the removal of proteolytic digestion sites such that the polypeptide exhibits increased protease resistance where the amino acid modifications are chosen from any one or more of Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F411, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, I90Q, I90H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192IH, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     In a particular example, a method of increasing protein stability involves the step of introducing one or more amino acid modification that leads to the removal of proteolytic digestion sites recognized by elastase such that the polypeptide exhibits increased protease resistance to elastase where the amino acid modifications are chosen from any one or more of S3Q, S3H, S3N, G4Q, G4H, G4N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V 197Q, V197H, V197N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
    
    
     DETAILED DESCRIPTION 
     Outline 
     A. Definitions 
     B. Hemostasis overview
         1. Clot initiation   2. Signal Amplification and the clotting cascade   3. Termination of coagulation and fibrinolysis       

     C. Factor IX (FIX)
         1. FIX structure and function   2. FIX polypeptides   3. FIX as a biopharmaceutical       

     D. Exemplary methods for modifying FIX
         1. Non-Restricted Rational Mutagenesis One-Dimensional (1D)-Scanning   2. Two dimensional (2D) rational scanning (restricted rational mutagenesis)
           a. Identifying in-silico HITS   b. Identifying replacing amino acids   c. Construction of mutant proteins and biological assays   
           3. Three dimensional (3D) scanning
           a. Homology   b. 3D-scanning (structural homology) methods   
           4. Super-LEADs and additive directional mutagenesis (ADM)   5. Multi-overlapped primer extensions       

     E. Modified FIX polypeptides exhibiting increased protein stability
         1. Protease resistance
           a. Serine Proteases   b. Matrix Metalloproteinases   c. Increased resistance to proteolysis by removal of proteolytic sites   d. Modified FIX polypeptides exhibiting increased protease resistance
               i. Modified FIX polypeptides exhibiting increased protease resistance to elastase only   
               e. Assessment of FIX variants with increased resistance to proteolysis   
           2. Super-LEADs   3. Other FIX modifications
           a. Immunogenicity   b. Glycosylation   c. Additional modifications   
               

     F. Production of FIX polypeptides
         1. Expression systems
           a. Prokaryotic expression   b. Yeast   c. Insects and insect cells   d. Mammalian cells   e. Plants   
           2. Purification   3. Fusion proteins   4. Polypeptide modification   5. Nucleotide sequences       

     G. Assessing modified FIX polypeptide properties and activities
         1. In vitro assays   2. Non-human animal models   3. Clinical Assays       

     H. Formulation/Packaging/Administration
         1. Administration of modified FIX polypeptides
           a. Oral administration   
           2. Administration of nucleic acids encoding modified FIX polypeptides (gene therapy)       

     I. Therapeutic Uses
         1. Hemophilia   2. Thrombotic diseases and conditions
           a. Arterial thrombosis   b. Venous thrombosis and thromboembolism
               i. Ischemic stroke   
               
           3. Acquired coagulation disorders
           a. Disseminated intravascular coagulation (DIC)   b. Bacterial infection and periodontitis   c. Liver disease   d. Warfarin and Heparin-induced bleeding   e. Intracerebral hemorrhage   
               

     J. Combination Therapies 
     K. Articles of manufacture and kits 
     L. EXAMPLES 
     A. DEFINITIONS 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information. 
     As used herein, a “factor IX” polypeptide (abbreviated herein as FIX) refers to any factor IX polypeptide, including but not limited to, a recombinantly produced polypeptide, a synthetically produced polypeptide and a factor IX polypeptide extracted or isolated from cells or tissues including, but not limited to, liver and blood. Alternative names that are used interchangeably for factor IX include Factor 9, Christmas factor, plasma thromboplastin component (PTC), coagulation factor IX, and serum factor IX. Abbreviations for factor IX include FIX and F9. Factor IX includes related polypeptides from different species including, but not limited to animals of human and non-human origin. Human factor DC (hFIX) includes factor IX, allelic variant isoforms, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof. Exemplary unmodified mature human factor IX polypeptides include, but are not limited to, unmodified and wild-type native factor IX polypeptide (such as the polypeptide containing a sequence set forth in SEQ ID NO: 2) and the unmodified and wild-type precursor factor IX polypeptide that includes a propeptide and/or a signal peptide (such as, the precursor FIX polypeptide that has the sequence set forth in SEQ ID NO: 1), and a polymorphic wild-type native factor IX polypeptide that has a mutation at T148A (SEQ ID NO: 892; or its precursor containing a sequence of amino acids set forth in SEQ ID NO: 893). One of skill in the art would recognize that the referenced positions of the mature factor IX polypeptide (SEQ ID NO: 2) differ by 46 amino acid residues when compared to the precursor FIX polypeptide SEQ ID NO: 1, which is the factor IX polypeptide containing the signal peptide and propeptide sequences Thus, the first amino acid residue of SEQ ID NO: 2 “corresponds to” the forty-seventh (47 th ) amino acid residue of SEQ ID NO: 1. The term “factor IX” also encompasses the active form of the factor IX polypeptide, also called factor IXa (FIXa), containing FIX light chain (SEQ ID NO: 894) and FIX heavy chain (SEQ ID NO: 896) linked by a disulfide bond between residues 132C and 289C. FIXa is produced from a mature FIX polypeptide (SEQ ID NO: 2) by proteolytic cleavage after amino acid residues R145 and R180. Proteolytic cleavage can be carried out, for example, by activated factor XI (FXIa) or the tissue factor/activated factor VII complex. The FIX polypeptides provided herein can be further modified, such as by chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. 
     Factor IX includes factor IX from any species, including human and non-human species. FIX polypeptides of non-human origin include, but are not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, and other primate factor IX polypeptides. Exemplary FIX polypeptides of non-human origin include, for example, chimpanzee ( Pan troglodytes , SEQ ID NO: 904), rhesus macaque ( Macaca mulatta , SEQ ID NO: 912), mouse ( Mus musculus , SEQ ID NO: 903), rat ( Rattus norvegicus , SEQ ID NO: 907), Guinea pig ( Cavia porcellus , SEQ ID NO: 900), pig ( Sus scrofa , SEQ ID NO: 905), dog ( Canis familiaris , SEQ ID NO: 899), cat ( Fells catus , SEQ ID NO: 902), rabbit ( Oryctolagus cuniculus , SEQ ID NO: 906), chicken ( Gallus gallus , SEQ ID NO: 901), cow ( Bos Taurus , SEQ ID NO: 898), sheep ( Ovis aries , SEQ ID NO: 908), frog ( Xenopus tropicalis , SEQ ID NO: 909), zebrafish ( Danio rerio , SEQ ID NO: 910), Japanese pufferfish ( Takifugu rubripes , SEQ ID NO: 911). Exemplary non-human FIX sequences that include a signal sequence and propeptide sequence include SEQ ID NOS: 899, 901-904, 909-912. Exemplary mature non-human FIX sequences that do not include a signal sequence and propeptide sequence include SEQ ID NOS: 898 and 905. Exemplary fragments of non-human FIX sequences include SEQ ID NOS: 900 and 906-908. 
     Human and non-human FIX polypeptides include FIX polypeptides, allelic variant isoforms, tissue-specific isoforms and allelic variants thereof, synthetic molecules prepared by translation of nucleic acids, proteins isolated from human and non-human tissue and cells, chimeric FIX polypeptides and modified forms thereof. Human and non-human FIX also include fragments or portions of FIX that are of sufficient length or include appropriate regions to retain at least one activity of the full-length mature polypeptide. Human and non-human FIX polypeptides also can include FIX polypeptides that are of sufficient length to inhibit one or more activities of a full-length mature FIX polypeptide (such as acts as an anticoagulant). 
     As used herein, corresponding residues refer to residues compared among or between two polypeptides that are allelic or species variants or other isoforms. One of skill in the art can readily identify residues that correspond between or among such polypeptides. For example, by aligning the sequences of factor IX polypeptides, one of skill in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. For example, Y1 of SEQ ID NO: 2 (mature factor IX) corresponds to Y47 of SEQ ID NO: 1 (precursor factor IX with signal peptide and propeptide sequences). In other instances, corresponding regions can be identified. For example, the gla domain Y1 through V46 of SEQ ID NO: 2 (mature factor IX) corresponds to Y47 through V92 of SEQ ID NO: 1 (precursor factor 1× with signal peptide and propeptide sequences). One skilled in the art also can employ conserved amino acid residues as guides to find corresponding amino acid residues between and among human and non-human sequences. For example, residues R145 and R180 of SEQ ID NO: 2 (human) correspond to R191 and R226 of SEQ ID NO: 904 (chimpanzee) or R192 and R236 of SEQ ID NO: 903 (mouse). 
     As used herein, an “active portion or fragment of a factor IX polypeptide” refers to a portion of a human or non-human FIX polypeptide that includes at least one modification provided herein and exhibits an activity, such as one or more activities of a full-length FIX polypeptide or possesses another activity. Such activities include, but are not limited to peptidase activity, any coagulant (also referred to as procoagulant) activity, anticoagulant activity or other activity. Activity can be any percentage of activity (more or less) of the full-length polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to the full polypeptide. Assays to determine function or activity of modified forms of FIX include those known to those of skill in the art, and exemplary assays are included herein. Assays include, for example, activated partial thromboplastin time (aPTT). In one such assay, coagulation activity is measured as the time required for formation of a fibrin clot. Activity also includes activities possessed by a fragment or modified form that are not possessed by the full length polypeptide or unmodified polypeptide. 
     As used herein, “mature factor IX” refers to a FIX polypeptide that lacks a signal sequence and a propeptide sequence. Typically, a signal sequence targets a protein for secretion via the endoplasmic reticulum (ER)-golgi pathway and is cleaved following insertion into the ER during translation. A propeptide sequence typically functions in post-translational modification of the protein and is cleaved prior to secretion of the protein from the cell. Thus, a mature FIX polypeptide is typically a secreted protein. In one example, a mature human FIX polypeptide is set forth in SEQ ID NO: 2. The amino acid sequence set forth in SEQ ID NO: 2 differs from that of the precursor polypeptide set forth in SEQ ID NO: 1 in that SEQ ID NO: 2 is lacking the signal sequence, which includes residues 1-21 of SEQ ID NO: 1, and the propeptide sequence, which includes residues 29-46 of SEQ ID NO: 1. 
     As used herein, “native factor IX” refers to a FIX polypeptide encoded by a naturally occurring FIX gene that is present in an organism in nature, including a human or other animal. Included among native FIX polypeptides are the encoded precursor polypeptide, fragments thereof, and processed forms thereof, such as a mature form lacking the signal peptide as well as any pre- or post-translationally processed or modified form thereof. For example, humans express FIX. Exemplary native human FIX sequences are set forth in SEQ ID NOS: 1 (precursor FIX with a signal peptide and propeptide), 2 (mature FIX lacking a signal peptide), 893 (precursor FIX T148A allelic variant), or 892 (mature FIX T148A allelic variant). Also included among native FIX polypeptides are those that are post-translationally modified, including, but not limited to, modification by glycosylation, carboxylation, hydroxylation, sulfation, and phosphorylation. Native FIX polypeptides also include those that have been activated by proteolytic cleavage, typically at R145 or R180 of SEQ ID NO: 2. Other animals produce native FIX, and include, but are not limited to, chimpanzees, mice, rats, pigs, dogs, cats, rabbits, chickens, cows, sheep, frogs, and fish. Exemplary native FIX sequences from other animals are provided in SEQ ID NOS: 898-912. 
     As used herein, “activated Factor IX” or “FIXa” refers to a FIX polypeptide that has been proteolytically cleaved to activate the peptidase activity of the FIX polypeptide. Typically, proteolytic cleavage occurs at two sites in the mature FIX polypeptide, after R145 and R180 (SEQ ID NO: 2). Cleavage is typically carried out by activated factor XI (FXIa) in the presence of vitamin K. The cleavage products, FIX heavy chain and FIX light chain, which are held together by disulfide bonds, form the active FIX enzyme. 
     As used herein, a “zymogen” refers to any compound, such as a polypeptide, that is an inactive precursor of an enzyme and requires some change, such as proteolysis of the polypeptide, to become active. For example, FIX polypeptides exist in the blood plasma as zymogens until activation of the coagulation cascade, whereby the FIX polypeptides are cleaved by activated FXI. 
     As used herein, “unmodified target protein,” “unmodified protein,” “unmodified polypeptide,” “unmodified coagulation factor,” “unmodified FIX” and grammatical variations thereof refer to a starting polypeptide that is selected for modification as provided herein. The starting target polypeptide can be a naturally-occurring, wild-type form of a polypeptide. In addition, the starting target polypeptide can be altered or mutated, such that it differs from a native wild type isoform but is nonetheless referred to herein as a starting unmodified target protein relative to the subsequently modified polypeptides produced herein. Thus, existing proteins known in the art that have been modified to have a desired increase or decrease in a particular activity or property compared to an unmodified reference protein can be selected and used as the starting unmodified target protein. For example, a protein that has been modified from its native form by one or more single amino acid changes and possesses either an increase or decrease in a desired property, such as reduced immunogenicity (see such as, US-2004-0254106) or a change in an amino acid residue or residues to alter glycosylation, can be a target protein, referred to herein as unmodified, for further modification of either the same or a different property. Exemplary modified FIX polypeptides known in the art include any FIX polypeptide described in, for example, U.S. Pat. Nos. 6,277,618, 6,315,995, and 6,531,298 and U.S. Patent Publication Nos. 2004-0102388, 2004-0110675, 2004-0254106, 2005-0100982, and 2006-0040856. 
     Existing proteins known in the art that previously have been modified to have a desired alteration, such as an increase or decrease, in a particular biological activity or property compared to an unmodified or reference protein can be selected and used as provided herein for identification of structurally homologous loci on other structurally homologous target proteins. For example, a protein that has been modified by one or more single amino acid changes and possesses either an increase or decrease in a desired property or activity, such as for example resistance to proteolysis, can be utilized with the methods provided herein to identify on structurally homologous target proteins, corresponding structurally homologous loci that can be replaced with suitable replacing amino acids and tested for either an increase or decrease in the desired activity. 
     As used herein, an “activity” or a “functional activity” of a FIX polypeptide refers to any activity exhibited by a factor IX polypeptide. Such activities will depend on the form of the FIX polypeptide. For example, active FIX polypeptide typically exhibits coagulant or procoagulant activity, whereas a zymogen or other inactive form of a FIX polypeptide typically has anticoagulant activity. Activities of a FIX polypeptide can be tested in vitro and/or in vivo and include, but are not limited to, coagulation activity, anticoagulation activity, enzymatic activity, and peptidase activity. Activity can be assessed in vitro or in vivo using recognized assays, for example, by measuring coagulation in vitro or in vivo. The results of such assays that indicate that a polypeptide exhibits an activity can be correlated to activity of the polypeptide in vivo, in which in vivo activity can be referred to as biological activity. Activity can be any level of percentage of activity of the polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more of activity compared to the full polypeptide. Assays to determine functionality or activity of modified forms of FIX are known to those of skill in the art. Exemplary assays to assess the functional activity of a FIX polypeptide include activated partial thromboplastin time (aPTT) assays, and are described in Example 3. 
     As used herein, “exhibits at least one activity” or “retains at least one activity” refers to the activity exhibited by a modified FIX polypeptide as compared to an unmodified FIX polypeptide of the same form and under the same conditions. For example, a modified FIX polypeptide that has been activated by cleavage into its heavy and light chain form is compared with an unmodified FIX polypeptide that also has been activated by cleavage into its heavy and light chain form, under the same experimental conditions, where the only difference between the two polypeptides is the modification under study. In another example, a modified FIX polypeptide in a zymogen form is compared with an unmodified FIX polypeptide in a zymogen form, under the same experimental conditions, where the only difference between the two polypeptides is the modification under study. Generally, a modified FIX polypeptide that retains an activity of an unmodified FIX polypeptide either improves or maintains the requisite biological activity (such as, coagulation activity) of an unmodified FIX polypeptide. In some instances, a modified FIX polypeptide can retain an activity that is increased compared to an unmodified FIX polypeptide. In some cases, a modified FIX polypeptide can retain an activity that is decreased compared to an unmodified FIX polypeptide. Activity of a modified FIX polypeptide can be any level of percentage of activity of the unmodified polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to the unmodified polypeptide. For example, a modified FIX polypeptide retains at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% of the activity of the wild-type FIX polypeptide. In other embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times greater than unmodified FIX. Activity can be measured, for example, using assays such as those described in the Examples below. 
     As used herein, a “property” of a factor IX polypeptide refers to any property exhibited by a factor IX polypeptide. Such properties include, but are not limited to, protein stability, resistance to proteolysis, conformational stability, thermal tolerance, and tolerance to pH conditions. Changes in properties can alter an “activity” of the polypeptide. 
     As used herein, “protein stability” refers to increased protein-half-life under one or more conditions including, but not limited to, exposure to proteases, increased temperature, particular pH conditions and/or exposure to denaturing ingredients. Increased protein stability exhibited by a FIX polypeptide can be manifested as increased protease resistance, or increased conformational stability such as increased tolerance to temperature, pH, or tolerance to other denaturing ingredients. A modified polypeptide that exhibits increased protein stability in vitro or in vivo is, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more stable than an unmodified polypeptide. The protein stability of a polypeptide, for example, can be assessed in assays of protease resistance or conformational stability (i.e. resistance to temperature) to determine if an activity of the polypeptide is altered, such as is described in the Examples below. For example, the resistance of the modified FIX polypeptides compared to wild-type FIX against enzymatic cleavage by proteases (such as, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, and trypsin) can be empirically tested by treating the polypeptides with proteases over time and then testing the polypeptides for residual functional activity such as for example, coagulation activities. 
     As used herein, “serum stability” refers to protein stability in serum. 
     As used herein, “resistance to proteolysis” refers to any amount of decreased cleavage of polypeptide by a proteolytic agent, such as a protease such that the resulting polypeptide exhibits a changed therapeutic property, such as increase half-life, increased bioavailability, particularly upon oral administration or other such properties. With respect to FIX polypeptides for which therapeutic activity requires activation, the proteases to which resistance is increased do not include required to activate the polypeptide, such as a protease that cleaves a zymogen into its active form. For example, FIX and modified FIX polypeptides provided herein when present in an inactive zymogen form require cleavage by factor XI (FXIa) or the tissue factor/activated factor VII complex to generate active Factor IX. Hence, reference to a FIX polypeptide that exhibits resistance to proteolysis or increased resistance to proteolysis refers to a polypeptide that exhibits resistance to one or more proteases, including, but not limited to, proteases of the gastrointestinal tract, digestive system, bloodstream, and serum, but retains the ability to be cleaved by factor XI (FXIa) or the tissue factor/activated factor VII complex 
     Resistance to proteolysis can be achieved by modifying particular amino acid residues that are susceptible to cleavage by a particular protease to render them less susceptible to cleavage compared to cleavage by the same protease under the same conditions. A modified polypeptide that exhibits increased resistance to proteolysis exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistance to proteolysis than an unmodified polypeptide. 
     As used herein, a FIX polypeptide that is resistant to proteolysis or is resistant to protease cleavage or similar terminology, is a modified FIX polypeptide that exhibits increased resistance to a protease or proteases compared to a FIX polypeptide that does not include such modification. Modifications refer to changes in the primary amino acid sequence, unless noted otherwise. 
     As used herein, “conformational stability” refers to any amount of increased tolerance of a polypeptide to denaturation. This can be achieved by modifying particular amino acid residues that are susceptible to denaturation conditions to render them less susceptible to denaturation under the same conditions. Conformational stability can be determined by assessing the resistance or susceptibility of a polypeptide to denaturation conditions, such as resistance to temperature or pH. A modified polypeptide that exhibits increased conformational stability exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistance to denaturation than an unmodified polypeptide. 
     As used herein, “denaturation” refers to any noncovalent change in the structure of a protein. This change can alter the secondary, tertiary and/or quaternary structure of the polypeptide molecule. Denaturation of a polypeptide can occur by, for example but not limited to, exposure to chaotropic agents such as urea and guanidine hydrochloride, detergents, temperature, pH, and reagents which cleave disulfide bridges such as dithiothreitol or dithiothreitol. 
     As used herein, “thermal tolerance” refers to any temperature affected or dependent change in the stability of a protein. For example, a change, such as an increased thermal tolerance, can be reflected in a decreased amount of denaturation of a polypeptide after exposure to altered (particularly increased) temperatures compared to the unmodified protein under the same conditions. A modified polypeptide that exhibits increased thermal tolerance exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more stability at varied temperatures compared to the unmodified polypeptide. For example, a modified polypeptide can exhibit increased thermal tolerance in vivo when administered to a subject compared to an unmodified polypeptide, and thereby exhibit increased serum half-life. 
     As used herein, “EC 50 ” refers to the effective concentration of FIX necessary to give one-half of a maximum response. For purposes herein, the response measured is any activity of a FIX polypeptide, such as but not limited to, activity in a coagulation activity assay. 
     As used herein, “half-life” refers to the time required for a measured parameter, such the potency, activity and effective concentration of a polypeptide molecule to fall to half of its original level, such as half of its original potency, activity, or effective concentration at time zero. Thus, the parameter, such as potency, activity, or effective concentration of a polypeptide molecule is generally measured over time. For purposes herein, half-life can be measured in vitro or in vivo. For example, the half-life of FIX or a modified FIX polypeptide can be measured in vitro by assessing its activity (i.e. coagulation activity) following incubation over increasing time under certain conditions, such as for example, after exposure to proteases, or denaturing conditions such as temperature of pH. In another example, the half-life of FIX or a modified FIX polypeptide can be measured in vivo following administration (i.e. intravenous, subcutaneous, intraduodenal, oral) of the polypeptide to a human or other animal, followed by sampling of the blood over time to determine the remaining effective concentration and/or activity of the polypeptide in the blood sample. 
     As used herein, formulated for oral administration refers to a formulation for ingestion into the digestive system. 
     As used herein, mucosal delivery refers to delivery via the mucosa and, while mucosal administration can be into the mouth, its delivery does not involve the digestive system. 
     As used herein, “proteases,” “proteinases” or “peptidases” are interchangeably used to refer to enzymes that catalyze the hydrolysis of covalent peptidic bonds. Proteases include, for example, serine proteases and matrix metalloproteinases. Serine protease or serine endopeptidases constitute a class of peptidases, which are characterized by the presence of a serine residue in the active center of the enzyme. Serine proteases participate in a wide range of functions in the body, including blood clotting, inflammation as well as digestive enzymes in prokaryotes and eukaryotes. The mechanism of cleavage by “serine proteases,” is based on nucleophilic attack of a targeted peptidic bond by a serine. Cysteine, threonine, or water molecules associated with aspartate or metals also can play this role. Aligned side chains of serine, histidine, and aspartate form a catalytic triad common to most serine proteases. The active site of serine proteases is shaped as a cleft where the polypeptide substrate binds. Amino acid residues are labeled from N to C termini of a polypeptide substrate (Pi, . . . , P3, P2, P1, P1′, P2′, P3′, . . . , Pj). The respective binding sub-sites are labeled (Si, . . . , S3, S2, S1, S1′, S2′, S3′, . . . , Sj). The cleavage is catalyzed between P1 and P1′. 
     As used herein, a matrix metalloproteinases (MMP) refers to any of a family of metal-dependent, such as Zn 2+ -dependent, endopeptidases that degrade components of the extracellular matrix (ECM). MMPs include four classes: collagenases, stromelysin, membrane-type metalloproteinases, and gelatinases. Proteolytic activities of MMPs and plasminogen activators, and their inhibitors, are important for maintaining the integrity of the ECM. Cell-ECM interactions influence and mediate a wide range of processes including proliferation, differentiation, adhesion, and migration of a variety of cell types. MMPs also process a number of cell-surface cytokines, receptors and other soluble proteins and are involved in tissue remodeling processes such as wound healing, pregnancy and angiogenesis. Under physiological conditions in vivo, MMPs are synthesized as inactive precursors (zymogens) and are cleaved to produce an active form. Additionally, the enzymes are specifically regulated by endogenous inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs). 
     As used herein, “factor IX-mediated disease or disorder” refers to any disease or disorder in which treatment with a factor IX (or modified factor IX) ameliorates any symptom or manifestation of the disease or disorder. Exemplary factor IX-mediated diseases and disorders include, but are not limited to, hemorrhagic disorders, such as hemophilia or thrombotic disorders, such as thromboembolism. Accordingly, a disease or condition that is treated by administration of FIX includes any disease or condition for which factor IX (or modified factor IX) is employed for treatment, including, but not limited to, hemorrhagic disorders, such as hemophilia or thrombotic disorders, such as thromboembolism. 
     As used herein, “procoagulant” refers to any substance that promotes blood coagulation. 
     As used herein, “anticoagulant” refers to any substance that inhibits blood coagulation. 
     As used herein, “hemophilia” refers to a bleeding disorder caused by or involving a deficiency in blood clotting factors. Hemophilia can be the result, for example, of absence, reduced expression, or reduced function of a clotting factor. The most common type of hemophilia is hemophilia A, which results from a deficiency in factor VIII. The second most common type of hemophilia is hemophilia B, which results from a deficiency in factor IX. Another, more rare form of hemophilia is hemophilia C, which results from a deficiency in factor XI. 
     As used herein, “congenital hemophilia” refers to types of hemophilia that are inherited. Congenital hemophilia results from mutation, deletion, insertion, or other modification of a clotting factor gene in which the production of the clotting factor is absent, reduced, or non-functional. For example, hereditary mutations in clotting factor genes, such as factor VIII and factor IX result in the congenital hemophilias, Hemophilia A and B, respectively. 
     As used herein, a “hereditary mutation” is a gene alteration that is passed from parent to offspring. 
     As used herein, “acquired hemophilia” refers to hemophilia that results from clotting factor deficiencies caused by conditions such as liver disease, vitamin K deficiency, or coumadin (warfarin) or other anti-coagulant therapy. 
     As used herein, “hemostasis” refers to the stopping of bleeding or blood flow in an organ or body part. The term hemostasis can encompass the entire process of blood clotting to prevent blood loss following blood vessel injury to subsequent dissolution of the blot clot following tissue repair. 
     As used herein fibrinolysis refers to the dissolution of a blood clot. 
     As used herein, thrombosis refers to the formation of a clot, or thrombus, inside a blood vessel, obstructing the flow of blood. The vessel can be any vein or artery as, for example, in a deep vein thrombosis or a coronary artery thrombosis. 
     As used herein, a thromboembolism is a clot that breaks loose and travels through the bloodstream. 
     As used herein, “thrombotic disease or condition” or “thrombolytic disease or disorder” are used interchangeably to refer to a disease characterized by the overproduction of blot clots. The clots can often break off and travel through the blood stream, causing damage to tissues, such as the lungs (pulmonary embolism), brain (ischemic stroke, transient ischemic attack), heart (heart attack/myocardial infarction, unstable angina), skin (purpura fulminans), and adrenal gland (hemorrhage). The clots also can cause deep vein thrombosis, low blood pressure (hypotension), and salt loss. 
     As used herein, “treating” a subject with a disease or condition means that the subject&#39;s symptoms are partially or totally alleviated, or remain static following treatment. 
     As used herein, treatment encompasses prophylaxis, therapy, and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. For example, treatment encompasses any pharmaceutical use of a modified factor IX and compositions provided herein. 
     As used herein, prevention refers to absolute prevention of a particular disease disorder. Since it generally is not possible to ascertain whether a disease or disorder never developed, prevention also includes reduction in the risk of developing or having a disease or disorder. 
     As used herein, a composition that does not include exogenously added proteases is a composition formulated without the addition of proteases, other than FIX or other active ingredient. Any additional proteases present in the composition would originate from the method of formulation. 
     As used herein, FIX is modified to be protease resistant by changes in primary structure. Among these are changes that do not result in changes in post-translational modification at that site. 
     As used herein, “therapeutically effective amount” or “therapeutically effective dose” refers to an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect. 
     As used herein, “subject” to be treated includes humans and human or non-human animals. Mammals include, primates, such as humans, chimpanzees, gorillas and monkeys; domesticated animals, such as dogs, horses, cats, pigs, goats, cows, and rodents, such as mice, rats, hamsters and gerbils. 
     As used herein, a patient is a human subject. 
     As used herein, “a directed evolution method” refers to methods that “adapt” either proteins, including natural proteins, synthetic proteins or protein domains to have changed proportions, such as the ability to act in different or existing natural or artificial chemical or biological environments and/or to elicit new functions and/or to increase or decrease a given activity, and/or to modulate a given feature. Exemplary directed evolution methods include, among others, rational directed evolution methods described in U.S. Published Application Nos. US 2003-0134351 A1 and US-2004-0132977-A1. 
     As used herein, “two dimensional rational mutagenesis scanning (2-D scanning)” refers to the processes provided herein in which two dimensions of a particular protein sequence are scanned: (1) one dimension is to identify specific amino acid residues along the protein sequence to replace with different amino acids, referred to as is-HIT target positions, and (2) the second dimension is the amino acid type selected for replacing the particular is-HIT target, referred to as the replacing amino acid. 
     As used herein, “in silico” refers to research and experiments performed using a computer. In silico methods include, but are not limited to, molecular modeling studies and biomolecular docking experiments. 
     As used herein, “is-HIT” refers to an in silico identified amino acid position along a target protein sequence that has been identified based on i) the particular protein properties to be evolved, ii) the protein&#39;s sequence of amino acids, and/or iii) the known properties of the individual amino acids. These is-HIT loci on the protein sequence are identified without use of experimental biological methods. For example, once the protein feature(s) to be optimized is (are) selected, diverse sources of information or previous knowledge (i.e., protein primary, secondary or tertiary structures, literature, patents) are exploited to determine those amino acid positions that are amenable to improved protein fitness by replacement with a different amino acid. This step uses protein analysis “in silico.” All possible candidate amino acid positions along a target protein&#39;s primary sequence that might be involved in the feature being evolved are referred to herein as “in silico HITs” (“is-HITs”). The collection (library), of all is-HITs identified during this step represents the first dimension (target residue position) of the two-dimensional scanning methods provided herein. 
     As used herein, “amenable to providing the evolved predetermined property or activity” in the context of identifying is-HITs refers to an amino acid position on a protein that is contemplated, based on in silico analysis, to possess properties or features that when replaced result in the desired activity being evolved. The phrase “amenable to providing the evolved predetermined property or activity” in the context of identifying replacement amino acids refers to a particular amino acid type that is contemplated, based on in silico analysis, to possess properties or features that when used to replace the original amino acid in the unmodified starting protein result in the evolution of a desired or preselected activity. 
     As used herein, “high-throughput screening” (HTS) refers to processes that test a large number of samples, such as samples of test proteins or cells containing nucleic acids encoding the proteins of interest to identify structures of interest or to identify test compounds that interact with the variant proteins or cells containing them. HTS operations are amenable to automation and are typically computerized to handle sample preparation, assay procedures, and the subsequent processing of large volumes of data. 
     As used herein, the term “restricted,” in the context of the identification of is-HIT amino acid positions along the amino acid residues in a protein selected for amino acid replacement and/or the identification of replacing amino acids, means that fewer than all amino acids on the protein-backbone are selected for amino acid replacement and/or fewer than all of the remaining 19 amino acids available to replace the original amino acid present in the unmodified starting protein are selected for replacement. In particular embodiments of the methods provided herein, the is-HIT amino acid positions are restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement. In other embodiments, the replacing amino acids are restricted such that fewer than all of the remaining 19 amino acids available to replace the native amino acid present in the unmodified starting protein are selected as replacing amino acids. In an exemplary embodiment, both of the scans to identify is-HIT amino acid positions and the replacing amino acids are restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement and fewer than all of the remaining 19 amino acids available to replace the native amino acid are selected for replacement. 
     As used herein, “candidate LEADs” are mutant proteins that are designed to have an alteration in property, activity or other attribute, typically a predetermined or preselected property, activity or other attribute, such as a, chemical, physical or biological property or activity in which such alteration is sought. The alteration can add, alter, remove or otherwise change a property, activity or other attribute of a polypeptide. In the methods herein, candidate LEADs are generally generated by systematically replacing is-HITS loci in a protein or a domain thereof with typically a restricted subset, or all, of the remaining 19 amino acids, such as obtained using PAM analysis. Candidate LEADs can be generated by other methods known to those of skill in the art tested by the high throughput methods herein. 
     As used herein, “LEADs” are “candidate LEADs” whose property, activity or other attribute has been changed, optimized, improved, added or eliminated. For purposes herein a “LEAD” typically has activity with respect to a property or activity of interest in an unmodified polypeptide that exhibits such activity or property that differs by at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from the unmodified and/or wild type (native) protein. In certain embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times greater than the activity of the unmodified and/or wild type (native) protein. The desired alteration, which can be either an increase or a reduction in activity, depends upon the function or property of interest (such as, at least about or 10%, at least about or 20%, etc.). The LEADs can be further optimized by replacement of a plurality (2 or more) of “is-HIT” target positions on the same protein molecule to generate “super-LEADs.” 
     As used herein, the term “super-LEAD” refers to protein mutants (variants) obtained by adding the single mutations present in two or more of the LEAD molecules in a single protein molecule. Accordingly, in the context of the modified proteins provided herein, the phrase “proteins comprising one or more single amino acid replacements” encompasses addition of two or more of the mutations described herein for one respective protein. For example, the modified proteins provided herein containing one or more single amino acid replacements can have any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the amino acid replacements at the disclosed replacement positions. The collection of super-LEAD mutant molecules is generated, tested, and phenotypically characterized one-by-one in addressable arrays. Super-LEAD mutant molecules are molecules containing a variable number and type of LEAD mutation. Those molecules displaying further improved fitness for the particular feature being evolved, are referred to as super-LEADs. Super-LEADs can be generated by other methods known to those of skill in the art and tested by the high throughput methods herein. For purposes herein, a super-LEAD typically has activity with respect to the function of interest that differs from the altered activity (or the new activity or eliminated activity) of a LEAD by a desired amount, such as at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from the LEAD mutant from which it is derived. As with LEADs, the change in the activity for super-LEADs is dependent upon the activity that is being “evolved.” The desired alteration, which can be either an increase or a reduction in activity, depends upon the function or property of interest. The desired alteration also can be an addition or elimination of a property or activity. 
     As used herein, the phrase “altered loci” refers to the is-HIT amino acid positions in the LEADs or super-LEADs that are replaced with different replacing amino acids resulting in the desired altered phenotype or activity. 
     As used herein, an “exposed residue” presents more than 15% of its surface exposed to the solvent. 
     As used herein, the phrase “structural homology” refers to the degree of coincidence in space between two or more protein backbones. Protein backbones that adopt the same protein structure, fold and show similarity upon three-dimensional structural superposition in space can be considered structurally homologous. Structural homology is not based on sequence homology, but rather on three-dimensional homology. Two amino acids in two different proteins said to be homologous based on structural homology between those proteins do not necessarily need to be in sequence-based homologous regions. For example, protein backbones that have a root mean squared (RMS) deviation of less than 3.5, 3.0, 2.5, 2.0, 1.7 or 1.5 angstroms at a given space position or defined region between each other can be considered to be structurally homologous in that region and are referred to herein as having a “high coincidence” between their backbones. It is contemplated herein that substantially equivalent (such as, “structurally related”) amino acid positions that are located on two or more different protein sequences that share a certain degree of structural homology have comparable functional tasks; also referred to herein as “structurally homologous loci.” These two amino acids then can be said to be “structurally similar” or “structurally related” with each other, even if their precise primary linear positions on the sequences of amino acids, when these sequences are aligned, do not match with each other. Amino acids that are “structurally related” can be far away from each other in the primary protein sequences, when these sequences are aligned following the rules of classical sequence homology. 
     As used herein, a “structural homolog” is a protein that is generated by structural homology. Exemplary FIX structural homologs include many other vitamin K dependent plasma proteins, including, for example, prothrombin, factor VII, factor X, and protein C. 
     As used herein, “variant,” “factor IX variant,” “modified factor IX polypeptides” and “modified factor IX proteins” refers to a FIX polypeptide that has one or more mutations compared to an unmodified factor IX polypeptide. The one or more mutations can be one or amino acid replacements, insertions or deletions and any combination thereof. Typically, a modified FIX polypeptide has one or more modifications in its primary sequence compared to an unmodified FIX polypeptide. For example, a modified FIX polypeptide provided herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations compared to an unmodified FIX polypeptide. Modifications that confer a property (such as, increased protein stability or increased protease resistance) by virtue of a change in a primary amino acid sequence do not require a change in post-translational modification of the modified polypeptide to confer the property. Any length polypeptide is contemplated as long as the resulting polypeptide exhibits at least one FIX activity associated with a native FIX polypeptide or inhibits an activity at least one FIX activity associated with a native FIX polypeptide (such as acts as an anticoagulant). 
     As used herein, a “single amino acid replacement” refers to the replacement of one amino acid by another amino acid. The replacement can be by a natural amino acid or non-natural amino acids. When one amino acid is replaced by another amino acid in a protein, the total number of amino acids in the protein is unchanged. 
     As used herein, the phrase “only one amino acid replacement occurs on each target protein” refers to the modification of a target protein, such that it differs from the unmodified form of the target protein by a single amino acid change. For example, in one embodiment, mutagenesis is performed by the replacement of a single amino acid residue at only one is-HIT target position on the protein backbone (such as, “one-by-one” in addressable arrays), such that each individual mutant generated is the single product of each single mutagenesis reaction. The single amino acid replacement mutagenesis reactions are repeated for each of the replacing amino acids selected at each of the is-HIT target positions. Thus, a plurality of mutant protein molecules are produced, whereby each mutant protein contains a single amino acid replacement at only one of the is-HIT target positions. 
     As used herein, the phrase “pseudo-wild type,” in the context of single or multiple amino acid replacements, are those amino acids that, while different from the original (such as, such as native) amino acid at a given amino acid position, can replace the native one at that position without introducing any measurable change in a particular protein activity. A population (library) of sets of nucleic acid molecules encoding a collection of mutant molecules is generated and phenotypically characterized such that proteins with sequences of amino acids different from the original amino acid, but that still elicit substantially the same level (i.e., at least about or 10%, 50%, 80%, 90%, 95%, 100%, depending upon the protein) and type of desired activity as the original protein are selected. A library, contains three, four, five, 10, 50, 100, 500, 1000, 10 3 , 10 4  or more modified FIX polypeptides. 
     As used herein, “in a position or positions corresponding to an amino acid position” of a protein, refers to amino acid positions that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference protein. For example, in a position corresponding to an amino acid position of human FIX set forth as SEQ ID NO: 2 can be determined empirically by aligning the sequence of amino acids set forth in SEQ ID NO: 2 with a particular FIX polypeptide of interest. Corresponding positions can be determined by such alignment by one of skill in the art using manual alignments or by using the numerous alignment programs available (for example, BLASTP). Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. Recitation that amino acids of a polypeptide correspond to amino acids in a disclosed sequence refers to amino acids identified upon alignment of the polypeptide with the disclosed sequence to maximize identity or homology (where conserved amino acids are aligned) using a standard alignment algorithm, such as the GAP algorithm. 
     As used herein, “at a position corresponding to” refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. The position of interest to the position in another reference protein can be in, for example, a precursor protein, an allelic variant, a heterologous protein, an amino acid sequence from the same protein of another species, etc. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 95%, 96%, 97%, 98% or 99% or more. The position of interest is then given the number assigned in the reference nucleic acid molecule. 
     As used herein, the terms “homology” and “identity”” are used interchangeably, but homology for proteins can include conservative amino acid changes. In general to identify corresponding positions the sequences of amino acids are aligned so that the highest order match is obtained (see, such as:  Computational Molecular Biology , Lesk, A. M., ed., Oxford University Press, New York, 1988;  Biocomputing: Informatics and Genome Projects , Smith, D. W., ed., Academic Press, New York, 1993 ; Computer Analysis of Sequence Data, Part I , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994 ; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; and  Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988)  SIAM J Applied Math  48:1073). 
     As use herein, “sequence identity” refers to the number of identical amino acids (or nucleotide bases) in a comparison between a test and a reference polypeptide or polynucleotide. Homologous polypeptides refer to a pre-determined number of identical or homologous amino acid residues. Homology includes conservative amino acid substitutions as well identical residues. Sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Homologous nucleic acid molecules refer to a pre-determined number of identical or homologous nucleotides. Homology includes substitutions that do not change the encoded amino acid (i.e., “silent substitutions”) as well identical residues. Substantially homologous nucleic acid molecules hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid or along at least about 70%, 80%, or 90% of the full-length nucleic acid molecule of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. (For determination of homology of proteins, conservative amino acids can be aligned as well as identical amino acids; in this case, percentage of identity and percentage homology vary). Whether any two nucleic acid molecules have nucleotide sequences (or any two polypeptides have amino acid sequences) that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988)  Proc. Natl. Acad. Sci. USA  85: 2444 (other programs include the GCG program package (Devereux, J., et al. (1984)  Nucleic Acids Research  12(I): 387), BLASTP, BLASTN, FASTA (Atschul, S. F., et al. (1990)  J. Molec. Biol.  215:403 ; Guide to Huge Computers , Martin J. Bishop, ed., Academic Press, San Diego (1994), and Carillo et al. (1988)  SIAM J Applied Math  48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (such as, Needleman et al. (1970)  J. Mol. Biol.  48: 443, as revised by Smith and Waterman (1981)  Adv. Appl. Math.  2: 482. Briefly, a GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al. (1986)  Nucl. Acids Res.  14: 6745, as described by Schwartz and Dayhoff, eds. (1979)  Atlas of Protein Sequence and Structure , National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. 
     Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, such as, 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software. 
     As used herein, “corresponding structurally-related” positions on two or more polypeptides, such as FIX polypeptides and other coagulation factors, refer to those amino acid positions determined based upon structural homology to maximize tri-dimensional overlapping between or among polypeptides. 
     As used herein, the phrase “sequence-related proteins” refers to proteins that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity or homology with each other. 
     As used herein, families of non-related proteins or “sequence-non-related proteins” refer to proteins having less than 50%, less than 40%, less than 30%, less than 20% amino acid identity, or homology with each other. 
     As used herein, it also is understood that the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art. 
     As used herein, “a naked polypeptide chain” refers to a polypeptide that is not post-translationally modified or otherwise chemically modified, but contains only covalently linked amino acids. 
     As used herein, a polypeptide complex includes polypeptides produced by chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. 
     As used herein, “output signal” refers to parameters that can be followed over time and, optionally, quantified. For example, when a recombinant protein is introduced into a cell, the cell containing the recombinant protein undergoes a number of changes. Any such change that can be monitored and used to assess the transformation or transfection is an output signal, and the cell is referred to as a reporter cell; the encoding nucleic acid is referred to as a reporter gene; and the construct that includes the encoding nucleic acid is a reporter construct. Output signals include, but are not limited to, enzyme activity, fluorescence, luminescence, amount of product produced, and other such signals. Output signals include expression of a gene or gene product, including heterologous genes (transgenes) inserted into the plasmid virus. Output signals are a function of time (“t”) and are related to the amount of protein used in the composition. For higher concentrations of protein, the output signal can be higher or lower. For any particular concentration, the output signal increases as a function of time until a plateau is reached. Output signals also can measure the interaction between cells, expressing heterologous genes and biological agents. 
     As used herein, a population of sets of nucleic acid molecules encoding a collection (library) of mutants refers to a collection of plasmids or other vehicles that carry (encode) the gene variants. Thus, individual plasmids or other individual vehicles carry individual gene variants. Each element (member) of the collection is physically separated from the others in an appropriate addressable array and has been generated as the single product of an independent mutagenesis reaction. When a collection (library) of such proteins is contemplated, it will be so-stated. A library contains three, four, five, 10, 50, 100, 500, 1000, 10 3 , 10 4  or more modified FIX polypeptides. 
     As used herein, a “reporter cell” is the cell that undergoes the change in response to a condition. For example, in response to exposure to a protein or a virus or to a change in its external or internal environment, the reporter cell “reports” (i.e., displays or exhibits the change). 
     As used herein, “reporter” or “reporter moiety” refers to any moiety that allows for the detection of a molecule of interest, such as a protein expressed by a cell. Reporter moieties include, but are not limited to, fluorescent proteins (such as, red, blue, and green fluorescent proteins), LacZ and other detectable proteins and gene products. For expression in cells, nucleic acids encoding the reporter moiety can be expressed as a fusion protein with a protein of interest or under the control of a promoter of interest. 
     As used herein, phenotype refers to the physical, physiological, or other manifestation of a genotype (a sequence of a gene). In methods herein, phenotypes that result from alteration of a genotype are assessed. 
     As used herein, culture medium is any medium suitable for supporting the viability, growth, and/or differentiation of mammalian cells ex vivo. Any such medium is known to those of skill in the art. Examples of culture medium include, but are not limited to, X-Vivo15 (BioWhittaker), RPMI 1640, DMEM, Ham=s F12, McCoys 5A and Medium 199. The medium can be supplemented with additional ingredients including serum, serum proteins, growth suppressing, and growth promoting substances, such as mitogenic monoclonal antibodies and selective agents for selecting genetically engineered or modified cells. 
     As used herein, the amino acids that occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art. 
     As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide comprises two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids, and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain). 
     As used herein, the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (1972)  Biochem.  11: 1726). Each naturally occurring L-amino acid is identified by the standard three letter code (or single letter code) or the standard three letter code (or single letter code) with the prefix “L-;” the prefix “D-” indicates that the stereoisomeric form of the amino acid is D. 
     As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. “NH 2 ” refers to the free amino group present at the amino terminus of a polypeptide. “COOH” refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in (1969)  J. Biol. Chem.,  243: 3552-3559, and adopted 37 C.F.R. □§§ 1 . 821 -1.822, abbreviations for amino acid residues are shown in Table 1: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Table of Correspondence 
               
            
           
           
               
               
               
               
            
               
                   
                 SYMBOL 
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1-Letter 
                 3-Letter 
                 AMINO ACID 
               
               
                   
                   
               
               
                   
                 Y 
                 Tyr 
                 tyrosine 
               
               
                   
                 G 
                 Gly 
                 glycine 
               
               
                   
                 F 
                 Phe 
                 phenylalanine 
               
               
                   
                 M 
                 Met 
                 methionine 
               
               
                   
                 A 
                 Ala 
                 alanine 
               
               
                   
                 S 
                 Ser 
                 serine 
               
               
                   
                 I 
                 Ile 
                 isoleucine 
               
               
                   
                 L 
                 Leu 
                 leucine 
               
               
                   
                 T 
                 Thr 
                 threonine 
               
               
                   
                 V 
                 Val 
                 valine 
               
               
                   
                 P 
                 Pro 
                 proline 
               
               
                   
                 K 
                 Lys 
                 lysine 
               
               
                   
                 H 
                 His 
                 histidine 
               
               
                   
                 Q 
                 Gln 
                 glutamine 
               
               
                   
                 E 
                 Glu 
                 glutamic acid 
               
               
                   
                 Z 
                 Glx 
                 Glu and/or Gln 
               
               
                   
                 W 
                 Trp 
                 tryptophan 
               
               
                   
                 R 
                 Arg 
                 arginine 
               
               
                   
                 D 
                 Asp 
                 aspartic acid 
               
               
                   
                 N 
                 Asn 
                 asparagine 
               
               
                   
                 B 
                 Asx 
                 Asn and/or Asp 
               
               
                   
                 C 
                 Cys 
                 cysteine 
               
               
                   
                 X 
                 Xaa 
                 Unknown or other 
               
               
                   
                   
               
            
           
         
       
     
     All amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH 2  or to a carboxyl-terminal group such as COOH. 
     As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. 
     As used herein, the term “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non-natural amino acids are described herein and are known to those of skill in the art. 
     As used herein, nucleic acids include DNA, RNA, and analogs thereof, including protein nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single- or double-stranded. When referring to probes or primers (optionally labeled with a detectable label, such as, a fluorescent or a radiolabel), single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 10, 15, 20, 25, or 30 contiguous nucleic acid bases of sequence complementary to, or identical to, a gene of interest. Probes and primers can be 5, 6, 7, 8, 9, 10, or more, 20 or more, 30 or more, 50 or more, 100, or more nucleic acids long. 
     As used herein, heterologous or foreign nucleic acid, such as DNA and RNA, are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it occurs or is found at a locus or loci in a genome that differs from that in which it occurs in nature. Heterologous nucleic acid includes nucleic acid not endogenous to the cell into which it is introduced, but that has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Heterologous DNA herein encompasses any DNA or RNA that one of skill in the art recognizes or considers as heterologous or foreign to the cell or locus in or at which it is expressed. Heterologous DNA and RNA also can encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes. Examples of heterologous nucleic acid include, but are not limited to, nucleic acid that encodes traceable marker proteins (such as, a protein that confers drug resistance), nucleic acid that encodes therapeutically effective substances (such as, anti-cancer agents), enzymes and hormones, and DNA that encodes other types of proteins (such as, antibodies). Hence, herein heterologous DNA or foreign DNA includes a DNA molecule not present in the exact orientation and position as the counterpart DNA molecule found in the genome. It also can refer to a DNA molecule from another organism or species (i.e., exogenous). 
     As used herein, “isolated with reference to a nucleic acid molecule or polypeptide or other biomolecule” means that the nucleic acid or polypeptide has separated from the genetic environment from which the polypeptide or nucleic acid were obtained. It also can mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated,” as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated. Also intended as an “isolated polypeptide” or an “isolated polynucleotide” are polypeptides or polynucleotides that have been partially or substantially purified from a recombinant host cell or from a native source. For example, a recombinantly produced version of a compound can be substantially purified by the one-step method described in Smith et al. (1988)  Gene,  67:31-40. The terms isolated and purified can be used interchangeably. 
     Thus, by “isolated” it is meant that the nucleic acid is free of coding sequences of those genes that, in the naturally-occurring genome of the organism (if any), immediately flank the gene encoding the nucleic acid of interest. Isolated DNA can be single-stranded or double-stranded, and can be genomic DNA, cDNA, recombinant hybrid DNA or synthetic DNA. It can be identical to a starting DNA sequence or can differ from such sequence by the deletion, addition, or substitution of one or more nucleotides. 
     “Purified” preparations made from biological cells or hosts mean at least the purity of a cell extracts containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques, and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures can include, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation, and electrophoresis. 
     A preparation of DNA or protein that is “substantially pure” or “isolated” refers to a preparation substantially free from naturally-occurring materials with which such DNA or protein is normally associated in nature and generally contains 5% or less of the other contaminants. 
     A cell extract that contains the DNA or protein of interest refers to a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest. The term “cell extract” is intended to include culture medium, especially spent culture medium from which the cells have been removed. 
     As used herein, “a targeting agent” refers to any molecule that can bind another target-molecule, such as an antibody, receptor, or ligand. 
     As used herein, “receptor” refers to a biologically active molecule that specifically binds to (or with) other molecules. The term “receptor protein” can be used to more specifically indicate the proteinaceous nature of a specific receptor. 
     As used herein, “recombinant” refers to any progeny formed as the result of genetic engineering. 
     As used herein, a “promoter region” refers to the portion of DNA of a gene that controls transcription of the DNA to which it is operatively linked. The promoter region includes specific sequences of DNA sufficient for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is referred to as the “promoter”. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. Promoters, depending upon the nature of the regulation, can be constitutive or regulated by cis acting or trans acting factors. 
     As used herein, the phrase “operatively linked” with reference to a nucleic acid molecule generally means the sequences or segments have been covalently joined into one piece of DNA, whether in single- or double-stranded form, whereby control or regulatory sequences on one segment control or permit expression or replication or other such control of other segments. The two segments are not necessarily contiguous. For gene expression, a DNA sequence and a regulatory sequence(s) are connected in such a way to control or permit gene expression when the appropriate molecular, such as, transcriptional activator proteins, are bound to the regulatory sequence(s). 
     As used herein, “production by recombinant means by using recombinant DNA methods” means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA, including cloning expression of genes and methods. 
     As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA. 
     As used herein, a composition refers to any mixture of two or more products or compounds (such as, agents, modulators, regulators, etc.). It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous formulations or any combination thereof. 
     As used herein, a combination refers to any association between two or more items. 
     As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass pharmaceutical compositions of modified FIX polypeptides and/or nucleic acids as described herein contained in articles of packaging. 
     As used herein, a “kit” refers to a combination of modified FIX polypeptides or nucleic acid molecules as described herein provided in pharmaceutical compositions and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of an activity or property of the polypeptides described herein. Kits, optionally, include instructions for use. 
     As used herein, “substantially identical to a product” means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product. 
     As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of exemplary vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Exemplary vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked; such vectors typically include origins of replication. Vectors also can be designed for integration into host chromosomes. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Expression vectors are often in the form of “plasmids,” which refer generally to circular double-stranded DNA loops which, in their vector form are not bound to the chromosome. “Plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vectors. Other such forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto. 
     As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells. 
     As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof among a population. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation. Typically, allelic variants, have at least 80%, 90%, 95% or greater amino acid identity with a wild-type and/or predominant form from the same species. 
     As used herein, the terms “gene” or “recombinant gene” refer to a nucleic acid molecule containing an open reading frame and including at least one exon and, optionally, an intron-encoding sequence. A gene can be either RNA or DNA. Genes can include regions preceding and following the coding region (leader and trailer). 
     As used herein, “intron” refers to a DNA fragment that occurs in a gene, but that is spliced out during mRNA maturation. 
     As used herein, “nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1” refers to the nucleotide sequence of the complementary strand of a nucleic acid strand encoding a polypeptide that includes an amino acid sequence having the particular SEQ ID NO: 1. An exemplary nucleic acid sequence that encodes an amino acid sequence having the particular SEQ ID NO: 1 is set forth in SEQ ID NO: 897. 
     The term “complementary strand” is used herein interchangeably with the term “complement.” The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand. When referring to double-stranded nucleic acids, the complement of a nucleic acid encoding a polypeptide containing amino acid residues having a sequence set forth in a particular SEQ ID NO: 1 refers to the complementary strand of the strand encoding the amino acid sequence set forth in the particular SEQ ID NO: 1 or to any nucleic acid molecule containing the nucleotide sequence of the complementary strand of the particular nucleic acid sequence. When referring to a single-stranded nucleic acid molecule containing a nucleotide sequence, the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of the particular nucleic acid sequence. 
     As used herein, the term “coding sequence” refers to that portion of a gene that encodes a sequence of amino acids present in a protein. 
     As used herein, the term “sense strand” refers to that strand of a double-stranded nucleic acid molecule that has the sequence of the mRNA that encodes the sequence of amino acids encoded by the double-stranded nucleic acid molecule. 
     As used herein, the term “antisense strand” refers to that strand of a double-stranded nucleic acid molecule that is the complement of the sequence of the mRNA that encodes the sequence of amino acids encoded by the double-stranded nucleic acid molecule. 
     As used herein, an “array” refers to a collection of elements, such as nucleic acid molecules, containing three or more members. An addressable array is one in which the members of the array are identifiable, typically by position on a solid phase support or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e., RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label. In certain embodiments, the members of the array are immobilized to discrete identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface. 
     As used herein, a “support” (such as, a matrix support, a matrix, an insoluble support or solid support, etc.) refers to any solid or semisolid or insoluble support to which a molecule of interest (such as, a biological molecule, organic molecule or biospecific ligand) is linked or contacted. Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The matrix herein can be particulate or can be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials. When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller. Such particles, referred collectively herein as “beads,” are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical “beads,” particularly microspheres that can be used in the liquid phase, also are contemplated. The “beads” can include additional components, such as magnetic or paramagnetic particles (see, for example, Dynabeads (Dynal, Oslo, Norway)) for separation using magnets as long as the additional components do not interfere with the methods and analyses herein. 
     As used herein, matrix or support particles refer to matrix materials that are in the form of discrete particles. The particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less and typically have a size that is 100 mm 3  or less, 50 mm 3  or less, 10 mm 3  or less, 1 mm 3  or less, 100 μm 3  or less and can be order of cubic microns. Such particles are collectively called “beads.” 
     As used herein, the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (1972)  Biochem.,  11: 942-944. 
     B. HEMOSTASIS OVERVIEW 
     Provided herein are modified FIX polypeptides. These modified polypeptides have a variety of uses and applications. To appreciate the biological role of FIX polypeptides and some of their uses, an understanding of their role function in vivo is helpful. The following discussion provides such background. 
     The process by which the body responds to vascular injury involves a series of dependent signaling events that ultimately leads to the formation of a blot clot. Effective coagulation of blood is needed to prevent excessive blood loss at the site of injury. Once a blood vessel is injured or ruptured, vascular constriction, which limits blood flow to the area of the injury, accompanies the initial physiologic response. The rupture exposes highly thrombogenic subendothelial connective tissue that is composed of fibrillar collagen, which binds and activates platelets and stimulates platelet aggregation at the injury site. The activated platelets secrete factors which stimulate additional platelet activation, and other molecules, such as serotonin, phospholipids, and lipoproteins, which are important mediators of the clotting process. Fibrinogen, a component of the blood plasma also binds platelets and aids in clumping of the activated platelets. The activated platelets in turn undergo changes in cell shape to form a loose platelet plug. A clotting cascade of peptidases is simultaneously initiated that generates activated molecules such as thrombin (from cleavage of prothrombin), which further activates platelets, and fibrin (from cleavage of fibrinogen), which forms a cross-linked polymer around the platelet plug to stabilize the clot. During clot formation, coagulation factor inhibitors also circulate through the blood to prevent clot formation beyond the injury site. 
     Following repair of the injured tissue, the body must efficiently dissolve the clot in order to restore normal blood flow. Fibrinolysis is the process by which the clotting cascade is reduced and the clot is eliminated. Dissolution of the clot is mediated by the protein plasmin. 
     The processes of clot initiation, signal amplification, and fibrinolysis, and the factors involved are presented in further detail below. 
     1. Clot Initiation 
     Under normal conditions, vascular endothelium is resistant to clot formation. This resistance is supported by mechanisms that enhance vasodilation, inhibit platelet adhesion and activation, suppress coagulation, and promote fibrin cleavage. Vascular endothelial cells secrete molecules such as nitrous oxide (NO) and prostacylin, which inhibit platelet aggregation and dilate blood vessels. Release of these molecules activates soluble guanylate cyclases (sGC) and cGMP-dependent protein kinase I (cGKI) and increases cyclic guanosine monophosphate (cGMP) levels, which cause relaxation of the smooth muscle in the vessel wall. Furthermore, endothelial cells express cell-surface ADPases, such as CD39, which control platelet activation and aggregation by converting ADP released from platelets into adenine nucleotide platelet inhibitors. Other membrane proteins expressed on the surface of vascular endothelial cells include heparin sulfate proteoglycans that function as cofactors for antithrombin III, which inhibits thrombin and other coagulation factors. In addition, vascular endothelial cells secrete plasminogen to promote fibrinolysis, which lyses and clears clots. 
     Upon blood vessel injury, the damaged vessel wall exposes the subendothelial connective tissue to the circulating blood. In contrast to normal endothelial cells, this tissue is extremely thrombogenic. The layer is composed of proteins such as von Willebrand factor (vWF) and fibrillar collagen, which bind to circulating platelets via glycoprotein receptors expressed on the surface of the platelets. The platelet glycoprotein receptor complex GPIb-V-IX binds vWF, whereby vWF can bridge the platelets to the collagen fibrils. vWF is a multimeric glycoprotein, which contributes to its ability to aggregate platelets. vWF also functions to stabilize coagulation factors, such as factor VIII, and promotes their survival in the blood stream. Furthermore, the platelet integrin receptors α 2 β 1  and GPVI can bind to collagen directly. Binding of collagen to α 2 β 1  can facilitate binding to the lower affinity GPVI receptor. The collagen interactions with α 2 β 1  and GPVI contribute to platelet adherence to the site of injury and furthermore promote platelet activation through activation of intracellular signaling cascades. 
     2. Signal Amplification and the Clotting Cascade 
     During clot formation, positive feedback loops are activated that enhance the maturation of a clot. Following platelet activation, expression of the platelet surface integrin α IIb β 3  is stimulated. α IIb β 3  can bind to vWF and fibrinogen. Binding of GPIb-V-IX to vWF can enhance the affinity of α IIb β 3  for vWF to promote platelet adhesion. Further, one fibrinogen molecule can bind to multiple α IIb β 3  integrin molecules from different cells, thus, enhancing platelet aggregation. 
     Platelet activation stimulates the release of platelet alpha and dense granules. The released contents of the granules include factors, such as vWF and ADP, which further contribute to aggregation and platelet activation. ADP helps stimulate modification of the platelet membrane that allows binding of fibrinogen to α IIb β 3 , and vWF contributes to adhesion and aggregation as discussed above. 
     In addition to ADP, activated platelets also secrete factors such as serotonin and thromboxane A 2  (TXA 2 ) which also positively regulate platelet activation. Signal transduction cascades activated in response to thrombin binding control release of these factors. During platelet activation, thrombin binds to a G-protein coupled receptor on the surface of platelets. The receptor becomes stimulated, and the activated G-protein activates phospholipase C-γ 2 (PLCγ2), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3  stimulates release of intracellular calcium (Ca 2+ ) stores from the endoplasmic reticulum by binding to IP 3  receptors. The released calcium in combination with collagen binding to platelets leads to the activation of phospholipase A2 (PLA 2 ). PLA 2  then hydrolyzes membrane phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), to generate arachidonic acid, which in turn stimulates the production and release of TXA 2 . 
     The production of DAG by activated platelets stimulates protein kinase C, which then phosphorylates a platelet specific 47 kDa protein (p47). The phosphorylated p47 protein induces the release of the platelet granules as discussed above. Intracellular Ca 2+  release also activates myosin light chain kinase (MLCK) that phosphorylates the light chain of myosin. The phosphorylated myosin then interacts with actin, bringing about a change in platelet morphology and motility, necessary for clot formation. 
     Platelet activation through collagen binding also elicits activation of a similar signaling cascade. When collagen binds to the GPVI integrin, it promotes clustering of GPVI with the Fc receptor γ-chain (FcγR). The clustering induces tyrosine phosphorylation of the FcγR by Src-family kinases Lyn and Fyn, which promote binding and activation of the tyrosine kinase Syk. Syk, in turn phosphorylates the transmembrane adapter protein LAT, which then assembles a signaling complex composed of proteins including phosphoinositide 3-kinase (PI3K), PLCγ2, adaptor proteins such as Gads, SLP-75, and SLAP-130, and the RhoGTP exchange factor Vav. PI3K can then regulate protein kinase B (PKB), phosphoinositide-dependent kinase (PDK1) and integrin-linked kinase (ILK), which can regulate integrin expression and signaling. PLCγ2 generates IP 3  and DAG to mobilize calcium and activate PKC as described above. 
     For stable clot formation, cross-linked fibrin polymers must be formed surrounding the activated platelet plug. Fibrin formation is generated though activation of coagulation factor cascades which occurs simultaneously with platelet activation, following vessel injury. Two converging pathways can be activated, the intrinsic and extrinsic pathway. Key factors that participate in the intrinsic and extrinsic coagulation cascades are listed in Tables 2 and 3. The high-molecular weight kininogen, prekallikrein, and factors XII, XI, IX, and VIII participate in the intrinsic pathway, while tissue factor and the factor VII complex contribute to the extrinsic pathway. The two pathways converge at the activation of factor X (to generate FXa), through proteolytic action of the activated factor IX (FIXa)/factor VIII (FVIIIa) tenase complex (intrinsic) or by the activated factor VII (FVIIa)/tissue factor (TF) complex (extrinsic). Activated factor X (FXa), with the help of factor V, calcium, and platelet phospholipid, generates thrombin from prothrombin. In turn, thrombin cleaves fibrinogen to produce fibrin and also activates factor XIII (transglutamidase), which cross-links fibrin polymers to form the stable clot. The surface of activated platelets facilitates the formation of the activated coagulation factor complexes and thus participates in amplification of the cascade. 
     The primary pathway for initiation is the extrinsic pathway; the intrinsic pathway functions to amplify the production of activated factor X. Following vascular injury, tissue factor (TF) in the vessel wall is exposed and generated. TF is expressed by endothelial cells, subendothelial tissue, and monocytes. When the vessel wall is disrupted TF produced by the subendothelial layer is exposed. In the case of endothelial cell damage in the absence of a vessel wall lesion, contact is made between the blood and TF expressed on the damaged endothelial layer in the cell wall. vWF aids in the interaction of platelets with the damaged endothelium. TF binds to VII and in turn catalyzes the activation of factor X. The thrombin that is generated by the extrinsic pathway then contributes to activation of factor XI of the intrinsic cascade, leading to the activation of factor IX. Factor IX also can be activated directly by the TF/factor VII complex. The intrinsic cascade also can be activated via factor XII-mediated activation of factor XI when prekallikrein is converted to kallikrein in response to collagen contact with the vessel surface (called the contact pathway), though this pathway plays a minimal role in coagulation as compared to the amplification effects of thrombin. The contact pathway plays a more important role in fibrinolysis since factor XII and kallikrein can convert plasminogen into plasmin and bradykinin, which is a potent vasodilator and stimulator for tissue plasminogen activator (tPA). 
     3. Termination of Coagulation and Fibrinolysis 
     During coagulation constitutive and stimulated processes inhibit further clot formation. Antithrombin III and tissue factor pathway inhibitor (TFPI) work constitutively to inhibit factors in the coagulation cascade. Specifically, antithrombin III inhibits thrombin, FIXa, and FXa, where as TFPI inhibits FXa and FVIIa/TF complex. Protein C, which is stimulated via platelet activation, proteolytically inactivates FVa and FVIIIa. Further, another factor which contributes to coagulation inhibition is the integral membrane protein thrombomodulin, which is produced by vascular endothelial cells and binds thrombin. Complex formation inhibits thrombin procoagulant activities and also contributes to protein C activation. 
     Clearance of the fibrin clot is carried out by the serine protease plasmin, which can digest fibrin. Prior to activation of this enzyme, it circulates in the blood in the form of the proenzyme plasminogen. Plasminogen can bind to fibrin and fibrinogen, which allows it to be incorporated into the clot as it is formed. Cleavage of plasminogen into active plasmin is primarily carried out by activated tPA, which is secreted by endothelial cells following injury, through stimulation of tPA activity. Production of plasmin also can be initiated and enhanced by the actions of factor XII as discussed above. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Coagulation Factors 
               
            
           
           
               
               
               
               
            
               
                 Factor 
                 Common Name 
                 Pathway 
                 Characteristic 
               
               
                   
               
               
                 I 
                 Fibrinogen 
                 Both 
                 — 
               
               
                 II 
                 Prothrombin 
                 Both 
                 Contains N-terminal 
               
               
                   
                   
                   
                 gla segment 
               
               
                 III 
                 Tissue Factor 
                 Extrinsic 
                 — 
               
               
                 IV 
                 Calcium 
                 Both 
                 — 
               
               
                 V 
                 Proaccelerin, labile factor, 
                 Both 
                 Protein cofactor 
               
               
                   
                 Accelerator globulin 
               
               
                 VI 
                 Accelerin 
                 — 
                 (Redundant to factor V) 
               
               
                 (Va) 
               
               
                 VII 
                 Proconvertin, serum prothrombin 
                 Extrinsic 
                 Endopeptidase with 
               
               
                   
                 conversion accelerator (SPCA) 
                   
                 gla residues 
               
               
                   
                 cothromboplastin 
               
               
                 VIII 
                 Antihemophiliac factor A, 
                 Intrinsic 
                 Protein cofactor 
               
               
                   
                 antihemophiliac globulin (AHG) 
               
               
                 IX 
                 Christmas factor, antihemophiliac 
                 Intrinsic 
                 Endopeptidase with 
               
               
                   
                 factor B, plasma thromboplastin 
                   
                 gla residues 
               
               
                   
                 component (PTC) 
               
               
                 X 
                 Stuart-prower factor 
                 Both 
                 Endopeptidase with 
               
               
                   
                   
                   
                 gla residues 
               
               
                 XI 
                 Plasma thromboplastin 
                 Intrinsic 
                 Endopeptidase 
               
               
                   
                 antecedent (PTA) 
               
               
                 XII 
                 Hageman factor 
                 Intrinsic 
                 Endopeptidase 
               
               
                 XIII 
                 Protransglutamidase, fibrin 
                 Both 
                 Transpeptidase 
               
               
                   
                 stabilizing factor (FSF), 
               
               
                   
                 fibrinoligase 
               
               
                   
               
               
                 *Table adapted from M. W. King (2006) med.unibs.it/~marchesi/blood.html 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Coagulation Factor Zymogens and Cofactors 
               
            
           
           
               
               
            
               
                 Name of Factor 
                 Activity 
               
               
                   
               
            
           
           
               
            
               
                 Zymogens of Serine Proteases 
               
            
           
           
               
               
            
               
                 Factor XII 
                 Binds exposed collagen at site of vessel 
               
               
                   
                 wall injury, activated by high-MW 
               
               
                   
                 kininogen and kallikrein 
               
               
                 Factor XI 
                 Activated by factor XIIa 
               
               
                 Factor IX 
                 Activated by factor XIa + Ca 2+   
               
               
                 Factor VII 
                 Activated by thrombin + Ca 2+   
               
               
                 Factor X 
                 Activated on platelet surface by tenase 
               
               
                   
                 complex; 
               
               
                   
                 Also activated by factor VIIa + tissue 
               
               
                   
                 factor + Ca 2+   
               
               
                 Factor II 
                 Activated on platelet surface by 
               
               
                   
                 prothrombinase complex; 
               
            
           
           
               
            
               
                 Cofactors 
               
            
           
           
               
               
            
               
                 Factor VIII 
                 Activated by thrombin; factor VIIa acts 
               
               
                   
                 as cofactor for factor IXa in activation of 
               
               
                   
                 factor X 
               
               
                 Factor V 
                 Activated by thrombin; factor Va acts as 
               
               
                   
                 cofactor for factor Xa in activation of 
               
               
                   
                 prothrombin 
               
               
                 Factor III (Tissue factor) 
                 Acts as cofactor for factor VII 
               
            
           
           
               
            
               
                 Fibrinogen 
               
            
           
           
               
               
            
               
                 Factor I 
                 Cleaved by thrombin to form fibrin 
               
            
           
           
               
            
               
                 Transglutaminase 
               
            
           
           
               
               
            
               
                 Factor XIII 
                 Activated by thrombin + Ca 2+ ; promotes 
               
               
                   
                 covalent cross-linking of fibrin 
               
            
           
           
               
            
               
                 Regulatory and other proteins 
               
            
           
           
               
               
            
               
                 von Willebrand factor (vWF) 
                 Acts as bridge between GPIb-V-IX 
               
               
                   
                 complex and collagen 
               
               
                 Protein C 
                 Activated by thrombin bound to 
               
               
                   
                 thrombomodulin; Ca degrades factors 
               
               
                   
                 VIIIa and Va 
               
               
                 Protein S 
                 Acts as cofactor of protein C 
               
               
                 Thrombomodulin 
                 Endothelial cell surface protein; binds 
               
               
                   
                 thrombin, which activates protein C 
               
               
                 Antithrombin III 
                 Coagulation inhibitor of thrombin, and 
               
               
                   
                 factors IXa, Xa, XIa, and XIIa. 
               
               
                   
               
               
                 *Table adapted from M. W. King (2006) med.unibs.it/~marchesi/blood.html 
               
            
           
         
       
     
     C. FACTOR IX (FIX) 
     Coagulation Factor IX (FIX) is a vitamin-K dependent serine protease produced in animals, including mammals, that plays a pivotal role in the blood clotting cascade that functions to seal injured blood vessels. FIX shows a high degree of homology to other vitamin-K dependent serine proteases, including factor VII, factor X, and protein C (Furie et al., 1988). FIX is synthesized in the liver as an inactive zymogen that is secreted into the blood stream. During the coagulation process, FIX is cleaved via the intrinsic pathway by active factor XIa to generate an active serine protease, FIXa. FIXa also is generated via the extrinsic pathway by factor VIIa/tissue factor (FVIIa/TF). FIXa binds to activated Factor VIII (FVIIIa) and catalyzes the conversion of factor X to its active form, factor Xa, which hydrolyzes prothrombin into active thrombin 
     Coagulation factors, including FIX, are used as therapeutic agents. Treatment with recombinant FIX is an established therapy. Recombinant FIX is used, for example, as a therapeutic for treatment of diseases such as hemophilia, in particular, hemophilia B. Patients receiving FIX are subject to frequent, repeated applications of the drug due to instability of FIX in the blood stream and under storage conditions. Hence, improved FIX stability (half-life) in serum or following oral administration and/or in in vitro applications can improve its activity and efficiency as a drug. Accordingly, provided herein are modified FIX polypeptides that display improved protein stability, such as by increased resistance to proteases, resulting in increased protein half-life. The modified polypeptides can possess increased stability in the bloodstream or following oral administration (in vivo) and/or under storage conditions (in vitro). 
     1. Factor IX Structure and Function 
     The human coagulation FIX gene is located on chromosome X at Xq27.1 and contains 8 exons of varying length from 25 base pairs to about 2000 base pairs. The mRNA for FIX is about 3 kilobases in length, comprising 205 bases of 5′ UTR (untranslated region), 1386 bases encoding the FIX polypeptide, and 1392 bases of 3′ UTR. The FIX mRNA encodes for a 461 amino acid precursor FIX polypeptide. The nucleotide coding sequence and the encoded precursor polypeptide are provided in SEQ ID NOS: 897 and 1, respectively. 
     The precursor FIX polypeptide comprises the following segments and domains: a hydrophobic signal peptide (aa 1-28 of SEQ ID NO: 1), a propeptide (aa 29-46 of SEQ ID NO: 1), a gla domain (aa 47-92 of SEQ ID NO: 1), a type B epidermal growth factor domain (EGF-like 1, aa 93-129 of SEQ ID NO: 1), a type A epidermal growth factor domain (EGF-like 2, aa 130-171 of SEQ ID NO: 1), an activation peptide (aa 192-226 of SEQ ID NO: 1), and a serine protease domain (aa 227-461 of SEQ ID NO: 1). The mature form of the FIX polypeptide lacks the signal peptide and propeptide sequences (SEQ ID NO: 2). In the mature form of the FIX polypeptide the corresponding amino acid positions for the above mentioned domains are as follows: gla domain (aa 1-46 of SEQ ID NO: 2), EGF-like 1 (aa 47-83 of SEQ ID NO: 2), EGF-like 2 (aa 84-125 of SEQ ID NO: 2), activation peptide (aa 146-180 of SEQ ID NO: 2), and serine protease domain (aa 181-415 of SEQ ID NO: 2). Amino acid positions for FIX polypeptides provided herein are given with respect to the mature form of the FIX polypeptide (SEQ ID NO: 2), unless otherwise noted. 
     The signal peptide at the N-terminus of the precursor FIX polypeptide (SEQ IS NO: 1) is 28 amino acids in length and functions to target the FIX polypeptide to the cellular secretory pathway by insertion into the endoplasmic reticulum (ER) during translation. The signal sequence is cleaved by a signal peptidase within the ER lumen. Once inside the ER lumen, the FIX polypeptide is post-translationally modified by N-linked and O-linked glycosylation, β-hydroxylation, and γ-carboxylation. β-hydroxylation and γ-carboxylation are completed in the ER, while glycosylation commences in the ER and finishes in the Golgi complex. 
     The propeptide, which is 18 amino acids in length, provides a binding site for a vitamin K-dependent carboxylase. The recognition element for the carboxylase is located in the N-terminal portion of the propeptide, which forms a 10 residue amphipathic α-helix. After binding, the carboxylase γ-carboxylates 12 glutamic acid residues within the gla domain of the FIX polypeptide, producing γ-carboxyglutamyl residues at positions E53, E54, E61, E63, E66, E67, E72, E73, E76, E79, E82, and E86 relative to the FIX precursor amino acid sequence set forth in SEQ ID NO: 1. These positions correspond to positions E7, E8, E15, E17, E20, E21, E26, E27, E30, E33, E36, and E40 relative to the mature FIX polypeptide set forth in SEQ ID NO: 2. The γ-carboxyglutamyl modifications aid in the correct folding of the mature FIX polypeptide and in the ability to FIX to bind calcium (Ca 2+ ) and phospholipids. γ-Carboxyglutamyl residues located at amino acid positions 17, 21, and 27, relative to the mature FIX polypeptide (SEQ ID NO: 2), form ion pairs with the amino terminal alanine in prothrombin, which stabilize the structure of the gla domain and promote interaction with phospholipid membranes (Soriano-Garcia et al. (1992)  Biochemistry  31:2554-2566). The C-terminal end of the gla domain also contains a conserved hydrophobic domain containing a Phe-Trp-X-X-Tyr motif, located at amino acid positions 41-45 relative to the mature FIX polypeptide (SEQ ID NO: 2), which is required for Ca 2+  dependent changes in protein structure and for formation of a membrane binding site with the gla domain. In addition, proper folding of the gla domain enables binding of cofactor FVIIIa to FIXa, in order to form the complex which catalyzes the conversion of factor X into its active form, FXa. With respect to the other post-translational modifications of FIX, β-hydroxylation occurs at D64 to form β-hydroxyaspartic acid, N-linked glycosylation at N157 and N167, O-linked glycosylation at S53, S61, T159, T169, T172, and T179; sulfation at Y155; and phosphorylation of S68 and S158, relative to the mature FIX polypeptide, SEQ ID NO: 2 (Furie et al. (1988)  Cell  53:505-518 and Kaufman et al. (1998)  Thromb. Haemost  79:1068-1079). 
     After post-translation modification and prior to secretion, the propeptide is cleaved by a propeptidase to generate the mature FIX polypeptide, which is 415 amino acids in length (SEQ ID NO: 2). The recognition element for binding of the propeptidase is proposed to be located on the carboxyl side of the propeptide, closer to the site of cleavage. PACE/furin is a subtilisin-like calcium-dependent serine propeptidase localized to the trans Golgi compartment that has the ability to cleave the FIX propeptide into its mature form (Wasley et al. (1993)  J Biol Chem.  268(12):8458-65). The name PACE is an acronym for Paired basic Amino acid Cleaving Enzyme, which describes the characteristic of the enzyme to cleave after a pair of basic amino acids, namely K45-R46 in FIX (positions relative to precursor FIX polypeptide as set forth in SEQ ID NO: 1; K-2 and R-1 relative to the mature FIX polypeptide set forth in SEQ ID NO: 2). The consensus motif for PACE cleavage is Arg-X-(Lys/Arg)-Arg. PACE/furin also functions to process the propeptides of other proteins, such as pro-von Willebrand factor, pro-nerve growth factor proalbumin, and complement pro-C3. PACE/furin is ubiquitously expressed and is likely to process additional proteins, including growth factors, receptors, and other coagulation factors. Other members of the PACE family of peptidases include PC1/3, PC2, PACE4, PC4, PC5/6 and PC7/SPC7/LPC/PC8, though functional and expression data suggest that PACE/furin is most likely the FIX propeptidase in vivo. Mutation of the tyrosine residue at position 1 of the mature FIX polypeptide (Y47 in the precursor FIX polypeptide) has been shown to increase the efficiency of cleavage of the propeptide sequence (Meulian et al. (1990)  Protein Eng.  3(7):629-33). 
     The first EGF-like domain (EGF-like 1, EGF 1) is a type B EGF domain and contains the β-hydroxylation site at D64. β-Hydroxylation aids in the binding of additional Ca 2+ , which promotes binding of cofactor VIII to the domain. Two O-linked glycosylation sites are located within this domain at S53 and S61 (relative to the mature FIX polypeptide, SEQ ID NO: 2). 
     The second EGF-like domain (EGF-like 2, EGF2) is a type A EGF domain, which lacks β-hydroxylated residues compared to the type B domain. The EGF2 domain is responsible for binding to platelets and for assembly of the factor X activating complex (Wilkinson et al. (2002)  J. Biol. Chem.,  277(8):5734-5741). Formation of this complex on the surface of activated platelets promotes efficient hydrolysis of factor X. 
     The activation peptide contains the two cleavage sites, R145-A146 and R180-V181 (relative to mature FIX polypeptide, SEQ ID NO: 2) which are hydrolyzed by FXIa in the presence of Ca 2+ , or by FVIIa in the presence of TF, phospholipid, and Ca 2+  to generate the activated form, FIXa. The activation peptide also contains two of the N-linked glycosylation sites at N157 and N167 and four of the O-linked glycosylation sites at T159, T169, T172, and T179. Upon cleavage, the activation peptide (SEQ ID NO: 895) is excised from the polypeptide, leaving the activated FIX polypeptide, containing of a light chain and a heavy chain that are attached via cysteine disulfide bonds. The light chain includes the amino terminal portion of the FIX polypeptide, comprising the gla, EGF1, and EGF2 domains (SEQ ID NO: 894). The heavy chain comprises the FIX serine protease domain (SEQ ID NO: 896). 
     The serine protease domain is a catalytic domain that is homologous to known serine proteases, such as chymotrypsin, and contains an analogous catalytic core of residues at H221, D269, and S365 (relative to the mature FIX polypeptide, SEQ ID NO: 2). The protease domain catalyzes the hydrolysis of factor X in the presence of Ca 2+ , phospholipids, and FVIIIa by cleaving the R53-I53 bond of mature factor X. 
     2. Factor IX Polypeptides 
     Factor IX (FIX) polypeptides are heterogeneous polypeptides, are made of varying amino acid sequence lengths and include, but are not limited to, recombinantly produced polypeptide, synthetically produced polypeptide and FIX extracted from cells as tissues such as, for example, liver. FIX includes related polypeptides from different species including, but not limited to, animals of human and non-human origin, allelic variant isoforms, species isoforms, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof. Exemplary unmodified mature FIX polypeptides include, but are not limited to, unmodified and wild-type native FIX polypeptide (such as the polypeptide containing a sequence set forth in SEQ ID NO: 2), the unmodified and wild-type precursor FIX polypeptide that includes a signal peptide and a propeptide (such as the polypeptide that has the sequence set forth in SEQ ID NO: 1), a polymorphic wild-type variant of native factor IX polypeptide that has alanine at amino acid position T148 (SEQ ID NO: 892; or its precursor containing a sequence of amino acids set forth in SEQ ID NO: 893), and chimeric variants of FIX polypeptides such as set forth in SEQ ID NO: 1035. Unmodified FIX polypeptides also include FIX polypeptides that are modified by the cellular machinery and include, for example, glycosylation, carboxylation, β-hydroxylation, sulfation, and phosphorylation. 
     Based on alignment of FIX with other coagulation factor family members, such as factor VII (FVII) or factor X (FX), homologous domains among the family members are readily identified. Chimeric variants of FIX polypeptides can be constructed where one or more amino acids or entire domains are replaced in the FIX amino acid sequence using the amino acid sequence of the corresponding family member. Such chimeric proteins can be used as the unmodified FIX polypeptide for the methods described herein. For example, amino acid residues of the gla domain can be replaced with residues from the analogous FVII or FX gla domains (Lin et al. (1990)  J. Biol. Chem.  265(1):144-150 and Cheung et al. (1991)  J. Biol. Chem.  266(14):8797-880). A chimeric FIX protein can then be further modified by amino acid replacement for increased protein stability using any the methods described herein. Exemplary modified chimeric FIX polypeptides have amino acid sequences set forth in SEQ ID NOS: 1036-2044. 
     FIX is functionally active following cleavage by FXIa in the presence of Ca 2+  or by FVIIa in the presence of TF, phospholipid, and Ca 2+ . Hydrolysis of mature FIX polypeptides generates heavy and light chains of FIX that are connected by disulfide bridges. FIX polypeptides therefore also include FIX heavy chain and FIX light chain polypeptides (SEQ ID NOS: 896 and 894, respectively) and combinations thereof. 
     The full structure of coagulation FIX is formed from six FIX molecules. Modified polypeptides can be stabilized by intra- (within FIX polypeptide) and inter- (between heavy and light chains of FIX polypeptide or between subunits) subunit interactions. Amino acids within the helices of each FIX polypeptide chain can interact with each other, thereby contributing to protein stability within a FIX polypeptide (i.e., “intrastability”). For example, cysteine residues form disulfide bonds between the FIX heavy and light polypeptide chains. In addition, interhelical contacts occur between helices of different subunits thereby contributing to protein stability between the monomers (i.e., “interstability”). For example, amino acid residues in helices on monomer A can interact with amino acid residues in helices on monomer B. 
     Contemplated herein are modified FIX polypeptides that differ from unmodified or wild-type FIX polypeptides with respect to a property or an activity. Modified FIX polypeptides provided herein can have increased stability, as manifested as increased serum half-life or increased resistance to proteases as compared to unmodified FIX polypeptides. 
     Generally, FIX is produced as a larger polypeptide (461 aa, SEQ ID NO: 1, for example) that is matured to a smaller polypeptide upon cleavage of the signal sequence (amino acids 1-28 of SEQ ID NO: 1, for example). FIX polypeptides are typically about 433 amino acids in length after cleavage of the signal sequence, and typically about 415 amino acids in length following further cleavage of the propeptide sequence, resulting in a mature FIX polypeptide (SEQ ID NO: 2, for example). 
     FIX is a secreted, glycosylated protein, including O- and N-linked glycosylation sites. The extent of glycosylation of the FIX polypeptides provided herein can contribute to the stability of the protein and to protease resistance. Glycosylation of FIX polypeptides provided herein also can contribute to the amount of protein expression, secretion, and potency of the FIX polypeptide. FIX polypeptides provided herein can be γ-carboxylated, β-hydroxylated, tyrosine sulfated, and/or phosphorylated. 
     FIX polypeptides from different species share conserved sequences of amino acids (see, for example, mouse, rat, guinea pig, cow, sheep, dog, cat, chicken, pig, rabbit, fish, and chimpanzee FIX polypeptides). Such sequences can be used for modification using the methods described herein. 
     Active and inactive forms of FIX polypeptides are known in the art. Active peptidase forms of FIX can be used in the treatment of hemophilias, where FIX polypeptides can function to promote blood coagulation. FIX polypeptides that have an inactive protease domain can be used in the treatment of thrombotic diseases by inhibiting blood coagulation via interference of the coagulation cascade. 
     Modes of administration include, but are not limited to, FIX polypeptide injection or the nucleic acid molecules encoding modified FIX polypeptides provided herein and also can include additional treatments administered similarly or through other routes of administration, including oral administration and inhalation, stem cell engraftment, and systemic administration of viral vectors encoding a FIX polypeptide. FIX also can be targeted for delivery into specific cell types. For example, adenoviral vectors encoding FIX polypeptides can be used for stable expression in nondividing cells, such as liver cells, and skeletal muscle cells (Arruda et al. (2001)  Blood  97(1): 130-138 and Yao et al. (1992)  PNAS  89: 3357-3361). In another example, viral or nonviral vectors encoding FIX polypeptides can be transduced into isolated cells for subsequent delivery. Additional cell types for expression and delivery of FIX are known in the art and include but are not limited to, fibroblasts and endothelial cells (Palmer et al. (1989)  Blood  73: 438-445; Yao et al. (1991)  PNAS  88: 8101-8105). 
     3. FIX as a Pharmaceutical 
     Activities induced by FIX include, but are not limited to, blood coagulation. Administration of FIX has been shown to promote blood clotting in patients suffering from hemophilia, particularly hemophilia B. In man, treatment with doses of FIX have been found to be safe and well-tolerated. 
     FIX can be administered orally, systemically, buccally, transdermally, intravenously, intramuscularly and subcutaneously and, typically, multiple administrations are used in treatment regimens. The formulations are typically stored in refrigerated (2-8° C.) conditions to ensure retention of activity. Hence, improved FIX stability (half-life) in administered conditions (in vivo), such as stability in serum, and in vitro (such as, during production, purification and storage conditions) can improve its utility and efficiency as a drug. 
     Provided herein are variants of the FIX polypeptide that display improved stability as assessed by resistance to proteases (blood, intestinal, etc) and/or increased thermal tolerance and/or pH conditions, wherein the mutant variants exhibit increased protein half-life. The mutant variants that exhibit improved stability possess increased stability in administration conditions such as in the bloodstream, gastrointestinal tract, under low pH conditions (such as, the stomach), mouth, throat, and/or under storage conditions. 
     D. EXEMPLARY METHODS FOR MODIFYING FIX 
     Provided herein are methods for increasing stability and half-life of a FIX polypeptide by increasing resistance to proteolysis. Provided herein are methods of modifying FIX polypeptides to increase resistance to proteolysis by proteases (blood, serum, gastrointestinal, etc.), whereby the modified polypeptide exhibits increased half-life in vitro and/or in vivo. Provided herein are modified FIX polypeptides in which the primary amino acid sequence is modified to confer increased protein stability. Among the amino acid modifications provided herein are such modifications including replacement of amino acids in the primary sequence of the FIX polypeptide in order to decrease proteolytic cleavage of the FIX polypeptide. Further modifications of the modified FIX polypeptide can be included, such as, but not limited to, addition of carbohydrate, phosphate, sulfur, hydroxyl, carboxyl, and polyethylene glycol (PEG) moieties. Thus, the modified FIX polypeptides provided herein can be further modified, for example, by glycosylation, phosphorylation, sulfation, hydroxylation, carboxylation, and/or PEGylation. Such modifications can be performed in vivo or in vitro. 
     Provided herein are methods of modifying polypeptides to increase resistance to proteolysis by proteases and contacting proteolytic enzymes with peptide inhibitors, thereby inhibiting activity of the proteases. Also provided herein are the modified polypeptides generated by said methods. Provided herein are FIX polypeptides that display improved stability as assessed by resistance to proteases; the modified FIX polypeptides exhibiting these properties possess, thereby, increased protein half-life in vitro or in vivo. 
     The modified FIX polypeptides (also referred to herein as variants) are more stable compared to unmodified FIX polypeptides. Increasing stability (i.e., the half-life of proteins in vivo) can result in a decrease in the frequency of injections needed to maintain a sufficient drug level in serum, thus leading to: i) higher comfort to, and acceptance by, treated subjects, particularly human subjects, ii) lower doses necessary to achieve comparable biological effects, and iii) as a consequence, an attenuation of the (dose-dependent) secondary effects. 
     Increased stability of FIX can be achieved, for example, by destruction of protease target residues or sequences and/or by modification of residues that contribute to conformational stability and are susceptible to denaturation by temperature, pH, or other denaturing agent. Modification of FIX to increase stability can be accomplished while keeping activity unchanged compared to the unmodified or wild-type FIX. Alternatively, modification of FIX stability can be accomplished while increasing activity compared to the unmodified or wild-type FIX. Any methods known in the art can be used to create modified FIX polypeptides. In the methods described herein, modifications are chosen using the method of 2D-scanning mutagenesis as described, for example, in PCT published applications WO 2004/022747 and WO 2004/022593. 
     In principle, there are several general approaches described for protein-directed evolution based on mutagenesis. Any of these, alone or in combination can be used to modify a FIX polypeptide to achieve increased stability and/or resistance to proteolysis. Such methods include random mutagenesis, where the amino acids in the starting protein sequence are replaced by all (or a group) of the 20 amino acids either in single or multiple replacements at different amino acid positions are generated on the same molecule, at the same time. Another method, restricted random mutagenesis, introduces either all of the 20 amino acids or DNA-biased residues. The bias is based on the sequence of the DNA and not on that of the protein in a stochastic or semi-stochastic manner, respectively, within restricted or predefined regions of the protein known in advance to be involved in the activity being “evolved.” Additionally, methods of rational mutagenesis including 1D-scanning, 2D-scanning, and 3D-scanning can be used alone or in combination to construct modified FIX variants. 
     1. Non-Restricted Rational Mutagenesis One-Dimensional (1D)-Scanning 
     Rational mutagenesis, also termed 1D-scanning, is a two-step process, and is described in co-pending U.S. application Ser. No. 10/022,249 (U.S. Publication No. 2003/0134351-A1). 1D-scanning can be used to modify FIX polypeptides and, additionally, to identify positions for further modification by other methods such as 2D- and 3D-scanning. Briefly, in the first step, full-length amino acid scanning is performed where all and each amino acid in the starting FIX polypeptide sequence (for example SEQ ID NO: 2) is replaced by a designated reference amino acid (such as, alanine). Only a single amino acid is replaced on each protein molecule at a time. A collection of protein molecules having a single amino acid replacement is generated such that molecules differ from each other by the amino acid position at which the replacement has taken place. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and such that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant nucleic acid molecules also are physically separated from each other, such as by formatting in addressable arrays. Activity assessment on each protein molecule allows for the identification of those amino acid positions that result in a drop in activity when replaced, thus indicating the involvement of that particular amino acid position in the protein&#39;s biological activity and/or conformation that leads to fitness of the particular feature being evolved. Those amino acid positions are referred to as HITs. 
     At the second step, a new collection of molecules is generated such that each molecule differs from each of the others by the amino acid present at the individual HIT positions identified in step 1. All 20 amino acids (19 remaining) are introduced at each of the HIT positions identified in step 1; while each individual molecule contains, in principle, one and only one amino acid replacement. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and such that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant DNA molecules also are physically separated from each other, such as by formatting in addressable arrays. Activity assessment then is individually performed on each individual mutant molecule. The newly generated mutants that lead to a desired alteration (such as an improvement) in a protein activity are referred to as LEADs. This method permits an indirect search for property or activity alteration, such as improved stability and/or improved resistance to proteases or thermal conditions based on one rational amino acid replacement and sequence change at a single amino acid position at a time, in search of a new, unpredicted amino acid sequence at some unpredicted regions along a protein to produce a protein that exhibits a desired activity or altered activity, such as better performance than the starting protein. 
     In this approach, neither the amino acid position nor the replacing amino acid type are restricted. Full length protein scanning is performed during the first step to identify HIT positions, and then all 20 amino acids are tested at each of the HIT positions, to identify LEAD sequences; while, as a starting point, only one amino acid at a time is replaced on each molecule. The selection of the target region (HITs and surrounding amino acids) for the second step is based upon experimental data on activity obtained in the first step. Thus, no prior knowledge of protein structure and/or function is necessary. Using this approach, LEAD sequences have been found on proteins that are located at regions of the protein not previously known to be involved in the particular biological activity being modified; thus emphasizing the power of this approach to discover unpredictable regions (HITs) as targets for fitness improvement. 
     2. Two Dimensional (2D) Rational Scanning (Restricted Rational Mutagenesis) 
     The 2D-scanning (or restricted rational mutagenesis) methods for protein rational evolution (see, co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747) are based on scanning over two dimensions. The first dimension is the amino acid position along the protein sequence, in order to identify is-HIT target positions. The second dimension is scanning the amino acid type selected for replacing a particular is-HIT amino acid position. An advantage of the 2D-scanning methods provided herein is that at least one, and typically the amino acid position and/or the replacing amino acid, can be restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement; and/or fewer than all of the remaining 19 amino acids available to replace an original, such as native, amino acid are selected for replacement. 
     In particular embodiments, based on i) the particular protein properties to be evolved (such as, resistance to proteolysis), ii) sequence of amino acids of the protein, and iii) the known properties of the individual amino acids, a number of target positions along the protein sequence are selected, in silico, as “is-HIT target positions.” This number of is-HIT target positions is as large as reasonably possible such that all reasonably possible target positions for the particular feature being evolved are included. In particular, embodiments where a restricted number of is-HIT target positions are selected for replacement, the amino acids selected to replace the is-HIT target positions on the particular protein being optimized can be either all of the remaining 19 amino acids or, more frequently, a more restricted group comprising selected amino acids that are contemplated to have the desired effect on protein activity. In another embodiment, so long as a restricted number of replacing amino acids are used, all of the amino acid positions along the protein backbone can be selected as is-HIT target positions for amino acid replacement. Mutagenesis then is performed by the replacement of single amino acid residues at specific is-HIT target positions on the protein backbone (such as, “one-by-one,” such as in addressable arrays), such that each individual mutant generated is the single product of each single mutagenesis reaction. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant DNA molecules also are physically separated from each other, such as by formatting in addressable arrays. Thus, a plurality of mutant protein molecules is produced. Each mutant protein contains a single amino acid replacement at only one of the is-HIT target positions. Activity assessment is then individually performed on each individual protein mutant molecule, following protein expression and measurement of the appropriate activity. An example of practice of this method is shown in the Examples in which mutant FIX molecules are produced. 
     The newly generated proteins that lead to altered, typically improved, target protein activity are referred to as LEADs. This method relies on an indirect search for protein improvement for a particular activity (such as increased resistance to proteolysis), based on amino acid replacement and sequence change at single or, in another embodiment, a limited number of amino acid positions at a time. As a result, optimized proteins, which have modified sequences of amino acids at some regions along the protein that perform better (at a particular target activity or other property) than or different from the starting protein, are identified and isolated. 
     2D-scanning on FIX was used to generate variants improved in protein stability, including improved resistance to proteolysis. To effect such modifications, amino acid positions were selected using in silico analysis of FIX. 
     a. Identifying in-Silico HITs 
     The 2D-scanning method for directed evolution of proteins includes identifying and selecting (using in silico analysis) specific amino acids and amino acid positions (referred to herein as is-HITs) along the protein sequence that are contemplated to be directly or indirectly involved in the feature being evolved. As noted, the 2D-scanning methods provided include the following two steps. The first step is an in silico search of a target sequence of amino acids of the protein to identify all possible amino acid positions that can be targets for the activity being evolved. This is effected, for example, by assessing the effect of amino acid residues on the property or properties to be altered on the protein, using any known standard software. The particulars of the in silico analysis is a function of the property to be modified. 
     Once identified, these amino acid positions or target sequences are referred to as “is-HITs” (in silico HITs). In silico HITs are defined as those amino acid positions (or target positions) that potentially are involved in the “evolving” feature, such as increased resistance to proteolysis. The discrimination of the is-HITs among all the amino acid positions in a protein sequence can be made based on the amino acid type at each position in addition to the information on the protein secondary or tertiary structure. In silico HITs constitute a collection of mutant molecules such that all possible amino acids, amino acid positions or target sequences potentially involved in the evolving feature are represented. No strong theoretical discrimination among amino acids or amino acid positions is made at this stage. In silico HIT positions are spread over the full length of the protein sequence. Single or a limited number of is-HIT amino acids are replaced at a time on the target FIX polypeptide. 
     A variety of parameters can be analyzed to determine whether or not a particular amino acid on a protein might be involved in the evolving feature, typically a limited number of initial premises (typically no more than 2) are used to determine the in silico HITs. For example, as described herein, to increase the stability of FIX, the first condition is the nature of the amino acids linked to stability of the molecule such as its participation in directing proteolytic cleavage. A second premise, for example, can be related to the specific position of those amino acids along the protein structure. 
     During the first step of identification of is-HITs according to the methods provided herein, each individual amino acid along the protein sequence is considered individually to assess whether it is a candidate for is-HIT. This search is done one-by-one and the decision on whether the amino acid is considered to be a candidate for a is-HIT is based on (1) the amino acid type; (2) the position in the protein and protein structure if known; and (3) the predicted interaction between that amino acid and its neighbors in sequence and space. 
     Is-HITs were identified for a number of properties of FIX that contribute to protein stability, such as removal/modification of protease sensitive sites. Such modifications contribute to protein stability and thereby, to increasing the half-life of a FIX polypeptide in vitro, in vivo or ex vivo. 
     b. Identifying Replacing Amino Acids 
     Once the is-HITs target positions are selected, the next step is identifying those amino acids that will replace the original, such as native, amino acid at each is-HIT position to alter the activity level for the particular feature being evolved. The set of replacing amino acids to be used to replace the original, such as native, amino acid at each is-HIT position can be different and specific for the particular is-HIT position. The choice of the replacing amino acids takes into account the need to preserve the physicochemical properties such as hydrophobicity, charge and polarity of essential (such as, catalytic, binding, etc.) residues and alter some other property of the protein (such as, protein stability). The number of replacing amino acids of the remaining 19 non-native (or non-original) amino acids that can be used to replace a particular is-HIT target position ranges from 1 up to about 19, and anywhere in between, depending on the properties for the particular modification. 
     Numerous methods of selecting replacing amino acids (also referred to herein as “replacement amino acids”) are well known in the art. Protein chemists determined that certain amino acid substitutions commonly occur in related proteins from different species. As the protein still functions with these substitutions, the substituted amino acids are compatible with protein structure and function. Often, these substitutions are to a chemically similar amino acid, but other types of changes, although relatively rare, also can occur. 
     Knowing the types of changes that are most and least common in a large number of proteins can assist with predicting alignments and amino acid substitutions for any set of protein sequences. Amino acid substitution matrices are used for this purpose. A number of matrices are available. A detailed presentation of such matrices can be found in the co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747, each of which is incorporated herein in their entirety herein. Such matrices also are known and available in the art, for example in the reference listed below. 
     In amino acid substitution matrices, amino acids are listed horizontally and vertically, and each matrix position is filled with a score that reflects how often one amino acid would have been paired with the other in an alignment of related protein sequences. The probability of changing amino acid “A” into amino acid “B” is assumed to be identical to the reverse probability of changing “B” into “A”. This assumption is made because, for any two sequences, the ancestor amino acid in the phylogenetic tree is usually not known. Additionally, the likelihood of replacement should depend on the product of the frequency of occurrence of the two amino acids and on their chemical and physical similarities. A prediction of this model is that amino acid frequencies will not change over evolutionary time (Dayhoff et al.,  Atlas of Protein Sequence and Structure,  5(3): 345-352, 1978). Several exemplary amino acid substitution matrices, including, but not limited to block substitution matrix (BLOSUM) (Henikoff et al. (1992)  Proc. Natl. Acad. Sci. USA,  89: 10915-10919), Jones et al. (1992)  Comput. Appl. Biosci.,  8: 275-282, Gonnet et al. (1992)  Science,  256: 1433-1445, Fitch (1966)  J. Mol. Evol.,  16(1): 9-16, Feng et al. (1985)  J. Mol. Evol.,  21: 112-125, McLachlan (1971)  J. Mol. Biol.,  61: 409-424, Grantham (1974)  Science,  185: 862-864, Miyata (1979)  J. Mol. Evol.,  12: 219-236, Rao (1987)  J. Pept. Protein Res.,  29: 276-281, Risler (1988)  J. Mol. Biol.,  204: 1019-1029, Johnson et al. (1993)  J. Mol. Biol.,  233: 716-738, and Point Accepted Mutation (PAM) (Dayhoff et al. (1978)  Atlas of Protein Sequence and Structure  5: 345-352. 
     Dayhoff and coworkers developed a model of protein evolution that resulted in the development of a set of widely used replacement matrices (Dayhoff et al., (1978)  Atlas of Protein Sequence and Structure  5(3):345-352) termed percent accepted mutation matrices (PAM). In deriving these matrices, each change in the current amino acid at a particular site is assumed to be independent of previous mutational events at that site. Thus, the probability of change of any amino acid A to amino acid B is the same, regardless of the previous changes at that site and also regardless of the position of amino acid A in a protein sequence. 
     In the Dayhoff approach, replacement rates are derived from alignments of protein sequences that are at least 85% identical; this constraint ensures that the likelihood of a particular mutation being the result of a set of successive mutations is low. Because these changes are observed in closely related proteins, they represent amino acid substitutions that do not significantly change the function of the protein. Hence, they are called “accepted mutations,” as defined as amino acid changes that are accepted by natural selection. 
     The outcome of the two steps set forth above, which is performed in silico is that: (1) the amino acid positions that are the target for mutagenesis are identified (referred to as is-HITs); and (2) the replacing amino acids for the original, such as native, amino acids at the is-HITs are identified, to provide a collection of candidate LEAD mutant molecules that are expected to perform differently from the native molecule. These are assayed for a desired optimized (or improved or altered) activity. 
     c. Construction of Modified Proteins and Biological Assays 
     Once is-HITs are selected as set forth above, replacing amino acids are introduced. Mutant proteins typically are prepared using recombinant DNA methods and assessed in appropriate biological assays for the particular activity (feature) optimized. An exemplary method of preparing the mutant proteins is by mutagenesis of the original, such as native, gene using methods well known in the art. Mutant molecules are generated one-by-one, such as in addressable arrays, such that each individual mutant generated is the single product of each single and independent mutagenesis reaction. Individual mutagenesis reactions are conducted separately, such as in addressable arrays where they are physically separated from each other. Once a population of sets of nucleic acid molecules encoding the respective mutant proteins is prepared, each is separately introduced one-by-one into appropriate cells for the production of the corresponding mutant proteins. This also can be performed, for example, in addressable arrays where each set of nucleic acid molecules encoding a respective mutant protein is introduced into cells confined to a discrete location, such as in a well of a multi-well microtiter plate. Each individual mutant protein is individually phenotypically characterized and performance is quantitatively assessed using assays appropriate for the feature being optimized (i.e., feature being evolved). Again, this step can be performed in addressable arrays. Those mutants displaying a desired increased or decreased performance compared to the original, such as native molecules are identified and designated LEADs. From the beginning of the process of generating the mutant DNA molecules up through the readout and analysis of the performance results, each candidate LEAD mutant is generated, produced and analyzed individually, such as from its own address in an addressable array. The process is amenable to automation. 
     3. Three Dimensional (3D) Scanning 
     3D scanning, as described in co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published PCT applications WO 2004/022747 and WO 2004/022593, is an additional method of rational evolution of proteins based on the identification of potential target sites for mutagenesis (is-HITs). The method uses comparison of patterns of protein backbone folding between structurally related proteins, irrespective of the underlying sequences of the compared proteins. Once the structurally related amino acid positions are identified on the protein of interest, then suitable amino acid replacement criteria, such as PAM analysis, can be employed to identify candidate LEADs for construction and screening. 
     For example, analysis of “structural homology” between and among a number of related vitamin K-dependent proteases can be used to identify on various members of the vitamin K-dependent protease family, those amino acid positions and residues that are structurally similar or structurally related. For example, 3D scanning can be used to identify amino acid positions on FIX that are structurally similar or structurally related to those found in factor VII mutants, for example, that have been modified for improved stability. Exemplary vitamin K-dependent proteases include, but are not limited to, prothrombin, factor VII, factor X and protein C. 
     Using the 3D-scanning methods described herein, once one protein within a family of proteins (such as, FIX within the vitamin-K dependent protease family) is modified using the methods provided herein for generating LEAD mutants, is-HITs can be identified for other or all proteins within a particular family by identifying the corresponding amino acid positions therein using structural homology analysis (based upon comparisons of the 3D structures of the family members with original protein to identify corresponding residues for replacement) as described hereinafter. The is-HITs for the family members identified in this manner then can be subjected to the next step of identifying replacing amino acids and further assayed to obtain LEADs or super-LEADs as described herein. Similarly, information from 2D-scanning performed on other vitamin K-dependent proteases such as prothrombin, factor VII, factor X or protein C, for example, can be used to optimize FIX polypeptides. 
     This method can be applied to any desired phenotype using any protein, such as a vitamin K-dependent protease, as the starting material to which an evolution procedure, such as the rational directed evolution procedure of U.S. Published Application No. US 2003-0134351 A1 or the 2-dimensional scanning method described herein. The structurally corresponding residues are then altered on members of the family to produce additional vitamin K-dependent proteases with similar phenotypic alterations. 
     a. Homology 
     Typically, homology between proteins is compared at the level of their amino acid sequences, based on the percent or level of coincidence of individual amino acids, amino acid per amino acid, when sequences are aligned starting from a reference, generally the residue encoded by the start codon. For example, two proteins are said to be “homologous” or to bear some degree of homology whenever their respective amino acid sequences show a certain degree of matching upon alignment comparison. Comparative molecular biology is primarily based on this approach. From the degree of homology or coincidence between amino acid sequences, conclusions can be made on the evolutionary distance between or among two or more protein sequences and biological systems. 
     The concept of “convergent evolution” is applied to describe the phenomena by which phylogenetically-unrelated organisms or biological systems have evolved to share features related to their anatomy, physiology and structure as a response to common forces, constraints and evolutionary demands from the surrounding environment and living organisms. Alternatively, “divergent evolution,” is applied to describe the phenomena by which strongly phylogenetically related organisms or biological systems have evolved to diverge from identity or similarity as a response to divergent forces, constraints and evolutionary demands from the surrounding environment and living organisms. 
     In the typical traditional analysis of homologous proteins there are two conceptual biases corresponding to: i) “convergent evolution,” and ii) “divergent evolution.” Whenever the aligned amino acid sequences of two proteins do not match well with each other, these proteins are considered “not related” or “less related” with each other and have different phylogenetic origins. There is no (or low) homology between these proteins and their respective genes are not homologous (or show little homology). If these two “non-homologous” proteins under study share some common functional features (such as, interaction with other specific molecules, activity), they are determined to have arisen by “convergent evolution,” i.e., by evolution of their non-homologous amino acid sequences, in such a way that they end up generating functionally “related” structures. 
     On the other hand, whenever the aligned amino acid sequences of two proteins do match with each other to a certain degree, these proteins are considered to be “related” and to share a common phylogenetic origin. A given degree of homology is assigned between these two proteins and their respective genes likewise share a corresponding degree of homology. During the evolution of their initial highly homologous amino acid sequence, enough changes can be accumulated in such a way that they end up generating “less-related” sequences and less related function. The divergence from perfect matching between these two “homologous” proteins under study is said come from “divergent evolution.” 
     b. 3D-Scanning (Structural Homology) Methods 
     Structural homology refers to homology between the topology and three-dimensional structure of two proteins. Structural homology is not necessarily related to “convergent evolution” or to “divergent evolution,” nor is it related to the underlying amino acid sequence. Rather, structural homology is likely driven (through natural evolution) by the need of a protein to fit specific conformational demands imposed by its environment. Particular structurally homologous “spots” or “loci” would not be allowed to structurally diverge from the original structure, even when its own underlying sequence does diverge. This structural homology is exploited herein to identify loci for mutation. 
     Within the amino acid sequence of a protein resides the appropriate biochemical and structural signals to achieve a specific spatial folding in either an independent or a chaperon-assisted manner. Indeed, this specific spatial folding ultimately determines protein traits and activity. Proteins interact with other proteins and molecules in general through their specific topologies and spatial conformations. In principle, these interactions are not based solely on the precise amino acid sequence underlying the involved topology or conformation. If protein traits, activity (behavior and phenotypes) and interactions rely on protein topology and conformation, then evolutionary forces and constraints acting on proteins can be expected to act on topology and conformation. Proteins sharing similar functions will share comparable characteristics in their topology and conformation, despite the underlying amino acid sequences that create those topologies and conformations. 
     4. Super-LEADs and Additive Directional Mutagenesis (ADM) 
     Modification of FIX polypeptides also can include combining two or more mutations. For example, Additive Directional Mutagenesis (ADM) can be used to assemble on a single mutant protein multiple mutations present on the individual LEAD molecules, so as to generate super-LEAD mutant proteins (see co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published PCT applications WO 2004/022747 and WO 2004/022593). ADM is a repetitive multi-step process where at each step after the creation of the first LEAD mutant protein a new LEAD mutation is added onto the previous LEAD mutant protein to create successive super-LEAD mutant proteins. ADM is not based on genetic recombination mechanisms, nor on shuffling methodologies; instead it is a simple one-mutation-at-a-time process, repeated as many times as necessary until the total number of desired mutations is introduced on the same molecule. To avoid the exponentially increasing number of all possible combinations that can be generated by putting together on the same molecule a given number of single mutations, a method is provided herein that, although it does not cover all the combinatorial possible space, still captures a big part of the combinatorial potential. “Combinatorial” is used herein in its mathematical meaning (i.e., subsets of a group of elements, containing some of the elements in any possible order) and not in the molecular biological or directed evolution meaning (i.e., generating pools, or mixtures, or collections of molecules by randomly mixing their constitutive elements). 
     A population of sets of nucleic acid molecules encoding a collection of new super-LEAD mutant molecules is generated tested and phenotypically characterized one-by-one in addressable arrays. Super-LEAD mutant molecules are such that each molecule contains a variable number and type of LEAD mutations. Those molecules displaying further improved fitness for the particular feature being evolved are referred to as super-LEADs. Super-LEADs can be generated by other methods known to those of skill in the art and tested by the high throughput methods herein. For purposes herein a super-LEAD typically has activity with respect to the function or biological activity of interest that differs from the improved activity of a LEAD by a desired amount, such as at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from at least one of the LEAD mutants from which it is derived. In yet other embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more greater than at least one of the LEAD molecules from which it is derived. As with LEADs, the change in the activity for super-LEADs is dependent upon the property that is being “evolved.” The desired alteration, which can be either an increase or a reduction in a feature or property, will depend upon the function or property of interest. 
     In one embodiment, the ADM method employs a number of repetitive steps, such that at each step a new mutation is added on a given molecule. Although numerous different ways are possible for combining each LEAD mutation onto a super-LEAD protein, an exemplary way the new mutations (such as, mutation 1 (m1), mutation 2 (m2), mutation 3 (m3), mutation 4 (m4), mutation 5 (m5), mutation n (mn)) can be added corresponds to the following diagram: 
     m1 
     m1+m2 
     m1+m2+m3 
     m1+m2+m3+m4 
     m1+m2+m3+m4+m5 
     m1+m2+m3+m4+m5+ . . . +nm 
     m1+m2+m4 
     m1+m2+m4+m5 
     m1+m2+m4+m5+ . . . +nm 
     m1+m2+m5 
     m1+m2+m5+ . . . +nm 
     m2 
     m2+m3 
     m2+m3+m4 
     m2+m3+m4+m5 
     m2+m3+m4+m5+ . . . +nm 
     m2+m4 
     m2+m4+m5 
     m2+m4+m5+ . . . +nm 
     m2+m5 
     m2+m5+ . . . +nm 
     . . . , etc. . . . 
     5. Multi-Overlapped Primer Extensions 
     Another method that can be employed to generate combinations of two or more mutations is using oligonucleotide-mediated mutagenesis referred to as “multi overlapped primer extensions”. This method can be used for the rational combination of mutant LEADs to form super-LEADS. This method allows the simultaneous introduction of several mutations throughout a small protein or protein-region of known sequence. Overlapping oligonucleotides of typically around 70 bases in length (since longer oligonucleotides lead to increased error) are designed from the DNA sequence (gene) encoding the mutant LEAD proteins in such a way that they overlap with each other on a region of typically around 20 bases. Although typically about 70 bases are used to create the overlapping oligonucleotides, the length of additional overlapping oligonucleotides for use can range from about 30 bases up to about 100 bases. Likewise, although typically the overlapping region of the overlapping oligonucleotides is about 20 bases, the length of other overlapping regions for use herein can range from about 5 bases up to about 40 bases. These overlapping oligonucleotides (including or not point mutations) act as template and primers in a first step of PCR (using a proofreading polymerase, such as, Pfu DNA polymerase, to avoid unexpected mutations) to create small amounts of full-length gene. The full-length gene resulting from the first PCR is then selectively amplified in a second step of PCR using flanking primers, each one tagged with a restriction site in order to facilitate subsequent cloning. One multi overlapped extension process yields a full-length (multi-mutated) nucleic acid molecule encoding a candidate super-LEAD protein having multiple mutations therein derived from LEAD mutant proteins. 
     E. MODIFIED FIX POLYPEPTIDES EXHIBITING INCREASED PROTEIN STABILITY, PARTICULARLY MODIFICATIONS THAT INCREASE RESISTANCE TO PROTEASES 
     Provided herein are modified FIX polypeptides (also referred to herein as variants of FIX) that display improved protein stability (i.e. increased protease resistance, or increased conformational stability that, for example, renders a polypeptide more resistant to denaturation by temperature or pH changes). A FIX polypeptide provided herein exhibiting increased protein stability can lead to an increased half-life of the polypeptide in vitro (such as, during production, purification and storage) or in vivo (such as, after administration to a subject). For example, increased half-life can occur following administration of the polypeptide to a subject, such as a human subject. The increased half-life of the modified FIX polypeptide can be increased by an amount that is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of the unmodified FIX polypeptide. In some examples, the increased half-life of the modified FIX polypeptide can be increased by an amount that is at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times when compared to the half-life of the unmodified FIX polypeptide. Hence, the modified FIX polypeptides provided herein offer FIX with advantages including a decrease in the frequency of injections needed to maintain a sufficient drug level in serum, thus leading to, for example, higher comfort and acceptance by subjects, lower doses necessary to achieve comparable biological effects and attenuation of secondary effects. 
     Provided herein are modified FIX polypeptides containing modifications that alter any one or more of the properties of FIX that contribute to increased protein stability, such as increased protease resistance, and any combinations of the modifications thereof. Increased protein stability can be accomplished by amino acid replacement, such that resistance to proteases by amino acid replacements can be achieved by direct destruction of the protease target residue or sequence. Generally, modified FIX polypeptides retain one or more activities of an unmodified FIX polypeptide. For example, the modified FIX polypeptides provided herein exhibit at least one activity that is substantially unchanged (less than 1%, 5% or 10% changed) compared to the unmodified or wild-type FIX. In other examples, the activity of a modified FIX polypeptide is increased or is decreased as compared to an unmodified FIX polypeptide. In another embodiment, the modified FIX polypeptides provided herein can inhibit an activity of the unmodified and/or wild-type native FIX polypeptide. Activity includes, for example, but not limited to blood coagulation, platelet binding, cofactor binding and protease activity. Activity can be assessed in vitro or in vivo and can be compared to the unmodified FIX polypeptide, such as for example, the mature, wild-type native FIX polypeptide (SEQ ID NO: 2), the wild-type precursor FIX polypeptide (SEQ ID NO: 1), or any other FIX polypeptide known to one of skill in the art that is used as the starting material. 
     Modified FIX polypeptides provided herein can be modified at one or more amino acid positions corresponding to amino acid positions of a mature FIX polypeptide, for example, a mature FIX polypeptide having an amino acid sequence set forth in SEQ ID NO: 2. FIX polypeptides can be modified compared to a mature or precursor FIX polypeptide having an amino acid sequence set forth in SEQ ID NO: 2 or 1, respectively. Modified FIX polypeptides provided herein include human FIX (hFIX) variants. An exemplary hFIX variant occurs at amino acid position 148 of the mature hFIX polypeptide, wherein the amino acid is Alanine or Threonine. The hFIX polypeptide can be of any human tissue or cell-type origin. Modified FIX polypeptides provided herein also include variants of FIX of non-human origin. Such alignments and selection of positions can be performed with any FIX polypeptide by aligning it with hFIX and selecting corresponding positions for modification. For example, modified FIX polypeptides can be variants of a non-human FIX, including, but not limited to, mouse, rat, guinea pig, cow, sheep, dog, cat, chicken, pig, rabbit, fish, and chimpanzee FIX. Exemplary unmodified non-human FIX polypeptides have amino acid sequences set forth in SEQ ID NOS: 898-912. Modified FIX polypeptides also include polypeptides that are hybrids of different FIX polypeptides and also synthetic FIX polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon known polypeptides. 
     Typically, modifications include replacement (substitution), addition, deletion or a combination thereof, of amino acid residues as described herein. Modified FIX polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. Generally, the modification results in increased stability without losing at least one activity, such as coagulant activity (L e. retains at least one activity as defined herein) of an unmodified FIX polypeptide. A modified FIX exhibiting increased protein stability containing a single amino acid change at an is-HIT position as compared to an unmodified FIX is called a LEAD. FIX polypeptide candidate LEAD polypeptides can include amino acid replacement or replacements at any one or more of the is-HIT positions selected using methods described herein or known in the art, such as obtained using PAM analysis. Exemplary amino acid modifications corresponding to amino acid positions of a mature FIX polypeptide that can contribute to an increase in protein stability with respect to protease resistance are set forth in Table 5. In Table 5 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution. 
     Also among the variants provided herein are modified FIX polypeptides with two or more modifications compared to native or wild-type FIX. Modified FIX polypeptides include those with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. The two or more modifications can include two or more modifications of the same property, such as, two modifications that modify resistance to proteases (blood, intestinal, etc.). In another embodiment, the two or more modifications include combinations of properties that each contribute to FIX stability. For example, a FIX variant can include one or more modifications that remove a protease sensitive site and one or more modifications that alter FIX conformational stability. FIX variants carrying replacements at more than one is-HIT sites and displaying improved stability are called super-LEADs. A FIX super-LEAD can for example, contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acid changes compared to wild-type or unmodified FIX. In one example, a modified FIX polypeptide candidate super-LEAD can contain two or more amino acid modifications set forth in Table 5. 
     1. Protease Resistance 
     The delivery of stable peptide and protein drugs to patients is a major challenge for the pharmaceutical industry. These types of drugs in the human body are constantly eliminated or taken out of circulation by different physiological processes including internalization, glomerular filtration and proteolysis. The latter is often the limiting process affecting the half-life of proteins used as therapeutic agents in per-oral administration and either intravenous or intramuscular injections. To solve this problem, therapeutic proteins that increase protein stability manifested as an increased resistance to digestion by proteases are provided. Among modifications for therapeutic proteins are those that increase protection against protease digestion without destroying or completely eliminating a therapeutic use or the therapeutic activity. Such changes result in longer-lasting therapeutic proteins, and, also permit oral administration of therapeutic proteins. 
     FIX polypeptides provided herein are modified to increase resistance to proteolysis compared to in the absence of the modification. The resulting FIX polypeptides exhibit increased the half-life of the modified FIX polypeptide in vitro (such as, for production, processing, storage and assays.) and/or in vivo (such as, serum stability) and/or other properties, such as increased bioavailability upon oral administration. The modifications that alter susceptibility to cleavage by a proteases, unless specifically desired, do not alter activation cleavage sites for conversion of the zymogen into an activated form. 
     Proteases, proteinases or peptidases catalyze the hydrolysis of covalent peptidic bonds. Modified FIX polypeptides provided herein exhibit increased resistance to proteolysis by proteases, including those that occur, for example, in body fluids and tissues, such as those that include, but are not limited to, saliva, blood, serum, intestinal, stomach, blood, cell lysates, cells and others. These include proteases of all types, such as, for example, serine proteases and matrix metalloproteinases. 
     Modifications of FIX polypeptides result in resistance to one or more proteases that include, but are not limited to, pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. 
     FIX polypeptides are cleaved by proteases and their activity reduced or inactivated. For example, human neutrophil elastase cleaves and inactivate FIX polypeptides after amino acid residues, such as T140, T144, I164, T172, and V181 (see, e.g., Takaki et al. (1983)  J. Clin. Invest.  72: 1706-1715 and Samis et al. (1998)  Blood  92(4): 1287-1296). In another example, thrombin cleaves and inactivates FIX polypeptides after amino acid residues, such as R318 and R327 (see, e.g., Kisiel et al.  Blood  66(6): 1302-1308 (1985). Plasmin cleaves and inactivates FIX polypeptides after amino acid residues (Samis et al. (2000)  Blood  95(3) 943-951). As shown herein, modifications of FIX polypeptides at sites, such as such as K43, R145, R180, K316, and R318 increase resistance of the FIX polypeptides to hydrolysis a protease. In a non-limiting example, modification of T140, T144, I164, T172 and/or V181 or other sites of a mature FIX polypeptide is shown herein to increase resistance to elastase. 
     Modified FIX polypeptides provided herein exhibit increased resistance to proteolysis, particularly by enzymes present in serum, blood, the gut, the mouth and other body fluids. Such increase in resistance can manifested as increased half-life of the FIX polypeptide by an amount that is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the unmodified or wild-type FIX polypeptide in either in vivo (such as in human blood, human serum, saliva, digestive fluid and the intestinal tract,) and/or an in vitro mixture containing one or more proteases. Increased resistance also is manifested by increased bioavailability upon oral administration. As described herein, therapeutic proteins are not generally amenable to oral administration by virtue of the activity of digestive and/or blood and serum proteases. The modified FIX polypeptides provided herein can be administered orally. Hence, as described herein, provided are oral compositions containing a modified FIX polypeptide. The oral compositions do not require additional protease inhibitors nor any additional modifications of the FIX polypeptides. Additional modifications of the FIX polypeptides are optional. 
     Typically, the half-life in vitro or in vivo of the modified FIX polypeptides provided herein is increased by an amount selected from at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more when compared to the half-life of unmodified or wild-type FIX in either blood, serum, or in an in vitro preparation or an in vitro mixture containing one or more proteases. 
     Typically, the modified FIX polypeptides provided herein exhibit at least one activity that is substantially unchanged (less than 1%, 5% or 10% changed) compared to the unmodified or wild-type FIX. In some examples, the activity is increased compared to the unmodified FIX. In other examples, the activity is decreased compared to the unmodified FIX polypeptide. The activity is retained at level so that the FIX polypeptide can be used therapeutically for the desired purpose. Activity includes, for example, blood coagulation activity, and can be compared to the unmodified polypeptide, such as for example, the mature, wild-type native FIX polypeptide (SEQ ID NO: 2), the wild-type precursor FIX polypeptide (SEQ ID NO: 1), or any other FIX polypeptide used as the starting material. In another embodiment the modified FIX polypeptide can inhibit one or more activities of an unmodified FIX polypeptide and/or act as an anticoagulant. 
     The FIX polypeptides can be modified to have resistance to a variety of proteases. These include, for example, members of the serine protease family and also metalloproteinases. 
     a. Serine Proteases 
     Serine proteases participate in a range of functions in the body, including blood clotting, inflammation as well as digestive enzymes in prokaryotes and eukaryotes. Serine proteases are sequence specific. While cascades of protease activations control blood clotting and complement, other proteases are involved in signaling pathways, enzyme activation and degradative functions in different cellular or extracellular compartments. 
     Serine proteases include, but are not limited, to chymotrypsin, trypsin, elastase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. Chymotrypsin, trypsin and elastase are synthesized by the pancreatic acinar cells, secreted in the small intestine and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. Each of these digestive serine proteases targets different regions of the polypeptide chain, based upon the amino acid residues and side chains surrounding the site of cleavage. The active site of serine proteases is shaped as a cleft where the polypeptide substrate binds. Amino acid residues are labeled from N to C term of the polypeptide substrate (Pi, . . . , P3, P2, P1, P1′, P2′, P3′, . . . , Pj) and their respective binding sub-sites (Si, . . . , S3, S2, S1, S1′, S2′, S3′, . . . , Sj). The cleavage is catalyzed between P1 and P1′. Chymotrypsin hydrolyzes peptide bonds flanked with bulky hydrophobic amino acid residues. Particular residues include phenylalanine, tryptophan and tyrosine, which fit into a snug hydrophobic pocket. Trypsin hydrolyzes peptide bonds flanked with positively charged amino acid residues. Instead of having the hydrophobic pocket of the chymotrypsin, trypsin possesses an aspartic acid residue at the back of the pocket, which can interact with positively charged residues such as arginine and lysine. Elastase hydrolyzes peptide bonds flanked with small neutral amino acid residues, such as alanine, glycine and valine. In contrast to trypsin and chymotrypsin, elastase contains a pocket that is lined with valine and threonine, rendering it a mere depression, which can accommodate the smaller amino acid residues. Serine proteases are ubiquitous in prokaryotes and eukaryotes and serve important and diverse biological functions such as hemostasis, fibrinolysis, complement formation and the digestion of dietary proteins. Coagulation factors, such as FIX, FVII and FX possess serine protease domains that hydrolyze specific members of the coagulation cascade. 
     Elastases that belong to the serine protease family display extensive sequence homology to other known serine proteases, including trypsin and chymotrypsin. Serine elastases preferentially cleave polypeptides adjacent to aliphatic amino acids residues, typically alanine, valine and methionine, and to a lesser extent, leucine and isoleucine. Humans have six elastase genes which encode the structurally similar proteins, elastase 1 (ELA-1, also known as pancreatic elastase, PE), elastase 2 (neutrophil elastase, NE, also known as PMN elastase, bone marrow serine protease, medullasin, human leukocyte elastase, HLE), elastase 2A (ELA-2A), elastase 2B (ELA-2B), elastase 3A (ELA-3A, elastase IIIA, Protease E), and elastase-3B (ELA-3B, elastase IIIB, protease E). Other serine proteases with elastase activity include, but are not limited to, proteinase-3 (PR-3), endogenous vascular elastase (EVE), and endothelial cell elastase (ECE). 
     Neutrophil primary azurophil granules carry NE (ELA-2) and PR-3, which are released upon neutrophil activation. NE is involved in degradation of the extracellular matrix and (ECM), including degradation of elastin, cartilage proteoglycans, collagens, and fibronectin, and digestion of material taken into the cell by phagocytosis. NE also helps in degradation of proteins, such as immunoglobulins and surfactant apoproteins. NE preferentially cleaves Val-X bonds and to a lesser extent Ala-X bonds. Abnormal or excessive release of NE has been linked to defects in connective tissue turnover, arthritis and inflammation. Like NE, PR-3 also functions to activate proenzymes, such as metalloproteinases, and cytokines, such as TNF-α, IL-1β, and interleukin-8 (IL-8). 
     Pancreatic elastase (ELA-1) preferentially cleaves Ala-X bonds and is expressed primarily in skin keratinocytes. Expression of ELA is not normally found in the adult pancreas though it is often expressed in and used as a marker for pancreatic cancers. Elastase activity of the normal pancreas is attributable to ELA-2A and ELA-2B. ELA-2A and ELA-2B preferentially cleaves Leu-X, Met-X and Phe-X bonds. 
     Some pathological conditions are result at least in part from an imbalance between the elastases and their endogenous inhibitors. Uncontrolled proteolytic degradation by neutrophil elastases, especially ELA-2 has been implicated in a number of pathological conditions like pulmonary emphysema, acute respiratory distress syndrome, septic shock, multiple organ failure, rheumatoid arthritis and cystic fibrosis. 
     High concentrations of elastases can be found in the gastrointestinal tract and blood stream. Hence, effective therapeutics to be administered via these routes can be achieved through modification of elastase cleavage sites. As mentioned above, FIX is known to be cleaved by human neutrophil elastase (HNE) at amino acid positions T140, T144, I164, T172 and V181 of a mature FIX polypeptide. Cleavage of FIX by HNE generates a FIX polypeptide without coagulant activity. Modification of FIX polypeptides at one or more elastase cleavage sites can increase resistance of FIX polypeptides to proteolytic degradation and thus, improve the therapeutic properties of the FIX polypeptide. 
     Increased levels of elastase are associated with coagulation disease states, such as disseminated intravascular coagulation (DIC), wherein there is a depletion of active coagulation factors. The decrease in pool of coagulation factors can result in excessive bleeding at sites of tissue damage due to the deficiency in clotting factors needed at the injury location. Modification of FIX polypeptides at elastase cleavage sites, such as the modified FIX polypeptides provided herein, can increase the resistance of FIX polypeptides to elevated levels of elastase in such disease states and thus, improve the therapeutic effectiveness of FIX polypeptides. 
     b. Matrix Metalloproteinases 
     Matrix metalloproteinases (MMPs) are a family of Zn 2+ - and calcium-dependent endopeptidases that degrade components of the extracellular matrix (ECM). In addition, MMPs also can process a number of cell-surface cytokines, receptors and other soluble proteins. They are involved in normal tissue remodeling processes such as wound healing, pregnancy and angiogenesis. Under physiological conditions, MMPs are made as inactive precursors (zymogens) and are processed to their active form. Additionally, the enzymes are specifically regulated by endogenous inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs). The proteolytic activity of MMPs acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. The excess synthesis and production of these proteins lead to accelerated degradation of the ECM which is associated with a variety of diseases and conditions such as, for example, bone homeostasis, arthritis, cancer, multiple sclerosis and rheumatoid arthritis. In the context of neuroinflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system (Leppert et al. (2001)  Brain Res. Rev.  36(2-3): 249-57; Borkakoti et al. (1998)  Prog. Biophys. Mol. Biol.  70(1): 73-94). 
     Members of the MMP family include collagenases, gelatinases, stromelysins, matrilysin and membrane-bound MMPs. Most MMPs are secreted in the inactive proenzyme form. The secreted proenzyme MMPs can be activated by several proinflammatory agents such as oxidants, proteinases including elastase, plasmin, and trypsin, and other MMPs (Cuzner and Opdenakker (1999)  J. Neuroimmunol.  94(1-2): 1-14). In tissues, physiological MMP activators include tissue or plasma proteinases or opportunistic bacterial proteinases. For example, the plasminogen activator/plasmin system, including ubiquitous plasminogen by urokinase (u-Pa) and tissue-type plasminogen activator (t-Pa), is an important activator of pro-MMP in pathological situations. MMP activity can be inhibited by tissue inhibitors of metalloproteinases (TIMPs), by serine proteinase inhibitors (serpins), and by nonspecific proteinase inhibitors, such as α2-macroglobulin. TIMPs inhibit the MMP activity through noncovalent binding of the active zinc-binding sites of MMPs. Proteolytic activities of MMPs and plasminogen activators, and their inhibitors, are important in maintaining the integrity of the ECM as cell-ECM interactions influence and mediate a wide range of processes including proliferation, differentiation, adhesion and migration of a variety of cell types. Excessive production of matrix metalloproteinases has been implicated in tissue damage and wound healing, inflammatory disorders, proliferative disorders and autoimmune diseases (St-Pierre et al. (2003)  Curr. Drug Targets Inflamm. Allergy  2(3): 206-215; Opdenakker (1997)  G. Verh. K. Acad. Geneeskd. Belg.  59(6): 489-514). 
     c. Increased Resistance to Proteolysis by Removal of Proteolytic Sites 
     As exemplified herein, the 2D-scanning methodology was used to identify the amino acids of hFIX that lead to an increase in stability when challenged with proteases (such as blood and/or intestinal proteins), blood lysate and/or serum. Increasing protein stability to proteases (such as blood, lysate and intestinal serum), provides a longer in vivo half-life for the particular protein molecules. This can reduce the frequency of administered doses and/or the total amount administered. 
     A first step in the design of hFIX mutants provided resistant to proteolysis included identifying sites vulnerable to proteolysis along the protein sequence. Based on a list of selected blood, intestinal or any other type of proteases considered (Table 4), the complete list of all amino acids and sequences of amino acids in hFIX that can be targeted by those proteases was first determined in silico. The protease targets (amino acids or sequences of amino acids along the hFIX polypeptide) are named in silico HITs (is-HITs). 
     The second step in the design of hFIX mutants that are resistant to proteolysis included identifying the appropriate replacing amino acids such that if they replaced the natural amino acids in hFIX at is-HITs, the protein (i) becomes resistant to proteolysis; and (ii) elicits a level of activity at least comparable to the wild-type hFIX polypeptide. In another embodiment the protein (i) becomes resistant to proteolysis; and (ii) inhibits an activity of the wild-type hFIX polypeptide and/or acts as an anticoagulant. The choice of the replacing amino acids included consideration of the broad target specificity of certain proteases and the need to preserve the physicochemical properties such as hydrophobicity, charge and polarity of essential (such as, catalytic, binding, etc.) residues in hFIX. 
     “Point Accepted Mutation” (PAM; Dayhoff et al., 1978) can be used as part of the 2D scanning approach. PAM values, originally developed to produce alignments between protein sequences, are available in the form of probability matrices that reflect an evolutionary distance between amino acids. Conservative substitutions of a residue in a reference sequence are those substitutions that are physically and functionally similar to the corresponding reference residues, i.e., that have a similar size, shape, electric charge, and/or chemical properties, including the ability to form covalent or hydrogen bonds and other such interactions. Conservative substitutions show the highest scores fitting with the PAM matrix criteria in the form of accepted point mutations. The PAM250 matrix is used in the frame of 2D-scanning to identify candidate replacing amino acids for the is-HITs in order to generate conservative mutations without affecting protein function. At least two amino acids with the highest values in PAM250 matrix corresponding to conservative substitutions or accepted point mutations were chosen for replacement at each is-HIT. The replacement of amino acids by cysteine residues is explicitly avoided since this change can lead to the formation of intermolecular disulfide bonds. 
     Briefly, using the algorithm PROTEOL (on-line at infobiogen.fr and at bioinfo.hku.hk/services/analyseq/cgi-bin/proteol_in.pl), a list of residues along the mature hFIX polypeptide of 415 amino acids (SEQ ID NO: 2), which can be recognized as substrate for proteases (blood, intestinal, etc.) in Table 4 was established. The algorithm generates a proteolytic digestion map based on a list of proteases, the proteolytic specificity of the proteases, and the polypeptide amino acid sequence that is entered. Table 4 shows the in silico identification of amino acid positions that are targets for proteolysis using selected proteases and chemical treatment. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                 Amino Acid 
                 Protease or Chemical 
               
               
                   
                 Abbreviation 
                 Position 
                 Treatment 
               
               
                   
                   
               
             
            
               
                   
                 AspN 
                 D 
                 Endoproteinase Asp-N 
               
               
                   
                 Chymo 
                 (F, W, Y, M, L)~P 
                 Chymotrypsin 
               
               
                   
                 Clos 
                 R 
                 Clostripain 
               
               
                   
                 CnBr 
                 M 
                 Cyanogen Bromide 
               
               
                   
                 IBzO 
                 W 
                 IodosoBenzoate 
               
               
                   
                 Myxo 
                 K 
                 Myxobacter 
               
               
                   
                 NH 2 OH 
                 N G 
                 Hydroxylamine 
               
               
                   
                 pH2.5 
                 D P 
                 pH 2.5 
               
               
                   
                 ProEn 
                 P 
                 Proline Endopeptidase 
               
               
                   
                 Staph 
                 E 
                 Staphylococcal Protease 
               
               
                   
                 Tryp 
                 (K, R)~P 
                 Trypsin 
               
               
                   
                 TrypK 
                 K~P 
                 Trypsin (Arg blocked) 
               
               
                   
                 TrypR 
                 R~P 
                 Trypsin (Lys blocked) 
               
               
                   
                   
               
            
           
         
       
     
     Is-HITS were identified and LEADS created for higher resistance to proteolysis of hFIX. The native amino acids at each of the is-HIT positions and replacing amino acids for increased resistance to proteolysis can include, but are not limited to replacing any of Y, A, L, S, T, I, V, F, Q and M by any of E, D, K, R, N, Q, S and T. Is-HITS and LEADs can include modifications at regions susceptible to proteolysis. 
     d. Modified FIX Polypeptides Exhibiting Increased Protease Resistance 
     Using such methods as outlined above, modified FIX proteins were generated. Sites, designated is-HIT positions, were identified and modified proteins prepared. The resulting proteins exhibited increased protease resistance. This was manifested as, for example, increased the methods described herein, the following is-HIT positions were identified to eliminate protease sensitive sites and increase protein stability of a FIX polypeptide. In addition, the modified FIX proteins can be administered orally and deliver therapeutically effective dosages to the blood. 
     Among the modified FIX polypeptides provided herein are those that included modifications at one or more loci. Typically modified FIX polypeptides include one, two, three, four or five such modifications. In addition, the background polypeptide that is modified can include other modifications known to alter desired properties, such as modification of sites to decrease immunogenicity and/or to increase glycosylation. The polypeptides can include further modifications, such as pegylation. 
     Provided herein are modified FIX polypeptides that include modifications of the amino acids at positions selected from among one or more of the following: 1, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 52, 53, 55, 57, 58, 59, 60, 61, 63, 64, 65, 66, 68, 69, 70, 72, 74, 75, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 168, 169, 171, 172, 174, 175, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217, 218, 219, 220, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 239, 240, 241, 242, 244, 245, 247, 248, 250, 251, 252, 253, 254, 255, 259, 261, 262, 263, 265, 266, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 283, 284, 285, 286, 287, 288, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 341, 342, 343, 344, 345, 348, 349, 351, 352, 353, 355, 356, 357, 358, 359, 360, 363, 364, 365, 366, 367, 368, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 407, 408, 409, 410, 411, 412, 413, 414, and 415. These positions (loci) are relative to the mature FIX polypeptide whose sequence is set forth in SEQ ID NO.: 2 or 1035 or corresponding loci in an allelic or species variant and/or in a background polypeptide (starting FIX polypeptide) including other modifications. The FIX polypeptides include precursor forms and mature forms, zymogens and activated forms. 
     In particular examples, amino acid replacement or replacements can be at any one or more positions corresponding to any of the following positions: Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, I66, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, 5141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, E294, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, T371, E372, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, L414, and T415 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or in corresponding loci in an allelic or species variant and/or in a background polypeptide including other modifications. 
     In one embodiment, positions are typically replaced as follows: replacement of D with N or Q, replacement of E with H, Q or N, replacement of F with I or V, replacement of K with Q or N, replacement of L with I or V, replacement of M with I or V, replacement of N with Q or S, replacement of P with A or S, replacement of R with H or Q, replacement of W with H or S, replacement of Y with I or H, replacement of A, G, I, S, T, or V with Q, H, or N. 
     In one embodiment, positions corresponding to hFIX are selected (is-HITS) and amino acid replacements are made (LEADs) with increased resistance to proteolysis that include, but are not limited to replacements corresponding to those set forth in Table 5, where the replacements are made compared to the sequence of amino acids set forth in SEQ ID NO: 2. Table 5 provides non-limiting examples of amino acid replacements, corresponding to amino acid positions of a mature FIX polypeptide, that increase resistance to proteolysis and, thereby, protein stability. The modified FIX polypeptides include precursor and mature forms, zymogen and active forms, allelic and species variants and/or modifications in a background FIX polypeptide including other modifications. 
     In referencing such mutants, the first amino acid (one-letter abbreviation) corresponds to the amino acid that is replaced, the number corresponds to position in the hFIX polypeptide sequence with reference to SEQ ID NO: 2, and the second amino acid (one-letter abbreviation) corresponds to the amino acid selected that replaces the first amino acid at that position. In Table 5 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution. The FIX polypeptides employed for modification can be any FIX polypeptide, including other mammalian FIX polypeptides. Corresponding positions, as assessed by appropriate alignment, are identified and modified as described herein. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 List of Human FIX Modifications to Increase Resistance to Proteolysis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Y1H (3) 
                 Y1I (4) 
                 K5N (5) 
                 K5Q (6) 
                 L6I (7) 
                 L6V (8) 
                 E7Q (9) 
               
               
                 E7H (10) 
                 E7N (11) 
                 E8Q (12) 
                 E8H (13) 
                 E8N (14) 
                 F9I (15) 
                 F9V (16) 
               
               
                 L14I (17) 
                 L14V (18) 
                 E15Q (19) 
                 E15H (20) 
                 E15N (21) 
                 R16H (22) 
                 R16Q (23) 
               
               
                 E17Q (24) 
                 E17H (25) 
                 E17N (26) 
                 M19I (27) 
                 M19V (28) 
                 E20Q (29) 
                 E20H (30) 
               
               
                 E20N (31) 
                 E21Q (32) 
                 E21H (33) 
                 E21N (34) 
                 K22N (35) 
                 K22Q (36) 
                 F25I (37) 
               
               
                 F25V (38) 
                 E26Q (39) 
                 E26H (40) 
                 E26N (41) 
                 E27Q (42) 
                 E27H (43) 
                 E27N (44) 
               
               
                 R29H (45) 
                 R29Q (46) 
                 E30Q (47) 
                 E30H (48) 
                 E30N (49) 
                 F32I (50) 
                 F32V (51) 
               
               
                 E33Q (52) 
                 E33H (53) 
                 E33N (54) 
                 E36Q (55) 
                 E36H (56) 
                 E36N (57) 
                 R37H (58) 
               
               
                 R37Q (59) 
                 E40Q (60) 
                 E40H (61) 
                 E40N (62) 
                 F41I (63) 
                 F41V (64) 
                 W42S (65) 
               
               
                 W42H (66) 
                 K43N (67) 
                 K43Q (68) 
                 Y45H (69) 
                 Y45I (70) 
                 D47N (71) 
                 D47Q (72) 
               
               
                 D49N (73) 
                 D49Q (74) 
                 E52Q (75) 
                 E52H (76) 
                 E52N (77) 
                 P55A (78) 
                 P55S (79) 
               
               
                 L57I (80) 
                 L57V (81) 
                 N58Q (82) 
                 N58S (83) 
                 K63N (84) 
                 K63Q (85) 
                 D64N (86) 
               
               
                 D64Q (87) 
                 D65N (88) 
                 D65Q (89) 
                 Y69H (90) 
                 Y69I (91) 
                 E70Q (92) 
                 E70H (93) 
               
               
                 E70N (94) 
                 W72S (95) 
                 W72H (96) 
                 P74A (97) 
                 P74S (98) 
                 F75I (99) 
                 F75V (100) 
               
               
                 F77I (101) 
                 F77V (102) 
                 E78Q (103) 
                 E78H (104) 
                 E78N (105) 
                 K80N (106) 
                 K80Q (107) 
               
               
                 E83Q (108) 
                 E83H (109) 
                 E83N (110) 
                 L84I (111) 
                 L84V (112) 
                 D85N (113) 
                 D85Q (114) 
               
               
                 K91N (115) 
                 K91Q (116) 
                 N92Q (117) 
                 N92S (118) 
                 R94H (119) 
                 R94Q (120) 
                 E96Q (121) 
               
               
                 E96H (122) 
                 E96N (123) 
                 F98I (124) 
                 F98V (125) 
                 K100N (126) 
                 K100Q (127) 
                 D104N (128) 
               
               
                 D104Q (129) 
                 K106N (130) 
                 K106Q (131) 
                 E113Q (132) 
                 E113H (133) 
                 E113N (134) 
                 Y115H (135) 
               
               
                 Y115I (136) 
                 R116H (137) 
                 R116Q (138) 
                 L117I (139) 
                 L117V (140) 
                 E119Q (141) 
                 E119H (142) 
               
               
                 E119N (143) 
                 K122N (144) 
                 K122Q (145) 
                 E125Q (146) 
                 E125H (147) 
                 E125N (148) 
                 P126A (149) 
               
               
                 P126S (150) 
                 P129A (151) 
                 P129S (152) 
                 P131A (153) 
                 P131S (154) 
                 R134H (155) 
                 R134Q (156) 
               
               
                 K142N (157) 
                 K142Q (158) 
                 L143I (159) 
                 L143V (160) 
                 R145H (161) 
                 R145Q (162) 
                 E147Q (163) 
               
               
                 E147H (164) 
                 E147N (165) 
                 P151A (166) 
                 P151S (167) 
                 D152N (168) 
                 D152Q (169) 
                 D154N (170) 
               
               
                 D154Q (171) 
                 Y155H (172) 
                 Y155I (173) 
                 E160Q (174) 
                 E160H (175) 
                 E160N (176) 
                 E162Q (177) 
               
               
                 E162H (178) 
                 E162N (179) 
                 L165I (180) 
                 L165V (181) 
                 D166N (182) 
                 D166Q (183) 
                 F175I (184) 
               
               
                 F175V (185) 
                 D177N (186) 
                 D177Q (187) 
                 F178I (188) 
                 F178V (189) 
                 R180H (190) 
                 R180Q (191) 
               
               
                 E185Q (192) 
                 E185H (193) 
                 E185N (194) 
                 D186N (195) 
                 D186Q (196) 
                 K188N (197) 
                 K188Q (198) 
               
               
                 P189A (199) 
                 P189S (200) 
                 P193A (201) 
                 P193S (202) 
                 W194S (203) 
                 W194H (204) 
                 L198I (205) 
               
               
                 L198V (206) 
                 N199Q (207) 
                 N199S (208) 
                 K201N (209) 
                 K201Q (210) 
                 D203N (211) 
                 D203Q (212) 
               
               
                 F205I (213) 
                 F205V (214) 
                 E213Q (215) 
                 E213H (216) 
                 E213N (217) 
                 K214N (218) 
                 K214Q (219) 
               
               
                 W215S (220) 
                 W215H (221) 
                 E224Q (222) 
                 E224H (223) 
                 E224N (224) 
                 K228N (225) 
                 K228Q (226) 
               
               
                 E235Q (227) 
                 E235H (228) 
                 E235N (229) 
                 E239Q (230) 
                 E239H (231) 
                 E239N (232) 
                 E240Q (233) 
               
               
                 E240H (234) 
                 E240N (235) 
                 E242Q (236) 
                 E242H (237) 
                 E242N (238) 
                 E245Q (239) 
                 E245H (240) 
               
               
                 E245N (241) 
                 K247N (242) 
                 K247Q (243) 
                 R248H (244) 
                 R248Q (245) 
                 R252H (246) 
                 R252Q (247) 
               
               
                 P255A (248) 
                 P255S (249) 
                 Y259H (250) 
                 Y259I (251) 
                 K265N (252) 
                 K265Q (253) 
                 Y266H (254) 
               
               
                 Y266I (255) 
                 D269N (256) 
                 D269Q (257) 
                 L272I (258) 
                 L272V (259) 
                 L273I (260) 
                 L273V (261) 
               
               
                 E274Q (262) 
                 E274H (263) 
                 E274N (264) 
                 L275I (265) 
                 L275V (266) 
                 D276N (267) 
                 D276Q (268) 
               
               
                 E277Q (269) 
                 E277H (270) 
                 E277N (271) 
                 P278A (272) 
                 P278S (273) 
                 L279I (274) 
                 L279V (275) 
               
               
                 L281I (276) 
                 L281V (277) 
                 Y284H (278) 
                 Y284I (279) 
                 P287A (280) 
                 P287S (281) 
                 D292N (282) 
               
               
                 D292Q (283) 
                 K293N (284) 
                 K293Q (285) 
                 E294Q (286) 
                 E294H (287) 
                 E294N (288) 
                 Y295H (289) 
               
               
                 Y295I (290) 
                 F299I (291) 
                 F299V (292) 
                 L300I (293) 
                 L300V (294) 
                 K301N (295) 
                 K301Q (296) 
               
               
                 F302I (297) 
                 F302V (298) 
                 Y306H (299) 
                 Y306I (300) 
                 W310S (301) 
                 W310H (302) 
                 R312H (303) 
               
               
                 R312Q (304) 
                 F314I (305) 
                 F314V (306) 
                 K316N (307) 
                 K316Q (308) 
                 R318H (309) 
                 R318Q (310) 
               
               
                 L321I (311) 
                 L321V (312) 
                 L323I (313) 
                 L323V (314) 
                 Y325H (315) 
                 Y325I (316) 
                 L326I (317) 
               
               
                 L326V (318) 
                 R327H (319) 
                 R327Q (320) 
                 P329A (321) 
                 P329S (322) 
                 L330I (323) 
                 L330V (324) 
               
               
                 D332N (325) 
                 D332Q (326) 
                 R333H (327) 
                 R333Q (328) 
                 L337I (329) 
                 L337V (330) 
                 R338H (331) 
               
               
                 R338Q (332) 
                 K341N (333) 
                 K341Q (334) 
                 F342I (335) 
                 F342V (336) 
                 Y345H (337) 
                 Y345I (338) 
               
               
                 M348I (339) 
                 M348V (340) 
                 F349I (341) 
                 F349V (342) 
                 F353I (343) 
                 F353V (344) 
                 E355Q (345) 
               
               
                 E355H (346) 
                 E355N (347) 
                 R358H (348) 
                 R358Q (349) 
                 D359N (350) 
                 D359Q (351) 
                 D364N (352) 
               
               
                 D364Q (353) 
                 P368A (354) 
                 P368S (355) 
                 E372Q (356) 
                 E372H (357) 
                 E372N (358) 
                 E374Q (359) 
               
               
                 E374H (360) 
                 E374N (361) 
                 F378I (362) 
                 F378V (363) 
                 L379I (364) 
                 L379V (365) 
                 W385S (366) 
               
               
                 W385H (367) 
                 E387Q (368) 
                 E387H (369) 
                 E387N (370) 
                 E388Q (371) 
                 E388H (372) 
                 E388N (373) 
               
               
                 M391I (374) 
                 M391V (375) 
                 K392N (376) 
                 K392Q (377) 
                 K394N (378) 
                 K394Q (379) 
                 Y395H (380) 
               
               
                 Y395I (381) 
                 Y398H (382) 
                 Y398I (383) 
                 K400N (384) 
                 K400Q (385) 
                 R403H (386) 
                 R403Q (387) 
               
               
                 Y404H (388) 
                 Y404I (389) 
                 W407S (390) 
                 W407H (391) 
                 K409N (392) 
                 K409Q (393) 
                 E410Q (394) 
               
               
                 E410H (395) 
                 E410N (396) 
                 K411N (397) 
                 K411Q (398) 
                 K413N (399) 
                 K413Q (400) 
                 L414I (401) 
               
               
                 L414V (402) 
                 S3N (403) 
                 S3Q (404) 
                 S3H (405) 
                 G4N (406) 
                 G4Q (407) 
                 G4H (408) 
               
               
                 V10N (409) 
                 V10Q (410) 
                 V10H (411) 
                 G12N (412) 
                 G12Q (413) 
                 G12H (414) 
                 S24N (415) 
               
               
                 S24Q (416) 
                 S24H (417) 
                 A28N (418) 
                 A28Q (419) 
                 A28H (420) 
                 V31N (421) 
                 V31Q (422) 
               
               
                 V31H (423) 
                 T35N (424) 
                 T35Q (425) 
                 T35H (426) 
                 T38N (427) 
                 T38Q (428) 
                 T38H (429) 
               
               
                 T39N (430) 
                 T39Q (431) 
                 T39H (432) 
                 V46N (433) 
                 V46Q (434) 
                 V46H (435) 
                 G48N (436) 
               
               
                 G48Q (437) 
                 G48H (438) 
                 S53N (439) 
                 S53Q (440) 
                 S53H (441) 
                 G59N (442) 
                 G59Q (443) 
               
               
                 G59H (444) 
                 G60N (445) 
                 G60Q (446) 
                 G60H (447) 
                 S61N (448) 
                 S61Q (449) 
                 S61H (450) 
               
               
                 I66N (451) 
                 I66Q (452) 
                 I66H (453) 
                 S68N (454) 
                 S68Q (455) 
                 S68H (456) 
                 G76N (457) 
               
               
                 G76Q (458) 
                 G76H (459) 
                 G79N (460) 
                 G79Q (461) 
                 G79H (462) 
                 V86N (463) 
                 V86Q (464) 
               
               
                 V86H (465) 
                 T87N (466) 
                 T87Q (467) 
                 T87H (468) 
                 I90N (469) 
                 I90Q (470) 
                 I90H (471) 
               
               
                 G93N (472) 
                 G93Q (473) 
                 G93H (474) 
                 S102N (475) 
                 S102Q (476) 
                 S102H (477) 
                 A103N (478) 
               
               
                 A103Q (479) 
                 A103H (480) 
                 V107N (481) 
                 V107Q (482) 
                 V107H (483) 
                 V108N (484) 
                 V108Q (485) 
               
               
                 V108H (486) 
                 S110N (487) 
                 S110Q (488) 
                 S110H (489) 
                 T112N (490) 
                 T112Q (491) 
                 T112H (492) 
               
               
                 G114N (493) 
                 G114Q (494) 
                 G114H (495) 
                 A118N (496) 
                 A118Q (497) 
                 A118H (498) 
                 S123N (499) 
               
               
                 S123Q (500) 
                 S123H (501) 
                 A127N (502) 
                 A127Q (503) 
                 A127H (504) 
                 V128N (505) 
                 V128Q (506) 
               
               
                 V128H (507) 
                 G133N (508) 
                 G133Q (509) 
                 G133H (510) 
                 V135N (511) 
                 V135Q (512) 
                 V135H (513) 
               
               
                 S136N (514) 
                 S136Q (515) 
                 S136H (516) 
                 V137N (517) 
                 V137Q (518) 
                 V137H (519) 
                 S138N (520) 
               
               
                 S138Q (521) 
                 S138H (522) 
                 T140N (523) 
                 T140Q (524) 
                 T140H (525) 
                 S141N (526) 
                 S141Q (527) 
               
               
                 S141H (528) 
                 T144N (529) 
                 T144Q (530) 
                 T144H (531) 
                 A146N (532) 
                 A146Q (533) 
                 A146H (534) 
               
               
                 T148N (535) 
                 T148Q (536) 
                 T148H (537) 
                 V149N (538) 
                 V149Q (539) 
                 V149H (540) 
                 V153N (541) 
               
               
                 V153Q (542) 
                 V153H (543) 
                 V156N (544) 
                 V156Q (545) 
                 V156H (546) 
                 S158N (547) 
                 S158Q (548) 
               
               
                 S158H (549) 
                 T159N (550) 
                 T159Q (551) 
                 T159H (552) 
                 A161N (553) 
                 A161Q (554) 
                 A161H (555) 
               
               
                 T163N (556) 
                 T163Q (557) 
                 T163H (558) 
                 I164N (559) 
                 I164Q (560) 
                 I164H (561) 
                 I168N (562) 
               
               
                 I168Q (563) 
                 I168H (564) 
                 T169N (565) 
                 T169Q (566) 
                 T169H (567) 
                 S171N (568) 
                 S171Q (569) 
               
               
                 S171H (570) 
                 T172N (571) 
                 T172Q (572) 
                 T172H (573) 
                 S174N (574) 
                 S174Q (575) 
                 S174H (576) 
               
               
                 T179N (577) 
                 T179Q (578) 
                 T179H (579) 
                 V181N (580) 
                 V181Q (581) 
                 V181H (582) 
                 V182N (583) 
               
               
                 V182Q (584) 
                 V182H (585) 
                 G183N (586) 
                 G183Q (587) 
                 G183H (588) 
                 G184N (589) 
                 G184Q (590) 
               
               
                 G184H (591) 
                 A187N (592) 
                 A187Q (593) 
                 A187H (594) 
                 G190N (595) 
                 G190Q (596) 
                 G190H (597) 
               
               
                 V196N (598) 
                 V196Q (599) 
                 V196H (600) 
                 V197N (601) 
                 V197Q (602) 
                 V197H (603) 
                 G200N (604) 
               
               
                 G200Q (605) 
                 G200H (606) 
                 V202N (607) 
                 V202Q (608) 
                 V202H (609) 
                 A204N (610) 
                 A204Q (611) 
               
               
                 A204H (612) 
                 G207N (613) 
                 G207Q (614) 
                 G207H (615) 
                 G208N (616) 
                 G208Q (617) 
                 G208H (618) 
               
               
                 S209N (619) 
                 S209Q (620) 
                 S209H (621) 
                 I210N (622) 
                 I210Q (623) 
                 I210H (624) 
                 V211N (625) 
               
               
                 V211Q (626) 
                 V211H (627) 
                 I216N (628) 
                 I216Q (629) 
                 I216H (630) 
                 V217N (631) 
                 V217Q (632) 
               
               
                 V217H (633) 
                 T218N (634) 
                 T218Q (635) 
                 T218H (636) 
                 A219N (637) 
                 A219Q (638) 
                 A219H (639) 
               
               
                 A220N (640) 
                 A220Q (641) 
                 A220H (642) 
                 V223N (643) 
                 V223Q (644) 
                 V223H (645) 
                 T225N (646) 
               
               
                 T225Q (647) 
                 T225H (648) 
                 G226N (649) 
                 G226Q (650) 
                 G226H (651) 
                 V227N (652) 
                 V227Q (653) 
               
               
                 V227H (654) 
                 I229N (655) 
                 I229Q (656) 
                 I229H (657) 
                 T230N (658) 
                 T230Q (659) 
                 T230H (660) 
               
               
                 V231N (661) 
                 V231Q (662) 
                 V231H (663) 
                 V232N (664) 
                 V232Q (665) 
                 V232H (666) 
                 A233N (667) 
               
               
                 A233Q (668) 
                 A233H (669) 
                 G234N (670) 
                 G234Q (671) 
                 G234H (672) 
                 I238N (673) 
                 I238Q (674) 
               
               
                 I238H (675) 
                 T241N (676) 
                 T241Q (677) 
                 T241H (678) 
                 T244N (679) 
                 T244Q (680) 
                 T244H (681) 
               
               
                 V250N (682) 
                 V250Q (683) 
                 V250H (684) 
                 I251N (685) 
                 I251Q (686) 
                 I251H (687) 
                 I253N (688) 
               
               
                 I253Q (689) 
                 I253H (690) 
                 I254N (691) 
                 I254Q (692) 
                 I254H (693) 
                 A261N (694) 
                 A261Q (695) 
               
               
                 A261H (696) 
                 A262N (697) 
                 A262Q (698) 
                 A262H (699) 
                 I263N (700) 
                 I263Q (701) 
                 I263H (702) 
               
               
                 I270N (703) 
                 I270Q (704) 
                 I270H (705) 
                 A271N (706) 
                 A271Q (707) 
                 A271H (708) 
                 V280N (709) 
               
               
                 V280Q (710) 
                 V280H (711) 
                 S283N (712) 
                 S283Q (713) 
                 S283H (714) 
                 V285N (715) 
                 V285Q (716) 
               
               
                 V285H (717) 
                 T286N (718) 
                 T286Q (719) 
                 T286H (720) 
                 I288N (721) 
                 I288Q (722) 
                 I288H (723) 
               
               
                 I290N (724) 
                 I290Q (725) 
                 I290H (726) 
                 A291N (727) 
                 A291Q (728) 
                 A291H (729) 
                 T296N (730) 
               
               
                 T296Q (731) 
                 T296H (732) 
                 I298N (733) 
                 I298Q (734) 
                 I298H (735) 
                 G303N (736) 
                 G303Q (737) 
               
               
                 G303H (738) 
                 S304N (739) 
                 S304Q (740) 
                 S304H (741) 
                 G305N (742) 
                 G305Q (743) 
                 G305H (744) 
               
               
                 V307N (745) 
                 V307Q (746) 
                 V307H (747) 
                 S308N (748) 
                 S308Q (749) 
                 S308H (750) 
                 G309N (751) 
               
               
                 G309Q (752) 
                 G309H (753) 
                 G311N (754) 
                 G311Q (755) 
                 G311H (756) 
                 V313N (757) 
                 V313Q (758) 
               
               
                 V313H (759) 
                 G317N (760) 
                 G317Q (761) 
                 G317H (762) 
                 S319N (763) 
                 S319Q (764) 
                 S319H (765) 
               
               
                 A320N (766) 
                 A320Q (767) 
                 A320H (768) 
                 V322N (769) 
                 V322Q (770) 
                 V322H (771) 
                 V328N (772) 
               
               
                 V328Q (773) 
                 V328H (774) 
                 V331N (775) 
                 V331Q (776) 
                 V331H (777) 
                 A334N (778) 
                 A334Q (779) 
               
               
                 A334H (780) 
                 T335N (781) 
                 T335Q (782) 
                 T335H (783) 
                 S339N (784) 
                 S339Q (785) 
                 S339H (786) 
               
               
                 T340N (787) 
                 T340Q (788) 
                 T340H (789) 
                 T343N (790) 
                 T343Q (791) 
                 T343H (792) 
                 I344N (793) 
               
               
                 I344Q (794) 
                 I344H (795) 
                 A351N (796) 
                 A351Q (797) 
                 A351H (798) 
                 G352N (799) 
                 G352Q (800) 
               
               
                 G352H (801) 
                 G356N (802) 
                 G356Q (803) 
                 G356H (804) 
                 G357N (805) 
                 G357Q (806) 
                 G357H (807) 
               
               
                 S360N (808) 
                 S360Q (809) 
                 S360H (810) 
                 G363N (811) 
                 G363Q (812) 
                 G363H (813) 
                 S365N (814) 
               
               
                 S365Q (815) 
                 S365H (816) 
                 G366N (817) 
                 G366Q (818) 
                 G366H (819) 
                 G367N (820) 
                 G367Q (821) 
               
               
                 G367H (822) 
                 V370N (823) 
                 V370Q (824) 
                 V370H (825) 
                 T371N (826) 
                 T371Q (827) 
                 T371H (828) 
               
               
                 V373N (829) 
                 V373Q (830) 
                 V373H (831) 
                 G375N (832) 
                 G375Q (833) 
                 G375H (834) 
                 T376N (835) 
               
               
                 T376Q (836) 
                 T376H (837) 
                 S377N (838) 
                 S377Q (839) 
                 S377H (840) 
                 T380N (841) 
                 T380Q (842) 
               
               
                 T380H (843) 
                 G381N (844) 
                 G381Q (845) 
                 G381H (846) 
                 I382N (847) 
                 I382Q (848) 
                 I382H (849) 
               
               
                 I383N (850) 
                 I383Q (851) 
                 I383H (852) 
                 S384N (853) 
                 S384Q (854) 
                 S384H (855) 
                 G386N (856) 
               
               
                 G386Q (857) 
                 G386H (858) 
                 A390N (859) 
                 A390Q (860) 
                 A390H (861) 
                 G393N (862) 
                 G393Q (863) 
               
               
                 G393H (864) 
                 G396N (865) 
                 G396Q (866) 
                 G396H (867) 
                 I397N (868) 
                 I397Q (869) 
                 I397H (870) 
               
               
                 T399N (871) 
                 T399Q (872) 
                 T399H (873) 
                 V401N (874) 
                 V401Q (875) 
                 V401H (876) 
                 S402N (877) 
               
               
                 S402Q (878) 
                 S402H (879) 
                 V405N (880) 
                 V405Q (881) 
                 V405H (882) 
                 I408N (883) 
                 I408Q (884) 
               
               
                 I408H (885) 
                 T412N (886) 
                 T412Q (887) 
                 T412H (888) 
                 T415N (889) 
                 T415Q (890) 
                 T415H (891) 
               
               
                 L165Q (1025) 
                 L165H (1026) 
                 F175H (1027) 
                 F178H (1028) 
                 F192H (1029) 
                 F192I (1030) 
                 F192V (1031) 
               
               
                 W194I (1032) 
                 L198Q (1033) 
                 L198H (1034) 
               
               
                   
               
            
           
         
       
     
     A modified FIX polypeptide provided herein that exhibits increased protease resistance can contain one or more amino acid modifications corresponding to any one or more modifications of Y1H (i.e., replacement of Y by H at a position corresponding to amino acid position 1 of mature human FIX (such as, SEQ ID NO: 2)), Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F91, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L14I, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L57I, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192IH, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, and T415N, where the modified polypeptide exhibits increased resistance to proteolysis. In some examples, the modifications are in an unmodified FIX polypeptide, such as a FIX having a sequence of amino acids set forth in SEQ ID NO: 2. Exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 3-891 or 1025-1034. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1036-1926 or 2035-2044. 
     Additionally, a modified FIX polypeptide as set forth above can contain a further modification compared to an unmodified FIX polypeptide. Generally, the resulting modified FIX polypeptide retains one or more activities of the unmodified FIX polypeptide. In some embodiments, the modified FIX polypeptide inhibits the activity of an unmodified or wild-type FIX polypeptide and/or acts as an anticoagulant. 
     i. Modified FIX Polypeptides Exhibiting Increased Protease Resistance to Elastase 
     Exemplary of modified FIX polypeptides exhibiting increased protease resistance are FIX polypeptides exhibiting increased resistance to elastase. Non-limiting modifications in a FIX polypeptide that confer increased resistance to elastase have been rationally determined herein based on the known substrate specificity of elastase. Polypeptides are cleaved by elastase at or near a sequence of small hydrophobic chains. Modifications that render polypeptides more resistant to hydrolysis by elastase are provided. Of the modifications disclosed in Table 5, some are more specific to resistance to protease digestion by elastase. The following is-HIT positions were identified to eliminate elastase sensitive sites and increase protein stability of FIX polypeptides: 3, 4, 10, 12, 24, 28, 31, 35, 38, 39, 46, 48, 53, 59, 60, 61, 66, 68, 76, 79, 86, 87, 90, 93, 102, 103, 107, 108, 110, 112, 114, 118, 123, 127, 128, 133, 135, 136, 137, 138, 140, 141, 144, 146; 148, 149, 153, 156, 158, 159, 161, 163, 164, 168, 169, 171, 172, 174, 179, 181, 182, 183, 184, 187, 190, 196, 197, 200, 202, 204, 207, 208, 209, 210, 211, 216, 217, 218, 219, 220, 223, 225, 226, 227, 229, 230, 231, 232, 233, 234, 238, 241, 244, 250, 251, 253, 254, 261, 262, 263, 270, 271, 280, 283, 285, 286, 288, 290, 291, 296, 298, 303, 304, 305, 307, 308, 309, 311, 313, 317, 319, 320, 322, 328, 331, 334, 335, 339, 340, 343, 344, 351, 352, 356, 357, 360, 363, 365, 366, 367, 370, 371, 373, 375, 376, 377, 380, 381, 382, 383, 384, 386, 390, 393, 396, 397, 399, 401, 402, 405, 408, 412, and 415, corresponding to amino acid positions in a mature FIX polypeptide set forth in SEQ ID NO: 2. Amino acid modifications can be at any one or more positions corresponding to any of the following positions: S3, G4, V10, G12, S24, A28, V31, T35, T38, T39, V46, G48, S53, G59, G60, S61, I66, S68, G76, G79, V86, T87, I90, G93, S102, A103, V107, V108, S110, T112, G114, A118, S123, A127, V128, G133, V135, S136, V137, S138, T140, S141, T144, A146, T148, V149, V153, V156, S158, T159, A161, T163, I164, 1168, T169, S171, T172, S174, T179, V181, V182, G183, G184, A187, G190, V196, V197, G200, V202, A204, G207, G208, S209, I210, V211, I216, V217, T218, A219, A220, V223, T225, G226, V227, I229, T230, V231, V232, A233, G234, I238, T241, T244, V250, I251, I253, I254, A261, A262, I263, I270, A271, V280, S283, V285, T286, I288, I290, A291, T296, I298, G303, S304, G305, V307, S308, G309, G311, V313, G317, S319, A320, V322, V328, V331, A334, T335, S339, T340, T343, I344, A351, G352, G356, G357, S360, G363, S365, G366, G367, V370, T371, V373, G375, T376, S377, T380, G381, I382, I383, S384, G386, A390, G393, I396, I397, T399, V401, S402, V405, I408, T412, and T415, of a mature FIX polypeptide set forth in SEQ ID NO: 2. Other candidate Leads can be obtained by replacement or replacements of amino acids at is-HIT positions such as, but not limited to, amino acid modifications as described for candidate LEADs in Table 6. Candidate leads can be empirically tested to determine those that confer resistance to elastase. Table 6 provides non-limiting examples of amino acid modifications that are more specific to resistance to protease digestion by elastase and, thereby, increase protein stability. Generally, a resulting modified FIX polypeptide retains one or more activities of a mature or unmodified FIX polypeptide. In some embodiments, the modified FIX polypeptide inhibits an activity of an unmodified or wild-type FIX polypeptide and/or acts as an anticoagulant. In Table 6 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution. 
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 List of Human FIX Modifications to Increase Resistance to Elastase Proteolysis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 S3N (403) 
                 S3Q (404) 
                 S3H (405) 
                 G4N (406) 
                 G4Q (407) 
                 G4H (408) 
                 V10N (409) 
               
               
                 V10Q (410) 
                 V10H (411) 
                 G12N (412) 
                 G12Q (413) 
                 G12H (414) 
                 S24N (415) 
                 S24Q (416) 
               
               
                 S24H (417) 
                 A28N (418) 
                 A28Q (419) 
                 A28H (420) 
                 V31N (421) 
                 V31Q (422) 
                 V31H (423) 
               
               
                 T35N (424) 
                 T35Q (425) 
                 T35H (426) 
                 T38N (427) 
                 T38Q (428) 
                 T38H (429) 
                 T39N (430) 
               
               
                 T39Q (431) 
                 T39H (432) 
                 V46N (433) 
                 V46Q (434) 
                 V46H (435) 
                 G48N (436) 
                 G48Q (437) 
               
               
                 G48H (438) 
                 S53N (439) 
                 S53Q (440) 
                 S53H (441) 
                 G59N (442) 
                 G59Q (443) 
                 G59H (444) 
               
               
                 G60N (445) 
                 G60Q (446) 
                 G60H (447) 
                 S61N (448) 
                 S61Q (449) 
                 S61H (450) 
                 I66N (451) 
               
               
                 I66Q (452) 
                 I66H (453) 
                 S68N (454) 
                 S68Q (455) 
                 S68H (456) 
                 G76N (457) 
                 G76Q (458) 
               
               
                 G76H (459) 
                 G79N (460) 
                 G79Q (461) 
                 G79H (462) 
                 V86N (463) 
                 V86Q (464) 
                 V86H (465) 
               
               
                 T87N (466) 
                 T87Q (467) 
                 T87H (468) 
                 I90N (469) 
                 I90Q (470) 
                 I90H (471) 
                 G93N (472) 
               
               
                 G93Q (473) 
                 G93H (474) 
                 S102N (475) 
                 S102Q (476) 
                 S102H (477) 
                 A103N (478) 
                 A103Q (479) 
               
               
                 A103H (480) 
                 V107N (481) 
                 V107Q (482) 
                 V107H (483) 
                 V108N (484) 
                 V108Q (485) 
                 V108H (486) 
               
               
                 S110N (487) 
                 S110Q (488) 
                 S110H (489) 
                 T112N (490) 
                 T112Q (491) 
                 T112H (492) 
                 G114N (493) 
               
               
                 G114Q (494) 
                 G114H (495) 
                 A118N (496) 
                 A118Q (497) 
                 A118H (498) 
                 S123N (499) 
                 S123Q (500) 
               
               
                 S123H (501) 
                 A127N (502) 
                 A127Q (503) 
                 A127H (504) 
                 V128N (505) 
                 V128Q (506) 
                 V128H (507) 
               
               
                 G133N (508) 
                 G133Q (509) 
                 G133H (510) 
                 V135N (511) 
                 V135Q (512) 
                 V135H (513) 
                 S136N (514) 
               
               
                 S136Q (515) 
                 S136H (516) 
                 V137N (517) 
                 V137Q (518) 
                 V137H (519) 
                 S138N (520) 
                 S138Q (521) 
               
               
                 S138H (522) 
                 T140N (523) 
                 T140Q (524) 
                 T140H (525) 
                 S141N (526) 
                 S141Q (527) 
                 S141H (528) 
               
               
                 T144N (529) 
                 T144Q (530) 
                 T144H (531) 
                 A146N (532) 
                 A146Q (533) 
                 A146H (534) 
                 T148N (535) 
               
               
                 T148Q (536) 
                 T148H (537) 
                 V149N (538) 
                 V149Q (539) 
                 V149H (540) 
                 V153N (541) 
                 V153Q (542) 
               
               
                 V153H (543) 
                 V156N (544) 
                 V156Q (545) 
                 V156H (546) 
                 S158N (547) 
                 S158Q (548) 
                 S158H (549) 
               
               
                 T159N (550) 
                 T159Q (551) 
                 T159H (552) 
                 A161N (553) 
                 A161Q (554) 
                 A161H (555) 
                 T163N (556) 
               
               
                 T163Q (557) 
                 T163H (558) 
                 I164N (559) 
                 I164Q (560) 
                 I164H (561) 
                 I168N (562) 
                 I168Q (563) 
               
               
                 I168H (564) 
                 T169N (565) 
                 T169Q (566) 
                 T169H (567) 
                 S171N (568) 
                 S171Q (569) 
                 S171H (570) 
               
               
                 T172N (571) 
                 T172Q (572) 
                 T172H (573) 
                 S174N (574) 
                 S174Q (575) 
                 S174H (576) 
                 T179N (577) 
               
               
                 T179Q (578) 
                 T179H (579) 
                 V181N (580) 
                 V181Q (581) 
                 V181H (582) 
                 V182N (583) 
                 V182Q (584) 
               
               
                 V182H (585) 
                 G183N (586) 
                 G183Q (587) 
                 G183H (588) 
                 G184N (589) 
                 G184Q (590) 
                 G184H (591) 
               
               
                 A187N (592) 
                 A187Q (593) 
                 A187H (594) 
                 G190N (595) 
                 G190Q (596) 
                 G190H (597) 
                 V196N (598) 
               
               
                 V196Q (599) 
                 V196H (600) 
                 V197N (601) 
                 V197Q (602) 
                 V197H (603) 
                 G200N (604) 
                 G200Q (605) 
               
               
                 G200H (606) 
                 V202N (607) 
                 V202Q (608) 
                 V202H (609) 
                 A204N (610) 
                 A204Q (611) 
                 A204H (612) 
               
               
                 G207N (613) 
                 G207Q (614) 
                 G207H (615) 
                 G208N (616) 
                 G208Q (617) 
                 G208H (618) 
                 S209N (619) 
               
               
                 S209Q (620) 
                 S209H (621) 
                 I210N (622) 
                 I210Q (623) 
                 I210H (624) 
                 V211N (625) 
                 V211Q (626) 
               
               
                 V211H (627) 
                 I216N (628) 
                 I216Q (629) 
                 I216H (630) 
                 V217N (631) 
                 V217Q (632) 
                 V217H (633) 
               
               
                 T218N (634) 
                 T218Q (635) 
                 T218H (636) 
                 A219N (637) 
                 A219Q (638) 
                 A219H (639) 
                 A220N (640) 
               
               
                 A220Q (641) 
                 A220H (642) 
                 V223N (643) 
                 V223Q (644) 
                 V223H (645) 
                 T225N (646) 
                 T225Q (647) 
               
               
                 T225H (648) 
                 G226N (649) 
                 G226Q (650) 
                 G226H (651) 
                 V227N (652) 
                 V227Q (653) 
                 V227H (654) 
               
               
                 I229N (655) 
                 I229Q (656) 
                 I229H (657) 
                 T230N (658) 
                 T230Q (659) 
                 T230H (660) 
                 V231N (661) 
               
               
                 V231Q (662) 
                 V231H (663) 
                 V232N (664) 
                 V232Q (665) 
                 V232H (666) 
                 A233N (667) 
                 A233Q (668) 
               
               
                 A233H (669) 
                 G234N (670) 
                 G234Q (671) 
                 G234H (672) 
                 I238N (673) 
                 I238Q (674) 
                 I238H (675) 
               
               
                 T241N (676) 
                 T241Q (677) 
                 T241H (678) 
                 T244N (679) 
                 T244Q (680) 
                 T244H (681) 
                 V250N (682) 
               
               
                 V250Q (683) 
                 V250H (684) 
                 I251N (685) 
                 I251Q (686) 
                 I251H (687) 
                 I253N (688) 
                 I253Q (689) 
               
               
                 I253H (690) 
                 I254N (691) 
                 I254Q (692) 
                 I254H (693) 
                 A261N (694) 
                 A261Q (695) 
                 A261H (696) 
               
               
                 A262N (697) 
                 A262Q (698) 
                 A262H (699) 
                 I263N (700) 
                 I263Q (701) 
                 I263H (702) 
                 I270N (703) 
               
               
                 I270Q (704) 
                 I270H (705) 
                 A271N (706) 
                 A271Q (707) 
                 A271H (708) 
                 V280N (709) 
                 V280Q (710) 
               
               
                 V280H (711) 
                 S283N (712) 
                 S283Q (713) 
                 S283H (714) 
                 V285N (715) 
                 V285Q (716) 
                 V285H (717) 
               
               
                 T286N (718) 
                 T286Q (719) 
                 T286H (720) 
                 I288N (721) 
                 I288Q (722) 
                 I288H (723) 
                 I290N (724) 
               
               
                 I290Q (725) 
                 I290H (726) 
                 A291N (727) 
                 A291Q (728) 
                 A291H (729) 
                 T296N (730) 
                 T296Q (731) 
               
               
                 T296H (732) 
                 I298N (733) 
                 I298Q (734) 
                 I298H (735) 
                 G303N (736) 
                 G303Q (737) 
                 G303H (738) 
               
               
                 S304N (739) 
                 S304Q (740) 
                 S304H (741) 
                 G305N (742) 
                 G305Q (743) 
                 G305H (744) 
                 V307N (745) 
               
               
                 V307Q (746) 
                 V307H (747) 
                 S308N (748) 
                 S308Q (749) 
                 S308H (750) 
                 G309N (751) 
                 G309Q (752) 
               
               
                 G309H (753) 
                 G311N (754) 
                 G311Q (755) 
                 G311H (756) 
                 V313N (757) 
                 V313Q (758) 
                 V313H (759) 
               
               
                 G317N (760) 
                 G317Q (761) 
                 G317H (762) 
                 S319N (763) 
                 S319Q (764) 
                 S319H (765) 
                 A320N (766) 
               
               
                 A320Q (767) 
                 A320H (768) 
                 V322N (769) 
                 V322Q (770) 
                 V322H (771) 
                 V328N (772) 
                 V328Q (773) 
               
               
                 V328H (774) 
                 V331N (775) 
                 V331Q (776) 
                 V331H (777) 
                 A334N (778) 
                 A334Q (779) 
                 A334H (780) 
               
               
                 T335N (781) 
                 T335Q (782) 
                 T335H (783) 
                 S339N (784) 
                 S339Q (785) 
                 S339H (786) 
                 T340N (787) 
               
               
                 T340Q (788) 
                 T340H (789) 
                 T343N (790) 
                 T343Q (791) 
                 T343H (792) 
                 I344N (793) 
                 I344Q (794) 
               
               
                 I344H (795) 
                 A351N (796) 
                 A351Q (797) 
                 A351H (798) 
                 G352N (799) 
                 G352Q (800) 
                 G352H (801) 
               
               
                 G356N (802) 
                 G356Q (803) 
                 G356H (804) 
                 G357N (805) 
                 G357Q (806) 
                 G357H (807) 
                 S360N (808) 
               
               
                 S360Q (809) 
                 S360H (810) 
                 G363N (811) 
                 G363Q (812) 
                 G363H (813) 
                 S365N (814) 
                 S365Q (815) 
               
               
                 S365H (816) 
                 G366N (817) 
                 G366Q (818) 
                 G366H (819) 
                 G367N (820) 
                 G367Q (821) 
                 G367H (822) 
               
               
                 V370N (823) 
                 V370Q (824) 
                 V370H (825) 
                 T371N (826) 
                 T371Q (827) 
                 T371H (828) 
                 V373N (829) 
               
               
                 V373Q (830) 
                 V373H (831) 
                 G375N (832) 
                 G375Q (833) 
                 G375H (834) 
                 T376N (835) 
                 T376Q (836) 
               
               
                 T376H (837) 
                 S377N (838) 
                 S377Q (839) 
                 S377H (840) 
                 T380N (841) 
                 T380Q (842) 
                 T380H (843) 
               
               
                 G381N (844) 
                 G381Q (845) 
                 G381H (846) 
                 I382N (847) 
                 I382Q (848) 
                 I382H (849) 
                 I383N (850) 
               
               
                 I383Q (851) 
                 I383H (852) 
                 S384N (853) 
                 S384Q (854) 
                 S384H (855) 
                 G386N (856) 
                 G386Q (857) 
               
               
                 G386H (858) 
                 A390N (859) 
                 A390Q (860) 
                 A390H (861) 
                 G393N (862) 
                 G393Q (863) 
                 G393H (864) 
               
               
                 G396N (865) 
                 G396Q (866) 
                 G396H (867) 
                 I397N (868) 
                 I397Q (869) 
                 I397H (870) 
                 T399N (871) 
               
               
                 T399Q (872) 
                 T399H (873) 
                 V401N (874) 
                 V401Q (875) 
                 V401H (876) 
                 S402N (877) 
                 S402Q (878) 
               
               
                 S402H (879) 
                 V405N (880) 
                 V405Q (881) 
                 V405H (882) 
                 I408N (883) 
                 I408Q (884) 
                 I408H (885) 
               
               
                 T412N (886) 
                 T412Q (887) 
                 T412H (888) 
                 T415N (889) 
                 T415Q (890) 
                 T415H (891) 
               
               
                   
               
            
           
         
       
     
     A modified FIX polypeptide provided herein that exhibits increased protease resistance to elastase can contain one or more amino acid modifications corresponding to modifications selected from any of S3Q, S3H, S3N, G4Q, G4H, 04N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T14811, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V197Q, V197H, V19′7N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V21111, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2. In some examples, the modifications are in a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2. Exemplary modified FIX candidate LEAD polypeptides are set forth in any one of SEQ ID NOS: 3-891 or 1025-1034. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1036-1926 or 2035-2044. 
     e. Assessment of FIX Variants with Increased Resistance to Proteolysis 
     Increased resistance to proteolysis of FIX variants can be assessed by any methods known in the art to assess protein stability, thermal tolerance, protease sensitivity and resistance and/or FIX activity. In one example, protease resistance is measured by incubating a modified FIX polypeptide with one or more proteases and then assessing residual activity compared to an untreated control. A modified FIX can be compared with an unmodified and/or wild-type native FIX treated under similar conditions to determine if the particular variant retains more activity than the unmodified FIX. Activity can be assessed by any methods known in the art, for example by measuring coagulation activities. 
     Kinetic studies of protease resistance also can be used to assess a modified FIX polypeptide. For example, a modified FIX polypeptide is incubated with one or more proteases and samples are taken over a series of time-points. At each time point, the proteases are inactivated and the samples are then tested for FIX activity. In one embodiment, the modified polypeptide is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistant to proteolysis. 
     In one exemplary embodiment, FIX variants are assessed for protease resistance with a mixture of proteases and proteolytic conditions including pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. For example, a cytopathic effects (CPE) assay can be used to assess coagulation activity of modified FIX polypeptides compared to unmodified FIX polypeptides. Specifically, coagulation activity of FIX can determined by the capacity of the modified FIX polypeptides to induce formation of a clot in an assay, such as an activated partial thromboplastin time assay. The resistance of the modified FIX polypeptides compared to wild-type FIX against enzymatic cleavage can be analyzed by mixing FIX polypeptides with proteases. After exposure to proteases, coagulation activities can be assessed. 
     In one example, activity of modified hFIX is assessed in an assay by measuring the capacity of the modified hFIX to modulate coagulation when added to the sample. Prior to the measurement of activity, hFIX molecules can be challenged with proteases (blood, intestinal, etc.) including conditions mimicking administered conditions, such as serum, blood, saliva, or digestive assays (in vitro assays), and/or administered to a subject such as a mouse or human (in vivo assays) during different incubation or post-injection times. The activity measured, corresponds then to the residual activity following exposure to the proteolytic mixtures. Activity can be compared with an unmodified FIX as a measurement of the effect of the modification on protease stability and on the activity. In one example, the unmodified FIX is a wild-type native FIX. In another example, the unmodified FIX is a variant form of FIX that was used as a starting material to introduce further modifications. Modified FIX polypeptides also can be compared with any known FIX polypeptide in any assay known in the art to compare protease sensitivity, thermal tolerance and/or any other activity. 
     2. Super-LEADs 
     Modification of FIX polypeptides also can include combining two or more modifications as set forth above. Modified FIX super-LEAD polypeptides are a combination of single amino acid mutations present in two or more of the respective modified FIX LEAD polypeptides. Thus, modified FIX super-LEAD polypeptides have two or more of the single amino acid replacements derived from two or more of the respective modified FIX LEAD polypeptides. As described above and in detail below, modified FIX polypeptides provided herein exhibit increased protein stability manifested as an increased resistance to proteolysis. Typically, modified FIX LEAD polypeptides created are those whose performance has been optimized with respect to the unmodified polypeptide by modification of a single amino acid replacement at one is-HIT position. Modified FIX super-Lead polypeptides are created such that the polypeptide contains two or more FIX LEAD modifications, each at a different is-HIT position. Modifications that increase proteolysis resistance can be added to other modifications provided herein or known in the art to increase proteolysis resistance. In one example, modifications that increase stability can be added to other modifications provided herein or known in the art to increase protein stability. In another example, modifications that increase stability can be added to modifications provided herein or known in the art to increase proteolysis resistance. Modifications that increase protease resistance and/or stability also can be added to modifications to FIX that alter other functionalities including activity, modifications that affect post-translation protein modifications and any other known modifications in the art. 
     Once the modified LEAD polypeptides have been identified using, for example, 2D-scanning methods, super-LEADs can be generated by combining two or more individual LEADs using methods well known in the art, such as recombination, mutagenesis and DNA shuffling, and by methods such as additive directional mutagenesis, 3D-scanning, and multi-overlapped primer extensions, as provided above. 
     Exemplary modified FIX super-LEAD polypeptides exhibiting increased protein stability can include FIX polypeptides containing two or more amino acid modifications as compared to an unmodified FIX polypeptide. In some examples, a FIX polypeptide can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. Generally, the resulting FIX polypeptide exhibits increased protein stability and retains at least one activity of an unmodified FIX polypeptide. A modified FIX polypeptide can include any two or more amino acid modifications set forth in Table 5 above. For example, the modified FIX polypeptide can contain two or more amino acid modifications corresponding to any two or more modifications of Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L14I, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M19I, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F321, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y451, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F751, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192IH, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2. In some examples, the modifications are in an unmodified FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2. Provided herein are modified FIX Super-LEAD polypeptides containing two or more amino acid modifications and exhibiting increased protein stability having a sequence of amino acids set forth in any of SEQ ID NOS: 917-1024. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1927-2034. 
     In one non-limiting example, a FIX polypeptide can contain a modification at a position corresponding to F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2, and can contain further amino acid modifications, such as any modification set forth in Table 5. Exemplary super-LEAD FIX polypeptides are set forth in Table 7 with the sequence identifier (SEQ ID NO.) in parenthesis next to each substitution. Resulting super-LEADs can be tested for one or more parameters to assess protein stability (such as, increased resistance to proteases) using any of the assays described herein, such as is described in the Examples. 
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Exemplary Modified FIX Super-LEADs to Increase Protein Stability 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 T163H/F192I (917) 
                 I164Q/F192I (918) 
                 T169H/F192I (919) 
                 S171Q/F192I (920) 
               
               
                 D152N/D154N/F192I (921) 
                 S174Q/F192I (922) 
                 S174H/F192I (923) 
                 T179Q/F192I (924) 
               
               
                 T179H/F192I (925) 
                 T163N/I164N/F192I (926) 
                 D166Q/F192I (927) 
                 D166N/F192I (928) 
               
               
                 D186Q/F192I (929) 
                 D186N/F192I (930) 
                 T148A/D152N/ 
                 D154N/F192I (931) 
               
               
                 F192I/D203Q (932) 
                 F192I/D203N (933) 
                 D152N/F192I (934) 
                 D152Q/F192I (935) 
               
               
                 T148A/D152N/ 
                 F192I (936) 
                 D154N/F192I (937) 
                 D154Q/F192I (938) 
               
               
                 T163N/I164N (939) 
                 A187N/G190N (940) 
                 V196N/V197N (941) 
                 T140N/T144N (942) 
               
               
                 F175I/F178I (943) 
                 F192I/W194I (944) 
                 T148A/D152N (945) 
                 T148A/D152Q (946) 
               
               
                 T148A/D154N (947) 
                 T148A/D154Q (948) 
                 D152N/D154N (949) 
                 I168N/T169N/S171N/ 
               
               
                   
                   
                   
                 T172N/S174N (950) 
               
               
                 T179N/V181N/V182N/ 
                 D152N/D154N/T148A (952) 
                 T163H/D166Q/F192I (953) 
                 T163H/D166N/F192I (954) 
               
               
                 G183N/G184N (951) 
               
               
                 T163H/D186Q/F192I (955) 
                 T163H/D186N/F192I (956) 
                 T163H/F192I/D203Q (957) 
                 T163H/F192I/D203N (958) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/T163H/ 
                 I164Q/D166Q/F192I (961) 
                 I164Q/D166N/F192I (962) 
               
               
                 T163H/F192I (959) 
                 F192I (960) 
               
               
                 I164Q/D186Q/F192I (963) 
                 I164Q/D186N/F192I (964) 
                 I164Q/F192I/D203Q (965) 
                 I164Q/F192I/D203N (966) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/I164Q/ 
                 T163N/I164N/D166Q/ 
                 T163N/I164N/D166N/ 
               
               
                 I164Q/F192I (967) 
                 F192I (968) 
                 F192I (969) 
                 F192I (970) 
               
               
                 T163N/I164N/D186Q/ 
                 T163N/I164N/D186N/ 
                 T163N/I164N/F192I/ 
                 T163N/I164N/F192I/ 
               
               
                 F192I (971) 
                 F192I (972) 
                 D203Q (973) 
                 D203N (974) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/T163N/ 
                 D166Q/T169H/F192I (977) 
                 D166N/T169H/F192I (978) 
               
               
                 T163N/I164N/F192I (975) 
                 I164N/F192I (976) 
               
               
                 T169H/D186Q/F192I (979) 
                 T169H/D186N/F192I (980) 
                 T169H/F192I/D203Q (981) 
                 T169H/F192I/D203N (982) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/T169H/ 
                 D166Q/S171Q/F192I (985) 
                 D166N/S171Q/F192I (986) 
               
               
                 T169H/F192I (983) 
                 F192I (984) 
               
               
                 S171Q/D186Q/F192I (987) 
                 S171Q/D186N/F192I (988) 
                 S171Q/F192I/D203Q (989) 
                 S171Q/F192I/D203N (990) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/S171Q/ 
                 D166Q/S174Q/F192I (993) 
                 D166N/S174Q/F192I (994) 
               
               
                 S171Q/F192I (991) 
                 F192I (992) 
               
               
                 S174Q/D186Q/F192I (995) 
                 S174Q/D186N/F192I (996) 
                 S174Q/F192I/D203Q (997) 
                 S174Q/F192I/D203N (998) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/S174Q/ 
                 D166Q/S174H/F192I (1001) 
                 D166N/S174H/F192I (1002) 
               
               
                 S174Q/F192I (999) 
                 F192I (1000) 
               
               
                 S174H/D186Q/F192I (1003) 
                 S174H/D186N/F192I (1004) 
                 S174H/F192I/D203Q (1005) 
                 S174H/F192I/D203N (1006) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/S174H/ 
                 D166Q/T179Q/F192I (1009) 
                 D166N/T179Q/F192I (1010) 
               
               
                 S174H/F192I (1007) 
                 F192I (1008) 
               
               
                 T179Q/D186Q/F192I (1011) 
                 T179Q/D186N/F192I (1012) 
                 T179Q/F192I/D203Q (1013) 
                 T179Q/F192I/D203N (1014) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/T179Q/ 
                 D166Q/T179H/F192I (1017) 
                 D166N/T179H/F192I (1018) 
               
               
                 T179Q/F192I (1015) 
                 F192I (1016) 
               
               
                 T179H/D186Q/F192I (1019) 
                 T179H/D186N/F192I (1020) 
                 T179H/F192I/D203Q (1021) 
                 T179H/F192I/D203N (1022) 
               
               
                 T148A/D152N/D154N/ 
                 T148A/D154N/T179H/ 
               
               
                 T179H/F192I (1023) 
                 F192I (1024) 
               
               
                   
               
            
           
         
       
     
     Non-limiting examples of FIX SuperLEAD polypeptides containing two or more amino acid modifications and exhibiting increased resistance to proteolysis can include amino acid replacements at amino acid residues corresponding to T163H/F192I, I164Q/F192I, T169H/F192I, S171Q/F192I, D152N/D154N/F192I, S174Q/F192I, S174H/F192I, T179Q/F192I, T179H/F192I, T163N/I164N/F192I, D166Q/F192I, D166N/F192I, D186Q/F192I, D186N/F192I, T148A/D152N/D154N/F192I, F192I/D203Q, F192I/D203N, D152N/F192I, D152Q/F192I, T148A/D154N/F192I, D154N/F192I, D154Q/F192I, T163N/I164N, A187N/G190N, V196N/V197N, T140N/T144N, F175I/F178I, F192I/W194I, T148A/D152N, T148A/D152Q, T148A/D154N, T148A/D154Q, D152N/D154N, I168N/T169N/S171N/T172N/S174N, T179N/V181N/V182N/G183N/G184N, D152N/D154N/T148A, T163H/D166Q/F192I, T163H/D166N/F192I, T163H/D186Q/F192I, T163H/D186N/F192I, T163H/F192I/D203Q, T163H/F192I/D203N, T148A/D152N/D154N/T163H/F192I, T148A/D154N/T163H/F192I, I164Q/D166Q/F192I, I164Q/D166N/F192I, I164Q/D186Q/F192I, I164Q/D186N/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D152N/D154N/I164Q/F192I, T148A/D154N/I164Q/F192I, T163N/I164N/D166Q/F192I, T163N/I164N/D166N/F192I, T163N/I164N/D186Q/F192I, T163N/I164N/D186N/F192I, T163N/I164N/F192I/D203Q, T163N/I164N/F192I/D203N, T148A/D152N/D154N/T163N/I164N/F192I, T148A/D154N/T163N/I164N/F192I, D166Q/T169H/F192I, D166N/T169H/F192I, T169H/D186Q/F192I, T169H/D186N/F192I, T169H/F192I/D203Q, T169H/F192I/D203N, T148A/D152N/D154N/T169H/F192I, T148A/D154N/T169H/F192I, D166Q/S171Q/F192I, D166N/S171Q/F192I, S171Q/D186Q/F192I, S171Q/D186N/F192I, S171Q/F192I/D203Q, S171Q/F192I/D203N, T148A/D152N/D154N/S171Q/F192I, T148A/D154N/S171Q/F192I, D166Q/S174Q/F192I, D166N/S174Q/F192I, S174Q/D186Q/F192I, S174Q/D186N/F192I, S174Q/F192I/D203Q, S174Q/F192I/D203N, T148A/D152N/D154N/S174Q/F192I, T148A/D154N/S174Q/F192I, D166Q/S174H/F192I, D166N/S174H/F192I, S174H/D186Q/F192I, S174H/D186N/F192I, S174H/F192I/D203 Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T148A/D154N/S174H/F192I, D166Q/T179Q/F192I, D166N/T179Q/F192I, T179Q/D186Q/F192I, T179Q/D186N/F192I, T179Q/F192I/D203Q, T179Q/F192I/D203N, T148A/D152N/D154N/T179Q/F192I, T148A/D154N/T179Q/F192I, D166Q/T179H/F192I, D166N/T179H/F192I, T179H/D186Q/F192I, T179H/D186N/F192I, T179H/F192I/D203Q, T179H/F192I/D203N, T148A/D152N/D154N/T179H/F192I, and T148A/D154N/T179H/F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Exemplary modified FIX SuperLEAD polypeptides containing two or more amino acid modifications and exhibiting increased protease resistance have a sequence of amino acids set forth in any one of SEQ ID NOS: 917-1024 or 1927-2034. 
     3. Other FIX Modifications 
     In addition to any one or more amino acid modifications provided herein, a modified FIX polypeptide also can contain one or more other modifications, including those known to those of skill in the art, such as PEGylation, hyperglycosylation, deimmunization and others (see such as U.S. Pat. Nos. 6,277,618, 6,315,995, and 6,531,298 and U.S. Patent Publication Nos. 2004-0102388, 2004-0110675, 2004-0254106, 2005-0100982, and 2006-0040856). Generally, the modification results in increased stability without losing at least one activity, such as blood coagulation activity (i.e. retains at least one activity as defined herein) of an unmodified FIX polypeptide. For example, other further modifications in a FIX polypeptide include one or more additional amino acid modifications and/or one or more chemical modifications. Such modifications include, but are not limited to, those that alter the immunogenicity, glycosylation, activity, or any other known property of a FIX polypeptide. In another example, chemical modifications include post-translational modifications of a protein, such as for example, glycosylation by a carbohydrate moiety; acylation; methylation; phosphorylation; sulfation; prenylation; Vitamin C-dependent modifications such as for example, proline, aspartic acid, lysine hydroxylations and carboxy terminal amidation; Vitamin K-dependent modifications such as for example, carboxylation of glutamic acid residues (i.e. gla residue); and incorporation of selenium to form a selenocysteine. Other protein modifications of a FIX polypeptide include PEGylation. In addition, protein modifications also can include modification to facilitate the detection, purification and assay development of a polypeptide, such as for example, modification of a polypeptide with a Sulfo-NHS-LC-biotin for covalent attachment to a primary amine on a protein, or other similar modification for florescent, non-isotopic or radioactive labels. Exemplary further modifications in a FIX polypeptide are described below. Modified polypeptides that are conjugates and/or labeled also are provided. For example, provided herein are modified polypeptides that are conjugated to a PEG moiety or contain a carbohydrate moiety covalently linked to one or more glycosylation sites on the polypeptide. 
     In another embodiment, other known properties of a FIX polypeptide can be modified in addition to any one or more amino acid modifications provided herein. Such modifications include, but are not limited to, alteration of the peptidase activity, coagulant activity, esterase activity, protein binding activities (such as FVIII), cofactor binding activities, such as Ca 2+ , phospholipid or cell surface binding activities, and increased resistance to inhibitors, such as heparin and warfarin. Resulting modified FIX polypeptides can be tested for one or more parameters to assess polypeptide properties, such as protein stability (such as, increased resistance to proteases), or polypeptide activities, such as coagulant activity, using any of the assays described herein. 
     In yet another embodiment, known properties of a FIX polypeptide, as mentioned above, can be modified by generation of chimeric FIX polypeptides, in which all, or portions thereof, of FIX polypeptide domains are replaced by homologous domains of other coagulation factor family members. Such chimeric proteins are known in the art and can be combined with the methods for modification of FIX polypeptides provided herein (International PCT application No. WO 2006/018204 A1, Lin et al. (1990)  J. Biol. Chem.  265(1):144-150 and Cheung et al. (1991)  J. Biol. Chem.  266(14):8797-880). An exemplary chimeric protein has a sequence of amino acids as set forth in SEQ ID NO: 1035. 
     a. Immunogenicity 
     There are many instances where the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. An immune response to a therapeutic protein, such as FIX, proceeds via the MHC class II peptide presentation pathway. Here, exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells, amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell receptor on the surface of the T cell, together with the cross binding of certain other co-receptors, such as the CD4 molecule, can induce an activated state within the T cell. Activation leads to the release of cytokines, further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response. 
     The ability of a peptide (T cell epitope) to bind a given MHC class II molecule for presentation on the surface of an APC is dependent on a number of factors, most notably its primary sequence. This will influence its propensity for proteolytic cleavage and also its affinity for binding within the peptide binding cleft of the MHC class II molecule. The MHC class II/peptide complex on the APC surface presents a binding face to a particular T cell receptor (TCR) able to recognize determinants provided by exposed residues of the peptide and the MHC class II molecule. 
     Formation of inhibitory antibodies to therapeutic FIX polypeptides is known in the art (Herzog et al. (2004)  Semin. Thromb. Hemost.  30(2): 215-226). Hence, the combination modified FIX polypeptides provided herein with modifications to decrease overall immunogenicity of the modified FIX polypeptides can improve the therapeutic properties of the FIX polypeptide. The identification of T cell epitopes can be carried out according to methods known in the art (see such as, U.S. Patent Publication No. 20040254106) and can be used to identify the binding propensity of FIX peptides to an MHC class II molecule. 
     Further modifications to a modified FIX polypeptide provided herein can include modifications of at least one amino acid residue resulting in a substantial reduction in activity of or elimination of one or more T cell epitopes from the protein, i.e. deimmunization of the polypeptide. One or more amino acid modification at particular positions within any of the MHC class II ligands can result in a deimmunized FIX polypeptide with a reduced immunogenic when administered as a therapeutic to a host, such as for example, a human host. 
     Exemplary amino acid positions for modification of a T cell epitope, and thereby a deimmunized FIX polypeptide with a reduced immunogenic potential, include amino acid modifications at one or more positions corresponding to any of the following positions: Y1, S3, L6, F9, V10, Q11, G12, N13, L14, E15, R16, E17, C18, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, F41, W42, K43, Q44, Y45, V46, D47, G48, D49, Q50, C51, E52, S53, N54, L57, G60, C62, K63, D65, I66, Y69, C71, W72, F75, F77, L84, V86, I90, K91, N92, G93, R94, C95, E96, Q97, F98, K100, N101, A103, D104, K106, V107, V108, V108, S110, C111, T112, E113, G114, Y115, R116, L117, A118, N120, Q121, S123, V128, F130, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, F150, D152, V153, D154, Y155, V156, N157, S158, T159, E160, A161, E162, T163, I164, L165, 1168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, G190, F192, W194, Q195, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, N212, E213, K214, W215, I216, V217, A219, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, H236, N237, I238, E239, E240, V250, I251, I253, I254, P255, H256, H257, N258, Y259, N260, A261, A262, I263, N264, K265, Y266, N267, H268, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, I288, C289, I290, A291, D292, K293, Y295, T296, N297, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, W310, G311, V313, F314, H315, K316, G317, R318, S319, A320, L321, V322, L323, Q324, Y325, L326, R327, V328, L330, V331, D332, R333, A334, T335, C336, L337, R338, S339, K341, F342, T343, I344, Y345, N346, N347, M348, F349, C350, A351, G352, F353, H354, E355, G356, G357, R358, D359, S360, C361, V370, V373, E374, G375, S377, F378, L379, T380, G381, I382, I383, S384, W385, E387, E388, A390, M391, K392, G393, Y395, Y398, K400, V401, S402, R403, Y404, V405, N406, W407, I408, K409, E410, K411, K413, of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Exemplary amino acid modifications that can contribute to reduced immunogenicity of a FIX polypeptide include any one or more amino acid modifications corresponding to any one or more modifications set forth in Table 8 corresponding to amino acid positions of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 List of human FIX Modifications for Decreased Immunogenicity 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Y1A 
                 Y1C 
                 Y1D 
                 Y1E 
                 Y1G 
                 Y1H 
                 Y1K 
                 Y1N 
                 Y1P 
                 Y1Q 
                 Y1R 
                 Y1S 
               
               
                 Y1T 
                 S3T 
                 L6A 
                 L6C 
                 L6D 
                 L6E 
                 L6G 
                 L6H 
                 L6K 
                 L6N 
                 L6P 
                 L6Q 
               
               
                 L6R 
                 L6S 
                 L6T 
                 L6M 
                 F9A 
                 F9C 
                 F9D 
                 F9E 
                 F9G 
                 F9H 
                 F9K 
                 F9N 
               
               
                 F9P 
                 F9Q 
                 F9R 
                 F9S 
                 F9T 
                 F9I 
                 F9M 
                 F9W 
                 V10A 
                 V10C 
                 V10D 
                 V10E 
               
               
                 V10G 
                 V10H 
                 V10K 
                 V10N 
                 V10P 
                 V10Q 
                 V10R 
                 V10S 
                 V10T 
                 V10F 
                 V10I 
                 V10M 
               
               
                 V10W 
                 V10Y 
                 Q11A 
                 Q11C 
                 Q11G 
                 Q11P 
                 G12D 
                 G12E 
                 G12G 
                 G12H 
                 G12K 
                 G12N 
               
               
                 G12P 
                 G12Q 
                 G12R 
                 G12S 
                 G12T 
                 N13A 
                 N13C 
                 N13G 
                 N13H 
                 N13P 
                 N13T 
                 L14A 
               
               
                 L14C 
                 L14D 
                 L14E 
                 L14G 
                 L14H 
                 L14K 
                 L14N 
                 L14P 
                 L14Q 
                 L14R 
                 L14S 
                 L14T 
               
               
                 L14F 
                 L14I 
                 L14M 
                 L14V 
                 L14W 
                 L14Y 
                 E15D 
                 E15H 
                 E15P 
                 R16A 
                 R16C 
                 R16G 
               
               
                 R16P 
                 R16T 
                 E17A 
                 E17C 
                 E17G 
                 E17P 
                 E17T 
                 C18D 
                 C18E 
                 C18G 
                 C18H 
                 C18K 
               
               
                 C18N 
                 C18P 
                 C18Q 
                 C18R 
                 C18S 
                 C18T 
                 M19A 
                 M19C 
                 M19D 
                 M19E 
                 M19G 
                 M19H 
               
               
                 M19K 
                 M19N 
                 M19P 
                 M19Q 
                 M19R 
                 M19S 
                 M19T 
                 M19F 
                 M19I 
                 M19M 
                 M19V 
                 M19W 
               
               
                 M19Y 
                 E20A 
                 E20C 
                 E20G 
                 E20P 
                 E20T 
                 E21A 
                 E21C 
                 E21G 
                 E21P 
                 K22H 
                 K22P 
               
               
                 K22T 
                 S24H 
                 S24P 
                 F25A 
                 F25C 
                 F25D 
                 F25E 
                 F25G 
                 F25H 
                 F25K 
                 F25N 
                 F25P 
               
               
                 F25Q 
                 F25R 
                 F25S 
                 F25T 
                 F25I 
                 F25M 
                 F25W 
                 F25Y 
                 E26A 
                 E26C 
                 E26G 
                 E26P 
               
               
                 E27A 
                 E27C 
                 E27G 
                 E27H 
                 E27P 
                 E27S 
                 E27T 
                 A28C 
                 A28D 
                 A28E 
                 A28G 
                 A28H 
               
               
                 A28K 
                 A28N 
                 A28P 
                 A28Q 
                 A28R 
                 A28S 
                 A28T 
                 R29A 
                 R29C 
                 R29G 
                 R29P 
                 E30D 
               
               
                 E30H 
                 E30P 
                 V31A 
                 V31C 
                 V31D 
                 V31E 
                 V31G 
                 V31H 
                 V31K 
                 V31N 
                 V31P 
                 V31Q 
               
               
                 V31R 
                 V31S 
                 V31T 
                 V31F 
                 V31I 
                 V31W 
                 V31Y 
                 F32A 
                 F32C 
                 F32D 
                 F32E 
                 F32G 
               
               
                 F32H 
                 F32K 
                 F32N 
                 F32P 
                 F32Q 
                 F32R 
                 F32S 
                 F32T 
                 E33H 
                 E33N 
                 E33P 
                 E33Q 
               
               
                 E33S 
                 E33T 
                 T35A 
                 T35C 
                 T35G 
                 T35P 
                 F41A 
                 F41C 
                 F41D 
                 F41E 
                 F41G 
                 F41H 
               
               
                 F41K 
                 F41N 
                 F41P 
                 F41Q 
                 F41R 
                 F41S 
                 F41T 
                 F41M 
                 F41W 
                 F41Y 
                 W42A 
                 W42C 
               
               
                 W42D 
                 W42E 
                 W42G 
                 W42H 
                 W42K 
                 W42N 
                 W42P 
                 W42Q 
                 W42R 
                 W42S 
                 W42T 
                 K43A 
               
               
                 K43C 
                 K43G 
                 K43P 
                 Q44P 
                 Q44T 
                 Q44 
                 Y45A 
                 Y45C 
                 Y45D 
                 Y45E 
                 Y45G 
                 Y45H 
               
               
                 Y45K 
                 Y45N 
                 Y45P 
                 Y45Q 
                 Y45R 
                 Y45S 
                 Y45T 
                 V46A 
                 V46C 
                 V46D 
                 V46E 
                 V46G 
               
               
                 V46H 
                 V46K 
                 V46N 
                 V46P 
                 V46Q 
                 V46R 
                 V46S 
                 V46T 
                 V46F 
                 V46I 
                 V46M 
                 V46W 
               
               
                 V46Y 
                 D47A 
                 D47C 
                 D47G 
                 D47H 
                 D47P 
                 D47T 
                 G48D 
                 G48E 
                 G48P 
                 G48T 
                 D49H 
               
               
                 D49P 
                 D49Q 
                 D49T 
                 Q50A 
                 Q50C 
                 Q50D 
                 Q50G 
                 Q50H 
                 Q50P 
                 Q50T 
                 C51D 
                 C51E 
               
               
                 C51G 
                 C51H 
                 C51K 
                 C51N 
                 C51P 
                 C51Q 
                 C51R 
                 C51S 
                 C51T 
                 E52P 
                 E52T 
                 S53A 
               
               
                 S53C 
                 S53G 
                 S53H 
                 S53P 
                 S53T 
                 N54H 
                 N54P 
                 N54T 
                 L57A 
                 L57C 
                 L57D 
                 L57E 
               
               
                 L57G 
                 L57H 
                 L57K 
                 L57N 
                 L57P 
                 L57Q 
                 L57R 
                 L57S 
                 L57T 
                 L57F 
                 L57I 
                 L57M 
               
               
                 L57W 
                 L57Y 
                 G60C 
                 G60D 
                 G60H 
                 G60P 
                 G60T 
                 C62D 
                 C62H 
                 C62P 
                 K63T 
                 D65H 
               
               
                 D65T 
                 I66A 
                 I66C 
                 I66D 
                 I66E 
                 I66G 
                 I66H 
                 I66K 
                 I66N 
                 I66P 
                 I66Q 
                 I66R 
               
               
                 I66S 
                 I66T 
                 I66M 
                 I66W 
                 I66Y 
                 Y69A 
                 Y69C 
                 Y69D 
                 Y69E 
                 Y69G 
                 Y69H 
                 Y69K 
               
               
                 Y69N 
                 Y69P 
                 Y69Q 
                 Y69R 
                 Y69S 
                 Y69T 
                 C71H 
                 C71P 
                 W72A 
                 W72C 
                 W72D 
                 W72E 
               
               
                 W72G 
                 W72H 
                 W72K 
                 W72N 
                 W72P 
                 W72Q 
                 W72R 
                 W72S 
                 W72T 
                 W72I 
                 W72Y 
                 F75A 
               
               
                 F75C 
                 F75D 
                 F75E 
                 F75G 
                 F75H 
                 F75K 
                 F75N 
                 F75P 
                 F75Q 
                 F75R 
                 F75S 
                 F75T 
               
               
                 F77A 
                 F77C 
                 F77D 
                 F77E 
                 F77G 
                 F77H 
                 F77K 
                 F77N 
                 F77P 
                 F77Q 
                 F77R 
                 F77S 
               
               
                 F77T 
                 L84A 
                 L84C 
                 L84D 
                 L84E 
                 L84G 
                 L84H 
                 L84K 
                 L84N 
                 L84P 
                 L84Q 
                 L84R 
               
               
                 L84S 
                 L84T 
                 L84M 
                 L84W 
                 L84Y 
                 V86A 
                 V86C 
                 V86D 
                 V86E 
                 V86G 
                 V86H 
                 V86K 
               
               
                 V86N 
                 V86P 
                 V86Q 
                 V86R 
                 V86S 
                 V86T 
                 I90A 
                 I90C 
                 I90D 
                 I90E 
                 I90G 
                 I90H 
               
               
                 I90K 
                 I90N 
                 I90P 
                 I90Q 
                 I90R 
                 I90S 
                 I90T 
                 I90M 
                 I90W 
                 K91A 
                 K91C 
                 K91G 
               
               
                 K91P 
                 N92A 
                 N92C 
                 N92G 
                 N92P 
                 N92T 
                 G93D 
                 G93E 
                 G93H 
                 G93K 
                 G93N 
                 G93P 
               
               
                 G93Q 
                 G93R 
                 G93S 
                 G93T 
                 R94A 
                 R94C 
                 R94G 
                 R94P 
                 C95D 
                 C95E 
                 C95G 
                 C95H 
               
               
                 C95K 
                 C95N 
                 C95P 
                 C95Q 
                 C95R 
                 C95S 
                 C95T 
                 E96P 
                 E96T 
                 Q97A 
                 Q97C 
                 Q97G 
               
               
                 Q97P 
                 F98A 
                 F98C 
                 F98D 
                 F98E 
                 F98G 
                 F98H 
                 F98K 
                 F98N 
                 F98P 
                 F98Q 
                 F98R 
               
               
                 F98S 
                 F98T 
                 F98M 
                 F98W 
                 F98Y 
                 K100A 
                 K100C 
                 K100G 
                 K100P 
                 N101H 
                 N101T 
                 A103D 
               
               
                 A103E 
                 A103H 
                 A103K 
                 A103N 
                 A103P 
                 A103Q 
                 A103R 
                 A103S 
                 A103T 
                 D104T 
                 K106H 
                 K106P 
               
               
                 K106T 
                 V107A 
                 V107C 
                 V107D 
                 V107E 
                 V107G 
                 V107H 
                 V107K 
                 V107N 
                 V107P 
                 V107Q 
                 V107R 
               
               
                 V107S 
                 V107T 
                 V108A 
                 V108C 
                 V108D 
                 V108E 
                 V108G 
                 V108H 
                 V108K 
                 V108N 
                 V108P 
                 V108Q 
               
               
                 V108R 
                 V108S 
                 V108T 
                 V108F 
                 V108M 
                 V108W 
                 V108Y 
                 S110A 
                 S110C 
                 S110G 
                 S110P 
                 C111D 
               
               
                 C111E 
                 C111H 
                 C111K 
                 C111N 
                 C111P 
                 C111Q 
                 C111R 
                 C111S 
                 C111T 
                 T112A 
                 T112C 
                 T112G 
               
               
                 T112P 
                 E113D 
                 E113H 
                 E113P 
                 G114D 
                 G114E 
                 G114H 
                 G114K 
                 G114N 
                 G114P 
                 G114Q 
                 G114R 
               
               
                 G114S 
                 G114T 
                 Y115A 
                 Y115C 
                 Y115D 
                 Y115E 
                 Y115G 
                 Y115H 
                 Y115K 
                 Y115N 
                 Y115P 
                 Y115Q 
               
               
                 Y115R 
                 Y115S 
                 Y115T 
                 Y115M 
                 Y115W 
                 R116P 
                 R116T 
                 L117A 
                 L117C 
                 L117D 
                 L117E 
                 L117G 
               
               
                 L117H 
                 L117K 
                 L117N 
                 L117P 
                 L117Q 
                 L117R 
                 L117S 
                 L117T 
                 A118D 
                 A118E 
                 A118H 
                 A118K 
               
               
                 A118N 
                 A118P 
                 A118Q 
                 A118R 
                 A118S 
                 A118T 
                 N120D 
                 N120H 
                 N120P 
                 Q121T 
                 S123H 
                 S123T 
               
               
                 V128A 
                 V128C 
                 V128D 
                 V128E 
                 V128G 
                 V128H 
                 V128K 
                 V128N 
                 V128P 
                 V128Q 
                 V128R 
                 V128S 
               
               
                 V128T 
                 F130A 
                 F130C 
                 F130D 
                 F130E 
                 F130G 
                 F130H 
                 F130K 
                 F130N 
                 F130P 
                 F130Q 
                 F130R 
               
               
                 F130S 
                 F130T 
                 V135A 
                 V135C 
                 V135D 
                 V135E 
                 V135G 
                 V135H 
                 V135K 
                 V135N 
                 V135P 
                 V135Q 
               
               
                 V135R 
                 V135S 
                 V135T 
                 V135W 
                 V135Y 
                 V137A 
                 V137C 
                 V137D 
                 V137E 
                 V137G 
                 V137H 
                 V137K 
               
               
                 V137N 
                 V137P 
                 V137Q 
                 V137R 
                 V137S 
                 V137T 
                 V137M 
                 V137W 
                 V137Y 
                 S138H 
                 S138T 
                 T140D 
               
               
                 T140H 
                 S141T 
                 K142H 
                 K142P 
                 L143A 
                 L143C 
                 L143D 
                 L143E 
                 L143G 
                 L143H 
                 L143K 
                 L143N 
               
               
                 L143P 
                 L143Q 
                 L143R 
                 L143S 
                 L143T 
                 L143F 
                 L143I 
                 L143M 
                 L143V 
                 L143W 
                 L143Y 
                 R145H 
               
               
                 R145P 
                 R145T 
                 A146P 
                 A146T 
                 T148H 
                 T148P 
                 V149A 
                 V149C 
                 V149D 
                 V149E 
                 V149G 
                 V149H 
               
               
                 V149K 
                 V149N 
                 V149P 
                 V149Q 
                 V149R 
                 V149S 
                 V149T 
                 V149F 
                 V149I 
                 V149M 
                 V149W 
                 V149Y 
               
               
                 F150A 
                 F150C 
                 F150D 
                 F150E 
                 F150G 
                 F150H 
                 F150K 
                 F150N 
                 F150P 
                 F150Q 
                 F150R 
                 F150S 
               
               
                 F150T 
                 F150M 
                 F150W 
                 F150Y 
                 D152A 
                 D152C 
                 D152G 
                 D152P 
                 D152S 
                 D152T 
                 V153A 
                 V153C 
               
               
                 V153D 
                 V153E 
                 V153G 
                 V153H 
                 V153K 
                 V153N 
                 V153P 
                 V153Q 
                 V153R 
                 V153S 
                 V153T 
                 V153F 
               
               
                 V153I 
                 V153M 
                 V153W 
                 V153Y 
                 D154A 
                 D154C 
                 D154G 
                 D154P 
                 D154Q 
                 D154S 
                 Y155A 
                 Y155C 
               
               
                 Y155D 
                 Y155E 
                 Y155G 
                 Y155H 
                 Y155K 
                 Y155N 
                 Y155P 
                 Y155Q 
                 Y155R 
                 Y155S 
                 Y155T 
                 Y155M 
               
               
                 Y155V 
                 Y155W 
                 V156A 
                 V156C 
                 V156D 
                 V156E 
                 V156G 
                 V156H 
                 V156K 
                 V156N 
                 V156P 
                 V156Q 
               
               
                 V156R 
                 V156S 
                 V156T 
                 V156I 
                 V156M 
                 V156W 
                 V156Y 
                 N157A 
                 N157C 
                 N157G 
                 N157H 
                 N157P 
               
               
                 N157Q 
                 N157T 
                 S158H 
                 S158P 
                 S158T 
                 T159A 
                 T159C 
                 T159G 
                 T159P 
                 E160A 
                 E160C 
                 E160G 
               
               
                 E160P 
                 A161C 
                 A161D 
                 A161E 
                 A161H 
                 A161K 
                 A161N 
                 A161P 
                 A161Q 
                 A161R 
                 A161S 
                 A161T 
               
               
                 E162P 
                 E162T 
                 T163A 
                 T163C 
                 T163G 
                 T163P 
                 I164A 
                 I164C 
                 I164D 
                 I164E 
                 I164G 
                 I164H 
               
               
                 I164K 
                 I164N 
                 I164P 
                 I164Q 
                 I164R 
                 I164S 
                 I164T 
                 L165A 
                 L165C 
                 L165D 
                 L165E 
                 L165G 
               
               
                 L165H 
                 L165K 
                 L165N 
                 L165P 
                 L165Q 
                 L165R 
                 L165S 
                 L165T 
                 L165M 
                 L165W 
                 L165Y 
                 I168A 
               
               
                 I168C 
                 I168D 
                 I168E 
                 I168G 
                 I168H 
                 I168K 
                 I168N 
                 I168P 
                 I168Q 
                 I168R 
                 I168S 
                 I168T 
               
               
                 F175A 
                 F175C 
                 F175D 
                 F175E 
                 F175G 
                 F175H 
                 F175K 
                 F175N 
                 F175P 
                 F175Q 
                 F175R 
                 F175S 
               
               
                 F175T 
                 F178A 
                 F178C 
                 F178D 
                 F178E 
                 F178G 
                 F178H 
                 F178K 
                 F178N 
                 F178P 
                 F178Q 
                 F178R 
               
               
                 F178S 
                 F178T 
                 F178M 
                 F178W 
                 F178Y 
                 T179A 
                 T179C 
                 T179G 
                 T179P 
                 R180A 
                 R180C 
                 R180D 
               
               
                 R180G 
                 R180H 
                 R180P 
                 V181A 
                 V181C 
                 V181D 
                 V181E 
                 V181G 
                 V181H 
                 V181K 
                 V181N 
                 V181P 
               
               
                 V181Q 
                 V181R 
                 V181S 
                 V181T 
                 V181F 
                 V181I 
                 V181M 
                 V181W 
                 V181Y 
                 V182A 
                 V182C 
                 V182D 
               
               
                 V182E 
                 V182G 
                 V182H 
                 V182K 
                 V182N 
                 V182P 
                 V182Q 
                 V182R 
                 V182S 
                 V182T 
                 V182F 
                 V182I 
               
               
                 V182M 
                 V182W 
                 V182Y 
                 G183D 
                 G183E 
                 G183H 
                 G183K 
                 G183N 
                 G183P 
                 G183Q 
                 G183S 
                 G183T 
               
               
                 G184D 
                 G184E 
                 G184H 
                 G184K 
                 G184N 
                 G184P 
                 G184Q 
                 G184R 
                 G184S 
                 G184T 
                 E185A 
                 E185C 
               
               
                 E185G 
                 E185H 
                 E185P 
                 E185T 
                 D186A 
                 D186C 
                 D186G 
                 D186H 
                 D186P 
                 D186T 
                 A187C 
                 A187D 
               
               
                 A187E 
                 A187G 
                 A187H 
                 A187K 
                 A187N 
                 A187P 
                 A187Q 
                 A187R 
                 A187S 
                 A187T 
                 K188A 
                 K188C 
               
               
                 K188G 
                 K188H 
                 K188P 
                 K188T 
                 G190D 
                 G190E 
                 G190H 
                 G190K 
                 G190N 
                 G190P 
                 G190Q 
                 G190R 
               
               
                 G190S 
                 G190T 
                 F192A 
                 F192C 
                 F192D 
                 F192E 
                 F192G 
                 F192H 
                 F192K 
                 F192N 
                 F192P 
                 F192Q 
               
               
                 F192R 
                 F192S 
                 F192T 
                 F192W 
                 F192Y 
                 W194A 
                 W194C 
                 W194D 
                 W194E 
                 W194G 
                 W194H 
                 W194K 
               
               
                 W194N 
                 W194P 
                 W194Q 
                 W194R 
                 W194S 
                 W194T 
                 Q195H 
                 Q195P 
                 Q195T 
                 V196A 
                 V196C 
                 V196D 
               
               
                 V196E 
                 V196G 
                 V196H 
                 V196K 
                 V196N 
                 V196P 
                 V196Q 
                 V196R 
                 V196S 
                 V196T 
                 V196F 
                 V196I 
               
               
                 V196M 
                 V196W 
                 V196Y 
                 V197A 
                 V197C 
                 V197D 
                 V197E 
                 V197G 
                 V197H 
                 V197K 
                 V197N 
                 V197P 
               
               
                 V197Q 
                 V197R 
                 V197S 
                 V197T 
                 V197F 
                 V197I 
                 V197M 
                 V197W 
                 V197Y 
                 L198A 
                 L198C 
                 L198D 
               
               
                 L198E 
                 L198G 
                 L198H 
                 L198K 
                 L198N 
                 L198P 
                 L198Q 
                 L198R 
                 L198S 
                 L198T 
                 L198I 
                 L198Y 
               
               
                 N199A 
                 N199C 
                 N199G 
                 N199H 
                 N199P 
                 N199S 
                 N199T 
                 G200P 
                 G200T 
                 K201A 
                 K201C 
                 K201D 
               
               
                 K201E 
                 K201G 
                 K201H 
                 K201N 
                 K201P 
                 K201Q 
                 K201S 
                 K201T 
                 V202A 
                 V202C 
                 V202D 
                 V202E 
               
               
                 V202G 
                 V202H 
                 V202K 
                 V202N 
                 V202P 
                 V202Q 
                 V202R 
                 V202S 
                 V202T 
                 V202F 
                 V202I 
                 V202M 
               
               
                 V202W 
                 V202Y 
                 D203A 
                 D203C 
                 D203G 
                 D203P 
                 D203T 
                 A204C 
                 A204D 
                 A204E 
                 A204G 
                 A204H 
               
               
                 A204K 
                 A204N 
                 A204P 
                 A204Q 
                 A204R 
                 A204S 
                 A204T 
                 F205A 
                 F205C 
                 F205D 
                 F205E 
                 F205G 
               
               
                 F205H 
                 F205K 
                 F205N 
                 F205P 
                 F205Q 
                 F205R 
                 F205S 
                 F205T 
                 F205M 
                 F205V 
                 F205W 
                 F205Y 
               
               
                 G207H 
                 G207P 
                 G208C 
                 G208D 
                 G208E 
                 G208H 
                 G208K 
                 G208N 
                 G208P 
                 G208Q 
                 G208R 
                 G208S 
               
               
                 G208T 
                 S209A 
                 S209C 
                 S209G 
                 S209P 
                 I210A 
                 I210C 
                 I210D 
                 I210E 
                 I210G 
                 I210H 
                 I210K 
               
               
                 I210N 
                 I210P 
                 I210Q 
                 I210R 
                 I210S 
                 I210T 
                 I210F 
                 I210W 
                 I210Y 
                 V211A 
                 V211C 
                 V211D 
               
               
                 V211E 
                 V211G 
                 V211H 
                 V211K 
                 V211N 
                 V211P 
                 V211Q 
                 V211R 
                 V211S 
                 V211T 
                 V211F 
                 V211I 
               
               
                 V211M 
                 V211W 
                 N212A 
                 N212C 
                 N212G 
                 N212P 
                 E213H 
                 E213P 
                 E213S 
                 E213T 
                 K214T 
                 W215A 
               
               
                 W215C 
                 W215D 
                 W215E 
                 W215G 
                 W215H 
                 W215K 
                 W215N 
                 W215P 
                 W215Q 
                 W215R 
                 W215S 
                 W215T 
               
               
                 I216A 
                 I216C 
                 I216D 
                 I216E 
                 I216G 
                 I216H 
                 I216K 
                 I216N 
                 I216P 
                 1216Q 
                 I216R 
                 I216S 
               
               
                 I216T 
                 V217A 
                 V217C 
                 V217D 
                 V217E 
                 V217G 
                 V217H 
                 V217K 
                 V217N 
                 V217P 
                 V217Q 
                 V217R 
               
               
                 V217S 
                 V217T 
                 V217I 
                 V217Y 
                 A219H 
                 A219P 
                 A219T 
                 V223A 
                 V223C 
                 V223D 
                 V223E 
                 V223G 
               
               
                 V223H 
                 V223K 
                 V223N 
                 V223P 
                 V223Q 
                 V223R 
                 V223S 
                 V223T 
                 V223M 
                 V223W 
                 V223Y 
                 G226P 
               
               
                 V227A 
                 V227C 
                 V227D 
                 V227E 
                 V227G 
                 V227H 
                 V227K 
                 V227N 
                 V227P 
                 V227Q 
                 V227R 
                 V227S 
               
               
                 V227T 
                 V227F 
                 V227I 
                 V227M 
                 V227W 
                 V227Y 
                 K228A 
                 K228C 
                 K228G 
                 K228H 
                 K228P 
                 I229A 
               
               
                 I229C 
                 I229D 
                 I229E 
                 I229G 
                 I229H 
                 I229K 
                 I229N 
                 I229P 
                 I229Q 
                 I229R 
                 I229S 
                 I229T 
               
               
                 I229M 
                 I229W 
                 I229Y 
                 T230A 
                 T230C 
                 T230G 
                 T230P 
                 V231A 
                 V231C 
                 V231D 
                 V231E 
                 V231G 
               
               
                 V231H 
                 V231K 
                 V231N 
                 V231P 
                 V231Q 
                 V231R 
                 V231S 
                 V231T 
                 V232A 
                 V232C 
                 V232D 
                 V232E 
               
               
                 V232G 
                 V232H 
                 V232K 
                 V232N 
                 V232P 
                 V232Q 
                 V232R 
                 V232S 
                 V232T 
                 V232F 
                 V232I 
                 V232M 
               
               
                 V232W 
                 V232Y 
                 A233C 
                 A233D 
                 A233E 
                 A233G 
                 A233H 
                 A233K 
                 A233N 
                 A233P 
                 A233Q 
                 A233R 
               
               
                 A233S 
                 A233T 
                 A233V 
                 G234D 
                 G234E 
                 G234H 
                 G234K 
                 G234N 
                 G234P 
                 G234Q 
                 G234R 
                 G234S 
               
               
                 G234T 
                 E235H 
                 E235N 
                 E235P 
                 E235Q 
                 E235S 
                 E235T 
                 H236A 
                 H236C 
                 H236G 
                 H236P 
                 N237A 
               
               
                 N237C 
                 N237G 
                 N237P 
                 N237T 
                 I238A 
                 I238C 
                 I238D 
                 I238E 
                 I238G 
                 I238H 
                 I238K 
                 I238N 
               
               
                 I238P 
                 I238Q 
                 I238R 
                 I238S 
                 I238T 
                 E239A 
                 E239C 
                 E239G 
                 E239P 
                 E240H 
                 E240T 
                 V250A 
               
               
                 V250C 
                 V250D 
                 V250E 
                 V250G 
                 V250H 
                 V250K 
                 V250N 
                 V250P 
                 V250Q 
                 V250R 
                 V250S 
                 V250T 
               
               
                 V250M 
                 V250W 
                 V250Y 
                 I251A 
                 I251C 
                 I251D 
                 I251E 
                 I251G 
                 I251H 
                 I251K 
                 I251N 
                 I251P 
               
               
                 I251Q 
                 I251R 
                 I251S 
                 I251T 
                 I253A 
                 I253C 
                 I253D 
                 I253E 
                 I253G 
                 I253H 
                 I253K 
                 I253N 
               
               
                 I253P 
                 I253Q 
                 I253R 
                 I253S 
                 I253T 
                 I253M 
                 I253W 
                 I253Y 
                 I254A 
                 I254C 
                 I254D 
                 I254E 
               
               
                 I254G 
                 I254H 
                 I254K 
                 I254N 
                 I254P 
                 I254Q 
                 I254R 
                 I254S 
                 I254T 
                 P255H 
                 H256P 
                 H256T 
               
               
                 H257A 
                 H257C 
                 H257G 
                 H257P 
                 N258P 
                 N258T 
                 Y259A 
                 Y259C 
                 Y259D 
                 Y259E 
                 Y259G 
                 Y259H 
               
               
                 Y259K 
                 Y259N 
                 Y259P 
                 Y259Q 
                 Y259R 
                 Y259S 
                 Y259T 
                 Y259M 
                 Y259W 
                 N260A 
                 N260C 
                 N260G 
               
               
                 N260P 
                 A261D 
                 A261E 
                 A261H 
                 A261K 
                 A261N 
                 A261P 
                 A261Q 
                 A261R 
                 A261S 
                 A261T 
                 A262C 
               
               
                 A262D 
                 A262E 
                 A262G 
                 A262H 
                 A262K 
                 A262N 
                 A262P 
                 A262Q 
                 A262R 
                 A262S 
                 A262T 
                 I263A 
               
               
                 I263C 
                 I263D 
                 I263E 
                 I263G 
                 I263H 
                 I263K 
                 I263N 
                 I263P 
                 I263Q 
                 I263R 
                 I263S 
                 I263T 
               
               
                 I263M 
                 I263V 
                 I263W 
                 I263Y 
                 N264A 
                 N264C 
                 N264D 
                 N264G 
                 N264H 
                 N264P 
                 K265A 
                 K265C 
               
               
                 K265G 
                 K265H 
                 K265P 
                 Y266A 
                 Y266C 
                 Y266D 
                 Y266E 
                 Y266G 
                 Y266H 
                 Y266K 
                 Y266N 
                 Y266P 
               
               
                 Y266Q 
                 Y266R 
                 Y266S 
                 Y266T 
                 Y266M 
                 Y266W 
                 N267A 
                 N267C 
                 N267G 
                 N267H 
                 N267P 
                 N267T 
               
               
                 H268P 
                 D269A 
                 D269C 
                 D269E 
                 D269G 
                 D269H 
                 D269N 
                 D269P 
                 D269Q 
                 D269S 
                 D269T 
                 I270A 
               
               
                 I270C 
                 I270D 
                 I270E 
                 I270G 
                 I270H 
                 I270K 
                 I270N 
                 I270P 
                 I270Q 
                 I270R 
                 I270S 
                 I270T 
               
               
                 I270M 
                 I270W 
                 A271C 
                 A271D 
                 A271E 
                 A271G 
                 A271H 
                 A271K 
                 A271N 
                 A271P 
                 A271Q 
                 A271R 
               
               
                 A271S 
                 A271T 
                 L272A 
                 L272C 
                 L272D 
                 L272E 
                 L272G 
                 L272H 
                 L272K 
                 L272N 
                 L272P 
                 L272Q 
               
               
                 L272R 
                 L272S 
                 L272T 
                 L272F 
                 L273A 
                 L273C 
                 L273D 
                 L273E 
                 L273G 
                 L273H 
                 L273K 
                 L273N 
               
               
                 L273P 
                 L273Q 
                 L273R 
                 L273S 
                 L273T 
                 L273F 
                 L273I 
                 L273M 
                 L273V 
                 L273W 
                 L273Y 
                 E274A 
               
               
                 E274C 
                 E274G 
                 E274P 
                 E274T 
                 L275A 
                 L275C 
                 L275D 
                 L275E 
                 L275G 
                 L275H 
                 L275K 
                 L275N 
               
               
                 L275P 
                 L275Q 
                 L275R 
                 L275S 
                 L275T 
                 L275W 
                 L275Y 
                 D276P 
                 D276S 
                 D276T 
                 E277A 
                 E277C 
               
               
                 E277G 
                 E277P 
                 P278T 
                 L279A 
                 L279C 
                 L279D 
                 L279E 
                 L279G 
                 L279H 
                 L279K 
                 L279N 
                 L279P 
               
               
                 L279Q 
                 L279R 
                 L279S 
                 L279T 
                 L279I 
                 L279Y 
                 V280A 
                 V280C 
                 V280D 
                 V280E 
                 V280G 
                 V280H 
               
               
                 V280K 
                 V280N 
                 V280P 
                 V280Q 
                 V280R 
                 V280S 
                 V280T 
                 V280F 
                 V280I 
                 V280W 
                 V280Y 
                 L281A 
               
               
                 L281C 
                 L281D 
                 L281E 
                 L281G 
                 L281H 
                 L281K 
                 L281N 
                 L281P 
                 L281Q 
                 L281R 
                 L281S 
                 L281T 
               
               
                 L281F 
                 L281I 
                 L281V 
                 L281W 
                 L281Y 
                 S283A 
                 S283C 
                 S283G 
                 S283P 
                 Y284A 
                 Y284C 
                 Y284D 
               
               
                 Y284E 
                 Y284G 
                 Y284H 
                 Y284K 
                 Y284N 
                 Y284P 
                 Y284Q 
                 Y284R 
                 Y284S 
                 Y284T 
                 Y284M 
                 V285A 
               
               
                 V285C 
                 V285D 
                 V285E 
                 V285G 
                 V285H 
                 V285K 
                 V285N 
                 V285P 
                 V285Q 
                 V285R 
                 V285S 
                 V285T 
               
               
                 V285M 
                 V285W 
                 V285Y 
                 T286A 
                 T286C 
                 T286G 
                 T286P 
                 I288A 
                 I288C 
                 I288D 
                 I288E 
                 I288G 
               
               
                 I288H 
                 I288K 
                 I288N 
                 I288P 
                 I288Q 
                 I288R 
                 I288S 
                 I288T 
                 C289D 
                 C289H 
                 C289P 
                 I290A 
               
               
                 I290C 
                 I290D 
                 I290E 
                 I290G 
                 I290H 
                 I290K 
                 I290N 
                 I290P 
                 I290Q 
                 I290R 
                 I290S 
                 I290T 
               
               
                 I290Y 
                 A291D 
                 A291E 
                 A291H 
                 A291K 
                 A291N 
                 A291P 
                 A291Q 
                 A291R 
                 A291S 
                 A291T 
                 D292A 
               
               
                 D292C 
                 D292G 
                 D292P 
                 D292T 
                 K293H 
                 K293P 
                 K293T 
                 Y295A 
                 Y295C 
                 Y295D 
                 Y295E 
                 Y295G 
               
               
                 Y295H 
                 Y295K 
                 Y295N 
                 Y295P 
                 Y295Q 
                 Y295R 
                 Y295S 
                 Y295T 
                 Y295W 
                 T296A 
                 T296C 
                 T296G 
               
               
                 T296P 
                 N297A 
                 N297C 
                 N297G 
                 N297P 
                 I298A 
                 I298C 
                 I298D 
                 I298E 
                 I298G 
                 I298H 
                 I298K 
               
               
                 I298N 
                 I298P 
                 I298Q 
                 I298R 
                 I298S 
                 I298T 
                 F299A 
                 F299C 
                 F299D 
                 F299E 
                 F299G 
                 F299H 
               
               
                 F299K 
                 F299N 
                 F299P 
                 F299Q 
                 F299R 
                 F299S 
                 F299T 
                 L300A 
                 L300C 
                 L300D 
                 L300E 
                 L300G 
               
               
                 L300H 
                 L300K 
                 L300N 
                 L300P 
                 L300Q 
                 L300R 
                 L300S 
                 L300T 
                 L300F 
                 L300I 
                 L300M 
                 L300V 
               
               
                 L300W 
                 L300Y 
                 K301A 
                 K301C 
                 K301G 
                 K301P 
                 K301T 
                 F302A 
                 F302C 
                 F302D 
                 F302E 
                 F302G 
               
               
                 F302H 
                 F302K 
                 F302N 
                 F302P 
                 F302Q 
                 F302R 
                 F302S 
                 F302T 
                 G303H 
                 G303P 
                 G303T 
                 S304A 
               
               
                 S304C 
                 S304G 
                 S304P 
                 S304T 
                 G305D 
                 G305E 
                 G305H 
                 G305N 
                 G305P 
                 G305Q 
                 G305S 
                 G305T 
               
               
                 Y306A 
                 Y306C 
                 Y306D 
                 Y306E 
                 Y306G 
                 Y306H 
                 Y306K 
                 Y306N 
                 Y306P 
                 Y306Q 
                 Y306R 
                 Y306S 
               
               
                 Y306T 
                 V307A 
                 V307C 
                 V307D 
                 V307E 
                 V307G 
                 V307H 
                 V307K 
                 V307N 
                 V307P 
                 V307Q 
                 V307R 
               
               
                 V307S 
                 V307T 
                 S308P 
                 S308T 
                 W310A 
                 W310C 
                 W310D 
                 W310E 
                 W310G 
                 W310H 
                 W310K 
                 W310N 
               
               
                 W310P 
                 W310Q 
                 W310R 
                 W310S 
                 W310T 
                 G311H 
                 V313A 
                 V313C 
                 V313D 
                 V313E 
                 V313G 
                 V313H 
               
               
                 V313K 
                 V313N 
                 V313P 
                 V313Q 
                 V313R 
                 V313S 
                 V313T 
                 F314A 
                 F314C 
                 F314D 
                 F314E 
                 F314G 
               
               
                 F314H 
                 F314K 
                 F314N 
                 F314P 
                 F314Q 
                 F314R 
                 F314S 
                 F314T 
                 F314M 
                 F314W 
                 F314Y 
                 H315A 
               
               
                 H315C 
                 H315G 
                 H315P 
                 K316A 
                 K316C 
                 K316G 
                 K316P 
                 G317C 
                 G317D 
                 G317E 
                 G317H 
                 G317K 
               
               
                 G317N 
                 G317P 
                 G317Q 
                 G317R 
                 G317S 
                 G317T 
                 R318A 
                 R318C 
                 R318G 
                 R318P 
                 S319D 
                 S319H 
               
               
                 S319N 
                 S319P 
                 S319Q 
                 A320C 
                 A320D 
                 A320E 
                 A320G 
                 A320H 
                 A320K 
                 A320N 
                 A320P 
                 A320Q 
               
               
                 A320R 
                 A320S 
                 A320T 
                 L321A 
                 L321C 
                 L321D 
                 L321E 
                 L321G 
                 L321H 
                 L321K 
                 L321N 
                 L321P 
               
               
                 L321Q 
                 L321R 
                 L321S 
                 L321T 
                 V322A 
                 V322C 
                 V322D 
                 V322E 
                 V322G 
                 V322H 
                 V322K 
                 V322N 
               
               
                 V322P 
                 V322Q 
                 V322R 
                 V322S 
                 V322T 
                 V322W 
                 V322Y 
                 L323A 
                 L323C 
                 L323D 
                 L323E 
                 L323G 
               
               
                 L323H 
                 L323K 
                 L323N 
                 L323P 
                 L323Q 
                 L323R 
                 L323S 
                 L323T 
                 L323F 
                 L323I 
                 L323M 
                 L323V 
               
               
                 L323W 
                 L323Y 
                 Q324A 
                 Q324C 
                 Q324G 
                 Q324P 
                 Y325A 
                 Y325C 
                 Y325D 
                 Y325E 
                 Y325G 
                 Y325H 
               
               
                 Y325K 
                 Y325N 
                 Y325P 
                 Y325Q 
                 Y325R 
                 Y325S 
                 Y325T 
                 Y325W 
                 L326A 
                 L326C 
                 L326D 
                 L326E 
               
               
                 L326G 
                 L326H 
                 L326K 
                 L326N 
                 L326P 
                 L326Q 
                 L326R 
                 L326S 
                 L326T 
                 L326F 
                 L326I 
                 L326M 
               
               
                 L326V 
                 L326W 
                 L326Y 
                 R327A 
                 R327C 
                 R327G 
                 R327H 
                 R327P 
                 V328A 
                 V328C 
                 V328D 
                 V328E 
               
               
                 V328G 
                 V328H 
                 V328K 
                 V328N 
                 V328P 
                 V328Q 
                 V328R 
                 V328S 
                 V328T 
                 V328F 
                 V328I 
                 V328M 
               
               
                 V328W 
                 V328Y 
                 L330A 
                 L330C 
                 L330D 
                 L330E 
                 L330G 
                 L330H 
                 L330K 
                 L330N 
                 L330P 
                 L330Q 
               
               
                 L330R 
                 L330S 
                 L330T 
                 L330F 
                 L330I 
                 L330V 
                 L330W 
                 L330Y 
                 V331A 
                 V331C 
                 V331D 
                 V331E 
               
               
                 V331G 
                 V331H 
                 V331K 
                 V331N 
                 V331P 
                 V331Q 
                 V331R 
                 V331S 
                 V331T 
                 V331F 
                 V331I 
                 V331M 
               
               
                 V331W 
                 V331Y 
                 D332A 
                 D332C 
                 D332G 
                 D332P 
                 R333A 
                 R333C 
                 R333D 
                 R333E 
                 R333G 
                 R333H 
               
               
                 R333N 
                 R333P 
                 R333Q 
                 R333R 
                 R333S 
                 R333T 
                 A334C 
                 A334D 
                 A334E 
                 A334G 
                 A334H 
                 A334K 
               
               
                 A334N 
                 A334P 
                 A334Q 
                 A334R 
                 A334S 
                 A334T 
                 T335A 
                 T335C 
                 T335G 
                 T335P 
                 C336D 
                 C336E 
               
               
                 C336H 
                 C336K 
                 C336N 
                 C336P 
                 C336Q 
                 C336R 
                 C336S 
                 C336T 
                 L337A 
                 L337C 
                 L337D 
                 L337E 
               
               
                 L337G 
                 L337H 
                 L337K 
                 L337N 
                 L337P 
                 L337Q 
                 L337R 
                 L337S 
                 L337T 
                 R338A 
                 R338C 
                 R338G 
               
               
                 R338P 
                 S339P 
                 S339T 
                 K341A 
                 K341C 
                 K341G 
                 K341P 
                 F342A 
                 F342C 
                 F342D 
                 F342E 
                 F342G 
               
               
                 F342H 
                 F342K 
                 F342N 
                 F342P 
                 F342Q 
                 F342R 
                 F342S 
                 F342T 
                 F342M 
                 F342W 
                 T343A 
                 T343C 
               
               
                 T343G 
                 T343P 
                 I344A 
                 I344C 
                 I344D 
                 I344E 
                 I344G 
                 I344H 
                 I344K 
                 I344N 
                 I344P 
                 I344Q 
               
               
                 I344R 
                 I344S 
                 I344T 
                 Y345A 
                 Y345C 
                 Y345D 
                 Y345E 
                 Y345G 
                 Y345H 
                 Y345K 
                 Y345N 
                 Y345P 
               
               
                 Y345Q 
                 Y345R 
                 Y345S 
                 Y345T 
                 Y345M 
                 Y345W 
                 N346A 
                 N346C 
                 N346G 
                 N346P 
                 N347H 
                 N347P 
               
               
                 M348A 
                 M348C 
                 M348D 
                 M348E 
                 M348G 
                 M348H 
                 M348K 
                 M348N 
                 M348P 
                 M348Q 
                 M348R 
                 M348S 
               
               
                 M348T 
                 F349A 
                 F349C 
                 F349D 
                 F349E 
                 F349G 
                 F349H 
                 F349K 
                 F349N 
                 F349P 
                 F349Q 
                 F349R 
               
               
                 F349S 
                 F349T 
                 F349I 
                 F349M 
                 F349W 
                 F349Y 
                 C350D 
                 C350H 
                 C350P 
                 C350T 
                 A351E 
                 A351H 
               
               
                 A351N 
                 A351P 
                 A351Q 
                 A351R 
                 A351S 
                 A351T 
                 G352A 
                 G352C 
                 G352P 
                 F353A 
                 F353C 
                 F353D 
               
               
                 F353E 
                 F353G 
                 F353H 
                 F353K 
                 F353N 
                 F353P 
                 F353Q 
                 F353R 
                 F353S 
                 F353T 
                 F353I 
                 F353M 
               
               
                 F353W 
                 H354A 
                 H354C 
                 H354G 
                 H354P 
                 E355A 
                 E355C 
                 E355D 
                 E355G 
                 E355H 
                 E355K 
                 E355N 
               
               
                 E355P 
                 E355Q 
                 E355S 
                 E355T 
                 G356D 
                 G356E 
                 G356H 
                 G356K 
                 G356N 
                 G356P 
                 G356Q 
                 G356R 
               
               
                 G356S 
                 G356T 
                 G357D 
                 G357E 
                 G357H 
                 G357K 
                 G357N 
                 G357P 
                 G357Q 
                 G357R 
                 G357S 
                 G357T 
               
               
                 R358D 
                 R358E 
                 R358H 
                 R358K 
                 R358N 
                 R358P 
                 R358Q 
                 R358R 
                 R358S 
                 R358T 
                 D359A 
                 D359C 
               
               
                 D359G 
                 D359P 
                 D359Q 
                 D359S 
                 D359T 
                 S360A 
                 S360C 
                 S360G 
                 S360P 
                 C361D 
                 C361E 
                 C361H 
               
               
                 C361K 
                 C361N 
                 C361P 
                 C361Q 
                 C361R 
                 C361S 
                 C361T 
                 V370A 
                 V370C 
                 V370D 
                 V370E 
                 V370G 
               
               
                 V370H 
                 V370K 
                 V370N 
                 V370P 
                 V370Q 
                 V370R 
                 V370S 
                 V370T 
                 V370W 
                 V370Y 
                 V373A 
                 V373C 
               
               
                 V373D 
                 V373E 
                 V373G 
                 V373H 
                 V373K 
                 V373N 
                 V373P 
                 V373Q 
                 V373R 
                 V373S 
                 V373T 
                 V373F 
               
               
                 V373I 
                 V373M 
                 V373W 
                 E374A 
                 E374C 
                 E374G 
                 E374P 
                 G375H 
                 S377A 
                 S377C 
                 S377G 
                 S377P 
               
               
                 F378A 
                 F378C 
                 F378D 
                 F378E 
                 F378G 
                 F378H 
                 F378K 
                 F378N 
                 F378P 
                 F378Q 
                 F378R 
                 F378S 
               
               
                 F378T 
                 F378W 
                 L379A 
                 L379C 
                 L379D 
                 L379E 
                 L379G 
                 L379H 
                 L379K 
                 L379N 
                 L379P 
                 L379Q 
               
               
                 L379R 
                 L379S 
                 L379T 
                 L379I 
                 L379M 
                 L379W 
                 L379Y 
                 T380A 
                 T380C 
                 T380G 
                 T380P 
                 G381D 
               
               
                 G381E 
                 G381H 
                 G381K 
                 G381N 
                 G381P 
                 G381Q 
                 G381R 
                 G381S 
                 G381T 
                 I382A 
                 I382C 
                 I382D 
               
               
                 I382E 
                 I382G 
                 I382H 
                 I382K 
                 I382N 
                 I382P 
                 I382Q 
                 I382R 
                 I382S 
                 I382T 
                 I382M 
                 I382W 
               
               
                 I382Y 
                 I383A 
                 I383C 
                 I383D 
                 I383E 
                 I383G 
                 I383H 
                 I383K 
                 I383N 
                 I383P 
                 I383Q 
                 I383R 
               
               
                 I383S 
                 I383T 
                 S384A 
                 S384C 
                 S384G 
                 S384P 
                 W385A 
                 W385C 
                 W385D 
                 W385E 
                 W385G 
                 W385H 
               
               
                 W385K 
                 W385N 
                 W385P 
                 W385Q 
                 W385R 
                 W385S 
                 W385T 
                 W385M 
                 E387A 
                 E387C 
                 E387G 
                 E387H 
               
               
                 E387P 
                 E387T 
                 E388H 
                 E388N 
                 E388P 
                 E388Q 
                 E388T 
                 A390C 
                 A390D 
                 A390E 
                 A390G 
                 A390H 
               
               
                 A390K 
                 A390N 
                 A390P 
                 A390Q 
                 A390R 
                 A390S 
                 M391A 
                 M391C 
                 M391D 
                 M391E 
                 M391G 
                 M391H 
               
               
                 M391K 
                 M391N 
                 M391P 
                 M391Q 
                 M391R 
                 M391S 
                 M391T 
                 M391F 
                 M391I 
                 M391W 
                 M391Y 
                 K392A 
               
               
                 K392C 
                 K392G 
                 K392P 
                 G393C 
                 G393D 
                 G393E 
                 G393H 
                 G393K 
                 G393N 
                 G393P 
                 G393Q 
                 G393R 
               
               
                 G393S 
                 G393T 
                 Y395A 
                 Y395C 
                 Y395D 
                 Y395E 
                 Y395G 
                 Y395H 
                 Y395K 
                 Y395N 
                 Y395P 
                 Y395Q 
               
               
                 Y395R 
                 Y395S 
                 Y395T 
                 Y398A 
                 Y398C 
                 Y398D 
                 Y398E 
                 Y398G 
                 Y398H 
                 Y398K 
                 Y398N 
                 Y398P 
               
               
                 Y398Q 
                 Y398R 
                 Y398S 
                 Y398T 
                 K400H 
                 V401A 
                 V401C 
                 V401D 
                 V401E 
                 V401G 
                 V401H 
                 V401K 
               
               
                 V401N 
                 V401P 
                 V401Q 
                 V401R 
                 V401S 
                 V401T 
                 V401F 
                 V401I 
                 V401M 
                 V401W 
                 V401Y 
                 S402A 
               
               
                 S402C 
                 S402G 
                 S402P 
                 R403A 
                 R403C 
                 R403G 
                 R403P 
                 R403T 
                 Y404A 
                 Y404C 
                 Y404D 
                 Y404E 
               
               
                 Y404G 
                 Y404H 
                 Y404K 
                 Y404N 
                 Y404P 
                 Y404Q 
                 Y404R 
                 Y404S 
                 Y404T 
                 V405A 
                 V405C 
                 V405D 
               
               
                 V405E 
                 V405G 
                 V405H 
                 V405K 
                 V405N 
                 V405P 
                 V405Q 
                 V405R 
                 V405S 
                 V405T 
                 V405W 
                 V405Y 
               
               
                 N406F 
                 N406H 
                 N406I 
                 N406L 
                 N406P 
                 N406W 
                 N406Y 
                 W407D 
                 W407E 
                 W407F 
                 W407H 
                 W407I 
               
               
                 W407K 
                 W407N 
                 W407P 
                 W407Q 
                 W407R 
                 W407S 
                 W407T 
                 W407Y 
                 I408D 
                 I408E 
                 I408H 
                 I408K 
               
               
                 I408N 
                 I408P 
                 I408Q 
                 I408R 
                 I408S 
                 I408T 
                 K409F 
                 K409H 
                 K409I 
                 K409P 
                 K409T 
                 K409V 
               
               
                 K409W 
                 K409Y 
                 E410H 
                 K411A 
                 K411C 
                 K411G 
                 K411I 
                 K411P 
                 K411T 
                 K411V 
                 K411W 
                 K411Y 
               
               
                 K413T 
               
               
                   
               
            
           
         
       
     
     b. Glycosylation 
     Many proteins with therapeutic activity contain one or more glycosylation sites, such as, amino acid sequences that are glycosylated by a eukaryotic cell. There have been various reports of attempts to increase the degree of glycosylation of therapeutic proteins in order to achieve 1) reduced immunogenicity; 2) less frequent administration of the protein; 3) increased protein stability such as increased serum half-life; and 4) reduction in adverse side effects such as inflammation. The glycosylation site(s) provides a site for attachment of a carbohydrate moiety on the subject polypeptide, such that when the subject polypeptide is produced in a eukaryotic cell capable of glycosylation, the subject polypeptide is glycosylated. The further glycosylation of a FIX polypeptide confers one or more advantages including increased serum half-life; reduced immunogenicity; increased functional in vivo half-life; reduced degradation by gastrointestinal tract conditions such as gastrointestinal tract proteases; and increased rate of absorption by gut epithelial cells. An increased rate of absorption by gut epithelial cells and reduced degradation by gastrointestinal tract conditions is important for enteral (such as oral) formulations of a FIX polypeptide. 
     Glycosylation of proteins results in the formation of glycoproteins due to the covalent attachment of oligosaccharides to a polypeptide. The carbohydrate modifications found in glycoproteins are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The N-glycosidic linkage is through the amide group of asparagines. The site of carbohydrate attachment to N-linked glycoproteins is found within a consensus sequence of amino acids, N-X-S/T, where X is any amino acid except proline. In N-linked glycosylation, the carbohydrate directly attached to the protein is GlcNAc. Since glycosylation is known to be highly host cell-dependent, the sugar chains associated with N-linked glycosylation of a protein can differ (Kagawa et al., (1988)  Journal of Biological Chemistry  263:17508-17515). The β-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. In Ser- and Thr-type O-linked glycoproteins, the carbohydrate directly attached to the protein is GalNAc. A number of O-linked glycosylation sites are known in the art and have been reported in the literature, see such as Ten Hagen et al. (1999)  Journal of Biological Chemistry,  274:27867-74; Hanisch et al. (2001)  Glycobiology,  11:731-740; and Ten Hagen et al., (2003)  Glycobiology,  13:1R-16R. 
     Modified FIX polypeptides provided herein can further be glycosylated (i.e. hyperglycosylated) as compared to an unmodified FIX polypeptide due to 1) a carbohydrate moiety covalently linked to at least one non-native glycosylation site not found in the unmodified FIX polypeptide or 2) a carbohydrate moiety covalently linked to at least one native glycosylation site found but not glycosylated in the unmodified FIX polypeptide. A hyperglycosylated FIX polypeptide can include O-linked glycosylation, N-linked glycosylation, and/or a combination thereof. In some examples, a hyperglycosylated FIX polypeptide can include 1, 2, 3, 4 or 5 carbohydrate moieties, each linked to different glycosylation sites. The glycosylation site can be a native glycosylation site. In other examples, the hyperglycosylated polypeptide can be glycosylated at a single non-native glycosylation site. In still other examples, the hyperglycosylated polypeptide can be glycosylated at more than one non-native glycosylation site, such as, the hyperglycosylated FIX polypeptide can be glycosylated at 2, 3 or 4 non-native glycosylation sites. 
     In some instances, a hyperglycosylated FIX polypeptide is glycosylated at a native glycosylation site. For example, FIX, such as for example human FIX having an amino acid sequence set forth in SEQ ID NO: 2, contains N-linked glycosylation sites at N157 and N167 and O-linked glycosylation sites at S53, S61, T159, T169, T172, and T179. The FIX polypeptide can be glycosylated at a single native glycosylation site, or at more than one native glycosylation site, such as, at 2, 3 or 4 native glycosylation sites. A hyperglycosylated FIX polypeptide also can be glycosylated at a native glycosylation site and a non-native glycosylation site. A hyperglycosylated FIX polypeptide also can be glycosylated at multiple native and non-native glycosylation sites. 
     Modified FIX polypeptides provided herein can have at least one additional carbohydrate moiety not found in the unmodified FIX polypeptide when each is synthesized in a eukaryotic cell that is capable of N- and/or O-linked protein glycosylation. Thus, e.g., compared to an unmodified FIX polypeptide, a hyperglycosylated modified FIX polypeptide can have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional carbohydrate moieties. For example, where an unmodified FIX polypeptide has one covalently linked carbohydrate moiety, a hyperglycosylated FIX polypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, or more covalently linked carbohydrate moieties. In some examples, a hyperglycosylated FIX polypeptide of a modified FIX polypeptide provided herein, lacks a carbohydrate moiety covalently linked to a non-native glycosylation site, and has instead at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional carbohydrate moieties attached to native glycosylation sites. In other examples, a hyperglycosylated FIX polypeptide lacks a carbohydrate moiety covalently linked to a native glycosylation site, and has instead at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more carbohydrate moieties attached to non-native glycosylation sites. In some examples, the compositions of the attached sugars can be modified, such as by increasing or decreasing sialic acid content of the attach sugar moieties. 
     Whether a subject FIX polypeptide has N-linked and/or O-linked glycosylation is readily determined using standard techniques, see such as, “Techniques in Glycobiology” R. Townsend and A. Hotchkiss, eds. (1997) Marcel Dekker; and “Glycoanalysis Protocols (Methods in Molecular Biology, Vol. 76)” E. Hounsell, ed. (1998) Humana Press. The change in electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation (such as, using endoglycosidases and/or exoglycosidases) is routinely used to determine the glycosylation status of a protein. Enzymatic deglycosylation can be carried out using any of a variety of enzymes, including, but not limited to, peptide-N4-(N-acetyl-β-D-glycosaminyl) asparagine amidase (PNGase F); endoglycosidase F1, endoglycosidase F2 and endoglycosidase F3, α(2→3,6,8,9) neuraminidase. For example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein, either pre-treated with PNGaseF or untreated with PNGaseF, is conducted. A marked decrease in band width and change in migration position after treatment with PNGaseF is considered diagnostic of N-linked glycosylation. The carbohydrate content of a glycosylated protein also can be detected using lectin analysis of protein blots (such as, proteins separated by SDS-PAGE and transferred to a support, such as a nylon membrane). Lectins, carbohydrate binding proteins from various plant tissues, have high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans (Cummings (1994)  Methods in Enzymol.  230:66-86). Lectins can be detectably labeled (either directly or indirectly), allowing detection of binding of lectins to carbohydrates on glycosylated proteins. For example, when conjugated with biotin or digoxigenin, a lectin bound to a glycosylated protein can be easily identified on membrane blots through a reaction utilizing avidin or anti-digoxigenin antibodies conjugated with an enzyme such as alkaline phosphatase, β-galactosidase, luciferase or horse radish peroxidase, to yield a detectable product. Screening with a panel of lectins with well-defined specificity provides considerable information about a glycoprotein&#39;s carbohydrate complement. 
     Exemplary amino acid positions contemplated herein for modification of a glycosylation site, for attachment of a carbohydrate moiety, include positions corresponding to positions N157 or N167 of a mature FIX polypeptide set forth in SEQ ID NO: 2. Amino acid replacement or replacements can correspond to any of the following positions: N157 or N167 of mature FIX. In a particular embodiment, the amino acid replacement or replacements contributing to hyperglycosylation of modified FIX polypeptides is (are) replacement of amino acids by asparagines (N) or threonine (T). Thus, provided herein are modified FIX polypeptides containing a further modification corresponding to any one or more of N157 or N167 of a mature FIX polypeptide set forth in SEQ ID NO: 2. 
     c. Additional Modifications 
     Additional modifications of polypeptides provided herein include chemical derivatization of polypeptides, including but not limited to, acetylation and carboxylation; changes in amino acid sequence that make the protein susceptible to PEGylation or other modification or that alter properties of the FIX polypeptide. Related moieties for modifying FIX polypeptides also are contemplated, including, but not limited to copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidine or polyproline (Abuchowski et al. (1981); Newmark et al. (1982); and Katre et al. (1987)). A modified FIX polypeptide provided herein can be modified with one or more polyethylene glycol moieties (PEGylated). Activated PEG derivatives can be used to interact directly with the FIX polypeptides, and include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups can be used for the modification of sulfhydryl groups, and PEG reagents containing hydrazine or hydrazide groups can be used to modify aldehydes generated by periodate oxidation of carbohydrate groups. In some instances, a modified FIX polypeptide provided herein can contain one or more non-naturally occurring pegylation sites that are engineered to provide PEG-derivatized polypeptides with reduced serum clearance (U.S. Published Patent Application No: 2006-0040856). 
     Also contemplated are modified FIX polypeptides sequences that have phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine. 
     Other suitable additional modifications of a modified FIX polypeptide provided herein are polypeptides that have been modified using standard chemical techniques and/or recombinant nucleic acid method as to increase their resistance to proteolytic degradation, to optimize solubility properties and/or to render them more suitable as a therapeutic agent. For example, the backbone of the peptide can be cyclized to enhance stability (see such as, Friedler et al. (2000)  J. Biol. Chem.  275:23783-23789). Analogs can be used that include residues other than naturally occurring L-amino acids, such as, D-amino acids or non-naturally occurring synthetic amino acids. 
     Modifications of FIX polypeptides provided herein also can be combined with modifications to improve post-translational processing of the FIX polypeptides. For example, FIX polypeptides can be modified to improve proteolytic cleavage of the signal or propeptides sequences (U.S. Pat. Application No. 2004-0102388), and/or to improve post-translational modifications such as carboxylation, phosphorylation, sulfation, and/or glycosylation, as discussed above. An exemplary modification that improves propeptide cleavage includes, modification of Y1, in particular Y1A (Meulien et al. (1990)  Protein. Eng.  3(7): 629-633 and U.S. Pat. Nos. 4,770,999 and 5,521,070). 
     Modifications of FIX polypeptides provided herein also can be combined with amino acid modifications to further improve stability, binding properties and serum half-life of the FIX polypeptides. A non-limiting example of such modification includes replacement of all or part of the FIX activation peptide (amino acid residues A146 through R180 of a mature FIX polypeptide, SEQ ID NO: 2) with an activation peptide of another vitamin K-dependent polypeptide, such as FVII, FX, or protein C. Another non-limiting example of such modification includes replacement of all or part of the gla domain (amino acid residues Y1 through V46 of a mature FIX polypeptide, SEQ ID NO: 2) with a gla domain of another vitamin K-dependent polypeptide, such as FVII, FX, or protein C. Such modifications and generation of chimeric proteins are described in, for example, published International PCT application No. WO 2006/018204 A1, Lin et al. (1990)  J. Biol. Chem.  265(1):144-150 and Cheung et al. (1991)  J. Biol. Chem.  266(14):8797-880. In another example, amino acid residues in FIX polypeptides that are involved in binding to collagen, in particular collagen IV in the liver, can be modified to increase circulating levels of the FIX polypeptides in the blood. For example, K5 and V 10 in the γ-carboxyglutamic acid (gla) domain can be modified, such as K5A and/or V10K (Cheung et al. (1992)  J. Biol. Chem.  267(29): 20529-20531, Gui et al. (2002)  Blood  100(1): 153-158, and Schuettrumpf et al. (2005)  Blood  105(6): 2316-2323). In yet another example modified FIX polypeptide can contain modifications to increase resistance to inhibition by heparin. One such modification is described in, for example, U.S. Pat. Application No. 2004-0110675. 
     Additional modifications that alter properties or activities of FIX polypeptides, such as, peptidase activity, coagulant activity, esterase activity, protein binding activities (such as to FVIII, FX, or FXI), cofactor binding activities, such as Ca2+, phospholipid or cell surface binding activities, and increased resistance to inhibitors, such as heparin and warfarin, are known in the art and can be combined with the modifications provided herein. Exemplary modifications of the peptidase activity include replacement of amino acid residues of the catalytic domain, H221, D269, S365 (U.S. Pat. No. 6,315,995) or replacement of 8338 to enhance clotting activity (U.S. Pat. No. 6,531,298 and Chang et al. (1998)  J. Biol. Chem.  273(20): 12089-12094). Peptidase activity also can be altered by modifying substrate binding sites or cofactor sites. For example, modifications of K316 can alter binding of factor X to modified FIX polypeptides (Kolkman and Mertens (2000)  Biochem. J.  350: 701-707). Exemplary modifications of FIX polypeptides with altered Ca 2+  binding include replacement of residues in the EGF-like domains, such as, but not limited to, G60S, or replacement of the EGF-1 domain of FIC with that of FVII (Chang et al. (1995)  Thromb and Haemost. Abst  73:1202). Exemplary modifications of FIX polypeptides that alter FVIIIa-dependent FIXa activity include mutations in the α-helical regions 333-339 and 301-333, which interact with FVIIIa. One or more properties of a FIX polypeptide may be altered as a result of a modification or combination of modifications. For example, modification of Ca 2+  binding may lead to a change in coagulation activity. 
     F. PRODUCTION OF FIX POLYPEPTIDES 
     1. Expression Systems 
     FIX polypeptides (modified and unmodified) can be produced by any methods known in the art for protein production, including the introduction of nucleic acid molecules encoding FIX into a host cell, host animal and expression from nucleic acid molecules encoding FIX in vitro. Expression hosts include  E. coli , yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification. 
     Expression in eukaryotic hosts can include expression in yeasts such as  Saccharomyces cerevisiae  and  Pichia Pastoria , insect cells such as  Drosophila  cells and lepidopteran cells, plants and plant cells such as tobacco, corn, rice, algae and lemna. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs. Transgenic animals for the production of wild-type FIX polypeptides are known in the art (U.S. Patent Publication Nos. 2002-0166130 and 2004-0133930) and can be adapted for production of modified FIX polypeptides provided herein. 
     Many expression vectors are available for the expression of FIX. The choice of expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells. 
     Methods of production of FIX polypeptides can include coexpression of one or more additional heterologous polypeptides that can aid in the generation of the FIX polypeptides. For example, such polypeptides can contribute to the post-translation processing of the FIX polypeptides. Exemplary polypeptides include, but are not limited to, peptidases that help cleave FIX precursor sequences, such as the propeptide sequence, and enzymes that participate in the modification of the FIX polypeptide, such as by glycosylation, hydroxylation, carboxylation or phosphorylation, for example. An exemplary peptidase that can be coexpressed with FIX is PACE/furin (or PACE-SOL), which aids in the cleavage of the FIX propeptide sequence (Harrison et al. (1998)  Semin Hematol.  35(2 Suppl 2): 4-10). An exemplary protein that aids in the carboxylation of the FIX polypeptide is the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR), which produces reduced vitamin K for utilization as a cofactor by the vitamin K-dependent γ-carboxylase (Wajih et al.,  J. Biol. Chem.  280(36)31603-31607). A subunit of this enzyme, VKORC1, can be coexpressed with the modified FIX polypeptide to increase the γ-carboxylation The one or more additional polypeptides can be expressed from the same expression vector as the FIX polypeptide or from a different vector. 
     a. Prokaryotic Expression 
     Prokaryotes, especially  E. coli , provide a system for producing large amounts of FIX (see, for example, Platis et al (2003)  Protein Exp. Purif.  31(2): 222-30; and Khalizzadeh et al. (2004)  J. Ind. Microbiol. Biotechnol.  31(2): 63-69). Transformation of  E. coli  is a simple and rapid technique well known to those of skill in the art. Expression vectors for  E. coli  can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λP L  promoter. 
     FIX can be expressed in the cytoplasmic environment of  E. coli . The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and β-mercaptoethanol and denaturants (such as, such as guanidine-HCl and urea) can be used to resolubilize the proteins. An alternative approach is the expression of FIX in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases leading to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility. Typically, temperatures between 25° C. and 37° C. are used. Mutations also can be used to increase solubility of expressed proteins. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells. 
     b. Yeast 
     Yeasts such as  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis , and  Pichia pastoris  are useful expression hosts for FIX (see for example, Skoko et al. (2003)  Biotechnol. Appl. Biochem.  38(Pt3):257-65). Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7, and GAL5 and metallothionein promoters such as CUP1. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble and co-expression with chaperonins, such as Bip and protein disulfide isomerase, can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from  Saccharomyces cerevisiae  and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the  Arxula adeninivorans  glucoamylase. A protease cleavage site (such as, the Kex-2 protease) can be engineered to remove the fused sequences from the polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs. 
     c. Insects and Insect Cells 
     Insects and insect cells, particularly using a baculovirus expression system, are useful for expressing polypeptide such as FIX (see, for example, Muneta et al. (2003)  J. Vet. Med. Sci.  65(2):219-23). Insect cells and insect larvae, including expression in the haemolymph, express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typically, expression vectors use a promoter such as the polyhedrin promoter of baculovirus for high level expression. Commonly used baculovirus systems include baculoviruses such as  Autographa californica  nuclear polyhedrosis virus (AcNPV), and the  bombyx mori  nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from  Spodoptera frugiperda, Pseudaletia unipuncta  (A7S) and  Danaus plexippus  (DpN1). For high level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines  Pseudaletia unipuncta  (A7S) and  Danaus plexippus  (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems. 
     An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells ( Drosophila melanogaster ) and C7 cells ( Aedes albopictus ) can be used for expression. The  Drosophila  metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin. 
     d. Mammalian Cells 
     Mammalian expression systems can be used to express FIX polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha 1-antitrypsin, beta-globin, myelin basic protein, myosin light chain-2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase and thymidine kinase. Fusion with cell surface signaling molecules such as TCR-ζ and Fc ε RI-γ can direct expression of the proteins in an active state on the cell surface. 
     Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include, but are not limited to, CHO, Balb/3T3, HeLa, MT2, mouse NS0 (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. One such example is the serum free EBNA-1 cell line (Pham et al., (2003)  Biotechnol. Bioeng.  84:332-42). Expression of recombinant wild-type FIX polypeptides exhibiting similar structure and post-translational modifications as plasma-derived FIX are known in the art (see, such as, Bon et al. (1998)  Semin Hematol.  35 (2 Suppl 2): 11-17). Methods of optimizing Factor IX expression are known. For example, supplementation of vitamin K in culture medium or co-expression of vitamin K-dependent γ-carboxylases (Wajih et al.,  J. Biol. Chem.  280(36)31603-31607) can aid in post-translational modification of FIX polypeptides. Multiple purification steps also can aid in enriched subpopulations of FIX polypeptides with desired post-translational modifications including, γ-carboxylation, β-hydroxylation, and glycosylation. 
     e. Plants 
     Transgenic plant cells and plants can be used for the expression of FIX. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with  agrobacterium -mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements, and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as  Arabidopsis  and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce proteins (see, for example, Mayfield et al. (2003)  PNAS  100:438-442). Because plants have different glycosylation patterns than mammalian cells, this can influence the choice to produce FIX in these hosts. 
     2. Purification 
     Methods for purification of FIX polypeptides from host cells depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary the proteins can be extracted and further purified using standard methods in the art. 
     FIX can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography and ionic exchange chromatography. Multiple chromatographic steps can be employed to improve selection of FIX polypeptides with particular posttranslational modifications. For example, multiple chromatographic steps can be used to separate γ-carboxylated FIX polypeptides (see such as, Cott et al. (2004)  Papers in Biotechnology  1-21). Such techniques also can be used to enrich for FIX polypeptides with or without modifications at particular sites, including modifications such as, but not limited to, γ-carboxylation of particular glutamic acid residues in the gla domain, phosphorylation (e.g., at S158), sulfation (such as, at Y155), and glycosylation (see such as, Kaufman et al. (1986)  J Biol Chem  261: 9622-8; Harrison et al. (1998)  Semin Hematol  35 (Suppl. 2): 4-10; Bond et al. (1998)  Semin Hematol  35 (Suppl. 2): 11-7). 
     Affinity purification techniques also can be used to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind FIX can be used in affinity purification. Expression constructs also can be engineered to add an affinity tag such as a myc epitope, GST fusion or His 6  and affinity purified with myc antibody, glutathione resin, and Ni-resin, respectively, to a protein. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques. 
     3. Fusion Proteins 
     Fusion proteins containing a targeting agent and a modified FIX polypeptide also are provided. Pharmaceutical compositions containing such fusion proteins formulated for administration by a suitable route are provided. Fusion proteins are formed by linking in any order the modified FIX polypeptide and an agent, such as an antibody or fragment thereof, growth factor, receptor, ligand, and other such agent for directing the mutant protein to a targeted cell or tissue. Linkage can be effected directly or indirectly via a linker. The fusion proteins can be produced recombinantly or chemically by chemical linkage, such as via heterobifunctional agents or thiol linkages or other such linkages. The fusion proteins can contain additional components, such as  E. coli  maltose binding protein (MBP) that aid in uptake of the protein by cells (see, International PCT application No. WO 01/32711). In another embodiment the modified FIX is fused to polypeptides that aid in stability, such as albumin (Sheffield et al. (2004)  Br. J. Haemotol.  126(4): 565-573). 
     4. Polypeptide Modification 
     Modified FIX polypeptides can be prepared as naked polypeptide chains or as a complex. For some applications, it can be desirable to prepare modified FIX in a “naked” form without post-translational or other chemical modifications. Naked polypeptide chains can be prepared in suitable hosts that do not post-translationally modify FIX. Such polypeptides also can be prepared in in vitro systems and using chemical polypeptide synthesis. For other applications, particular modifications can be desired including pegylation, albumination, glycosylation, carboxylation, hydroxylation, phosphorylation, or other known modifications. Modifications can be made in vitro or, for example, by producing the modified FIX in a suitable host that produces such modifications. 
     5. Nucleotide Sequences 
     Nucleic acid molecules encoding modified FIX polypeptides or the fusion protein operationally linked to a promoter, such as an inducible promoter for expression in mammalian cells also are provided. Such promoters include, but are not limited to, CMV and SV40 promoters; adenovirus promoters, such as the E2 gene promoter, which is responsive to the HPV E7 oncoprotein; a PV promoter, such as the PBV p89 promoter that is responsive to the PV E2 protein; and other promoters that are activated by the HIV or PV or oncogenes. 
     Modified FIX polypeptides provided herein also can be delivered to cells in gene transfer vectors. The transfer vectors can encode additional therapeutic agent(s) for treatment of diseases or disorders, such as treatments for hemophilia, inherited disorders and others for which FIX is administered. Transfer vectors encoding modified FIX polypeptides can be used systemically by administering the nucleic acid to a subject. For example, the transfer vector can be a viral vector, such as an adenoviral vector. Vectors encoding FIX also can be incorporated into stem cells and such stem cells administered to a subject, for example, by transplanting or engrafting the stem cells at sites for therapy. For example, mesenchymal stem cells (MSCs) can be engineered to express a modified FIX and such MSCs engrafted at a tumor site for therapy. 
     G. ASSESSING MODIFIED FIX POLYPEPTIDE PROPERTIES AND ACTIVITIES 
     FIX activities and properties can be assessed in vitro and/or in vivo. Assays for such assessment are known to those of skill in the art and are known to correlate tested activities and results to therapeutic and in vivo activities. In one example, FIX variants can be assessed in comparison to unmodified and/or wild-type FIX. In other examples, a modified FIX polypeptide can be assessed for biological activity following in vitro or in vivo exposure to protein stability-altering conditions (i.e. exposure to proteases, or denaturing agents such as temperature or pH). In vitro assays include any laboratory assay known to one of skill in the art, such as for example, cell-based assays including coagulation assays, protein assays, and molecular biology assays. In vivo assays include FIX assays in animal models as well as administration to humans. In some cases, activity of FIX in vivo can be determined by assessing blood, serum, or other bodily fluid for assay determinants. FIX variants also can be tested in vivo to assess an activity or property, such as stability (such as, half-life) and therapeutic effect. 
     1. In Vitro Assays 
     Exemplary in vitro assays include assays to assess polypeptide stability and activity. Stability assays include assays that assess protease resistance or thermal stability or other physical property indicative of stability of the polypeptide in vivo or in vitro. Stability also can be assessed by protein structure and conformational assays known in the art. Assays for activity include, but are not limited to, measurement of FIX interaction with other coagulation cofactors, such as factor VIII, enzymatic assays to determine proteolytic activity of FIX polypeptides, and cell-based assays to determine the effect of FIX polypeptides variants on coagulation. 
     Concentrations of modified FIX polypeptides can be assessed by methods well-known in the art, including but not limited to enzyme-linked immunosorbant assays (ELISA), SDS-PAGE; Bradford, Lowry, BCA methods; UV absorbance, and other quantifiable protein labeling methods, such as, but not limited to, immunological, radioactive and fluorescent methods and related methods. 
     Assessment of cleavage products of proteolysis reactions, including cleavage of FIX polypeptides or products produced by FIX protease activity, can be performed using methods including but not limited to, chromogenic substrate cleavage, HPLC, SDS-PAGE analysis, immunohistochemistry, immunoprecipitation, NH 2 -terminal sequencing, and protein labeling. 
     FIX polypeptides that have been activated via proteolytic cleavage after R145 and R180 can be prepared in vitro. The FIX polypeptides can be first prepared by any of the methods of production described herein, including, but not limited to, production in mammalian cells followed by purification. Cleavage of the FIX polypeptides into the active protease form of FIX can be accomplished by incubation with activated factor XI (FXIa). The activated polypeptides can be used in any of the assays to measure FIX activities described herein. 
     Modified FIX polypeptides can be tested for proteolytic activity. Activated forms of the modified FIX polypeptides (FIXa) can be used in the assay. Assays using a synthetic substrate, such as a CH 3 SO 2 -LGR-pNA peptide, can be employed to measure enzymatic cleavage activity of the FIXa polypeptides. Hydrolysis of CH 3 SO 2 -LGR-pNA in the presence of FIXa can be measured by assessing the production of p-nitroanaline (pNA) from the cleavage reaction sample. The amount of pNA in the sample is proportional to the absorbance of the sample at 405 nm and thus indicates the extent of proteolytic activity in the FIXa sample. Additional exemplary fluorogenic substrates that can be used to assess FIXa cleavage activity include, but are not limited to, Mes- D -CHD-Gly-Arg-AMC (Pefafluor FIXa10148) and H- D -Leu-PHG-Arg-AMC (Pefafluor FIXa3688), wherein cleavage is assessed by release of AMC. Molecules that enhance FIXa catalytic activity, such as ethylene glycol, can be employed in such assays (Sturzebecher et al. (1997)  FEBS Lett . (412) 295-300). 
     Proteolytic activity of FIXa also can be assessed by measuring the conversion of factor X (FX) into activated factor X (FXa). Modified FIXa polypeptides can be incubated in the presence of FX polypeptides in the presence of phospholipids vesicles (phosphatidylserine and/or phosphatidylcholine) and Ca 2+ , and cleavage of FX to produce FXa can be assayed using a chromogenic substrate, such as S2222 or S2765, which is specifically cleaved by FXa (Chromogenics AB, Molndal, Sweden). 
     Inhibition assays can be used to measure resistance of modified FIX polypeptides to FIX inhibitors. Exemplary inhibitors include, but are not limited to, antithrombin, p-aminobenzamidine, serine protease inhibitors, and FIX-specific antibodies. Inhibitors such as antithrombin which bind to the active site of FIXa can be used in inhibition assays, such as competition assays, wherein the ability of the inhibitor to compete with another substrate is measured. 
     FIX polypeptides can be tested for binding to other coagulation factors and inhibitors. For example, FIX direct and indirect interactions with proteins of the tenase complex, including factors VIIIa, IXa and X, and inhibitors, such as antithrombin III and heparin can be assessed using any binding assay known in the art, including, but not limited to, immunoprecipitation, column purification, non-reducing SDS-PAGE, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), isothermal titration calorimetry (ITC), circular dichroism (CD), protein fragment complementation assays (PCA), Nuclear Magnetic Resonance (NMR) spectroscopy, light scattering, sedimentation equilibrium, small-zone gel filtration chromatography, gel retardation, Far-western blotting, fluorescence polarization, hydroxyl-radical protein footprinting, phage display, and various two-hybrid systems. 
     FIX polypeptides can be tested for coagulation activity by using assays well known in the art. For example, some of the assays include, but are not limited to, a two stage clotting assay, (Leibman, H. A. et al. (1985)  Proc. Natl. Acad. Sci., USA,  82, 3879-3883); an assay based on the single stage activated partial thromboplastin time (“aPTT”), Smith, K. J. et al. (1988)  Blood,  72, 1269-1277; and assays which are modifications of the aPTT test, for example, Jenny, R. et al. (1986)  Preparative Biochemistry,  16, 227-245, whole blood partial thromboplastin time (PTT), activated clotting time (ACT), recalcified activated clotting time, or the Lee-White Clotting time. For the aPTT assay, coagulation activity of a FIX polypeptide can be assessed by using equal volumes of activated partial thromboplastin reagent, FIX-deficient plasma (obtained from either a patient with hemophilia B using sterile phlebotomy techniques well known in the art or by using FIX immunodepleted plasma), and normal pooled plasma as standard, or the sample. In this assay, one unit of activity is defined as that amount present in one milliliter of normal pooled plasma. Further, an assay for biological activity based on the ability of FIX to reduce the clotting time of plasma from FIX-deficient patients to normal can be performed as described in, for example, Proctor and Rapaport (1961)  Amer. J. Clin. Path.  36: 212. 
     FIX polypeptides also can be assessed for presence of post-translational modifications. Such assays are known in the art and include assays to measure glycosylation, hydroxylation, sulfation, carboxylation, and phosphorylation. In an exemplary assay for glycosylation, carbohydrate analysis can be performed, for example, with SDS page analysis of FIX polypeptides exposed to hydrazinolysis or endoglycosidase treatment. Hydrazinolysis releases N- and O-linked glycans from glycoproteins by incubation with anhydrous hydrazine, while endoglycosidase release involves PNGase F, which releases most N-glycans from glycoproteins. Hydrazinolysis or endoglycosidase treatment of FIX polypeptides generates a reducing terminus that can be tagged with a fluorophore or chromophore label. Labeled FIX polypeptides can be analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The fluorescent tag for glycans also can be used for monosaccharide analysis, profiling or fingerprinting of complex glycosylation patterns by HPLC. Exemplary HPLC methods include hydrophilic interaction chromatography, electronic interaction, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Exemplary glycan probes include, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA-Ac) and 2-aminobenzoic acid (2-AA). Carbohydrate moieties also can be detected through use of specific antibodies that recognize the glycosylated FIX polypeptide. 
     An exemplary assay to measure carboxylation comprises reverse phase HPLC analysis of FIX polypeptides that have been subjected to alkaline hydrolysis (Przysiecki et al. (1987)  PNAS  84: 7856-7860). Exemplary assays to measure phosphorylation include use of phosphospecific antibodies to phospho-serine and/or -tyrosine amino acid residues or to a serine-phosphorylated FIX polypeptide.  32 P metabolic labeling of cells that produce the FIX polypeptide also can be used to assess phosphorylation, wherein the labeled FIX polypeptide can be purified and analyzed for incorporation of radioactive phosphate. An exemplary assay for tyrosine sulfation includes  35 S labeling of cells that produce the FIX polypeptide. In such method, cells are incubated with either  35 S—S 2 SO 4  or  35 S-methionine and incorporation of the  35 S is determined by normalization to the  35 S-methionine sample. 
     Structural properties of modified FIX polypeptides also can be assessed. For example, X-ray crystallography, nuclear magnetic resonance (NMR), and cryoelectron microscopy (cryo-EM) of modified FIX polypeptides can be performed to assess three-dimensional structure of the FIX polypeptides and/or other properties of FIX polypeptides, such as Ca 2+ or cofactor binding. 
     Additionally, the presence and extent of FIX degradation can be measured by standard techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting of electrophoresed FIX-containing samples. FIX polypeptides that have been exposed to proteases also can be subjected to N-terminal sequencing to determine location or changes in cleavage sites if the modified FIX polypeptides. 
     2. Non-Human Animal Models 
     Non-human animal models can be used to assess activity and stability of modified FIX polypeptides. For example, non-human animals can be used as models for a disease or condition. Non-human animals can be injected with disease and/or phenotype-inducing substances prior to administration of FIX variants to monitor the effects on disease progression. Genetic models also are useful. Animals, such as mice, can be generated which mimic a disease or condition by the overexpression, underexpression or knock-out of one or more genes. Such animals can be generated by transgenic animal production techniques well-known in the art or using naturally-occurring or induced mutant strains. Examples of useful non-human animal models of diseases associated with FIX include, but are not limited to, models of bleeding disorders, in particular hemophilia, or thrombotic disease. These non-human animal models can be used to monitor activity of FIX variants compared to a wild type FIX polypeptide. 
     Animal models also can be used to monitor stability, half-life, and clearance of modified FIX polypeptides. Such assays are useful for comparing modified FIX polypeptides and for calculating doses and dose regimens for further non-human animal and human trials. For example, a modified FIX polypeptide can be injected into the tail vein of mice. Blood samples are then taken at time-points after injection (such as minutes, hours and days afterwards) and then the level of the modified FIX polypeptides in bodily samples including, but not limited to, serum or plasma can be monitored at specific time-points for example by ELISA or radioimmunoassay. Blood samples also can be tested for coagulation activity in methods, such as the aPTT assay. 
     Modified FIX polypeptides can be tested for immune tolerance using animal models. Animal models for immune tolerance of FIX are known in the art and include rhesus macaque, mouse, rat, and dog models to test long term expression of FIX via injection of polypeptides or gene transfer vectors (Lozier et al. (1999)  Blood  93(6) 1875-1881 and Keith et al., (1995)  Thromb. Haemost.  73(1): 101-105). 
     Modified FIX polypeptides can be tested for therapeutic effectiveness using animal models for hemophilia. In one non-limiting example, an animal model such as a mouse can be used. Mouse models of hemophilia are available in the art and include FIX deficient mice and mice expressing mutant FIX polypeptides, and can be employed to test modified FIX polypeptides (Wang et al. (1997)  PNAS  94:11563-11566, Lin et al. (1997)  Blood  90:3962-3966, Kundu et al. (1998)  Blood  92: 168-174, Sabatino et al. (2004)  Blood  104(9): 2767-2774 and Jin et al. (2004)  Blood  104:1733-1739) 
     Other models of FIX deficiencies include hemophilic dogs that express defective FIX or that have been hepatectomized (Evans et al. (1989)  PNAS  86:10095, Mauser et al. (1996)  Blood  88:3451, and Kay et al. (1994)  PNAS  91:2353-2357). 
     Modified FIX polypeptides can be tested for therapeutic effectiveness using animal models for thrombotic disease (Dodds (1987)  Ann N Y Acad. Sci.  516: 631-635). Modified FIX polypeptides that have antithrombotic activity can be tested in such models. In one non-limiting example, an animal model such as a rat or mouse model of ischemic stroke can be used to test the effectiveness of modified FIX polypeptides. Such animal models are known in the art (Beech, et al. (2001)  Brain Res  895: 18-24, Buchan et al.  Stroke  23: 273-9 (1992), Carmichael et al. (2005)  NeuroRx  2: 396-409, Chen et al. (1986)  Stroke  17: 738-43, Gerriets et al. (2003)  J Neurosci Methods  122: 201-11, Koizumi et al. (1986)  Jpn. J. Stroke  8: 1-8, Longa et al. (1989)  Stroke  20: 84-91, Mayzel-Oreg et al. (2004)  Magn Reson Med  51: 1232-8, Tamura, et al. (1981)  J Cereb Blood Flow Metab  1: 53-60, Watson et al. (1985)  Ann Neurol  17: 497-504, Zhang et al. (1997)  J Cereb Blood Flow Metab  17: 123-35). 
     3. Clinical Assays 
     Many assays are available to assess activity of FIX for clinical use. Such assays can include assessment of coagulation, protein stability, and half-life in vivo and phenotypic assays. Phenotypic assays and assays to assess the therapeutic effect of FIX treatment include assessment of blood levels of FIX (such as measurement of serum FIX prior to administration and time-points following administrations including, after the first administration, immediately after last administration, and time-points in between, correcting for the body mass index (BMI)), phenotypic response to FIX treatment including amelioration of symptoms over time compared to subjects treated with an unmodified and/or wild type FIX or placebo. Examples of clinical assays to assess FIX activity can be found such as in Franchini et al. (2005)  Thromb Haemost.  93(6): 1027-1035, Shapiro et al. (2005)  Blood  105(2): 518-525, and White et al. (1997)  Thromb Haemost.  78(1): 261-265. Patients can be monitored regularly over a period of time for routine or repeated administrations, following administration in response to acute events, such as hemorrhage, trauma, or surgical procedures. 
     H. FORMULATION/PACKAGING/ADMINISTRATION 
     Pharmaceutical compositions containing an optimized polypeptide produced using methods described herein, including FIX variant (modified) polypeptides, modified FIX fusion proteins or encoding nucleic acid molecules, can be formulated in any conventional manner by mixing a selected amount of the polypeptide with one or more physiologically acceptable carriers or excipients. Selection of the carrier or excipient is within the skill of the administering profession and can depend upon a number of parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical or any other mode) and disorder treated. The pharmaceutical compositions provided herein can be formulated for single dosage (direct) administration or for dilution or other modification. The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment. Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. 
     1. Administration of Modified FIX Polypeptides 
     The polypeptides can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients. The polypeptides can be targeted for delivery, such as by conjugation to a targeting agent, such as an antibody. Liposomal suspensions, including tissue-targeted liposomes, also can be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Pat. No. 4,522,811. Liposomal delivery also can include slow release formulations, including pharmaceutical matrices such as collagen gels and liposomes modified with fibronectin (see, for example, Weiner et al. (1985)  J Pharm Sci.  74(9): 922-5). 
     The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein. The active compounds can be administered by any appropriate route, for example, oral, nasal, pulmonary, parenteral, intravenous, intradermal, subcutaneous, or topical, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. In a particular embodiment, the FIX polypeptide is administered orally. FIX polypeptides can be formulated with additional coagulation factors. 
     The modified FIX and physiologically acceptable salts and solvates can be formulated for administration by inhalation (either through the mouth or the nose), oral, transdermal, pulmonary, parenteral, or rectal administration or injection. For administration by inhalation, the modified FIX can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer with the use of a suitable propellant, such as, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of such as, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of a therapeutic compound and a suitable powder base such as lactose or starch. 
     FIX polypeptides can be formulated as liquid or powder. In the case of a liquid, the modified polypeptides can be injected from a syringe or an auto-injector. In the case of a powder, the modified polypeptides can be reconstituted with a pharmaceutically acceptable excipient, such as pharmaceutically-acceptable saline, prior to administration. Administration can be by a medical professional or self-administration. 
     For pulmonary administration to the lungs, the modified FIX can be delivered in the form of an aerosol spray presentation from a nebulizer, turbonebulizer, or microprocessor-controlled metered dose oral inhaler with the use of a suitable propellant. Generally, the particle size is small, such as in the range of 0.5 to 5 microns. In the case of a pharmaceutical composition formulated for pulmonary administration, detergent surfactants are not typically used. Pulmonary drug delivery is a promising non-invasive method of systemic administration. The lungs represent an attractive route for drug delivery, mainly due to the high surface area for absorption, thin alveolar epithelium, extensive vascularization, lack of hepatic first-pass metabolism, and relatively low metabolic activity. 
     The modified FIX polypeptides exhibit increased resistance to proteolysis and half-life in the gastrointestinal tract. Thus, preparations for oral administration can be suitably formulated without the use of protease inhibitors, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat. 
     The modified FIX polypeptides can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. 
     The modified FIX can be formulated, for example, for parenteral administration by injection (such as, by bolus injection or continuous infusion). Formulations for injection can be presented in unit dosage form (such as, in ampoules or in multi-dose containers) with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder-lyophilized form for constitution with a suitable vehicle, such as, sterile pyrogen-free water, before use. 
     Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in a conventional manner. 
     The pharmaceutical compositions can be formulated for local or topical application, such as for topical application to the skin (transdermal) and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, can be formulated as 0.01%-10% isotonic solutions and pH about 5-7 with appropriate salts. The compounds can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma). 
     The concentration of active compound in the drug composition depends on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. As described further herein, dosages can be determined empirically using dosages known in the art for administration of unmodified FIX, and comparisons of properties and activities (such as, stability and activities) of the modified FIX compared to the unmodified and/or native FIX. 
     The compositions, if desired, can be presented in a package, in a kit or dispenser device, that can contain one or more unit dosage forms containing the active ingredient. The package, for example, contains metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The compositions containing the active agents can be packaged as articles of manufacture containing packaging material, an agent provided herein, and a label that indicates the disorder for which the agent is provided. 
     a. Oral Administration 
     Among the modified FIX polypeptides provided herein are FIX polypeptides modified to increase protein stability to conditions amendable to oral delivery. Oral delivery can include administration to the mouth and/or gastrointestinal tract. Such modifications can include increased protein-half life under one or more conditions such as exposure to saliva, exposure to proteases in the gastrointestinal tract, exposure to increased temperature, and exposure to particular pH conditions, such as the low pH of the stomach and/or pH conditions in the intestine. For example, modifications can include resistance to one or more proteases including pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, elastase, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. Modifications also can include increasing overall stability to potentially denaturing or conformation-altering conditions such as thermal tolerance, and tolerance to mixing and aeration (such as, chewing). 
     FIX polypeptides modified for suitability to oral delivery can be prepared using any of the methods described herein. For example, 2D- and 3D-scanning mutagenesis methods for protein rational evolution (see, co-pending U.S. Publication No. US 2005-0202438 A1 and U.S. Publication No. US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747) can be used to prepare modified FIX. Modification of FIX polypeptides for suitability for oral delivery can include removal of proteolytic digestion sites and/or increasing the overall stability of the protein structure. Such FIX variants exhibit increased protein half-life compared to an unmodified and/or wild-type native FIX in one or more conditions for oral delivery. For example, a modified FIX can have increased protein half-life and/or bioavailability in the mouth, throat (such as, through the mucosal lining), the gastrointestinal tract or systemically. 
     In one embodiment, the half-life of the modified FIX polypeptides provided herein is increased by an amount at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more, when compared to the half-life of a native FIX polypeptide exposed to one or more conditions (i.e. proteases, pH, temperature) for oral delivery. In other embodiments, the half-life in vitro or in vivo (protein stability) of the modified FIX polypeptides provided herein is increased by an amount of at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more, compared to the half-life of native FIX exposed to one or more conditions for oral delivery (i.e. proteases, pH, temperature). 
     In one example, half-life of the modified FIX polypeptide is assessed by increased half-life in the presence of one or more proteases. Proteases include, but are not limited to, proteases in blood, serum, the gastrointestinal tract, and the stomach. For example, proteases include, but are not limited to, pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, trypsin, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, elastase, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. The modified polypeptide can be mixed with one or more proteases and then assessed for activity and/or protein structure after a suitable reaction time. In one embodiment, the modified polypeptide is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% more resistant to proteolysis. 
     Assessment of half-life also can include exposure to increased temperature, such as the body temperature of a subject; exposure to gastric juices and/or simulated gastric juices; exposure to particular pH conditions and/or a combination of two or more conditions. Following exposure to one or more conditions, activity and/or assessment of protein structure can be used to assess the half-life of the modified FIX in comparison to an appropriate control (i.e., an unmodified and/or wild-type FIX polypeptide). 
     For oral administration, the pharmaceutical compositions can take the form of, for example, tablets, pills, liquid suspensions, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as, magnesium stearate, talc or silica); disintegrants (such as, potato starch or sodium starch glycolate); or wetting agents (such as, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically-acceptable saline, pharmaceutically acceptable additives such as suspending agents (such as, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as, lecithin or acacia); non-aqueous vehicles (such as, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (such as, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and sweetening agents as appropriate. 
     The modified FIX polypeptides can be formulated for oral administration, Oral formulations include tablets, capsules, liquids or other suitable vehicle for oral administration. In some examples, the capsules or tablets are formulated with an enteric coating to be gastro-resistant. Preparation of pharmaceutical compositions containing a modified FIX for oral delivery can include formulating modified FIX polypeptides with oral formulations known in the art and/or those described herein. The compositions as formulated do not require addition of protease inhibitors and/or other ingredients that are necessary for stabilization of unmodified (for protease resistance) and wild-type FIX polypeptides upon exposure to proteases, such as selecting pH and other conditions to minimize protease cleavage. For example, such compositions exhibit stability in the absence of compounds such as actinonin or epiactinonin and derivatives thereof; Bowman-Birk inhibitor and conjugates thereof; aprotinin and camostat. In other examples, the preparations for oral administration can include protease inhibitors. 
     Additionally, because modified FIX polypeptides provided herein exhibit increased protein stability, there is more flexibility in the administration of pharmaceutical compositions than their unmodified counterparts. Typically, orally ingested polypeptides are administered in the morning before eating (i.e., before digestive enzymes are activated). The modified polypeptides provided herein exhibit protease resistance to digestive enzymes and can offer the ability to administer pharmaceutical compositions containing a modified FIX polypeptide at other periods during the day and under conditions when digestive enzymes are present and active. 
     For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as, pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as, magnesium stearate, talc or silica); disintegrants (such as, potato starch or sodium starch glycolate); or wetting agents (such as, sodium lauryl sulphate). The active ingredient present in the capsule can be in, for example, liquid or lyophilized form. The tablets or capsules can be coated by methods well known in the art. Tablets and capsules can be coated, for example, with an enteric coating. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (such as, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as, lecithin or acacia); non aqueous vehicles (such as, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (such as, methyl or propyl-p hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and/or sweetening agents as appropriate. 
     Preparations for oral administration can be formulated to give controlled or sustained release or for release after passage through the stomach or in the small intestine of the active compound. For oral administration the compositions can take the form of tablets, capsules, liquids, lozenges and other forms suitable for oral administration. Formulations suitable for oral administration include lozenges and other formulations that deliver the pharmaceutical composition to the mucosa of the mouth, throat and/or gastrointestinal tract. Lozenges can be formulated with suitable ingredients including excipients for example, anhydrous crystalline maltose and magnesium stearate. As noted, modified polypeptides described herein exhibit resistance to blood or intestinal proteases. 
     The compositions for oral administration can be formulated, for example, as gastro-resistant capsules or tablets. Such gastro-resistant capsules are modified release capsules that are intended to resist the gastric fluid and to release their active ingredient or ingredients in the intestinal fluid. They are prepared by providing hard or soft capsules with a gastro-resistant shell (enteric capsules) or by filling capsules with granules or with particles covered with a gastro-resistant coating. 
     The enteric coating is typically, although not necessarily, a polymeric material. Enteric coating materials can contain bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers. The “coating weight,” or relative amount of coating material per capsule, generally dictates the time interval between ingestion and drug release. Any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating to achieve delivery of the active ingredient to the lower gastrointestinal tract. The selection of the specific enteric coating material will depend on the following properties: resistance to dissolution and disintegration in the stomach; impermeability to gastric fluids and drug/carrier/enzyme while in the stomach; ability to dissolve or disintegrate rapidly at the target intestine site; physical and chemical stability during storage; non-toxicity; ease of application as a coating (substrate friendly); and economical practicality. 
     Suitable enteric coating materials include, but are not limited to: cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, such as formed from acrylic acid, met acrylic acid, methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (such as, those copolymers sold under the trade name EUDRAGIT); vinyl polymers and copolymers, such as polyvinyl pyrrolidone (PVP), polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac). Combinations of different coating materials also can be used to coat a single capsule. Exemplary of such gastro-resistant capsules are hard gelatin capsules (sold by Torpac or Capsugel) size 9, coated with cellulose acetate phthalate (CAP) at 12% in acetone. 
     The enteric coating provides for controlled release of the active agent, such that drug release can be accomplished at some generally predictable location in the lower intestinal tract below the point at which drug release would occur without the enteric coating. The enteric coating also prevents exposure of the hydrophilic therapeutic agent and carrier to the epithelial and mucosal tissue of the buccal cavity, pharynx, esophagus, and stomach, and to the enzymes associated with these tissues. The enteric coating therefore helps to protect the active agent and a patient&#39;s internal tissue from any adverse event prior to drug release at the desired site of delivery. Furthermore, the coated capsules can permit optimization of drug absorption, active agent protection, and safety. Multiple enteric coatings targeted to release the active agent at various regions in the lower gastrointestinal tract would enable even more effective and sustained improved delivery throughout the lower gastrointestinal tract. 
     The coating optionally can contain a plasticizer to prevent the formation of pores and cracks that would permit the penetration of the gastric fluids. Suitable plasticizers include, but are not limited to, triethyl citrate (CITROFLEX 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (CITROFLEC A2), CARBOWAX 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, a coating comprised of an anionic carboxylic acrylic polymer will typically contain less than about 50% by weight, such as less than about 30%, 10% to about 25% by weight, based on the total weight of the coating, of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin. The coating also can contain other coating excipients, such as detackifiers, antifoaming agents, lubricants (such as, magnesium stearate), and stabilizers (such as, hydroxypropylcellulose, acids and bases) to solubilize or disperse the coating material, and to improve coating performance and the coated product. 
     The coating can be applied to the capsule or tablet using conventional coating methods and equipment. For example, an enteric coating can be applied to a capsule using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Detailed information concerning materials, equipment and processes for preparing coated dosage forms are described in  Pharmaceutical Dosage Forms: Tablets , eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and in Ansel et al.,  Pharmaceutical Dosage Forms and Drug Delivery Systems,  6 th  Edition (Media, Pa.: Williams &amp; Wilkins, 1995). The coating thickness, as noted above, must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the lower intestinal tract is reached. 
     Preparations for oral administration can be formulated to give controlled or sustained release or for release after passage through the stomach or in the small intestine of the active compound. For oral administration the compositions can take the form of tablets, capsules, liquids, lozenges and other forms suitable for oral administration Formulations suitable for oral administration include lozenges and other formulations that deliver the pharmaceutical composition to the mucosa of the mouth, throat and/or gastrointestinal tract. Lozenges can be formulated with suitable ingredients including excipients for example, anhydrous crystalline maltose and magnesium stearate. As noted, modified polypeptides herein exhibit resistance to blood or intestinal proteases and can exhibit increased half-life in the gastrointestinal tract. Thus, preparations of oral administration can be suitably formulated without additional protease inhibitors or other protective compounds, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat. Preparations for oral administration also can include a modified FIX resistant to proteolysis formulated with one or more additional ingredients that also confer proteases resistance, or confer stability in other conditions, such as particular pH conditions. 
     2. Administration of Nucleic Acids Encoding Modified FIX Polypeptides (Gene Therapy) 
     Also provided are compositions of nucleic acid molecules encoding the modified FIX polypeptides and expression vectors encoding them that are suitable for gene therapy. Rather than deliver the protein, nucleic acid can be administered in vivo, such as systemically or by other route, or ex vivo, such as by removal of cells, including lymphocytes, introduction of the nucleic therein, and reintroduction into the host or a compatible recipient. 
     Modified FIX polypeptides can be delivered to cells and tissues by expression of nucleic acid molecules. Modified FIX polypeptides can be administered as nucleic acid molecules encoding modified FIX polypeptides, including ex vivo techniques and direct in vivo expression. Nucleic acids can be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid sequences can be incorporated into vectors for further manipulation. 
     Methods for administering modified FIX polypeptides by expression of encoding nucleic acid molecules include administration of recombinant vectors. The vector can be designed to remain episomal, such as by inclusion of an origin of replication or can be designed to integrate into a chromosome in the cell. Modified FIX polypeptides also can be used in ex vivo gene expression therapy using non-viral vectors. For example, cells can be engineered to express a modified FIX polypeptide, such as by integrating a modified FIX polypeptide encoding-nucleic acid into a genomic location, either operatively linked to regulatory sequences or such that it is placed operatively linked to regulatory sequences in a genomic location. Such cells then can be administered locally or systemically to a subject, such as a patient in need of treatment. 
     Viral vectors, include, for example adenoviruses, adeno-associated viruses (AAV), poxviruses, herpes viruses, retroviruses and others designed for gene therapy can be employed. The vectors can remain episomal or can integrate into chromosomes of the treated subject. A modified FIX polypeptide can be expressed by a virus, which is administered to a subject in need of treatment. Viral vectors suitable for gene therapy include adenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses, vaccinia viruses and others noted above. For example, adenovirus expression technology is well-known in the art and adenovirus production and administration methods also are well known. Adenovirus serotypes are available, for example, from the American Type Culture Collection (ATCC, Rockville, Md.). Adenovirus can be used ex vivo, for example, cells are isolated from a patient in need of treatment, and transduced with a modified FIX polypeptide-expressing adenovirus vector. After a suitable culturing period, the transduced cells are administered to a subject, locally and/or systemically. Alternatively, modified FIX polypeptide-expressing adenovirus particles are isolated and formulated in a pharmaceutically-acceptable carrier for delivery of a therapeutically effective amount to prevent, treat or ameliorate a disease or condition of a subject. Typically, adenovirus particles are delivered at a dose ranging from 1 particle to 1014 particles per kilogram subject weight, generally between 106 or 108 particles to 1012 particles per kilogram subject weight. In some situations it is desirable to provide a nucleic acid source with an agent that targets cells, such as an antibody specific for a cell surface membrane protein or a target cell, or a ligand for a receptor on a target cell. FIX also can be targeted for delivery into specific cell types. For example, adenoviral vectors encoding FIX polypeptides can be used for stable expression in nondividing cells, such as liver cells, and skeletal muscle cells (Arruda et al. (2001)  Blood  97(1): 130-138 and Yao et al. (1992)  PNAS  89: 3357-3361). In another example, viral or nonviral vectors encoding FIX polypeptides can be transduced into isolated cells for subsequent delivery. Additional cell types for expression and delivery of FIX are known in the art and include but are not limited to, fibroblasts and endothelial cells (Palmer et al. (1989)  Blood  73: 438-445; Yao et al. (1991)  PNAS  88: 8101-8105). 
     The nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes, such as ACES (see, Lindenbaum et al.  Nucleic Acids Res.  2004 Dec. 7; 32(21):e172) can be engineered to encode and express the isoform. Briefly, mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells&#39; chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure using an ACE System. Using this approach, specific loading of one or two gene targets has been achieved in LMTK(−) and CHO cells. 
     Another method for introducing nucleic acids encoding the modified FIX polypeptides is a two-step gene replacement technique in yeast, starting with a complete adenovirus genome (Ad2; Ketner et al. (1994)  Proc. Natl. Acad. Sci. USA  91: 6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a plasmid containing adenovirus sequences to target a specific region in the YAC clone, an expression cassette for the gene of interest and a positive and negative selectable marker. YACs are of particular interest because they permit incorporation of larger genes. This approach can be used for construction of adenovirus-based vectors bearing nucleic acids encoding any of the described modified FIX polypeptides for gene transfer to mammalian cells or whole animals. 
     The nucleic acids can be encapsulated in a vehicle, such as a liposome, or introduced into cells, such as a bacterial cell, particularly an attenuated bacterium or introduced into a viral vector. For example, when liposomes are employed, proteins that bind to a cell surface membrane protein associated with endocytosis can be used for targeting and/or to facilitate uptake, such as capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. 
     For ex vivo and in vivo methods, nucleic acid molecules encoding the modified FIX polypeptide is introduced into cells that are from a suitable donor or the subject to be treated. Cells into which a nucleic acid can be introduced for purposes of therapy include, for example, any desired, available cell type appropriate for the disease or condition to be treated, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, such as, such as stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof. 
     For ex vivo treatment, cells from a donor compatible with the subject to be treated or the subject to be treated cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the subject. Treatment includes direct administration, such as, for example, encapsulated within porous membranes, which are implanted into the patient (see, such as, U.S. Pat. Nos. 4,892,538 and 5,283,187 each of which is herein incorporated by reference in its entirety). Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes and cationic lipids (such as, DOTMA, DOPE and DC-Chol) electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation methods. Methods of DNA delivery can be used to express modified FIX polypeptides in vivo. Such methods include liposome delivery of nucleic acids and naked DNA delivery, including local and systemic delivery such as using electroporation, ultrasound and calcium-phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer and spheroplast fusion. 
     In vivo expression of a modified FIX polypeptide can be linked to expression of additional molecules. For example, expression of a modified FIX polypeptide can be linked with expression of a cytotoxic product such as in an engineered virus or expressed in a cytotoxic virus. Such viruses can be targeted to a particular cell type that is a target for a therapeutic effect. The expressed modified FIX polypeptide can be used to enhance the cytotoxicity of the virus. 
     In vivo expression of a modified FIX polypeptide can include operatively linking a modified FIX polypeptide encoding nucleic acid molecule to specific regulatory sequences such as a cell-specific or tissue-specific promoter. Modified FIX polypeptides also can be expressed from vectors that specifically infect and/or replicate in target cell types and/or tissues. Inducible promoters can be used to selectively regulate modified FIX polypeptide expression. An exemplary regulatable expression system is the doxycycline-inducible gene expression system, which has been used to regulate recombinant FIX expression (Srour et al.  Thromb Haemost.  90(3): 398-405 (2003)). 
     Nucleic acid molecules, as naked nucleic acids or in vectors, artificial chromosomes, liposomes and other vehicles can be administered to the subject by systemic administration, topical, local and other routes of administration. When systemic and in vivo, the nucleic acid molecule or vehicle containing the nucleic acid molecule can be targeted to a cell. 
     Administration also can be direct, such as by administration of a vector or cells that typically targets a cell or tissue. For example, tumor cells and proliferating cells can be targeted cells for in vivo expression of modified FIX polypeptides. Cells used for in vivo expression of a modified FIX polypeptide also include cells autologous to the patient. Such cells can be removed from a patient, nucleic acids for expression of an modified FIX polypeptide introduced, and then administered to a patient such as by injection or engraftment. 
     Polynucleotides and expression vectors provided herein can be made by any suitable method. Further provided are nucleic acid vectors comprising nucleic acid molecules as described above, including a nucleic acid molecule comprising a sequence of nucleotides that encodes the polypeptide as set forth in any of SEQ ID NOS: 3-891, 917-1034, 1036-2044 or a fragment thereof. Further provided are nucleic acid vectors comprising nucleic acid molecules as described above and cells containing these vectors. 
     I. THERAPEUTIC USES 
     The modified FIX polypeptides and nucleic acid molecules provided herein can be used for treatment of any condition for which unmodified FIX is employed. Modified FIX polypeptides have therapeutic activity alone or in combination with other agents. The modified polypeptides provided herein are designed to retain therapeutic activity but exhibit modified properties, particularly increased stability. Such modified properties, for example, can improve the therapeutic effectiveness of the polypeptides and/or can provide for additional routes of administration, such as oral administration. The modified FIX polypeptides and encoding nucleic acid molecules provided herein can be used for treatment of any condition for which unmodified FIX is employed. This section provides exemplary uses of and administration methods. These described therapies are exemplary and do not limit the applications of modified FIX polypeptides. 
     The modified FIX polypeptides provided herein can be used in various therapeutic as well as diagnostic methods in which FIX is employed. Such methods include, but are not limited to, methods of treatment of physiological and medical conditions described and listed below. Modified FIX polypeptides provided herein can exhibit improvement of in vivo activities and therapeutic effects compared to wild-type FIX, including lower dosage to achieve the same effect, a more sustained therapeutic effect and other improvements in administration and treatment. 
     The modified FIX polypeptides described herein exhibit increased protein stability and improved half-life. Thus, modified FIX polypeptides can be used to deliver longer-lasting, more stable therapies. Examples of therapeutic improvements using modified FIX polypeptides include, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects and increased therapeutic effects. 
     In particular, modified FIX polypeptides, are intended for use in therapeutic methods in which FIX has been used for treatment. Such methods include, but are not limited to, methods of treatment of diseases and disorders, such as, but not limited to, blood coagulation disorders, hematologic diseases, hemorrhagic disorders, hemophilias, in particular hemophilia B, and acquired blood disorders, such as caused by liver disease. Modified FIX polypeptides also can be used in the treatment of additional bleeding diseases and disorders, such as, but not limited to, thrombocytopenia (such as, idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand&#39;s disease, hereditary platelet disorders (such as, storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation. In some embodiments, the bleedings to be treated by FIX polypeptides occur in organs such as the brain, inner ear region, eyes, liver, lung, tumor tissue, gastrointestinal tract. In other embodiments, the bleeding is diffuse, such as in haemorrhagic gastritis and profuse uterine bleeding. Patients with bleeding disorders are often at risk for hemorrhage and excessive bleeding during surgery or trauma. Such patients often have acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, (such as, muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from the renal tract), cerebral hemorrhage, surgery (such as, hepatectomy), dental extraction, and gastrointestinal bleedings (such as, UGI bleeds), that can be treated with modified FIX polypeptides. In one embodiment, the modified FIX polypeptides can be used to treat bleeding episodes due to trauma, or surgery, or lowered count or activity of platelets, in a subject. Exemplary methods for patients undergoing surgery include treatments to prevent hemorrhage and treatments before, during, or after surgeries such as, but not limited to, heart surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, dental surgery, or organ transplant surgery, including transplantation of heart, lung, pancreas, or liver. 
     FIX polypeptides lacking functional peptidase activity have been used in therapeutic methods to inhibit blood coagulation (U.S. Pat. No. 6,315,995). Modified FIX polypeptides provided herein that inhibit or antagonize blood coagulation can be used in anticoagulant methods of treatment for ischemic disorders, such as a peripheral vascular disorder, a pulmonary embolus, a venous thrombosis, deep vein thrombosis (DVT), superficial thrombophlebitis (SVT), arterial thrombosis, a myocardial infarction, a transient ischemic attack, unstable angina, a reversible ischemic neurological deficit, an adjunct thrombolytic activity, excessive clotting conditions, reperfusion injury, sickle cell anemia or stroke disorder. In patients with an increased risk of excessive clotting, such as DVT or SVT, during surgery, protease inactive modified FIX polypeptides provided herein can be administered to prevent excessive clotting in surgeries, such as, but not limited to heart surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, or organ transplant surgery, including transplantation of heart, lung, pancreas, or liver. In some cases treatment is performed with FIX alone. In some cases, FIX is administered in conjunction with additional coagulation or anticoagulation factors as required by the condition or disease to be treated. 
     Treatment of diseases and conditions with modified FIX polypeptides can be effected by any suitable route of administration using suitable formulations as described herein including, but not limited to, injection, pulmonary, oral and transdermal administration. If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, exemplary doses of recombinant and native FIX polypeptides can be used as a starting point to determine appropriate dosages. For example, a recombinant FIX polypeptide, BeneFIX®, has been administered to patients with hemophilia B, at a dosage of 50 I.U./kg over a 10 minute infusion, resulting in a mean circulating activity of 0.8±0.2 I.U./dL per I.U./kg infused with a mean half-life of 19.4±5.4 hours. Modified FIX polypeptides that are more stable and have an increased half-life in vivo, can be effective at reduced dosage amounts and or frequencies. For example, because of the improvement in properties such as serum stability, dosages can be lower than comparable amounts of unmodified FIX. Dosages for unmodified FIX polypeptides can be used as guidance for determining dosages for modified FIX polypeptides. Factors such as the level of activity and half-life of the modified FIX in comparison to the unmodified FIX can be used in making such determinations. Particular dosages and regimens can be empirically determined. 
     Dosage levels and regimens can be determined based upon known dosages and regimens, and, if necessary can be extrapolated based upon the changes in properties of the modified polypeptides and/or can be determined empirically based on a variety of factors. Such factors include body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient&#39;s disposition to the disease and the judgment of the treating physician. The active ingredient, the polypeptide, typically is combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form or multi-dosage form can vary depending upon the host treated and the particular mode of administration. 
     The effect of the FIX polypeptides on the clotting time of blood can be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time. 
     Upon improvement of a patient&#39;s condition, a maintenance dose of a compound or compositions can be administered, if necessary; and the dosage, the dosage form, or frequency of administration, or a combination thereof can be modified. In some cases, a subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms or based upon scheduled dosages. In other cases, additional administrations can be required in response to acute events such as hemorrhage, trauma, or surgical procedures. 
     The following are some exemplary conditions for which FIX has been used as a treatment agent alone or in combination with other agents. 
     1. Hemophilia 
     Hemophilia is a bleeding disorder that is caused by a deficiency in one or more blood coagulation factors. It is characterized by a decreased ability to form blood clots at sites of tissue damage. Congenital X-linked hemophilias include hemophilia A and hemophilia B, or Christmas disease, which are caused by deficiencies in FVIII and FIX, respectively. Hemophilia A occurs at a rate of 1 out of 10,0000 males, while hemophilia B occurs in 1 out of 50,000 males. 
     Patients with hemophilia suffer from recurring joint and muscle bleeds, which can be spontaneous or in response to trauma. The bleeding can cause severe acute pain, restrict movement, and lead to secondary complications including synovial hypertrophy. Furthermore, the recurring bleeding in the joints can cause chronic synovitis, which can cause joint damage, destroying synovium, cartilage, and bone. 
     Hemophilia B can be effectively managed with administration of FIX therapeutics. Patients can often achieve normal life span with proper treatment to control bleeding episodes. Administration of FIX can aid in controlling bleeding during surgery, trauma, during dental extraction, or to alleviate bleeding associated with hemarthroses, hematuria, mucocutaneous bleeding, such as epistaxis or gastrointestinal tract bleeding, cystic lesions in subperiosteal bone or soft tissue, or hematomas, which cause neurological complications such as intracranial bleeding, spinal canal bleeding. Death in patients with hemophilia is often the result of bleeding in the central nervous system. Other serious complications in hemophilic patients include development of inhibitors to coagulation factor therapeutics and disease, such as progressive hepatitis with hepatic failure or AIDS caused by use of contaminated blood products. 
     The most frequent alterations in the FIX gene are point mutations, in particular missense mutations. Most of the identified FIX mutations occur in amino acid residues in the coding region of the FIX gene, often affecting evolutionarily conserved amino acids. The severity of the hemophilia depends upon the nature of the mutation. Mutations in the coding region can affect a number of different properties or activities of the FIX polypeptide including alteration of protease activity, cofactor binding, signal peptide or propeptide cleavage, post-translational modifications, and inhibition of cleavage of FIX into its activated form. Other types of point mutations include nonsense mutations that produce an unstable truncated FIX polypeptide, and frameshift mutations (small deletions and insertions) that result in a terminally aberrant FIX molecule. In addition, FIX point mutations can be found in the promoter region, which can disrupt the recognition sequences for several specific gene regulatory proteins, resulting in reduced transcription of coagulation factor IX. Decreased FIX as a result of transcriptional abnormalities is called Hemophilia B Leyden. An exemplary mutation in the promoter region includes disruption of the HNF-4 binding site, which affect regulation of FIX transcription by the androgen receptor. The severity of this type of hemophilia is governed by the levels of androgen in the blood, which increase during puberty and partially alleviate the FIX transcriptional deficiency (Kurachi et al. (1995)). Other missense nucleotide changes affect the processing of factor IX primary RNA transcript. For example, some mutations occur at evolutionarily conserved donor-splice (GT), and acceptor-splice (AG) consensus sequences, which can create cryptic splice junctions and disrupt assembly of spliceosomes. Some severe cases of hemophilia (approximately 10%) present with large deletions in the FIX gene (Gianelli et al. (1998). Treatment of FIX deficiency most often involves administration of FIX in the form of purified plasma concentrates, purified FIX preparations, or recombinant forms of FIX. Occasionally patients with large deletions produce anti-FIX antibodies, or inhibitors, in response to replacement therapy (Gianelli et al. (1983)). Among purified FIX therapeutics available for the treatment of hemophilia are: MonoNine® (Aventis Behring), Alpha-nine-SD® (Alpha therapeutic), and BeneFix® (Genetics Institute). MonoNine® and Alpha-nine-SD® are FIX preparations derived from human plasma, while BeneFix® is a recombinant FIX generated from the native FIX polypeptide sequence. FIX complex therapeutics include Bebulin® VII, a purified concentrate of FIX with FX and low amounts of FVII; Konyne® 80 (Bayer), a purified concentrate of FIX, with FII, FX, and low levels of FVII; PROPLEX® T (Baxter International), a heat treated product prepared from pooled normal human plasma containing FIX with FII, FVII, and FX; and Profilnine SD® (Alpha Therapeutic Corporation), a solvent detergent treated, concentrate of FIX with FII, FX and low levels of FVII. All such products can be modified as described herein and/or replaced with modified FIX polypeptides provided herein. 
     The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for hemophilia, including treatment of bleeding conditions associated with hemophilia. The modified FIX polypeptides provided herein can be used, for example, to control or prevent spontaneous bleeding episodes or to control or prevent bleeding in response to trauma or surgical procedures. In one embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of hemophilia B. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for hemophilia. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example FIX-deficient mice, or any other known disease model for hemophilia, can be treated with modified FIX polypeptides. Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX. 
     The modified FIX polypeptide can be used to deliver longer lasting, more stable hemophilia B therapies. Thus, the modified FIX polypeptides provided herein can be administered at lower dosages and/or less frequently then unmodified or native FIX polypeptides, such as MonoNine® or Alpha-nine-SD®, or recombinant forms of FIX, such as BeneFix®, while retaining one or more therapeutic activities and/or having one or more fewer/decreased side effects. 
     2. Thrombotic Diseases and Conditions 
     Thrombotic diseases are characterized by hypercoagulation, or the deregulation of hemostasis in favor of development of blot clots. Exemplary thrombotic diseases and conditions include arterial thrombosis, venous thrombosis, venous thromboembolism, pulmonary embolism, deep vein thrombosis, stroke, ischemic stroke, myocardial infarction, unstable angina, atrial fibrillation, renal damage, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, artificial organs, shunts or prostheses, and other acquired thrombotic diseases, as discussed below. Typical therapies for thrombotic diseases involve anticoagulant therapies, including inhibition of the coagulation cascade. 
     The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in anticoagulant therapies for thrombotic diseases and conditions, including treatment of conditions involving intravascular coagulation. The modified FIX polypeptides provided herein that can inhibit blood coagulation can be used, for example, to control or prevent formation of thromboses. In a particular embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of an arterial thrombotic disorder. In another embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of a venous thrombotic disorder, such as deep vein thrombosis. In a particular embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of an ischemic disorder, such as stroke. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for thrombotic diseases and conditions. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example mouse models of ischemic stroke, or any other known disease model for a thrombotic disease or condition, can be treated with modified FIX polypeptides (Dodds (1987)  Ann NY Acad Sci  516: 631-635). Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX. 
     a. Arterial Thrombosis 
     Arterial thrombi form as a result of a rupture in the arterial vessel wall. Most often the rupture occurs in patients with vascular disease, such as atherosclerosis. The arterial thrombi usually form in regions of disturbed blood flow and at sites of rupture due to an atherosclerotic plaque, which exposes the thrombogenic subendothelium to platelets and coagulation proteins, which in turn activate the coagulation cascade. Plaque rupture may also produce further narrowing of the blood vessel due to hemorrhage into the plaque. Nonocclusive thrombi can become incorporated into the vessel wall and can accelerate the growth of atherosclerotic plaques. Formation arterial thrombi can result in ischemia either by obstructing flow or by embolism into the distal microcirculation. Anticoagulants and drugs that suppress platelet function and the coagulation cascade can be effective in the prevention and treatment of arterial thrombosis. Such classes of drugs are effective in the treatment of arterial thrombosis. Arterial thrombosis can lead to conditions of unstable angina and acute myocardial infarction. Modified FIX polypeptides provided herein that inhibit coagulation can be used in the treatment and/or prevention of arterial thrombosis and conditions, such as unstable angina and acute myocardial infarction. 
     b. Venous Thrombosis and Thromboembolism 
     Venous thrombosis is a condition in the which a blood clot forms in a vein due to imbalances in the signals for clot formation versus clot dissolution, especially in instances of low blood flow through the venous system. Results of thrombus formation can include damage to the vein and valves of the vein, though the vessel wall typically remains intact. The clots can often embolize, or break off, and travel through the blood stream where they can lodge into organ areas such as the lungs (pulmonary embolism), brain (ischemic stroke, transient ischemic attack), heart (heart attack/myocardial infarction, unstable angina), skin (purpura fulminans), and adrenal gland. In some instances the blockage of blood flow can lead to death. Patients with a tendency to have recurrent venous thromboembolism are characterized as having thrombophilia. Risk factors for developing thromboembolic disease include trauma, immobilization, malignant disease, heart failure, obesity, high levels of estrogens, leg paralysis, myocardial infarction, varicose veins, cancers, dehydration, smoking, oral contraceptives, and pregnancy. Genetic studies of families with thrombophilia have shown inheritable high levels coagulation factors, including FVIII, FIX, and FXI (Lavigne et al. (2003)  J. Thromb. Haemost.  1:2134-2130). 
     Deep vein thrombosis (DVT) refers the formation of venous blot clot in the deep leg veins. The three main factors that contribute to DVT are injury to the vein lining, increased tendency for the blood to clot and slowing of blood flow. Collectively, these factor are called Virchow&#39;s triad. Veins can become injured during trauma or surgery, or as a result of disease condition, such as Buerger&#39;s disease or DIC, or another clot. Other contributing factors to development of DVT are similar to that of more general thromboembolic diseases as discussed above. The clot that forms in DVT causes only minor inflammation, thus, allowing it to break loose into the blood stream more easily. Often the thrombus can break off as a result of minor contraction of the leg muscles. Once the thrombus becomes an embolus it can become lodged into vessels of the lungs where is can cause a pulmonary infarction. Patients with high levels of active FIX in their bloodstream are at an increased risk of developing deep vein thrombosis (Weltermann et al. (2003)  J. Thromb. Haemost.  1(1): 16-18). 
     Thromboembolic disease can be hereditary, wherein the disease is caused by hereditary abnormalities in clotting factors, thus leading to the imbalance in hemostasis. Several congenital deficiencies include antithrombin III, protein C, protein S, or plasminogen. Other factors include resistance to activated protein C (also termed APC resistance or Factor V leiden effect, in which a mutation in factor V makes it resistant to degradation by protein C), mutation in prothrombin, dysfibrinogemia (mutations confer resistance to fibrinolysis), and hyperhomocysteinemia. Development of thrombombolic disease in younger patients is most often due to the congenital defects described above and is called Juvenile Thrombophilia. 
     Treatments for venous thromboembolic disease and DVT typically involve anticoagulant therapy, in which oral doses of heparin and warfarin are administered. Heparin is usually infused into patients to control acute events, followed by longer term oral anticoagulant therapy with warfarin to control future episodes. Other therapies include direct thrombin inhibitors, inhibitors of platelet function, such as aspirin and dextran, and therapies to counteract venous stasis, including compression stockings and pneumatic compression devices. Modified FIX polypeptides provided herein that inhibit blood coagulation can be used in anticoagulant therapies for thromboembolic disease and/or DVT. In some embodiments, modified FIX polypeptides provided herein that inhibit blood coagulation can be used in prevention therapies for thromboembolic disease and/or DVT in patients exhibiting risk factors for thromboembolic disease and/or DVT. Modified FIX polypeptides provided herein that inhibit coagulation can be used in anticoagulation therapies for thromboembolic disease and/or DVT due to high levels of FIX in the blood stream. 
     i. Ischemic Stroke 
     Ischemic stroke occurs when the blood flow to the brain is interrupted, wherein the sudden loss of circulation to an area of the brain results in a corresponding loss of neurologic function. In contrast to a hemorrhagic stroke, which is characterized by intracerebral bleeding, an ischemic stroke is usually caused by thrombosis or embolism. Ischemic strokes account for approximately 80% of all strokes. In addition to the causes and risk factors for development a thromboembolism as discussed above, processes that cause dissection of the cerebral arteries (such as, trauma, thoracic aortic dissection, arteritis) can cause thrombotic stroke. Other causes include hypoperfusion distal to a stenotic or occluded artery or hypoperfusion of a vulnerable watershed region between 2 cerebral arterial territories. Treatments for ischemic stroke involve anticoagulant therapy for the prevention and treatment of the condition. Modified FIX polypeptides provided herein that inhibit coagulation can be used in the treatment and/or prevention or reduction of risk of ischemic stroke. 
     3. Acquired Coagulation Disorders 
     Acquired coagulation disorders are the result of conditions or diseases, such as vitamin K deficiency, liver disease, disseminated intravascular coagulation (DIC), or development of circulation anticoagulants. The defects in blood coagulation are the result of secondary deficiencies in clotting factors caused by the condition or disease. For example, production of coagulation factors from the liver is often impaired when the liver is in a diseased state. Along with decreased synthesis of coagulation factors, fibrinolysis becomes increased and thrombocytopenia (deficiency in platelets) is increased. Decreased production of coagulation factors by the liver also can result from fulminant hepatitis or acute fatty liver of pregnancy. Such conditions promote intravascular clotting which consumes available coagulation factors. Modified FIX polypeptides provided herein can be used in the treatment of acquired coagulation disorders in order to alleviate deficiencies in blood clotting factors. 
     a. Disseminated Intravascular Coagulation (DIC) 
     Disseminated intravascular coagulation (DIC) is a disorder characterized by a widespread and ongoing activation of coagulation. In DIC, there is a loss of balance between thrombin activation of coagulation and plasmin degradation of blot clots. Vascular or microvascular fibrin deposition as a result can compromise the blood supply to various organs, which can contribute to organ failure. Prolonged activation of the coagulation system impairs the synthesis of new coagulation factors and increases the degradation of existing coagulation factors, thus leading to decreased levels of procoagulant proteins. As a result, patients with DIC are at an increased risk of serious bleeding, especially during surgery and trauma situations. In acute, massive DIC, thrombocytopenia and depletion of coagulation factors, which promote severe bleeding, are accompanied by secondary fibrinolysis, wherein fibrin degradation products are generated that interfere with normal platelet function and impairs proper fibrin formation. The fibrinolysis is associated with high levels of plasmin which also contributes to the hemorrhagic state by degradation of coagulation factors, such as FIX, FVII, and FV. Treatments for acute DIC, therefore, aid in correcting the coagulation factor deficiencies in order to restore normal coagulation. In sub-acute or chronic DIC, patients present with a more hypercoagulatory phenotype, with thromboses from excess thrombin formation, and the symptoms and signs of venous thrombosis can be present. In contrast to acute DIC, sub-acute or chronic DIC is treated by methods of alleviating the hyperthrombosis, including heparin, anti-thrombin III and activated protein C treatment. 
     The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for acute DIC. In one embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used in combination with other coagulation factors to treat deficiencies in clotting factors caused by acute DIC. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for DIC. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example FIX-deficient mice, or any other known disease model for DIC, can be treated with modified FIX polypeptides. Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX. 
     b. Bacterial Infection and Periodontitis 
     Systemic infection with microorganisms, such as bacteria, are commonly associated with DIC. The upregulation of coagulation pathways can be mediated in part by cell membrane components of the microorganism (lipopolysaccharide or endotoxin) or bacterial exotoxins (such as staphylococcal alpha toxin) that cause inflammatory responses leading to elevated levels of cytokines. The cytokines, in turn, can influence induction of coagulation. 
     Bacterial pathogens, such as  Porphyrus gingivalis , are well-known as causative agents for adult periodontitis. The  Porphyrus gingivalis  bacterium produces arginine-specific cysteine proteinases that function as virulence factors (Grenier et al. (1987)  J. Clin. Microbiol.  25:738-740, Smalley et al. (1989)  Oral Microbiol. Immunol.  4:178-181, Marsh, et al. (1989)  FEMS Microbiol.  59:181-185, and Potempa et al. (1998)  J. Biol. Chem.  273:21648-21657.  Porphyrus gingivalis  generated two proteinases that are referred to as 50 kDa and 95 kDa gingipains R (RgpB and HRgpA, respectively). The protease can proteolytically cleave and hence activate coagulation factors, such as FIX. During bacterial infection release of the gingipains R into the blood stream can thus lead to uncontrolled activation of the coagulation cascade leading to overproduction of thrombin and increase the possibility of inducing disseminated intravascular coagulation (DIC). The large increases in thrombin concentrations can furthermore contribute alveolar bone resorption by osteoclasts at sites of periodontitis. 
     The modified FIX polypeptides provided herein that inhibit blood coagulation, and nucleic acids encoding modified FIX polypeptides can be used in treatment of periodontitis. The modified FIX polypeptides herein provide increased protein stability and improved half-life. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies. Examples of therapeutic improvements using modified FIX polypeptides include, for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness for airway responsiveness in periodontitis models. Such models are available in animals, such as nonhuman primates, dogs, mice, rats, hamsters, and guinea pigs (Weinberg and Bral (1999)  J. of Periodontology  26(6), 335-340). Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX. 
     c. Liver Disease 
     The liver is the site of production of most coagulation factors of the coagulation cascade. It also is responsible for clearing degraded and inactivated components of coagulation. As such, diseases of the liver can have a profound effect on hemostasis. In some cases, liver disease can be associated with hypersplenism and suppression of platelet production in the bone marrow. Liver disease can be characterized as either an acute or chronic condition. Acute hepatic necrosis is caused by factors, such as infection, shock, or toxins, wherein the necrosis of the liver leads to the inability to produce sufficient quantities of coagulation factors. Chronic liver disease is caused by factors, such as, but not limited to, alcohol, viral infection and autoimmune disease. Hemostatic defects in chronic liver disease include decreased coagulation factors due to decreased production, decreased platelet count due to sequestration in the spleen, and increased fibrin degradation products (FDP) due to decreased clearance, which can lead to conditions, such as DIC, as described above. 
     The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for liver disease, including treatment of deficiencies in coagulation factors. The modified FIX polypeptides provided herein can be used, for example, to control or prevent spontaneous bleeding episodes or to control or prevent bleeding in response to trauma or surgical procedures. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for liver disease. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example animal models of autoimmune liver disease and alcohol induced liver disease are available and can be treated with modified FIX polypeptides provided herein (such as Nanji et al. (2003)  Alcohol Res Health.  27(4):325-30; Heneghan and McFarlane (2005)  Hepatology  42(1): 149-55). Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX. 
     d. Warfarin and Heparin-Induced Bleeding 
     Patients undergoing anticoagulant therapies for the treatment of conditions, such as thromboembolism, can exhibit bleeding episodes upon acute administration of anticoagulants or develop hemorrhagic disorders as a result long term usage of such therapies. Treatments for bleeding episodes typically include administration of procoagulants, such as vitamin K, plasma, concentrates of FIX polypeptides, and protamines to neutralize heparin. The modified FIX polypeptides provided herein can be used in treatments to control bleeding episodes in patients with acquired bleeding disorders due to anticoagulant treatments. 
     e. Intracerebral Hemorrhage 
     Intracerebral hemorrhage (ICH) is a type of stroke characterized by spontaneous bleeding into the brain. In contrast to ischemic stroke, which can be treated effectively with anticoagulant therapies, the progressive bleeding induced in ICH is more difficult to treat. Mortality from hemorrhagic stroke at 30 days is seven times higher than ischemic strokes. The most common cause of ICH is degeneration and rupture of small arteries or arterioles due to sustained hypertension. In the elderly, cerebral amyloid angiopathy also can induce ICH Therapies for ICH involves the use of coagulation factor therapies, in particular recombinant factor VII. The modified FIX polypeptides provided herein, either alone or in combination with other coagulation factors, can be used in treatments to control bleeding episodes in patients with ICH. 
     J. COMBINATION THERAPIES 
     Any of the modified FIX polypeptides, and nucleic acid molecules encoding modified FIX polypeptides described herein can be administered in combination with, prior to, intermittently with, or subsequent to, other therapeutic agents or procedures including, but not limited to, other biologics, small molecule compounds and surgery. For any disease or condition, including all those exemplified above, for FIX is indicated or has been used and for which other agents and treatments are available, FIX can be used in combination therewith. Hence, the modified FIX polypeptides provided herein similarly can be used. Depending on the disease or condition to be treated, exemplary combinations include, but are not limited to combination with other plasma purified or recombinant coagulation factors, procoagulants, anticoagulants, anti-coagulation antibodies, glycosaminoglycans, heparins, heparinoids, heparin derivatives, heparin-like drugs, coumarins, such as warfarin and coumarin derivatives. Additional procoagulants that can be used in combination therapies with modified FIX polypeptides include, but are not limited to, vitamin K, vitamin K derivatives, and protein C inhibitors. Additional anticoagulants that can be used in combination therapies with modified FIX polypeptides include, but are not limited to, β2 adrenoreceptor antagonists, neuropeptide V2 antagonists, prostacyclin analogs, thromboxane synthase inhibitors, calcium agonists, elastase inhibitors, non-steroidal anti-inflammatory molecules, thrombin inhibitors, lipoxygenase inhibitors, FVIIa inhibitors, FXa inhibitors, phosphodiesterase III inhibitors, fibrinogen, vitamin K antagonists, and glucoprotein IIb/IIIa antagonists. 
     K. ARTICLES OF MANUFACTURE AND KITS 
     Pharmaceutical compounds of modified FIX polypeptides for nucleic acids encoding modified FIX polypeptides, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a FIX-mediated disease or disorder, and a label that indicates that modified FIX polypeptide or nucleic acid molecule is to be used for treating a FIX-mediated disease or disorder. 
     The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,033,252 and 5,052,558, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any FIX-mediated disease or disorder. 
     Modified FIX polypeptides and nucleic acid molecules also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration. For example a modified FIX can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of FIX or a FIX regulated system of a subject. 
     L. EXAMPLES 
     The following examples are included for illustrative purposes only and are not intended to limit the scope of the embodiments provided herein. 
     Example 1 
     Cloning and Generation FIX Mutants 
     A. Cloning of cDNA Encoding FIX and Insertion into a Mammalian Expression Vector 
     The nucleotide sequence comprising the coding sequence of FIX was cloned into the plasmid pCR-Blunt II-TOPO (Invitrogen, SEQ ID NO: 915) by polymerase chain reaction (PCR), using standard techniques known in the art, from plasmid pCMV5.8 (ATCC Number: 79829), using the following primers: 
     FIXHIND 5′-GCATTAGGATAAGCTTGGATGCAGCGCGTGAACATG-3′ (SEQ ID NO: 913) and 
     FIXXBA 5′-GCATCGGCGCAGATCTCTGTTAATTTTCAATTCC-3′ (SEQ ID NO: 914). After confirming the sequence of the FIX fragment contained in the pTOPO-PCR bluntII plasmid by sequencing, the FIX fragment was subcloned by restriction digest using EcoRI into plasmid pNaut to produce plasmid pNaut-FIX-EcoRI (SEQ ID NO: 916). The encoded mature form of the FIX polypeptide has a sequence of amino acids as set forth in SEQ ID NO: 1035. This sequence differs from SEQ ID NO: 2 at five positions: K39T, L40E, 143K, S44Q and S46V. These changes do not affect the clotting activity of the polypeptide. 
     B. Generation of FIX Mutants 
     A collection of pre-designed, targeted mutants was generated such that each individual mutant was created and processed individually, and physically separated from each other and in addressable arrays. 2D-scanning technology, described herein and also described in published U.S. Application Nos. US-2004-0132977-A1 and US 2005-0202438 A1) was used to design and obtain FIX mutants with improved resistance to proteolysis. Is-HITs were identified based upon (1) the protein property to be evolved (such as, resistance to proteolysis or stability); (2) the amino acid sequence; and (3) the properties of individual amino acids. 
     1. LEADS Created for Higher Resistance to Proteolysis of FIX 
     Variants were designed using 2D-scanning to identify positions conferring resistance to proteolysis. Positions selected (is-HITs) on human FIX (SEQ ID NO: 2) were (numbering corresponds to amino acid positions in the mature FIX polypeptide set forth in SEQ ID NO: 2) Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, 166, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, S141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, 1290, A291, D292, K293, E294, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, I327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, T371, E372, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, L414, and T415. The native amino acid at each of the is-HIT positions listed above was replaced by residues as listed in Table 9. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 Amino acid at 
                 Replacing 
               
               
                   
                 is-HIT 
                 ammo acids 
               
               
                   
                   
               
             
            
               
                   
                 R 
                 H, Q 
               
               
                   
                 E 
                 H, N, Q 
               
               
                   
                 K 
                 N, Q 
               
               
                   
                 D 
                 N, Q 
               
               
                   
                 M 
                 I, V 
               
               
                   
                 P 
                 A, S 
               
               
                   
                 Y 
                 I, H 
               
               
                   
                 F 
                 I, V 
               
               
                   
                 W 
                 H, S 
               
               
                   
                 L 
                 I, V 
               
               
                   
                 N 
                 Q, S 
               
               
                   
                 A 
                 H, N, Q 
               
               
                   
                 G 
                 H, N, Q 
               
               
                   
                 I 
                 H, N, Q 
               
               
                   
                 S 
                 H, N, Q 
               
               
                   
                 T 
                 H, N, Q 
               
               
                   
                 V 
                 H, N, Q 
               
               
                   
                   
               
            
           
         
       
     
     The FIX variants generated for testing increased resistance to proteolysis are listed in Table 10 (SEQ ID NOS: 3-891, 1025-1034, 1036-1926, and 2035-2044). 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 List of human FIX variants for testing increased resistance to proteolysis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Y1H 
                 Y1I 
                 S3Q 
                 S3H 
                 S3N 
                 G4Q 
                 G4H 
                 G4N 
                 K5N 
                 K5Q 
               
               
                 L6I 
                 L6V 
                 E7Q 
                 E7H 
                 E7N 
                 E8Q 
                 E8H 
                 E8N 
                 F9I 
                 F9V 
               
               
                 V10Q 
                 V10H 
                 V10N 
                 G12Q 
                 G12H 
                 G12N 
                 L14I 
                 L14V 
                 E15Q 
                 E15H 
               
               
                 E15N 
                 R16H 
                 R16Q 
                 E17Q 
                 E17H 
                 E17N 
                 M19I 
                 M19V 
                 E20Q 
                 E20H 
               
               
                 E20N 
                 E21Q 
                 E21H 
                 E21N 
                 K22N 
                 K22Q 
                 S24Q 
                 S24H 
                 S24N 
                 F25I 
               
               
                 F25V 
                 E26Q 
                 E26H 
                 E26N 
                 E27Q 
                 E27H 
                 E27N 
                 A28Q 
                 A28H 
                 A28N 
               
               
                 R29H 
                 R29Q 
                 E30Q 
                 E30H 
                 E30N 
                 V31Q 
                 V31H 
                 V31N 
                 F32I 
                 F32V 
               
               
                 E33Q 
                 E33H 
                 E33N 
                 T35Q 
                 T35H 
                 T35N 
                 E36Q 
                 E36H 
                 E36N 
                 R37H 
               
               
                 R37Q 
                 T38Q 
                 T38H 
                 T38N 
                 T39Q 
                 T39H 
                 T39N 
                 E40Q 
                 E40H 
                 E40N 
               
               
                 F41I 
                 F41V 
                 W42S 
                 W42H 
                 K43N 
                 K43Q 
                 Y45H 
                 Y45I 
                 V46Q 
                 V46H 
               
               
                 V46N 
                 D47N 
                 D47Q 
                 G48Q 
                 G48H 
                 G48N 
                 D49N 
                 D49Q 
                 E52Q 
                 E52H 
               
               
                 E52N 
                 S53Q 
                 S53H 
                 S53N 
                 P55A 
                 P55S 
                 L57I 
                 L57V 
                 N58Q 
                 N58S 
               
               
                 G59Q 
                 G59H 
                 G59N 
                 G60Q 
                 G60H 
                 G60N 
                 S61Q 
                 S61H 
                 S61N 
                 K63N 
               
               
                 K63Q 
                 D64N 
                 D64Q 
                 D65N 
                 D65Q 
                 I66Q 
                 I66H 
                 I66N 
                 S68Q 
                 S68H 
               
               
                 S68N 
                 Y69H 
                 Y69I 
                 E70Q 
                 E70H 
                 E70N 
                 W72S 
                 W72H 
                 P74A 
                 P74S 
               
               
                 F75I 
                 F75V 
                 G76Q 
                 G76H 
                 G76N 
                 F77I 
                 F77V 
                 E78Q 
                 E78H 
                 E78N 
               
               
                 G79Q 
                 G79H 
                 G79N 
                 K80N 
                 K80Q 
                 E83Q 
                 E83H 
                 E83N 
                 L84I 
                 L84V 
               
               
                 D85N 
                 D85Q 
                 V86Q 
                 V86H 
                 V86N 
                 T87Q 
                 T87H 
                 T87N 
                 I90Q 
                 I90H 
               
               
                 I90N 
                 K91N 
                 K91Q 
                 N92Q 
                 N92S 
                 G93Q 
                 G93H 
                 G93N 
                 R94H 
                 R94Q 
               
               
                 E96Q 
                 E96H 
                 E96N 
                 F98I 
                 F98V 
                 K100N 
                 K100Q 
                 S102Q 
                 S102H 
                 S102N 
               
               
                 A103Q 
                 A103H 
                 A103N 
                 D104N 
                 D104Q 
                 K106N 
                 K106Q 
                 V107Q 
                 V107H 
                 V107N 
               
               
                 V108Q 
                 V108H 
                 V108N 
                 S110Q 
                 S110H 
                 S110N 
                 T112Q 
                 T112H 
                 T112N 
                 E113Q 
               
               
                 E113H 
                 E113N 
                 G114Q 
                 G114H 
                 G114N 
                 Y115H 
                 Y115I 
                 R116H 
                 R116Q 
                 L117I 
               
               
                 L117V 
                 A118Q 
                 A118H 
                 A118N 
                 E119Q 
                 E119H 
                 E119N 
                 K122N 
                 K122Q 
                 S123Q 
               
               
                 S123H 
                 S123N 
                 E125Q 
                 E125H 
                 E125N 
                 P126A 
                 P126S 
                 A127Q 
                 A127H 
                 A127N 
               
               
                 V128Q 
                 V128H 
                 V128N 
                 P129A 
                 P129S 
                 P131A 
                 P131S 
                 G133Q 
                 G133H 
                 G133N 
               
               
                 R134H 
                 R134Q 
                 V135Q 
                 V135H 
                 V135N 
                 S136Q 
                 S136H 
                 S136N 
                 V137Q 
                 V137H 
               
               
                 V137N 
                 S138Q 
                 S138H 
                 S138N 
                 T140Q 
                 T140H 
                 T140N 
                 S141Q 
                 S141H 
                 S141N 
               
               
                 K142N 
                 K142Q 
                 L143I 
                 L143V 
                 T144Q 
                 T144H 
                 T144N 
                 R145H 
                 R145Q 
                 A146Q 
               
               
                 A146H 
                 A146N 
                 E147Q 
                 E147H 
                 E147N 
                 T148Q 
                 T148H 
                 T148N 
                 V149Q 
                 V149H 
               
               
                 V149N 
                 P151A 
                 P151S 
                 D152N 
                 D152Q 
                 V153Q 
                 V153H 
                 V153N 
                 D154N 
                 D154Q 
               
               
                 Y155H 
                 Y155I 
                 V156Q 
                 V156H 
                 V156N 
                 S158Q 
                 S158H 
                 S158N 
                 T159Q 
                 T159H 
               
               
                 T159N 
                 E160Q 
                 E160H 
                 E160N 
                 A161Q 
                 A161H 
                 A161N 
                 E162Q 
                 E162H 
                 E162N 
               
               
                 T163Q 
                 T163H 
                 T163N 
                 I164Q 
                 I164H 
                 I164N 
                 L165I 
                 L165V 
                 L165Q 
                 L165H 
               
               
                 D166N 
                 D166Q 
                 I168Q 
                 I168H 
                 I168N 
                 T169Q 
                 T169H 
                 T169N 
                 S171Q 
                 S171H 
               
               
                 S171N 
                 T172Q 
                 T172H 
                 T172N 
                 S174Q 
                 S174H 
                 S174N 
                 F175I 
                 F175V 
                 F175H 
               
               
                 D177N 
                 D177Q 
                 F178I 
                 F178V 
                 F178H 
                 T179Q 
                 T179H 
                 T179N 
                 R180H 
                 R180Q 
               
               
                 V181Q 
                 V181H 
                 V181N 
                 V182Q 
                 V182H 
                 V182N 
                 G183Q 
                 G183H 
                 G183N 
                 G184Q 
               
               
                 G184H 
                 G184N 
                 E185Q 
                 E185H 
                 E185N 
                 D186N 
                 D186Q 
                 A187Q 
                 A187H 
                 A187N 
               
               
                 K188N 
                 K188Q 
                 P189A 
                 P189S 
                 G190Q 
                 G190H 
                 G190N 
                 F192I 
                 F192V 
                 F192IH 
               
               
                 P193A 
                 P193S 
                 W194S 
                 W194H 
                 W194I 
                 V196Q 
                 V196H 
                 V196N 
                 V197Q 
                 V197H 
               
               
                 V197N 
                 L198I 
                 L198V 
                 L198Q 
                 L198H 
                 N199Q 
                 N199S 
                 G200Q 
                 G200H 
                 G200N 
               
               
                 K201N 
                 K201Q 
                 V202Q 
                 V202H 
                 V202N 
                 D203N 
                 D203Q 
                 A204Q 
                 A204H 
                 A204N 
               
               
                 F205I 
                 F205V 
                 G207Q 
                 G207H 
                 G207N 
                 G208Q 
                 G208H 
                 G208N 
                 S209Q 
                 S209H 
               
               
                 S209N 
                 I210Q 
                 I210H 
                 I210N 
                 V211Q 
                 V211H 
                 V211N 
                 E213Q 
                 E213H 
                 E213N 
               
               
                 K214N 
                 K214Q 
                 W215S 
                 W215H 
                 I216Q 
                 I216H 
                 I216N 
                 V217Q 
                 V217H 
                 V217N 
               
               
                 T218Q 
                 T218H 
                 T218N 
                 A219Q 
                 A219H 
                 A219N 
                 A220Q 
                 A220H 
                 A220N 
                 V223Q 
               
               
                 V223H 
                 V223N 
                 E224Q 
                 E224H 
                 E224N 
                 T225Q 
                 T225H 
                 T225N 
                 G226Q 
                 G226H 
               
               
                 G226N 
                 V227Q 
                 V227H 
                 V227N 
                 K228N 
                 K228Q 
                 I229Q 
                 I229H 
                 I229N 
                 T230Q 
               
               
                 T230H 
                 T230N 
                 V231Q 
                 V231H 
                 V231N 
                 V232Q 
                 V232H 
                 V232N 
                 A233Q 
                 A233H 
               
               
                 A233N 
                 G234Q 
                 G234H 
                 G234N 
                 E235Q 
                 E235H 
                 E235N 
                 I238Q 
                 I238H 
                 I238N 
               
               
                 E239Q 
                 E239H 
                 E239N 
                 E240Q 
                 E240H 
                 E240N 
                 T241Q 
                 T241H 
                 T241N 
                 E242Q 
               
               
                 E242H 
                 E242N 
                 T244Q 
                 T244H 
                 T244N 
                 E245Q 
                 E245H 
                 E245N 
                 K247N 
                 K247Q 
               
               
                 R248H 
                 R248Q 
                 V250Q 
                 V250H 
                 V250N 
                 I251Q 
                 I251H 
                 I251N 
                 R252H 
                 R252Q 
               
               
                 I253Q 
                 I253H 
                 I253N 
                 I254Q 
                 I254H 
                 I254N 
                 P255A 
                 P255S 
                 Y259H 
                 Y259I 
               
               
                 A261Q 
                 A261H 
                 A261N 
                 A262Q 
                 A262H 
                 A262N 
                 I263Q 
                 I263H 
                 I263N 
                 K265N 
               
               
                 K265Q 
                 Y266H 
                 Y266I 
                 D269N 
                 D269Q 
                 I270Q 
                 I270H 
                 I270N 
                 A271Q 
                 A271H 
               
               
                 A271N 
                 L272I 
                 L272V 
                 L273I 
                 L273V 
                 E274Q 
                 E274H 
                 E274N 
                 L275I 
                 L275V 
               
               
                 D276N 
                 D276Q 
                 E277Q 
                 E277H 
                 E277N 
                 P278A 
                 P278S 
                 L279I 
                 L279V 
                 V280Q 
               
               
                 V280H 
                 V280N 
                 L281I 
                 L281V 
                 S283Q 
                 S283H 
                 S283N 
                 Y284H 
                 Y284I 
                 V285Q 
               
               
                 V285H 
                 V285N 
                 T286Q 
                 T286H 
                 T286N 
                 P287A 
                 P287S 
                 I288Q 
                 I288H 
                 I288N 
               
               
                 I290Q 
                 I290H 
                 I290N 
                 A291Q 
                 A291H 
                 A291N 
                 D292N 
                 D292Q 
                 K293N 
                 K293Q 
               
               
                 E294Q 
                 E294H 
                 E294N 
                 Y295H 
                 Y295I 
                 T296Q 
                 T296H 
                 T296N 
                 I298Q 
                 I298H 
               
               
                 I298N 
                 F299I 
                 F299V 
                 L300I 
                 L300V 
                 K301N 
                 K301Q 
                 F302I 
                 F302V 
                 G303Q 
               
               
                 G303H 
                 G303N 
                 S304Q 
                 S304H 
                 S304N 
                 G305Q 
                 G305H 
                 G305N 
                 Y306H 
                 Y306I 
               
               
                 V307Q 
                 V307H 
                 V307N 
                 S308Q 
                 S308H 
                 S308N 
                 G309Q 
                 G309H 
                 G309N 
                 W310S 
               
               
                 W310H 
                 G311Q 
                 G311H 
                 G311N 
                 R312H 
                 R312Q 
                 V313Q 
                 V313H 
                 V313N 
                 F314I 
               
               
                 F314V 
                 K316N 
                 K316Q 
                 G317Q 
                 G317H 
                 G317N 
                 R318H 
                 R318Q 
                 S319Q 
                 S319H 
               
               
                 S319N 
                 A320Q 
                 A320H 
                 A320N 
                 L321I 
                 L321V 
                 V322Q 
                 V322H 
                 V322N 
                 L323I 
               
               
                 L323V 
                 Y325H 
                 Y325I 
                 L326I 
                 L326V 
                 R327H 
                 R327Q 
                 V328Q 
                 V328H 
                 V328N 
               
               
                 P329A 
                 P329S 
                 L330I 
                 L330V 
                 V331Q 
                 V331H 
                 V331N 
                 D332N 
                 D332Q 
                 R333H 
               
               
                 R333Q 
                 A334Q 
                 A334H 
                 A334N 
                 T335Q 
                 T335H 
                 T335N 
                 L337I 
                 L337V 
                 R338H 
               
               
                 R338Q 
                 S339Q 
                 S339H 
                 S339N 
                 T340Q 
                 T340H 
                 T340N 
                 K341N 
                 K341Q 
                 F342I 
               
               
                 F342V 
                 T343Q 
                 T343H 
                 T343N 
                 I344Q 
                 I344H 
                 I344N 
                 Y345H 
                 Y345I 
                 M348I 
               
               
                 M348V 
                 F349I 
                 F349V 
                 A351Q 
                 A351H 
                 A351N 
                 G352Q 
                 G352H 
                 G352N 
                 F353I 
               
               
                 F353V 
                 E355Q 
                 E355H 
                 E355N 
                 G356Q 
                 G356H 
                 G356N 
                 G357Q 
                 G357H 
                 G357N 
               
               
                 R358H 
                 R358Q 
                 D359N 
                 D359Q 
                 S360Q 
                 S360H 
                 S360N 
                 G363Q 
                 G363H 
                 G363N 
               
               
                 D364N 
                 D364Q 
                 S365Q 
                 S365H 
                 S365N 
                 G366Q 
                 G366H 
                 G366N 
                 G367Q 
                 G367H 
               
               
                 G367N 
                 P368A 
                 P368S 
                 V370Q 
                 V370H 
                 V370N 
                 T371Q 
                 T371H 
                 T371N 
                 E372Q 
               
               
                 E372H 
                 E372N 
                 V373Q 
                 V373H 
                 V373N 
                 E374Q 
                 E374H 
                 E374N 
                 G375Q 
                 G375H 
               
               
                 G375N 
                 T376Q 
                 T376H 
                 T376N 
                 S377Q 
                 S377H 
                 S377N 
                 F378I 
                 F378V 
                 L379I 
               
               
                 L379V 
                 T380Q 
                 T380H 
                 T380N 
                 G381Q 
                 G381H 
                 G381N 
                 I382Q 
                 I382H 
                 I382N 
               
               
                 I383Q 
                 I383H 
                 I383N 
                 S384Q 
                 S384H 
                 S384N 
                 W385S 
                 W385H 
                 G386Q 
                 G386H 
               
               
                 G386N 
                 E387Q 
                 E387H 
                 E387N 
                 E388Q 
                 E388H 
                 E388N 
                 A390Q 
                 A390H 
                 A390N 
               
               
                 M391I 
                 M391V 
                 K392N 
                 K392Q 
                 G393Q 
                 G393H 
                 G393N 
                 K394N 
                 K394Q 
                 Y395H 
               
               
                 Y395I 
                 G396Q 
                 G396H 
                 G396N 
                 I397Q 
                 I397H 
                 I397N 
                 Y398H 
                 Y398I 
                 T399Q 
               
               
                 T399H 
                 T399N 
                 K400N 
                 K400Q 
                 V401Q 
                 V401H 
                 V401N 
                 S402Q 
                 S402H 
                 S402N 
               
               
                 R403H 
                 R403Q 
                 Y404H 
                 Y404I 
                 V405Q 
                 V405H 
                 V405N 
                 W407S 
                 W407H 
                 I408Q 
               
               
                 I408H 
                 I408N 
                 K409N 
                 K409Q 
                 E410Q 
                 E410H 
                 E410N 
                 K411N 
                 K411Q 
                 T412Q 
               
               
                 T412H 
                 T412N 
                 K413N 
                 K413Q 
                 L414I 
                 L414V 
                 T415Q 
                 T415H 
                 T415N 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Production of Native and Modified Human FIX Polypeptides (Proteins) in Mammalian Cells and Yield Determination 
     Human fetal kidney 293 HEK EBNA fibroblast cells were grown in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 2 mM glutamine and 10% SVF. Cells were plated at 5×10 5  cells per well in 6-well plate for 2 days, washed two times with PBS, and then switched to serum free medium. Cells were transfected with 2 μg of DNA (FIX variants in pNaut-FIX plasmid) using transfection reagent according to the instructions of the manufacturer (Perfectin, GTS). Cell medium was replaced after 4 hours with serum free media containing 50 μg/ml of vitamin K and 1×-ITS (insulin, transferrin, selenium) mixture. Cell medium was collected 72 hours after transfection and stored at −80° C. 
     ASSERACHROM® IX: Ag ELISA (Diagnostica Stago, France) was used to determine concentration of FIX in supernatants from cell production according to the instructions of the manufacturer. Samples were diluted 20-fold using kit dilution reagent. Absorbance at 492 nm was measured using a spectrophotometer (Spectramax, Molecular Devices). Results were obtained by interpolation of the absorbance values from the calibration curve obtained from patients&#39; plasma dilutions and plotted in log-log graph. FIX level at 100% corresponded approximately to 4 mg/l. Yields of FIX native and mutant proteins produced in 293 HEK cells are shown in Table 11. The data are the results from two productions. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Yield of FIX native and mutant proteins 
               
            
           
           
               
               
               
            
               
                 Nemo 
                   
                   
               
               
                 Code # 
                 Mutation 
                 FIX (mg/L) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Native FIX 
                 9.2 
               
               
                 2 
                 T163N 
                 14.1 
               
               
                 3 
                 T163Q 
                 11.2 
               
               
                 4 
                 T163H 
                 10.1 
               
               
                 5 
                 I164N 
                 8.9 
               
               
                 6 
                 I164Q 
                 13.3 
               
               
                 7 
                 I164H 
                 14.9 
               
               
                 8 
                 T163N/I164N 
                 10.6 
               
               
                 9 
                 I168N 
                 11.9 
               
               
                 10 
                 I168Q 
                 11.9 
               
               
                 11 
                 I168H 
                 12.6 
               
               
                 12 
                 T169N 
                 7.5 
               
               
                 13 
                 T169Q 
                 9.1 
               
               
                 14 
                 T169H 
                 9.7 
               
               
                 15 
                 S171N 
                 11.3 
               
               
                 16 
                 S171Q 
                 11.7 
               
               
                 17 
                 S171H 
                 10.2 
               
               
                 18 
                 T172N 
                 17.9 
               
               
                 19 
                 T172Q 
                 12.6 
               
               
                 20 
                 T172H 
                 16.3 
               
               
                 21 
                 S174N 
                 14.3 
               
               
                 22 
                 S174Q 
                 11.5 
               
               
                 23 
                 S174H 
                 12.1 
               
               
                 24 
                 I168N/T169N/S171N/T172N/S174N 
                 12.9 
               
               
                 25 
                 T179N 
                 10.0 
               
               
                 26 
                 T179Q 
                 13.3 
               
               
                 27 
                 T179H 
                 8.3 
               
               
                 28 
                 V181N 
                 9.8 
               
               
                 29 
                 V181Q 
                 20.3 
               
               
                 30 
                 V181H 
                 13.2 
               
               
                 31 
                 V182N 
                 7.5 
               
               
                 32 
                 V182Q 
                 7.6 
               
               
                 33 
                 V182H 
                 12.1 
               
               
                 34 
                 G183N 
                 14.1 
               
               
                 35 
                 G183Q 
                 15.4 
               
               
                 36 
                 G183H 
                 11.6 
               
               
                 37 
                 G184N 
                 7.6 
               
               
                 38 
                 G184Q 
                 10.7 
               
               
                 39 
                 G184H 
                 8.9 
               
               
                 40 
                 T179N/V181N/V182N/G183N/G184N 
                 4.9 
               
               
                 41 
                 A187N 
                 1.3 
               
               
                 42 
                 A187Q 
                 3.0 
               
               
                 43 
                 A187H 
                 1.6 
               
               
                 44 
                 G190N 
                 1.1 
               
               
                 45 
                 G190Q 
                 0.4 
               
               
                 46 
                 G190H 
                 5.4 
               
               
                 47 
                 A187N/G190N 
                 1.1 
               
               
                 48 
                 V196N 
                 0.5 
               
               
                 49 
                 V196Q 
                 0.1 
               
               
                 50 
                 V196H 
                 0.1 
               
               
                 51 
                 V197N 
                 0.6 
               
               
                 52 
                 V197Q 
                 1.4 
               
               
                 53 
                 V197H 
                 3.1 
               
               
                 54 
                 V196N/V197N 
                 0.1 
               
               
                 55 
                 R180H 
                 9.9 
               
               
                 56 
                 R180Q 
                 8.1 
               
               
                 57 
                 K188N 
                 10.5 
               
               
                 58 
                 K188Q 
                 13.4 
               
               
                 59 
                 K201N 
                 8.3 
               
               
                 60 
                 K201Q 
                 6.7 
               
               
                 61 
                 L165I 
                 7.0 
               
               
                 62 
                 L165V 
                 11.3 
               
               
                 63 
                 F175I 
                 7.0 
               
               
                 64 
                 F175V 
                 9.3 
               
               
                 65 
                 F178I 
                 10.3 
               
               
                 66 
                 F178V 
                 8.7 
               
               
                 67 
                 F192I 
                 2.9 
               
               
                 68 
                 F192V 
                 1.7 
               
               
                 69 
                 W194H 
                 0.1 
               
               
                 70 
                 W194I 
                 0.1 
               
               
                 71 
                 L198I 
                 6.5 
               
               
                 72 
                 L198V 
                 9.8 
               
               
                 73 
                 D166Q 
                 7.6 
               
               
                 74 
                 D166N 
                 6.8 
               
               
                 75 
                 D177Q 
                 8.4 
               
               
                 76 
                 D177N 
                 7.7 
               
               
                 77 
                 D186Q 
                 9.7 
               
               
                 78 
                 D186N 
                 14.6 
               
               
                 79 
                 D203Q 
                 4.5 
               
               
                 80 
                 D203N 
                 1.0 
               
               
                 81 
                 T140N 
                 12.9 
               
               
                 82 
                 T140Q 
                 11.4 
               
               
                 83 
                 T140H 
                 7.8 
               
               
                 84 
                 T144N 
                 9.3 
               
               
                 85 
                 T144Q 
                 1.8 
               
               
                 86 
                 T144H 
                 9.3 
               
               
                 87 
                 T140N/T144N 
                 6.3 
               
               
                 88 
                 L165Q 
                 13.9 
               
               
                 89 
                 L165H 
                 8.3 
               
               
                 90 
                 F175L/F178I 
                 8.1 
               
               
                 91 
                 F175H 
                 10.7 
               
               
                 92 
                 F178H 
                 4.5 
               
               
                 93 
                 F192I/W194I 
                 &lt;0.1 
               
               
                 94 
                 F192H 
                 0.8 
               
               
                 95 
                 L198Q 
                 0.1 
               
               
                 96 
                 L198H 
                 1.5 
               
               
                 97 
                 D152N/D154N 
                 7.2 
               
               
                 98 
                 D152N 
                 11.8 
               
               
                 99 
                 D152Q 
                 7.0 
               
               
                 100 
                 D154N 
                 4.4 
               
               
                 101 
                 D154Q 
                 6.7 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Determination of Specific Activity of Human FIX by Coagulation Assay 
     The collection of human FIX mutants was tested for coagulation activity. Coagulation activity (expressed as clotting time T 50 ) was determined as the concentration needed for formation of a fibrin clot. Clotting activity of native and mutant FIX molecules was measured by the one stage activated partial thromboplastin time (aPTT) clotting assay (Cephascreen, Diagnostica Stago, France) using FIX immunodepleted human plasma (Deficient IX, Diagnostica Stago, France). 
     Briefly, 20 μl of production supernatant containing 60 ng of native FIX or mutant FIX was diluted with 60 μl of immunodepleted plasma. 80 μl of Cephascreen reagent containing cephalin (platelet substitute) prepared from rabbit cerebral tissues and a polyphenolic activator in a buffered medium was added to diluted FIX samples and incubated at 37° C. for 15 minutes. 80 μl of pre-warmed CaCl 2  (0.025M, Diagnostica Stago, France) was added to mixture of FIX/Cephascreen reagent samples, and the time required for clot formation was immediately monitored using spectrophotometer (Spectramax, Molecular devices) at 350 nm. Serial dilution of normal human pooled plasma representing 100 to 120% of FIX activity (System control N, Diagnostica Stago, France) was used as the standard curve in order to validate linearity of clotting time detection. Clotting activity of FIX native and mutant proteins by clotting assay is shown in Tables 12a and 12b. The T 50  values are displayed in seconds and represent the time required to achieved 50% clotting. The data are the result from two assays done in duplicate. Tables 12a and 12b below depict two separate experiments measuring the coagulation activity of exemplary non-limiting modified FIX polypeptides and native FIX. Table 12c depicts the protease specificity groups as indicated for each mutant. 
     
       
         
           
               
             
               
                 TABLE 12a 
               
             
            
               
                   
               
               
                 Clotting activity of FIX native and mutant proteins 
               
            
           
           
               
               
               
            
               
                   
                   
                 Bioactivity 
               
               
                 Nemo 
                   
                 Clotting Assay 
               
               
                 Code # 
                 Mutation 
                 T50 (sec) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Native FIX 
                 116 
               
               
                 2 
                 T163N 
                 111 
               
               
                 3 
                 T163Q 
                 117 
               
               
                 4 
                 T163H 
                 108 
               
               
                 5 
                 I164N 
                 105 
               
               
                 6 
                 I164Q 
                 113 
               
               
                 7 
                 I164H 
                 118 
               
               
                 8 
                 T163N/I164N 
                 108 
               
               
                 9 
                 I168N 
                 112 
               
               
                 10 
                 I168Q 
                 119 
               
               
                 11 
                 I168H 
                 112 
               
               
                 12 
                 T169N 
                 104 
               
               
                 13 
                 T169Q 
                 104 
               
               
                 14 
                 T169H 
                 111 
               
               
                 15 
                 S171N 
                 116 
               
               
                 16 
                 S171Q 
                 112 
               
               
                 17 
                 S171H 
                 110 
               
               
                 18 
                 T172N 
                 133 
               
               
                 19 
                 T172Q 
                 111 
               
               
                 20 
                 T172H 
                 111 
               
               
                 21 
                 S174N 
                 113 
               
               
                 22 
                 S174Q 
                 115 
               
               
                 23 
                 S174H 
                 113 
               
               
                 24 
                 I168N/T169N/S171N/T172N/S174N 
                 112 
               
               
                 25 
                 T179N 
                 115 
               
               
                 26 
                 T179Q 
                 112 
               
               
                 27 
                 T179H 
                 110 
               
               
                 28 
                 V181N 
                 178 
               
               
                 29 
                 V181Q 
                 141 
               
               
                 30 
                 V181H 
                 179 
               
               
                 31 
                 V182N 
                 174 
               
               
                 32 
                 V182Q 
                 156 
               
               
                 33 
                 V182H 
                 176 
               
               
                 34 
                 G183N 
                 146 
               
               
                 35 
                 G183Q 
                 155 
               
               
                 36 
                 G183H 
                 170 
               
               
                 37 
                 G184N 
                 177 
               
               
                 38 
                 G184Q 
                 178 
               
               
                 39 
                 G184H 
                 179 
               
               
                 40 
                 T179N/V181N/V182N/G183N/G184N 
                 nd 
               
               
                 41 
                 A187N 
                 180 
               
               
                 42 
                 A187Q 
                 169 
               
               
                 43 
                 A187H 
                 201 
               
               
                 44 
                 G190N 
                 148 
               
               
                 45 
                 G190Q 
                 162 
               
               
                 46 
                 G190H 
                 120 
               
               
                 47 
                 A187N/G190N 
                 194 
               
               
                 48 
                 V196N 
                 183 
               
               
                 49 
                 V196Q 
                 176 
               
               
                 50 
                 V196H 
                 183 
               
               
                 51 
                 V197N 
                 161 
               
               
                 52 
                 V197Q 
                 157 
               
               
                 53 
                 V197H 
                 123 
               
               
                 54 
                 V196N/V197N 
                 190 
               
               
                 55 
                 R180H 
                 191 
               
               
                 56 
                 R180Q 
                 186 
               
               
                 57 
                 K188N 
                 124 
               
               
                 58 
                 K188Q 
                 130 
               
               
                 59 
                 K201N 
                 119 
               
               
                 60 
                 K201Q 
                 118 
               
               
                 61 
                 L165I 
                 109 
               
               
                 62 
                 L165V 
                 120 
               
               
                 63 
                 F175I 
                 116 
               
               
                 64 
                 F175V 
                 118 
               
               
                 65 
                 F178I 
                 140 
               
               
                 66 
                 F178V 
                 122 
               
               
                 67 
                 F192I 
                 134 
               
               
                 68 
                 F192V 
                 145 
               
               
                 69 
                 W194H 
                 183 
               
               
                 70 
                 W194I 
                 168 
               
               
                 71 
                 L198I 
                 124 
               
               
                 72 
                 L198V 
                 168 
               
               
                 73 
                 D166Q 
                 119 
               
               
                 74 
                 D166N 
                 118 
               
               
                 75 
                 D177Q 
                 118 
               
               
                 76 
                 D177N 
                 182 
               
               
                 77 
                 D186Q 
                 127 
               
               
                 78 
                 D186N 
                 117 
               
               
                 79 
                 D203Q 
                 109 
               
               
                 80 
                 D203N 
                 113 
               
               
                 81 
                 T140N 
                 118 
               
               
                 82 
                 T140Q 
                 143 
               
               
                 83 
                 T140H 
                 119 
               
               
                 84 
                 T144N 
                 151 
               
               
                 85 
                 T144Q 
                 nd 
               
               
                 86 
                 T144H 
                 117 
               
               
                 87 
                 T140N/T144N 
                 133 
               
               
                 88 
                 L165Q 
                 117 
               
               
                 89 
                 L165H 
                 118 
               
               
                 90 
                 F175I/F178I 
                 155 
               
               
                 91 
                 F175H 
                 153 
               
               
                 92 
                 F178H 
                 127 
               
               
                 93 
                 F192I/W194I 
                 175 
               
               
                 94 
                 F192H 
                 154 
               
               
                 95 
                 L198Q 
                 183 
               
               
                 96 
                 L198H 
                 178 
               
               
                 97 
                 D152N/D154N 
                 125 
               
               
                 98 
                 D152N 
                 148 
               
               
                 99 
                 D152Q 
                 126 
               
               
                 100 
                 D154N 
                 119 
               
               
                 101 
                 D154Q 
                 150 
               
               
                   
               
               
                 nd = not determined 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12b 
               
             
            
               
                   
               
               
                 Clotting activity of FIX native and mutant proteins 
               
            
           
           
               
               
               
            
               
                   
                   
                 Bioactivity 
               
               
                 Nemo 
                   
                 Clotting Assay 
               
               
                 Code # 
                 Mutation 
                 T50 (sec) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Native FIX 
                 114 
               
               
                 2 
                 T163N 
                 113 
               
               
                 3 
                 T163Q 
                 118 
               
               
                 4 
                 T163H 
                 110 
               
               
                 5 
                 I164N 
                 107 
               
               
                 6 
                 I164Q 
                 114 
               
               
                 7 
                 I164H 
                 115 
               
               
                 8 
                 T163N/I164N 
                 110 
               
               
                 9 
                 I168N 
                 111 
               
               
                 10 
                 I168Q 
                 119 
               
               
                 11 
                 I168H 
                 111 
               
               
                 12 
                 T169N 
                 110 
               
               
                 13 
                 T169Q 
                 108 
               
               
                 14 
                 T169H 
                 111 
               
               
                 15 
                 S171N 
                 116 
               
               
                 16 
                 S171Q 
                 111 
               
               
                 17 
                 S171H 
                 110 
               
               
                 18 
                 T172N 
                 143 
               
               
                 19 
                 T172Q 
                 112 
               
               
                 20 
                 T172H 
                 113 
               
               
                 21 
                 S174N 
                 113 
               
               
                 22 
                 S174Q 
                 115 
               
               
                 23 
                 S174H 
                 112 
               
               
                 24 
                 I168N/T169N/S171N/T172N/S174N 
                 113 
               
               
                 25 
                 T179N 
                 116 
               
               
                 26 
                 T179Q 
                 114 
               
               
                 27 
                 T179H 
                 115 
               
               
                 28 
                 V181N 
                 200 
               
               
                 29 
                 V181Q 
                 120 
               
               
                 30 
                 V181H 
                 196 
               
               
                 31 
                 V182N 
                 180 
               
               
                 32 
                 V182Q 
                 165 
               
               
                 33 
                 V182H 
                 180 
               
               
                 34 
                 G183N 
                 150 
               
               
                 35 
                 G183Q 
                 162 
               
               
                 36 
                 G183H 
                 182 
               
               
                 37 
                 G184N 
                 195 
               
               
                 38 
                 G184Q 
                 187 
               
               
                 39 
                 G184H 
                 192 
               
               
                 40 
                 T179N/V181N/V182N/G183N/G184N 
                 169 
               
               
                 41 
                 A187N 
                 200 
               
               
                 42 
                 A187Q 
                 163 
               
               
                 43 
                 A187H 
                 202 
               
               
                 44 
                 G190N 
                 148 
               
               
                 45 
                 G190Q 
                 158 
               
               
                 46 
                 G190H 
                 123 
               
               
                 47 
                 A187N/G190N 
                 201 
               
               
                 48 
                 V196N 
                 191 
               
               
                 49 
                 V196Q 
                 183 
               
               
                 50 
                 V196H 
                 191 
               
               
                 51 
                 V197N 
                 155 
               
               
                 52 
                 V197Q 
                 148 
               
               
                 53 
                 V197H 
                 130 
               
               
                 54 
                 V196N/V197N 
                 203 
               
               
                 55 
                 R180H 
                 202 
               
               
                 56 
                 R180Q 
                 200 
               
               
                 57 
                 K188N 
                 124 
               
               
                 58 
                 K188Q 
                 130 
               
               
                 59 
                 K201N 
                 121 
               
               
                 60 
                 K201Q 
                 118 
               
               
                 61 
                 L165I 
                 109 
               
               
                 62 
                 L165V 
                 121 
               
               
                 63 
                 F175I 
                 119 
               
               
                 64 
                 F175V 
                 120 
               
               
                 65 
                 F178I 
                 166 
               
               
                 66 
                 F178V 
                 122 
               
               
                 67 
                 F192I 
                 113 
               
               
                 68 
                 F192V 
                 134 
               
               
                 69 
                 W194H 
                 192 
               
               
                 70 
                 W194I 
                 188 
               
               
                 71 
                 L1981 
                 128 
               
               
                 72 
                 L198V 
                 175 
               
               
                 73 
                 D166Q 
                 118 
               
               
                 74 
                 D166N 
                 116 
               
               
                 75 
                 D177Q 
                 118 
               
               
                 76 
                 D177N 
                 195 
               
               
                 77 
                 D186Q 
                 125 
               
               
                 78 
                 D186N 
                 116 
               
               
                 79 
                 D203Q 
                 112 
               
               
                 80 
                 D203N 
                 110 
               
               
                 81 
                 T140N 
                 116 
               
               
                 82 
                 T140Q 
                 124 
               
               
                 83 
                 T140H 
                 117 
               
               
                 84 
                 T144N 
                 132 
               
               
                 85 
                 T144Q 
                 123 
               
               
                 86 
                 T144H 
                 114 
               
               
                 87 
                 T140N/T144N 
                 128 
               
               
                 88 
                 L165Q 
                 117 
               
               
                 89 
                 L165H 
                 119 
               
               
                 90 
                 F175I/F178I 
                 147 
               
               
                 91 
                 F175H 
                 131 
               
               
                 92 
                 F178H 
                 122 
               
               
                 93 
                 F192I/W194I 
                 193 
               
               
                 94 
                 F192H 
                 154 
               
               
                 95 
                 L198Q 
                 198 
               
               
                 96 
                 L198H 
                 199 
               
               
                 97 
                 D152N/D154N 
                 121 
               
               
                 98 
                 D152N 
                 131 
               
               
                 99 
                 D152Q 
                 121 
               
               
                 100 
                 D154N 
                 112 
               
               
                 101 
                 D154Q 
                 134 
               
               
                 102 
                 D152N 
                 123 
               
               
                 103 
                 D152Q 
                 121 
               
               
                 104 
                 D154N 
                 120 
               
               
                 105 
                 D154Q 
                 118 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12c 
               
             
            
               
                   
               
               
                 Protease specificity groups for FIX mutant proteins 
               
            
           
           
               
               
               
            
               
                 Nemo 
                   
                 Specificity 
               
               
                 Code # 
                 Mutation 
                 Group 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Native FIX 
                 — 
               
               
                 2 
                 T163N 
                 E 
               
               
                 3 
                 T163Q 
                 E 
               
               
                 4 
                 T163H 
                 E 
               
               
                 5 
                 I164N 
                 E 
               
               
                 6 
                 I164Q 
                 E 
               
               
                 7 
                 I164H 
                 E 
               
               
                 8 
                 T163N/I164N 
                 E 
               
               
                 9 
                 I168N 
                 E 
               
               
                 10 
                 I168Q 
                 E 
               
               
                 11 
                 I168H 
                 E 
               
               
                 12 
                 T169N 
                 E 
               
               
                 13 
                 T169Q 
                 E 
               
               
                 14 
                 T169H 
                 E 
               
               
                 15 
                 S171N 
                 E 
               
               
                 16 
                 S171Q 
                 E 
               
               
                 17 
                 S171H 
                 E 
               
               
                 18 
                 T172N 
                 E 
               
               
                 19 
                 T172Q 
                 E 
               
               
                 20 
                 T172H 
                 E 
               
               
                 21 
                 S174N 
                 E 
               
               
                 22 
                 S174Q 
                 E 
               
               
                 23 
                 S174H 
                 E 
               
               
                 24 
                 I168N/T169N/S171N/T172N/S174N 
                 E 
               
               
                 25 
                 T179N 
                 E 
               
               
                 26 
                 T179Q 
                 E 
               
               
                 27 
                 T179H 
                 E 
               
               
                 28 
                 V181N 
                 E 
               
               
                 29 
                 V181Q 
                 E 
               
               
                 30 
                 V181H 
                 E 
               
               
                 31 
                 V182N 
                 E 
               
               
                 32 
                 V182Q 
                 E 
               
               
                 33 
                 V182H 
                 E 
               
               
                 34 
                 G183N 
                 E 
               
               
                 35 
                 G183Q 
                 E 
               
               
                 36 
                 G183H 
                 E 
               
               
                 37 
                 G184N 
                 E 
               
               
                 38 
                 G184Q 
                 E 
               
               
                 39 
                 G184H 
                 E 
               
               
                 40 
                 T179N/V181N/V182N/G183N/G184N 
                 E 
               
               
                 41 
                 A187N 
                 E 
               
               
                 42 
                 A187Q 
                 E 
               
               
                 43 
                 A187H 
                 E 
               
               
                 44 
                 G190N 
                 E 
               
               
                 45 
                 G190Q 
                 E 
               
               
                 46 
                 G190H 
                 E 
               
               
                 47 
                 A187N/G190N 
                 E 
               
               
                 48 
                 V196N 
                 E 
               
               
                 49 
                 V196Q 
                 E 
               
               
                 50 
                 V196H 
                 E 
               
               
                   
                   
                 E 
               
               
                 51 
                 V197N 
                 E 
               
               
                 52 
                 V197Q 
                 E 
               
               
                 53 
                 V197H 
                 E 
               
               
                 54 
                 V196N/V197N 
                 E 
               
               
                 55 
                 R180H 
                 T 
               
               
                 56 
                 R180Q 
                 T 
               
               
                 57 
                 K188N 
                 T 
               
               
                 58 
                 K188Q 
                 T 
               
               
                 59 
                 K201N 
                 T 
               
               
                 60 
                 K201Q 
                 T 
               
               
                 61 
                 L165I 
                 CT 
               
               
                 62 
                 L165V 
                 CT 
               
               
                 63 
                 F175I 
                 CT 
               
               
                 64 
                 F175V 
                 CT 
               
               
                 65 
                 F178I 
                 CT 
               
               
                 66 
                 F178V 
                 CT 
               
               
                 67 
                 F192I 
                 CT 
               
               
                 68 
                 F192V 
                 CT 
               
               
                 69 
                 W194H 
                 CT 
               
               
                 70 
                 W194I 
                 CT 
               
               
                 71 
                 L198I 
                 CT 
               
               
                 72 
                 L198V 
                 CT 
               
               
                 73 
                 D166Q 
                 EAsp 
               
               
                 74 
                 D166N 
                 EAsp 
               
               
                 75 
                 D177Q 
                 EAsp 
               
               
                 76 
                 D177N 
                 EAsp 
               
               
                 77 
                 D186Q 
                 EAsp 
               
               
                 78 
                 D186N 
                 EAsp 
               
               
                 79 
                 D203Q 
                 EAsp 
               
               
                 80 
                 D203N 
                 EAsp 
               
               
                 81 
                 T140N 
                 CT 
               
               
                 82 
                 T140Q 
                 CT 
               
               
                 83 
                 T140H 
                 CT 
               
               
                 84 
                 T144N 
                 CT 
               
               
                 85 
                 T144Q 
                 CT 
               
               
                 86 
                 T144H 
                 CT 
               
               
                 87 
                 T140N/T144N 
                 CT 
               
               
                 88 
                 L165Q 
                 CT 
               
               
                 89 
                 L165H 
                 CT 
               
               
                 90 
                 F175I/F178I 
                 CT 
               
               
                 91 
                 F175H 
                 CT 
               
               
                 92 
                 F178H 
                 CT 
               
               
                 93 
                 F192I/W194I 
                 CT 
               
               
                 94 
                 F192H 
                 CT 
               
               
                 95 
                 L198Q 
                 CT 
               
               
                 96 
                 L198H 
                 CT 
               
               
                 97 
                 D152N/D154N 
                 EAsp 
               
               
                 98 
                 D152N 
                 EAsp 
               
               
                 99 
                 D152Q 
                 EAsp 
               
               
                 100 
                 D154N 
                 EAsp 
               
               
                 101 
                 D154Q 
                 EAsp 
               
               
                 102 
                 D152N 
                 EAsp 
               
               
                 103 
                 D152Q 
                 EAsp 
               
               
                 104 
                 D154N 
                 EAsp 
               
               
                 105 
                 D154Q 
                 EAsp 
               
               
                   
               
               
                 E = elastase; T = trypsin; CT = chymotrypsin; EAsp = Endoproteinase AspN 
               
            
           
         
       
     
     Example 4 
     Resistance to Proteolysis 
     The collection of human FIX mutants was tested for their resistance to proteolysis as measured by retention of coagulation activity following treatment with proteases. Equal amounts of FIX native or mutant proteins (60 ng per assays) were treated with selected proteases (1.5% w/w trypsin, chymotrypsin, endoproteinase AspN, and endoproteinase GluC; and 6% w/w elastase). Aliquots of the samples were taken at different time points between 0.083 and 8 hrs and the proteolysis reaction was stopped by adding antiprotease cocktail (Roche Cat No. 11836170001). Samples were conserved at −20° C. until determination of clotting time could be performed. Clotting activity of the treated samples was assayed as described in Example 3 to determine residual clotting activity at each time point. Resistance to proteases for exemplary non-limiting modified FIX polypeptides is displayed in Table 13 as either no change in (N) or increased (+) resistance to proteases as compared to the residual coagulation activity of native FIX under the same protease treatment conditions. 
     The data are not meant to be representative of all proteases, but are exemplary data showing the resistance to proteolysis to an exemplary protease cocktail containing the proteases as described above. Thus, the data are not comprehensive and are not meant to be indicative that other FIX polypeptides do not exhibit protease resistance. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Resistance to proteolysis of FIX native and mutant proteins 
               
            
           
           
               
               
               
            
               
                 Nemo 
                   
                 Resistance 
               
               
                 Code # 
                 Mutation 
                 to proteases 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Native FIX 
                 — 
               
               
                 2 
                 T163N 
                 N 
               
               
                 3 
                 T163Q 
                 N 
               
               
                 4 
                 T163H 
                 + 
               
               
                 5 
                 I164N 
                 N 
               
               
                 6 
                 I164Q 
                 + 
               
               
                 7 
                 I164H 
                 + 
               
               
                 8 
                 T163N/I164N 
                 + 
               
               
                 9 
                 I168N 
                 N 
               
               
                 10 
                 I168Q 
                 + 
               
               
                 11 
                 I168H 
                 + 
               
               
                 12 
                 T169N 
                 N 
               
               
                 13 
                 T169Q 
                 N 
               
               
                 14 
                 T169H 
                 + 
               
               
                 15 
                 S171N 
                 + 
               
               
                 16 
                 S171Q 
                 + 
               
               
                 17 
                 S171H 
                 N 
               
               
                 18 
                 T172N 
                 N 
               
               
                 19 
                 T172Q 
                 + 
               
               
                 20 
                 T172H 
                 N 
               
               
                 21 
                 S174N 
                 N 
               
               
                 22 
                 S174Q 
                 + 
               
               
                 23 
                 S174H 
                 + 
               
               
                 24 
                 I168N/T169N/S171N/T172N/S174N 
                 N 
               
               
                 25 
                 T179N 
                 + 
               
               
                 26 
                 T179Q 
                 + 
               
               
                 27 
                 T179H 
                 + 
               
               
                 28 
                 V181N 
                 N 
               
               
                 29 
                 V181Q 
                 + 
               
               
                 30 
                 V181H 
                 N 
               
               
                 31 
                 V182N 
                 + 
               
               
                 32 
                 V182Q 
                 N 
               
               
                 33 
                 V182H 
                 N 
               
               
                 34 
                 G183N 
                 N 
               
               
                 35 
                 G183Q 
                 N 
               
               
                 36 
                 G183H 
                 N 
               
               
                 37 
                 G184N 
                 + 
               
               
                 38 
                 G184Q 
                 N 
               
               
                 39 
                 G184H 
                 N 
               
               
                 40 
                 T179N/V181N/V182N/G183N/G184N 
                 N 
               
               
                 41 
                 A187N 
                 N 
               
               
                 42 
                 A187Q 
                 + 
               
               
                 43 
                 A187H 
                 N 
               
               
                 44 
                 G190N 
                 N 
               
               
                 45 
                 G190Q 
                 N 
               
               
                 46 
                 G190H 
                 N 
               
               
                 47 
                 A187N/G190N 
                 N 
               
               
                 48 
                 V196N 
                 N 
               
               
                 49 
                 V196Q 
                 N 
               
               
                 50 
                 V196H 
                 N 
               
               
                 51 
                 V197N 
                 N 
               
               
                 52 
                 V197Q 
                 N 
               
               
                 53 
                 V197H 
                 + 
               
               
                 54 
                 V196N/V197N 
                 N 
               
               
                 55 
                 R180H 
                 N 
               
               
                 56 
                 R180Q 
                 N 
               
               
                 57 
                 K188N 
                 N 
               
               
                 58 
                 K188Q 
                 N 
               
               
                 59 
                 K201N 
                 N 
               
               
                 60 
                 K201Q 
                 N 
               
               
                 61 
                 L1651 
                 N 
               
               
                 62 
                 L165V 
                 N 
               
               
                 63 
                 F175I 
                 N 
               
               
                 64 
                 F175V 
                 N 
               
               
                 65 
                 F178I 
                 N 
               
               
                 66 
                 F178V 
                 N 
               
               
                 67 
                 F192I 
                 + 
               
               
                 68 
                 F192V 
                 N 
               
               
                 69 
                 W194H 
                 N 
               
               
                 70 
                 W194I 
                 N 
               
               
                 71 
                 L198I 
                 + 
               
               
                 72 
                 L198V 
                 + 
               
               
                 73 
                 D166Q 
                 + 
               
               
                 74 
                 D166N 
                 + 
               
               
                 75 
                 D177Q 
                 + 
               
               
                 76 
                 D177N 
                 + 
               
               
                 77 
                 D186Q 
                 + 
               
               
                 78 
                 D186N 
                 + 
               
               
                 79 
                 D203Q 
                 + 
               
               
                 80 
                 D203N 
                 + 
               
               
                 81 
                 T140N 
                 N 
               
               
                 82 
                 T140Q 
                 N 
               
               
                 83 
                 T140H 
                 N 
               
               
                 84 
                 T144N 
                 N 
               
               
                 85 
                 T144Q 
                 N 
               
               
                 86 
                 T144H 
                 N 
               
               
                 87 
                 T140N/T144N 
                 + 
               
               
                 88 
                 L165Q 
                 N 
               
               
                 89 
                 L165H 
                 N 
               
               
                 90 
                 F175I/F178I 
                 + 
               
               
                 91 
                 F175H 
                 + 
               
               
                 92 
                 F178H 
                 N 
               
               
                 93 
                 F192I/W194I 
                 N 
               
               
                 94 
                 F192H 
                 N 
               
               
                 95 
                 L198Q 
                 N 
               
               
                 96 
                 L198H 
                 N 
               
               
                 97 
                 D152N/D154N 
                 + 
               
               
                 98 
                 D152N 
                 N 
               
               
                 99 
                 D152Q 
                 + 
               
               
                 100 
                 D154N 
                 + 
               
               
                 101 
                 D154Q 
                 N 
               
               
                   
               
               
                 N = no change; + = Increased resistance to proteolysis 
               
            
           
         
       
     
     In one exemplary assay, native FIX had a large increase in its T 50  clotting time from about 140 seconds to about 200 seconds after only 0.5 hours of incubation with proteases, whereas exemplary modified FIX polypeptides with increased resistance to proteases, such as D203Q, had an increase in T 50  clotting time from about 150 seconds to about 160 seconds after 0.5 hours of incubation with proteases. After 2 hours of incubation with proteases the T 50  clotting time for D203Q only increased to about 173 seconds, and after 3 hours, 190 seconds. Similarly, exemplary modified FIX polypeptides D154N, and D152N/D154N exhibited T 50  clotting times of 160 seconds and 170 seconds, respectively, after 0.5 hours of incubation with proteases, and 170 seconds and 178 seconds, after 2 hours of incubation with proteases. The results demonstrate that exemplary modified FIX polypeptides are more resistant to protease treatment than native FIX. Thus, the modified FIX polypeptides indicated to have an “increase” protease resistance as compared to native FIX have a greater half-life and are more stable than wild-type FIX polypeptides following incubation with one or more proteases. 
     Example 5 
     SuperLEAD Clotting Activity and Resistance to Proteolysis 
     Based on the data obtained above for protease resistance of FIX LEAD polypeptides, selected FIX SuperLEAD polypeptides were generated and tested for their clotting activity and resistance to proteolysis as measured by retention of coagulation activity following treatment with proteases. A schematic representation of the combinations used to generate triple SuperLEADs is shown in Table 14. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Generation of SuperLEAD Combinations 
               
            
           
           
               
               
            
               
                   
                 2 nd  mutation 
               
               
                 NEMO code 
                 (Endoproteinase AspN) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 67 
                 73 
                 74 
                 77 
                 78 
                 79 
                 80 
                 97 
                 100 
                 ← 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 4 
                 141 
                 142 
                 143 
                 144 
                 145 
                 146 
                 147 
                 148 
                   
               
               
                 6 
                 149 
                 150 
                 151 
                 152 
                 153 
                 154 
                 155 
                 156 
               
               
                 8 
                 157 
                 158 
                 159 
                 160 
                 161 
                 162 
                 163 
                 164 
               
               
                 14 
                 165 
                 166 
                 167 
                 168 
                 169 
                 170 
                 171 
                 172 
               
               
                 16 
                 173 
                 174 
                 175 
                 176 
                 177 
                 178 
                 179 
                 180 
               
               
                 22 
                 181 
                 182 
                 183 
                 184 
                 185 
                 186 
                 187 
                 188 
               
               
                 23 
                 189 
                 190 
                 191 
                 192 
                 193 
                 194 
                 195 
                 196 
               
               
                 26 
                 197 
                 198 
                 199 
                 200 
                 201 
                 202 
                 203 
                 204 
               
               
                 27 
                 205 
                 206 
                 207 
                 208 
                 209 
                 210 
                 211 
                 212 
               
               
                 ↑ 
               
               
                 3 rd   
               
               
                 mutation 
               
               
                 (Elastase) 
               
               
                   
               
            
           
         
       
     
     Clotting activity of exemplary non-limiting FIX SuperLEAD polypeptides was assayed as described above in Example 3, and the data is shown in Table 14b. The T 50  values are displayed in seconds and represent the time required to achieved 50% clotting. 
     
       
         
           
               
             
               
                 TABLE 14b 
               
             
            
               
                   
               
               
                 Clotting activity of FIX SuperLEAD mutant proteins 
               
            
           
           
               
               
               
            
               
                   
                   
                 Bioactivity 
               
               
                 Nemo 
                   
                 Clotting Assay 
               
               
                 Code # 
                 Mutation 
                 T 50  (sec) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 107 
                 T163H/F192I 
                 99 
               
               
                 108 
                 I164Q/F192I 
                 101 
               
               
                 109 
                 T163N/I164N/F192I 
                 106 
               
               
                 110 
                 T169H/F192I 
                 94 
               
               
                 111 
                 S171Q/F192I 
                 105 
               
               
                 112 
                 S174Q/F192I 
                 99 
               
               
                 113 
                 S174H/F192I 
                 101 
               
               
                 114 
                 T179Q/F192I 
                 94 
               
               
                 115 
                 T179H/F192I 
                 101 
               
               
                 116 
                 D166Q/F192I 
                 93 
               
               
                 117 
                 D166N/F192I 
                 104 
               
               
                 118 
                 D186Q/F192I 
                 113 
               
               
                 119 
                 D186N/F192I 
                 111 
               
               
                 120 
                 F192I/D203Q 
                 90 
               
               
                 121 
                 F192I/D203N 
                 181 
               
               
                 122 
                 T148A/D152N/D154N/F192I 
                 90 
               
               
                 123 
                 T148A/D154N/F192I 
                 105 
               
               
                 141 
                 T163H/D166Q/F192I 
                 97 
               
               
                 142 
                 T163H/D166N/F192I 
                 89 
               
               
                 143 
                 T163H/D186Q/F192I 
                 123 
               
               
                 144 
                 T163H/D186N/F192I 
                 103 
               
               
                 145 
                 T163H/F192I/D203Q 
                 90 
               
               
                 146 
                 T163H/F192I/D203N 
                 89 
               
               
                 147 
                 T148A/D152N/D154N/T163H/F192I 
                 101 
               
               
                 148 
                 T148A/D154N/T163H/F192I 
                 92 
               
               
                 149 
                 I164Q/D166Q/F192I 
                 ND 
               
               
                 150 
                 I164Q/D166N/F192I 
                 ND 
               
               
                 151 
                 I164Q/D186Q/F192I 
                 109 
               
               
                 152 
                 I164Q/D186N/F192I 
                 101 
               
               
                 153 
                 I164Q/F192I/D203Q 
                 96 
               
               
                 154 
                 I164Q/F192I/D203N 
                 87 
               
               
                 155 
                 T148A/D152N/D154N/I164Q/F192I 
                 97 
               
               
                 156 
                 T148A/D154N/I164Q/F192I 
                 95 
               
               
                 157 
                 T163N/I164N/D166Q/F192I 
                 103 
               
               
                 158 
                 T163N/I164N/D166N/F192I 
                 100 
               
               
                 159 
                 T163N/I164N/D186Q/F192I 
                 128 
               
               
                 160 
                 T163N/I164N/D186N/F192I 
                 102 
               
               
                 161 
                 T163N/I164N/F192I/D203Q 
                 101 
               
               
                 162 
                 T163N/I164N/F192I/D203N 
                 95 
               
               
                 163 
                 T148A/D152N/D154N/T163N/I164N/F192I 
                 100 
               
               
                 164 
                 T148A/D154N/T163N/I164N/F192I 
                 97 
               
               
                 165 
                 D166Q/T169H/F192I 
                 99 
               
               
                 166 
                 D166N/T169H/F192I 
                 96 
               
               
                 167 
                 T169H/D186Q/F192I 
                 114 
               
               
                 168 
                 T169H/D186N/F192I 
                 100 
               
               
                 169 
                 T169H/F192I/D203Q 
                 86 
               
               
                 170 
                 T169H/F192I/D203N 
                 97 
               
               
                 171 
                 T148A/D152N/D154N/T169H/F192I 
                 95 
               
               
                 172 
                 T148A/D154N/T69H/F192I 
                 87 
               
               
                 173 
                 D166Q/S171Q/F192I 
                 104 
               
               
                 174 
                 D166N/S171Q/F192I 
                 97 
               
               
                 175 
                 S171Q/D186Q/F192I 
                 126 
               
               
                 176 
                 S171Q/D186N/F192I 
                 116 
               
               
                 177 
                 S171Q/F192I/D203Q 
                 90 
               
               
                 178 
                 S171Q/F192I/D203N 
                 90 
               
               
                 179 
                 T148A/D152N/D154N/S171Q/F192I 
                 ND 
               
               
                 180 
                 T148A/D154N/S171Q/F192I 
                 104 
               
               
                 181 
                 D166Q/S174Q/F192I 
                 93 
               
               
                 182 
                 D166N/S174Q/F192I 
                 95 
               
               
                 183 
                 S174Q/D186Q/F192I 
                 129 
               
               
                 184 
                 S174Q/D186N/F192I 
                 109 
               
               
                 185 
                 S174Q/F192I/D203Q 
                 95 
               
               
                 186 
                 S174Q/F192I/D203N 
                 96 
               
               
                 187 
                 T148A/D152N/D154N/S171Q/F192I 
                 97 
               
               
                 188 
                 T148A/D154N/S171Q/F192I 
                 91 
               
               
                 189 
                 D166Q/S174H/F192I 
                 93 
               
               
                 190 
                 D166N/S174H/F192I 
                 102 
               
               
                 191 
                 S174H/D186Q/F192I 
                 110 
               
               
                 192 
                 S174H/D186N/F192I 
                 102 
               
               
                 193 
                 S174H/F192I/D203Q 
                 98 
               
               
                 194 
                 S174H/F192I/D203N 
                 99 
               
               
                 195 
                 T148A/D152N/D154N/S174H/F192I 
                 98 
               
               
                 196 
                 T148A/D154N/S174H/F192I 
                 102 
               
               
                 197 
                 D166Q/T179Q/F192I 
                 100 
               
               
                 198 
                 D166N/T179Q/F192I 
                 89 
               
               
                 199 
                 T179Q/D186Q/F192I 
                 120 
               
               
                 200 
                 T179Q/D186N/F192I 
                 107 
               
               
                 201 
                 T179Q/F192I/D203Q 
                 92 
               
               
                 202 
                 T179Q/F192I/D203N 
                 94 
               
               
                 203 
                 T148A/D152N/D154N/T179Q/F192I 
                 106 
               
               
                 204 
                 T148A/D154N/T179Q/F192I 
                 98 
               
               
                 205 
                 D166Q/T179H/F192I 
                 103 
               
               
                 206 
                 D166N/T179H/F192I 
                 97 
               
               
                 207 
                 T179H/D186Q/F192I 
                 130 
               
               
                 208 
                 T179H/D186N/F192I 
                 113 
               
               
                 209 
                 T179H/F192I/D203Q 
                 94 
               
               
                 210 
                 T179H/F192I/D203N 
                 96 
               
               
                 211 
                 T148A/D152N/D154N/T179H/F192I 
                 103 
               
               
                 212 
                 T148A/D154N/T179H/F192I 
                 102 
               
               
                   
               
            
           
         
       
     
     Resistance to proteolysis of FIX SuperLEADs was assayed as described above in Example 4. For testing of FIX SuperLEADs, the concentration of selected proteases (trypsin, chymotrypsin, endoproteinase AspN, endoproteinase GluC, and elastase) was increased to 6% w/w. Resistance to proteases for exemplary non-limiting modified FIX SuperLEAD polypeptides is displayed in Table 15a. The data is expressed as relative resistance to proteases among the samples tested: (+), (++), or (+++), with (+++) indicating the highest resistance to proteases. 
     
       
         
           
               
             
               
                 TABLE 15a 
               
             
            
               
                   
               
               
                 Resistance to proteolysis of FIX SuperLEAD mutant proteins 
               
            
           
           
               
               
               
            
               
                 Nemo 
                   
                 Resistance 
               
               
                 Code # 
                 Mutation 
                 to proteases 
               
               
                   
               
               
                 107 
                 T163H/F192I 
                 (+) 
               
               
                 108 
                 I164Q/F192I 
                 (+) 
               
               
                 109 
                 T163N/I164N/F192I 
                 (+) 
               
               
                 110 
                 T169H/F192I 
                 (+) 
               
               
                 111 
                 S171Q/F192I 
                 (+) 
               
               
                 112 
                 S174Q/F192I 
                 (+) 
               
               
                 113 
                 S174H/F192I 
                 (+) 
               
               
                 114 
                 T179Q/F192I 
                 (+) 
               
               
                 115 
                 T179H/F192I 
                 (+) 
               
               
                 116 
                 D166Q/F192I 
                 (+) 
               
               
                 117 
                 D166N/F192I 
                 (+) 
               
               
                 118 
                 D186Q/F192I 
                 (++) 
               
               
                 119 
                 D186N/F192I 
                 (+) 
               
               
                 120 
                 F192I/D203Q 
                 (+) 
               
               
                 121 
                 F192I/D203N 
                 (+) 
               
               
                 122 
                 T148A/D152N/D154N/ 
                 (++) 
               
               
                   
                 F192I 
               
               
                 123 
                 T148A/D154N/F192I 
                 (+) 
               
               
                 141 
                 T163H/D166Q/F192I 
                 (++) 
               
               
                 142 
                 T163H/D166N/F192I 
                 (+) 
               
               
                 143 
                 T163H/D186Q/F192I 
                 (+) 
               
               
                 144 
                 T163H/D186N/F192I 
                 (+) 
               
               
                 145 
                 T163H/F192I/D203Q 
                 (++) 
               
               
                 146 
                 T163H/F192I/D203N 
                 (+) 
               
               
                 147 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 T163H/F192I 
               
               
                 148 
                 T148A/D154N/T163H/ 
                 (++) 
               
               
                   
                 F192I 
               
               
                 149 
                 I164Q/D166Q/F192I 
                 (+) 
               
               
                 150 
                 I164Q/D166N/F192I 
                 (+) 
               
               
                 151 
                 I164Q/D186Q/F192I 
                 (+) 
               
               
                 152 
                 I164Q/D186N/F192I 
                 (+) 
               
               
                 153 
                 I164Q/F192I/D203Q 
                 (++) 
               
               
                 154 
                 I164Q/F192I/D203N 
                 (++) 
               
               
                 155 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 I164Q/F192I 
               
               
                 156 
                 T148A/D154N/I164Q/ 
                 (+) 
               
               
                   
                 F192I 
               
               
                 157 
                 T163N/I164N/D166Q/ 
                 (+) 
               
               
                   
                 F192I 
               
               
                 158 
                 T163N/I164N/D166N/ 
                 (+) 
               
               
                   
                 F192I 
               
               
                 159 
                 T163N/I164N/D186Q/ 
                 (+) 
               
               
                   
                 F192I 
               
               
                 160 
                 T163N/I164N/D186N/ 
                 (+) 
               
               
                   
                 F192I 
               
               
                 161 
                 T163N/I164N/F192I/ 
                 (+) 
               
               
                   
                 D203Q 
               
               
                 162 
                 T163N/I164N/F192I/ 
                 (+) 
               
               
                   
                 D203N 
               
               
                 163 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 T163N/I164N/F192I 
               
               
                 164 
                 T148A/D154N/T163N/ 
                 (++) 
               
               
                   
                 I164N/F192I 
               
               
                 165 
                 D166Q/T169H/F192I 
                 (+) 
               
               
                 166 
                 D166N/T169H/F192I 
                 (+) 
               
               
                 167 
                 T169H/D186Q/F192I 
                 (+) 
               
               
                 168 
                 T169H/D186N/F192I 
                 (+) 
               
               
                 169 
                 T169H/F192I/D203Q 
                 (+) 
               
               
                 170 
                 T169H/F192I/D203N 
                 (++) 
               
               
                 171 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 T169H/F192I 
               
               
                 172 
                 T148A/D154N/T169H/F192I 
                 (+) 
               
               
                 173 
                 D166Q/S171Q/F192I 
                 (+) 
               
               
                 174 
                 D166N/S171Q/F192I 
                 (+) 
               
               
                 175 
                 S171Q/D186Q/F192I 
                 (+) 
               
               
                 176 
                 S171Q/D186N/F192I 
                 (+) 
               
               
                 177 
                 S171Q/F192I/D203Q 
                 (+) 
               
               
                 178 
                 S171Q/F192I/D203N 
                 (+) 
               
               
                 179 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 S171Q/F192I 
               
               
                 180 
                 T148A/D154N/S171Q/F192I 
                 (+) 
               
               
                 181 
                 D166Q/S174Q/F192I 
                 (+) 
               
               
                 182 
                 D166N/S174Q/F192I 
                 (+) 
               
               
                 183 
                 S174Q/D186Q/F192I 
                 (+) 
               
               
                 184 
                 S174Q/D186N/F192I 
                 (+) 
               
               
                 185 
                 S174Q/F192I/D203Q 
                 (+++) 
               
               
                 186 
                 S174Q/F192I/D203N 
                 (+) 
               
               
                 187 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 S174Q/F192I 
               
               
                 188 
                 T148A/D154N/S174Q/F192I 
                 (+) 
               
               
                 189 
                 D166Q/S174H/F192I 
                 (+) 
               
               
                 190 
                 D166N/S174H/F192I 
                 (+) 
               
               
                 191 
                 S174H/D186Q/F192I 
                 (+) 
               
               
                 192 
                 S174H/D186N/F192I 
                 (+++) 
               
               
                 193 
                 S174H/F192I/D203Q 
                 (+) 
               
               
                 194 
                 S174H/F192I/D203N 
                 (+) 
               
               
                 195 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 S174H/F192I 
               
               
                 196 
                 T148A/D154N/S174H/F192I 
                 (+) 
               
               
                 197 
                 D166Q/T179Q/F192I 
                 (+) 
               
               
                 198 
                 D166N/T179Q/F192I 
                 (+) 
               
               
                 199 
                 T179Q/D186Q/F192I 
                 (+) 
               
               
                 200 
                 T179Q/D186N/F192I 
                 (+) 
               
               
                 201 
                 T179Q/F192I/D203Q 
                 (+++) 
               
               
                 202 
                 T179Q/F192I/D203N 
                 (+) 
               
               
                 203 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 T179Q/F192I 
               
               
                 204 
                 T148A/D154N/T179Q/F192I 
                 (+) 
               
               
                 205 
                 D166Q/T179H/F192I 
                 (+) 
               
               
                 206 
                 D166N/T179H/F192I 
                 (+) 
               
               
                 207 
                 T179H/D186Q/F192I 
                 (+) 
               
               
                 208 
                 T179H/D186N/F192I 
                 (+) 
               
               
                 209 
                 T179H/F192I/D203Q 
                 (+) 
               
               
                 210 
                 T179H/F192I/D203N 
                 (+) 
               
               
                 211 
                 T148A/D152N/D154N/ 
                 (+) 
               
               
                   
                 T179H/F192I 
               
               
                 212 
                 T148A/D154N/T179H/F192I 
                 (+) 
               
               
                   
               
            
           
         
       
     
     All variants tested exhibited increased protease resistance relative to the native FIX polypeptide. Several FIX SuperLEAD polypeptides exhibited particularly high resistance to proteases including D186Q/F 192I, Ti 48A/D 152N/D 154N/F 192I, T163H/D166Q/F192I, T163H/F192I/D203Q, T148A/D154N/T163H/F192I, I164Q/F192I/D203 Q, I164Q/F192I/D203N, T148A/D154N/T163N/I164N/F192I, T169H/F 192I/D203N, S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203 Q. 
     In a separate experiment, protease resistance of selected SuperLEADs was assayed by the same methods as described above. In this experiment, the data for resistance to proteases was compared in terms of t 1/2  clotting time fold increase at 50% of T 0  compared to native Factor IX. Data for this analysis is shown in Table 15b. 
     
       
         
           
               
             
               
                 TABLE 15b 
               
             
            
               
                   
               
               
                 Resistance to proteolysis of FIX SuperLEAD mutant proteins 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 protease resistance 
               
               
                 Nemo 
                   
                 protease specificity 
                 clotting time 
                 (50% t 1/2  clotting fold- 
               
               
                 code 
                 mutations 
                 of leads 
                 (seconds) 
                 increase versus native) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 161 
                 T163N/I164N/F192I/D203Q 
                 EndAsp + CT + E 
                 101 
                 4.2 
               
               
                 194 
                 S174H/F192I/D203N 
                 EndAsp + CT + E 
                 99 
                 4.2 
               
               
                 195 
                 T148A/D152N/D154N/S174H/F192I 
                 EndAsp + CT + E 
                 98 
                 4 
               
               
                 145 
                 T163H/F192I/D203Q 
                 EndAsp + CT + E 
                 90 
                 3.3 
               
               
                 107 
                 F192I-T163H 
                 EndAsp + CT 
                 99 
                 3.2 
               
               
                 120 
                 F192I-D203Q 
                 EndAsp + CT 
                 90 
                 3.2 
               
               
                 147 
                 T148A/D152N/D154N/T163H/F192I 
                 EndAsp + CT + E 
                 101 
                 3 
               
               
                 170 
                 T169H/F192I/D203N 
                 EndAsp + CT + E 
                 97 
                 2.8 
               
               
                 177 
                 S171Q/F192I/D203Q 
                 EndAsp + CT + E 
                 90 
                 2.8 
               
               
                 203 
                 T148A/D152N/D154N/T179Q/F192I 
                 EndAsp + CT + E 
                 106 
                 2.8 
               
               
                 153 
                 I164Q/F192I/D203Q 
                 EndAsp + CT + E 
                 96 
                 2.7 
               
               
                 196 
                 T148A/D154N/S174H/F192I 
                 EndAsp + CT + E 
                 102 
                 2.7 
               
               
                 157 
                 T163N/I164N/D166Q/F192I 
                 EndAsp + CT + E 
                 103 
                 2.5 
               
               
                 180 
                 T148A/D154N/S171Q/F192I 
                 EndAsp + CT + E 
                 104 
                 2.5 
               
               
                 193 
                 S174H/F192I/D203Q 
                 EndAsp + CT + E 
                 98 
                 2.5 
               
               
                 204 
                 T148A/D154N/T179Q/F192I 
                 EndAsp + CT + E 
                 98 
                 2.5 
               
               
                 146 
                 T163H/F192I/D203N 
                 EndAsp + CT + E 
                 89 
                 2.3 
               
               
                 169 
                 T169H/F192I/D203Q 
                 EndAsp + CT + E 
                 86 
                 2.3 
               
               
                 179 
                 T148A/D152N/D154N/S171Q/F192I 
                 EndAsp + CT + E 
                 97 
                 2.3 
               
               
                 192 
                 S174H/D186N/F192I 
                 EndAsp + CT + E 
                 102 
                 2.3 
               
               
                 172 
                 T148A/D154N/T169H/F192I 
                 EndAsp + CT + E 
                 87 
                 2.2 
               
               
                 173 
                 D166Q/S171Q/F192I 
                 EndAsp + CT + E 
                 104 
                 2.2 
               
               
                 202 
                 T179Q/F192I/D203N 
                 EndAsp + CT + E 
                 94 
                 2.2 
               
               
                 185 
                 S174Q/F192I/D203Q 
                 EndAsp + CT + E 
                 95 
                 2 
               
               
                 187 
                 T148A/D152N/D154N/S174Q/F192I 
                 EndAsp + CT + E 
                 97 
                 1.8 
               
               
                 190 
                 D166N/S174H/F192I 
                 EndAsp + CT + E 
                 102 
                 1.8 
               
               
                 119 
                 F192I-D186N 
                 EndAsp + CT 
                 111 
                 1.7 
               
               
                 205 
                 D166Q/T179H/F192I 
                 EndAsp + CT + E 
                 103 
                 1.7 
               
               
                 148 
                 T148A/D154N/T163H/F192I 
                 EndAsp + CT + E 
                 92 
                 1.5 
               
               
                 175 
                 S171Q/D186Q/F192I 
                 EndAsp + CT + E 
                 126 
                 1.5 
               
               
                 155 
                 T148A/D152N/D154N/I164Q/F192I 
                 EndAsp + CT + E 
                 97 
                 1.3 
               
               
                 native FIX 
                   
                   
                 114 
                 1 
               
               
                   
               
            
           
         
       
     
     All variants tested exhibited increased protease resistance relative to the native FIX polypeptide. Exemplary FIX SuperLEAD polypeptides that exhibited particularly high resistance to proteases in this analysis included T163N/I164N/F192I/D203Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T163H/F192I/D203Q, T163H/F192I, F192I/D203Q, T148A/D152N/D154N/T163H/F192I, T169H/F192I/D203N, S171Q/F192I/D203Q, and T148A/D152N/D154N/T179Q/F192I. 
     The data above for the protease resistance are exemplary showing the resistance to proteolysis to an exemplary protease cocktail containing the proteases as described above. Thus, the data are not comprehensive and are not meant to be indicative that other FIX polypeptides do not exhibit protease resistance. 
     Example 6 
     SuperLEAD Clotting Activity and Resistance to Proteolysis Using an Alternative FIX Polypeptide Background 
     As mentioned in Example 1, the starting FIX polypeptide on which the FIX variants were generated is shown in SEQ ID NO: 1035. This sequence differs from another wild-type FIX polypeptide shown in SEQ ID NO: 2 at five positions: K39T, L40E, I43K, S44Q and S46V. These changes, however, do not affect the clotting activity of the FIX polypeptide. In order to compare the activity of the FIX variants between the two polypeptide backgrounds, site-directed mutagenesis was performed to convert the SuperLEAD variants to the alternative FIX background (BFIX-T148). The mutants that were generated are shown in Table 16. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 FIX SuperLEAD Variants Produced on BFIX-T148 Background 
               
            
           
           
               
               
               
            
               
                 NFIX code 
                 BFIX-T148 code 
                 mutations 
               
               
                   
               
            
           
           
               
               
               
            
               
                 161 
                 249 
                 T163N/I164N/F192I/D203Q 
               
               
                 194 
                 250 
                 S174H/F192I/D203N 
               
               
                 195 
                 251 
                 T148A/D152N/D154N/S174H/F192I 
               
               
                 145 
                 252 
                 T163H/F192I/D203Q 
               
               
                 107 
                 253 
                 T163H/F192I 
               
               
                 120 
                 254 
                 F192I/D203Q 
               
               
                 147 
                 255 
                 T148A/D152N/D154N/T163H/F192I 
               
               
                 170 
                 256 
                 T169H/F192I/D203N 
               
               
                 177 
                 257 
                 S171Q/F192I/D203Q 
               
               
                 203 
                 258 
                 T148A/D152N/D154N/T179Q/F192I 
               
               
                 153 
                 259 
                 I164Q/F192I/D203Q 
               
               
                 196 
                 260 
                 T148A/D154N/S174H/F192I 
               
               
                 157 
                 261 
                 T163N/I164N/D166Q/F192I 
               
               
                 180 
                 262 
                 T148A/D154N/S171Q/F192I 
               
               
                 193 
                 263 
                 S174H/F192I/D203Q 
               
               
                 204 
                 264 
                 T148A/D154N/T179Q/F192I 
               
               
                 146 
                 265 
                 T163H/F192I/D203N 
               
               
                 169 
                 266 
                 T169H/F192I/D203Q 
               
               
                 179 
                 267 
                 T148A/D152N/D154N/S171Q/F192I 
               
               
                 192 
                 268 
                 S174H/D186N/F192I 
               
               
                 172 
                 269 
                 T148A/D154N/T169H/F192I 
               
               
                 173 
                 270 
                 D166Q/S171Q/F192I 
               
               
                 202 
                 271 
                 T179Q/F192I/D203N 
               
               
                 185 
                 272 
                 S174Q/F192I/D203Q 
               
               
                 187 
                 273 
                 T148A/D152N/D154N/S174Q/F192I 
               
               
                 190 
                 274 
                 D166N/S174H/F192I 
               
               
                 119 
                 275 
                 D186N/F192I 
               
               
                 205 
                 276 
                 D166Q/T179H/F192I 
               
               
                 148 
                 277 
                 T148A/D154N/T163H/F192I 
               
               
                 175 
                 278 
                 S171Q/D186Q/F192I 
               
               
                 155 
                 279 
                 T148A/D152N/D154N/I164Q/F192I 
               
               
                   
               
            
           
         
       
     
     Human fetal kidney 293 HEK EBNA fibroblast cell line was grown in Dulbecco&#39;s modified eagle medium supplemented with 2 mM of glutamine and 10% of fetal bovine serum (FBS). Cells were plated at 5×10 5  cells per well in 6-well plates for 2 days, washed twice with PBS and then placed in serum-free medium. Cells were transfected with 2 μg of DNA using transfection reagent (PEI, NaCl). The cell medium was replaced after 4 hours by serum-free medium containing 50 μg/mL of K vitamin and 1×-ITS (Insulin, Transferrin, Selenium) mixture. The cell medium was collected 72 h after transfection and stored at −80° C. 
     Asserachrom® IX:Ag Elisa kit (Diagnostica Stago, France) was used to determine the concentration of the 31 selected mutant cell productions according to the instructions of the manufacturer. Samples were diluted 20-fold using the dilution reagent. Absorbance at 492 nm was measured using a spectrophotometer (Spectramax, Molecular Devices). Results were obtained by interpolation of the absorbance values from the calibration curve obtained from patients&#39; plasma dilutions and plotted in a log-log graph. Factor IX level at 100% corresponds approximately to 4 mg/L. Data are presented in Table 17. 
     
       
         
           
               
             
               
                 TABLE 17 
               
               
                   
               
               
                 Yields for BFIX T148 Super LEAD variants. 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Mean of production corresponds to 3 separated productions (except for mutants marked with * = only one production). Lower produced mutants are highlighted in grey. 
               
            
           
         
       
     
     In order to determine the bioactivity of each mutant, the clotting activity was measured by the one-stage activated partial thromboplastin time (aPTT) clotting assay (Cephascreen, Diagnostica Stago, France) using Factor IX immunodepleted human plasma (Deficient IX, Diagnostica Stago, France). Supernatants containing 60 ng of native or mutant Factor IX molecules from transient transfection production were diluted with 60 μL of immunodepleted plasma. 80 μL of Cephascreen reagent containing cephalin (platelet substitute) prepared from rabbit cerebral tissues and a polyphenolic activator in a buffered medium was added to dilute Factor IX samples and incubated at 37° C. for 15 minutes. Thus, 80 μL of pre-warmed CaCl 2  (0.025M, Diagnostica Stago, France) was added to Factor IX samples/Cephascreen reagent mix. The time required for clot formation was immediately monitored using a spectrophotometer (Spectramax, Molecular devices) at 350 nm. A serial dilution of normal human pooled plasma representing 100 to 120% of Factor IX activity (System control N, Diagnostica Stago, France) was used as the standard curve in order to validate the linearity of the clotting time detection. Data are presented in Table 18. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Clotting activity of BFIX T148 SuperLEAD variants 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 mutant number 
                 Production 1 
                 Production 2 
                 Production 3 
                 MEAN OF T50 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 in BFIX-T148 
                 T50 (clotting) 
                 conversion 
                 T50 (clotting) 
                 conversion 
                 T50 (clotting) 
                 conversion 
                 (clotting) 
               
               
                 Rank 
                 matrix 
                 (seconds) 
                 (μg/l) 
                 (seconds) 
                 (μg/l) 
                 (seconds) 
                 (μg/l) 
                 (seconds) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 249 
                 122 
                 38.2 
                 100 
                 46.7 
                 110 
                 22.9 
                 111 
               
               
                 2 
                 250 
                 121 
                 34.0 
                 114 
                 24.0 
                 99 
                 40.2 
                 111 
               
               
                 3 
                 251 
                 131 
                 21.2 
                 132 
                 10.7 
                 99 
                 40.2 
                 121 
               
               
                 4 
                 252 
                 105 
                 33.5 
                 122 
                 18.7 
                 106 
                 43.9 
                 111 
               
               
                 5 
                 253 
                 113 
                 22.8 
                 128 
                 13.8 
                 136 
                 12.4 
                 125 
               
               
                 6 
                 254 
                 114 
                 21.7 
                 128 
                 13.4 
                 130 
                 15.6 
                 124 
               
               
                 7 
                 255 
                 137 
                 18.9 
                 103 
                 39.1 
                 110 
                 23.9 
                 117 
               
               
                 8 
                 256 
                 140 
                 17.1 
                 107 
                 32.4 
                 108 
                 26.2 
                 118 
               
               
                 9 
                 257 
                 nd 
                 nd 
                 nd 
                 nd 
                 114 
                 30.4 
                  114* 
               
               
                 10 
                 258 
                 121 
                 32.2 
                 116 
                 24.1 
                 98 
                 42.5 
                 112 
               
               
                 11 
                 259 
                 nd 
                 nd 
                 109 
                 29.6 
                 130 
                 15.7 
                 119 
               
               
                 12 
                 260 
                 128 
                 23.4 
                 120 
                 17.9 
                 101 
                 36.7 
                 117 
               
               
                 13 
                 261 
                 144 
                 14.9 
                 115 
                 21.8 
                 110 
                 23.1 
                 123 
               
               
                 14 
                 262 
                 nd 
                 nd 
                 nd 
                 nd 
                 133 
                 13.9 
                  133* 
               
               
                 15 
                 263 
                 118 
                 23.3 
                 100 
                 51.3 
                 115 
                 18.7 
                 111 
               
               
                 16 
                 264 
                 118 
                 37.7 
                 108 
                 34.9 
                 109 
                 39.2 
                 111 
               
               
                 17 
                 265 
                 nd 
                 nd 
                 106 
                 34.1 
                 120 
                 23.0 
                 113 
               
               
                 18 
                 266 
                 138 
                 19.6 
                 115 
                 21.5 
                 100 
                 39.6 
                 118 
               
               
                 19 
                 267 
                 115 
                 21.3 
                 123 
                 16.7 
                 128 
                 16.5 
                 122 
               
               
                 20 
                 268 
                 nd 
                 nd 
                 nd 
                 nd 
                 126 
                 18.5 
                  126* 
               
               
                 21 
                 269 
                 115 
                 24.9 
                 110 
                 28.8 
                 105 
                 30.5 
                 110 
               
               
                 22 
                 270 
                 120 
                 19.8 
                 118 
                 19.8 
                 116 
                 17.5 
                 118 
               
               
                 23 
                 271 
                 133 
                 11.4 
                 106 
                 34.2 
                 115 
                 18.1 
                 118 
               
               
                 24 
                 272 
                 125 
                 15.5 
                 116 
                 21.8 
                 120 
                 14.9 
                 120 
               
               
                 25 
                 273 
                 nd 
                 nd 
                 125 
                 14.6 
                 128 
                 16.8 
                 126 
               
               
                 26 
                 274 
                 118 
                 21.7 
                 102 
                 45.3 
                 109 
                 24.4 
                 109 
               
               
                 27 
                 275 
                 nd 
                 nd 
                 135 
                 10.5 
                 147 
                 8.1 
                 141 
               
               
                 28 
                 276 
                 nd 
                 nd 
                 123 
                 17.1 
                 135 
                 12.9 
                 129 
               
               
                 29 
                 277 
                 127 
                 14.8 
                 116 
                 21.6 
                 102 
                 34.5 
                 115 
               
               
                 30 
                 278 
                 136 
                 17.8 
                 129 
                 12.6 
                 135 
                 12.6 
                 133 
               
               
                 31 
                 279 
                 128 
                 28.4 
                 102 
                 39.9 
                 113 
                 20.7 
                 114 
               
               
                   
                 BFIX 
                 107 
                 48     
                 103 
                 44     
                   
                   
                 105 
               
               
                   
                 BFIX-T148 
                 116 
                 32     
                 104 
                 42     
                 120 
                 15.0 
                 113 
               
               
                   
                 NFIX 
                 128 
                 19     
                 117 
                 21     
                   
                   
                 123 
               
               
                   
               
               
                 Mean of production corresponds to 3 separated clotting time assays (except for mutants marked with * = only one production). Conversion in μg/L was based from standard curves obtained from normal human sera. 
               
            
           
         
       
     
     All selected mutants in BFIX-T148 background were active by clotting time assays and were similar to BFIX-T148 native sequence (t 1/2  between 111 sec and 133 sec). Only mutant 275 exhibited a clotting time higher than 140 sec. 
     Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.