Patent Application: US-12595002-A

Abstract:
disclosed are compositions for treating blood coagulation disorders and allows for manipulation of the blood coagulation cascade . more particularly the invention , relates to compositions for altering bleeding that include a mixture of at least one blood coagulation factor in a low dose and phospholipid vesicles . the invention has a variety of important uses including controlling bleeding in a mammal that has or is suspected of having a potentially life - threatening blood coagulation disorder .

Description:
as discussed , this invention relates to the therapeutic treatment of patients with bleeding disorders involving administering a mixture of phospholipids with serine protease - activated blood coagulation factors . preferred invention compositions act synergistically to boost activity of activated blood coagulation factors , preferably fviia such as recombinant forms thereof typical of many commercial preparations . as also discussed , preferred practice of the invention provides substantial advantages including reducing treatment costs , decreasing factor dosages , reducing risk of an immune reaction , such as anaphylaxis , and also decreases the risk of antibody formation specific for the blood coagulation factors . furthermore , inhibitors of blood coagulation factors are not produced , thus therapy is simplified . preferred therapeutic compositions of the present invention include between from about 0 . 1 nm to about 10 nm of activated blood coagulation factors , typically recombinant factor viia ( rfviia ) and selected phospholipid vesicles . as discussed previously , particular phospholipid vesicles comprise anionic phospholipids at about physiological ph , preferably phosphatidylcholine and phosphatidylserine ( pcps ). the preferred weight to volume ( w / v ) of pcps , is comprised between from about 50 % ( w / v ) to about 75 % ( w / v ) phosphatidylcholine and between from about 10 % ( w / v ) to about 25 % ( w / v ) phosphatidylserine . more preferably , the weight to volume ( w / v ) of ( pcps ), is comprised between from about 75 % ( w / v ) to about 90 % ( w / v ) phosphatidylcholine and between from about 25 % ( w / v ) to about 50 % ( w / v ) phosphatidylserine . most preferably , the weight to volume ( w / v ) of ( pcps ), is comprised between from about 50 % ( w / v ) to about 90 % ( w / v ) phosphatidylcholine and between from about 10 % ( w / v ) to about 50 % ( w / v ) phosphatidylserine . preferred use of the composition involves administration of a coagulation - effective amount usually between from about 10 to about 1000 fold less the amount of the same factor used in conventional therapies . although it will be appreciated that precise dosage and treatment regimens will vary according to recognized parameters including , but not limited to , patient health , age , sex , etc ., a preferred unit dose of the pcps is between from about 1 arbitrary unit to about 300 arbitrary units per kg body weight , more preferably the unit dose of the pcps is between from about 300 arbitrary units to about 1 , 500 arbitrary units per kg body weight of the mammal , most preferably the unit dose of the pcps is between from about 1 arbitrary units to about 3 , 000 arbitrary units per kg body weight of the mammal . as used herein , 1 arbitrary unit is defined as 0 . 005 mg per kg body weight . the therapeutic compositions of the present invention , more particularly include an effective mixture of pcps and blood coagulation factor , typically recombinant factor viia . a preferred unit dose range of the amount of recombinant factor viia is between from about 18 units to about 180 units per kg of body weight of the mammal . a more preferred unit dose range of the amount of recombinant factor viia is between from about 180 units to about 900 units per kg of body weight of the mammal . a most preferred unit dose range of the amount of recombinant factor viia is between from about 18 units to about less than 1 , 800 units per kg of body weight of the mammal . a preferred weight ratio of the pcps to the blood coagulation factor for most invention applications is between from about 0 . 15 : 1 to about 10 , 000 : 1 . a more preferred weight ratio of the pcps to the blood coagulation factor administered to a patient is between from about 0 . 15 : 1 to about 20 , 000 : 1 . a most preferred weight ratio of the pcps to the blood coagulation factor administered to a patient is between from about 0 . 15 : 1 to about 30 , 000 : 1 . in order to produce the desired therapeutic effect , as embodied in this invention , the composition is desirably administered by a continuous intravenous infusion , using isotonic saline for injection to keep the vein open . the treatment may consist of a single dose or a plurality of doses over a period of time . an advantageous treatment schedule requires administration of the above - described therapeutic composition with an interval between administrations calculated on a per patient basis . as already discussed briefly , factors to consider in the treatment regimen would be weight , age , bleeding disorder , severity of bleeding etc . those who are skilled in the art may modify the regimen according to the individual patient . “ synthetic plasma ” as used herein refers to a composition of highly purified natural and recombinant proteins involved in the coagulation and its regulation and synthetic phospholipid membranes or platelets . “ synthetic plasma test ” as used herein refers to the use of synthetic plasma wherein phospholipid combinations , concentrations , blood coagulation activity of blood coagulation factors , blood coagulation effective amounts , and the like , are determined by the most optimal compositions that cause coagulation . see for example k . g . mann , thrombosis and haemostasis , ( 1999 ), 82 ( 2 ): 165 - 174 and also examples 5 - 8 described infra . as used herein “ an effective blood coagulation amount ” is the minimum amount of blood coagulation factor required for blood clotting in a time less than ten minutes , as determined by the synthetic “ plasma ” test or blood coagulation experiments . see methods and examples 6 and 7 described infra . the preferred blood coagulation factors are factor viia , factor viiia , factor ixa , factor xia , more preferably , recombinant factor viia . an effective blood coagulation amount , for example for recombinant factor viia , is typically in the range between from about 0 . 1 nm to about 10 nm . ( about 18 units per kg body weight to about 1 , 800 units per kg body weight ). a “ pharmaceutically acceptable composition ” as used herein , is a therapeutic composition of the present invention employed in a physically discrete unit suitable as unitary dosages for a primate such as a human , each unit containing a pre - determined quantity of active material calculated to produce the desired therapeutic effect in association with the required phospholipid vehicle . precise coagulation - effective amounts of the therapeutic composition to be administered will be guided by the judgment of the practitioner , however , the unit dose will generally depend on the route of administration . “ pharmaceutically acceptable ” as used herein , refers to compositions that are not toxic to the patient and do not interfere with the desired activity of the blood coagulation factors , of the present invention . “ substantially pure ” as used herein , refers to the blood coagulation factors obtained as a result of the purification procedure described in van t &# 39 ; veer et al ., j . biol . chem ., ( 1997 ) 272 : 4367 - 4377 , the teachings of which are specifically incorporated herein . the term “ mammal ” as defined herein preferably refers to a primate such as a human being or an animal . as used herein , the term “ platelet activation ” refers to platelet release reactions ( e . g ., osteonectin , platelet factor 4 or β thrombomodulin ). however , platelet activation can also refer to the occurrence of one or more of the following events : platelet aggregation , platelet adhesion , platelet agglutination , expression of platelet external receptors ( e . g ., gpiib / iiia or p - selectin ), or platelet interaction with other blood components ( e . g ., collagen or fibrinogen ) and cells ( e . g ., leukocytes ). as discussed , the invention is compatible with a variety of suitable phospholipid vesicle mixtures . in particular , preferred phospholipids can be provided in a number of forms but the preferred form is as phosphatidylcholine / phosphatidylserine vesicles ( pcps ). the pcps vesicle preparations and the method of administration of xa / pcps is described in giles , et al ., u . s . pat . no : 4 , 721 , 618 ( 1988 ), the teachings of which are specifically incorporated herein . for example , phospholipid vesicles or liposomal suspensions are also preferred as pharmaceutically acceptable carriers or delivery vehicles . these can be prepared according to methods known to those skilled in the art and can contain , for example , phosphatidylserine / phosphatidylcholine or other compositions of phospholipids or detergents . blood coagulation factors suitable for use with the invention can be prepared or purchased from commercial vendors as needed . for example , recombinant fviia can be purchased from novo biolabs ( danbury , conn .) and is the preferred source for purposes of this invention . the amount of recombinant factor viia in the dosage form is extremely small and allows for treatment of large numbers of hemophiliacs , in comparison to the high doses required for the provision of more conventional therapy . this has distinct advantages , apart from the obvious one of economy . the accidental transmission of infection is a major hazard of multiple transfusion practice in patients such as hemophiliacs . hepatitis and acquired immunodeficiency syndrome are major problems . there are other sources available to obtain factor vii , for example it can be purified from plasma and activated into factor viia by the methods described by broze and majerus , j . biol . chem . 255 ( 4 ) ( 1980 ) 1242 - 1247 and hedner and kisiel , j . clin . invest . 71 ( 1983 ) 1836 - 1841 . factor viia may also be produced by recombinant dna - technology by culturing in an appropriate medium mammalian cells transfected with a dna - sequence encoding factor vii , isolating the protein produced and activating said protein to factor viia ( european patent application no . 86302855 . 1 ). human coagulation factors , such as for example vii , x , ix , and prothrombin may also be isolated from fresh frozen plasma using the general methods of bajaj et al , ( prep . biochem ., 11 : 397 - 412 , 1981 ). fviia biological activity : the fviia biological activity is characterized by the mediation of blood coagulation via the synergistic mixture of a phospholipid and fviia . biological activity , as used herein , is a function or set of functions performed by a molecule in a biological context ( i . e . in an organism or an in vitro facsimile ). biological activities of proteins may be divided into catalytic and effector activities . catalytic activities of clotting factors generally involve the activation of other factors through the specified cleavage of precursors . effector activities including specific binding of the biologically active molecule in a calcium - dependent manner , to macromolecules such as proteins or to cells . effector activity frequently augments , or is essential to , catalytic activity under physiological conditions . catalytic and effector activities may , in some cases , reside within the same domain of a protein . the preparations according to the invention enable a safe and simple treatment of blood coagulation disorders , in which an effective onset of action can be observed within a very short time . other conditions that can be treated with this combination include surgical bleeding from the microvasculature , bleeding at skin graft harvest sites , postoperative bleeding , including following orthopedic surgery , brain surgery or brain trauma , bleeding secondary to thrombocytopenia , and platelet dysfunction . in a particularly preferred invention embodiment , compositions include synthetic phospholipids : 75 % phosphatidylcholine and 25 % phosphatidylserine , with 10 nm recombinant factor viia . phosphatidylcholine ( pc ) and phosphatidylserine ( ps ) are available commercially as semi - purified reagents and are prepared from egg yolks and bovine brain respectively . the pcps lipid vesicles may be prepared by a conventional and standardized protocol ( nesheim et al , j . biol . chem . 254 : 10952 , 1979 and barenholz et al ., biochem . j . 16 : 2806 , 1976 ) which produces single compartment vesicles of uniform dimension ( 325 - 350 å ) which may be stored at 4 ° c . for 2 to 3 weeks . the molar ratio of phosphatidylserine to phosphatidylcholine is about 1 : 3 , based on the relative amounts of these lipids used in the preparation of the vesicles . the recombinant factor viia - pcps mixture is freshly prepared by mixing recombinant factor viia and pcps in the desired ratio immediately prior to use . the dosage of recombinant factor viia / pcps is administered on a dose / kg body weight basis . the dose of pcps is unitized in arbitrary units . 1 arbitrary unit pcps equals 1 × 10 − 8 moles of phospholipid as assayed by an inorganic phosphorus assay . recombinant factor viia is unitized according to an internationally accepted classification in which 1 unit of factor vii is the amount present in 1 ml of normal plasma and 1 unit of factor viia is the amount of activity present when 1 unit of factor vii is fully activated . the assay is standardized by measuring activity in the test preparation against the activity in a normal pool plasma standard as described by suomela h ., et al ( thrombosis research 10 : 267 , 1977 ) as modified by giles a . r . et al ( thrombosis research 17 ; 353 , 1980 ). the activity of the purchased recombinant factor viia ( novo biolabs ) is 50 , 000 units per mg . for the methods of present invention , the preferred method for administration of the blood coagulation factors is intravenous administration . a continuous infusion is established via butterfly needle in the femoral vein using isotonic saline for injection to keep the vein open . in the case of internal bleeding or for systemic bleeding , the composition may be suitable for enteral application . the composition may be encapsulated in an acid - stable , base - labile coating ( e . g . an enteric pill which is coated with a substance such as salol which will not dissolve in the stomach or a hexylresorcinol pill which consists of hexylresorcinol covered with a rupture resistant coating that disintegrates in the digestive tract ). the pill would be stable in the acidic stomach , but would become “ activated ” as it enters the high ph small intestine . the preparations according to the invention enable a safe and simple treatment of blood coagulation disorders , in which an effective onset of action can be observed within a very short time . other conditions that can be treated with this combination include surgical bleeding from the microvasculature , bleeding at skin graft harvest sites , postoperative bleeding , including following orthopedic surgery , brain surgery or brain trauma , bleeding secondary to thrombocytopenia , and platelet dysfunction . it will be appreciated that the actual preferred amounts of blood coagulation factors used in a given therapy will vary according to the blood coagulation factors used or combination of blood coagulation factors being utilized , the phospholipid vesicle composition , the mode of application , the particular site of administration , etc . optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art . the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed , since these embodiments are intended as illustrations of several aspects of the invention . any equivalent embodiments are intended to be within the scope of this invention . indeed , various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description . such modifications are also intended to fall within the scope of the appended claims . various references are cited herein , the disclosure of which are incorporated by reference in their entireties . the present invention is further illustrated by the following examples . these examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof . the following materials and methods were used as needed to conduct the examples which follow . phosphatidylserine ( ps ), phosphatidylcholine ( pc ), and edta were purchased from sigma . phospholipid vesicles ( pcps ) composed of 25 % ps and 75 % pc were prepared as described ( 1 ). spectrozyme th was purchased from american diagnostica , inc . d - phe - pro - argch 2 c1 ( fprck ) was synthesized in home . elisa thrombin - at - iii ( tat ) kit ( enzygnost tat ) was purchased from behring . osteonectin elisa plates were coated in home . monoclonal anti - osteonectin and anti - factor ix antibodies were developed in the biochemistry antibody core laboratory ( university of vermont ). fibrinogen , fibrinopeptide a ( fpa ) and b ( fpb ), and solid clot analyses were performed as described previously ( 2 ). human coagulation factors vii , x , ix , and prothrombin , were isolated from fresh 10 l frozen plasma using the general methods of bajaj et al . ( 3 ), and were purged of trace contaminants and traces of active enzymes as described ( 4 ). recombinant factor viii and recombinant tissue factor ( tf ) ( residues 1 - 242 ) were provided as gifts from drs . shu len liu and roger lundblad ( hyland division , baxter healthcare corp ). recombinant human factor viia was purchased from novo pharmaceuticals . recombinant full - length tfpi produced in escherichia coli was purchased from american diagnostica . corn trypsin inhibitor was isolated from popcorn as described elsewhere ( 7 ). washed platelets were prepared by the procedure of mustard et al . ( 8 ). relipidation of tf was done as described elsewhere ( 7 ). human factor v and at - iii were isolated from freshly frozen plasma ( 5 , 6 ). briefly , human coagulation factor vii was isolated from the factor vii containing fractions eluted in the nacl gradient of the deae - sepharose chromatography , involved in the purification of vitamin k - dependent clotting factors . factor vii activity in the fractions was measured by incubating 25 μl of 1000 - fold diluted samples in 20 nmol / l tris , 150 nmol / l nacl , ph 7 . 4 ( tbs ) containing 0 . 1 % bovine serum albumin ( bsa ) with 50 μl of a solution containing 1 / 300 diluted rabbit thromboplastin ( thromboplastin - c , dade , baxter ), 160 nmol / l factor x , and 10 nmol / l cacl 2 for 30 minutes . generation of factor xa was monitored using the chromogenic substrate spectrozyme xa . factor vii eluted from the deae - sepharose before and with some overlap with the first part of the c4bp / protein s peak . c4bp / protein s - containing fractions were not included in the factor vii pool . the factor vii pool was diluted 1 . 5 times with water containing 1 nmol / l benzamidine and applied to a 20 ml q - sepharose ff column previously equilibrated in 17 nmol / l tris , 50 mmol / l nacl , and 1 mmol / l benzamidine , ph 7 . 4 . after application of the pool , the column was washed with 100 ml - equilibration buffer . factor vii activity was eluted with 20 mmol / l cacl 2 in the same buffer . fractions were collected in 1 / 10 of a volume of 0 . 4 mol / l edta ph 7 . 4 . factor vii containing fractions were pooled and precipitated by adding ammonium sulfate to 80 % saturation . this solution was kept overnight at 4 ° c ., then spun in an sw - 50 rotor at 40 000 rpm , 4 ° c . for 45 minutes . the protein pellet was resuspended in 50 % glycerol and stored at − 20 ° c . before use in the experiment , factor vii was dialyzed extensively against tbs at 4 ° c . factor vii prepared via this method appeared homogeneous on nonreduced and reduced sds - page and had a specific activity of 2000 u / mg measured in a single clotting assay . thrombin generation initiated by factor viia - tf was studied in a reconstituted coagulation model as described previously . tf was relipidated at the indicated concentrations in 400 μmol / l 75 % phosphatidylcholine , 25 % phosphatidylserine vesicles for 30 minutes at 37 ° c . in 20 mmol / l hepes , 150 mmol / l nacl , 2 mmol / l cacl 2 ph 7 . 4 ( hepes / ca 2 + ). factor v and factor viii were added to the relipidated tf mixture immediately before the reaction was started by the addition of a solution containing factor viia with or without factor vii and factor x , factor ix , and prothrombin . the zymogen solution was also prepared in hepes / ca 2 + and preheated at 37 ° c . for 3 minutes before addition to the tf , factor v , and factor viii mixture . when tfpi or at - iii were included , they were added to the factor x , factor ix , and prothrombin mixture . the final concentrations of the proteins in the reaction , chosen to represent mean plasma values , were 160 nmol / l factor x , 90 nmol / l factor ix , 0 . 7 nmol / l factor viii , 20 nmol / l factor v , 1 . 4 μmol / l prothrombin , 2 . 5 nmol / l tfpi , and 3 . 4 μmol / l at - iii . after starting the reaction , aliquots were withdrawn from the reaction mixture in time and quenched in 20 mmol / l edta / tbs ph 7 . 4 to assay for thrombin formation . assays for thrombin activity were performed using the chromogenic substrate spectrozyme th . the hydrolysis of the substrate was monitored by the change in absorbance at 405 nm . thrombin generation was calculated from a standard curve prepared by a serial dilution of α - thrombin . when at - iii was added to the reaction mixture , samples were withdrawn in 20 mmol / l edta / tbs , containing 0 . 4 mmol / l spectrozyme th , and assayed immediately for thrombin activity as described before , to obtain accurate thrombin measurements in the presence of at - iii . clotting in freshly drawn , non - anticoagulated whole blood was performed in 32 tubes ( two series per experiment — 16 tubes / series ). all tubes were loaded with corn trypsin inhibitor ( 100 μg / ml blood ), 12 . 5 pm relipidated tf / ml blood ( pcps : tf = 2 , 000 ) in hbs with 2 mm cacl 2 ( all tubes in each series except phlebotomy control tube ), 10 nm recombinant factor viia ( all tubes , experiment series only ), and equivalent volume factor viia dilution buffer ( hbs , ph 7 . 4 , all tubes control series only ). no more than 35 μl of reagents were loaded in each tube . the zero tube of each series was pretreated using 1 ml of 50 mm edta and 10 μl of 10 mm fprck ( diluted in 0 . 01 m hcl ). normal donor or hemophilia a patient ( factor viii & lt ; 1 %) blood was drawn by venipuncture , delivered into the reagent - loaded tubes , and the tubes were periodically quenched with edta and fprck . the clotting time was observed visually by two observers . tubes were centrifuged and the supernatants were aliquoted for further analyses . elisas for tat were performed . fpa and fpb release was evaluated by hplc , fibrinogen depletion by western blots . solid clots were solubilized and analyzed by gel electrophoresis . the results of this experiment are shown in fig3 a ( tat formation ), wherein (▪) represents normal control ; (▴) represents normal control with 10 nm factor viia addition ; () represents hemophilia a patient ; and (⋄) represents hemophilia a patient with 10 nm factor viia addition . in fig3 b , the upper lanes in each panel represent gels of soluble fibrinogen , lower parts represent gels of solubilized solid clots . treatment of induced hemophilia b blood with pcps and factor viia . clotting in freshly drawn , non - anticoagulated whole blood was performed in 32 tubes ( two series per experiment — 16 tubes / series ). all tubes were loaded with corn trypsin inhibitor ( 100 μg / ml blood ), 12 . 5 pm relipidated tf / ml blood ( pcps : tf = 2 , 000 ) and 50 μg / ml anti - factor ix antibody ( antibody was not loaded into one normal control tube ) in hbs with 2 mm cacl 2 , 10 nm recombinant factor viia and varying concentrations of pcps ( 0 - 200 μm ) ( all tubes , experiment series only ) and equivalent volume factor viia dilution buffer ( hbs , ph 7 . 4 ) and pcps ( all tubes control series only ). no more than 35 μl of reagents were loaded in each tube . normal donor blood was drawn by venipuncture , delivered into the reagent - loaded tubes , and the clotting time was observed visually by two observers . tubes were quenched with edta and fprck in 10 min after clot formation . tubes were centrifuged and the supernatants were aliquoted for tat analyses . the results are shown in fig3 c , wherein , () represents normal control ; (▪) represents induced hemophilia b with pcps addition ; and , (□) represents induced hemophilia b with 10 nm factor viia and pcps addition . factor viia at varying concentrations ( 0 - 100 nm ), 40 μg / ml cti , 50 μm pcps , and 30 mm cacl 2 were added to the factor viii deficient plasma pre - warmed to 37 ° c . clotting was initiated by the addition of 12 . 5 pm (▪) or 25 pm (♦) tf . dotted lines represent clotting time of normal plasma initiated by corresponding concentration of tf and without factor viia addition . the results are shown in fig4 . in the control experiment (▪), relipidated tf ( pcps : tf = 2 , 000 ) at a concentration of 50 pm was incubated in hbs , 2 mm cacl 2 for 10 min at 37 ° c . factor v ( 40 nm ), factor viii ( 1 . 4 nm ), and platelets ( 4 × 10 8 / ml ; 2 x mean physiological concentration ) were added to the relipidated tf , and thrombin generation was started by addition of an equal volume of zymogen - inhibitor mixture containing prothrombin ( 2 . 8 μm ), factors vii ( 20 nm ), viia ( 0 . 2 nm ), x ( 340 nm ) and ix ( 180 nm ), tfpi ( 5 nm ), and at - iii ( 6 . 8 μm ) prepared in hbs , 2 mm cacl 2 and preheated at 37 ° c . for 3 minutes . the final concentrations of the proteins and platelets in the reaction represent mean physiological values . final concentration of tf was 25 pm . following initiation of the reaction , at selected time points , 10 μl aliquots were withdrawn from the reaction mixture and quenched in 20 mm edta in hbs ( ph 7 . 4 ) containing 0 . 2 mm spectrozyme th and assayed immediately for thrombin activity . the hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a molecular devices v max spectrophotometer . thrombin generation was calculated from a standard curve prepared by serial dilutions of α - thrombin . results are shown in fig5 wherein factor viii was omitted ( hemophilia a situation ), and additional amounts of factor viia were added : () represents hemophilia control — no additional factor viia ; (▴) represents 10 rim factor viia ; and , (♦) represents 40 nm factor viia . thrombin generation in the synthetic “ plasma ” in the presence of2 μm pcps . in the control experiment (▪), relipidated tf ( pcps : tf = 2 , 000 ) at a concentration of 50 pm was incubated with 4 μm pcps in hbs , 2 mm cacl 2 for 10 min at 37 ° c . factor v ( 40 nm ) and factor viii ( 1 . 4 nm ) were added to tf , and thrombin generation was started by addition of an equal volume of zymogen - inhibitor mixture containing prothrombin ( 2 . 8 μm ), factors vii ( 20 nm ), viia ( 0 . 2 nm ), x ( 340 nm ) and ix ( 180 nm ), tfpi ( 5 nm ), and at - iii ( 6 . 8 μm ) prepared in hbs , 2 mm cacl 2 and preheated at 37 ° c . for 3 minutes . the final concentrations of the proteins in the reaction represent mean physiological values . final concentration of tf was 25 pm . final concentration of pcps was 2 μm . following initiation of the reaction , at selected time points , 10 μl aliquots were withdrawn from the reaction mixture and quenched in 20 mm edta in hbs ( ph 7 . 4 ) containing 0 . 2 mm spectrozyme th and assayed immediately for thrombin activity . the hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a molecular devices v max spectrophotometer . thrombin generation was calculated from a standard curve prepared by serial dilutions of α - thrombin . the results are shown in fig6 wherein factor viii was omitted ( hemophilia a situation ), and additional amounts of factor viia were added : () represents hemophilia control — no additional factor viia ; (▴) represents 10 nm factor viia ; and , (♦) represents 40 nm factor viia . thrombin generation in the synthetic “ plasma ” at varying pcps concentrations . in the control experiment ( fig7 a ; o ), relipidated tf ( pcps : tf = 2 , 000 ) at a concentration of 25 pm was incubated with 400 μm pcps in hbs , 2 mm cacl 2 for 10 min at 37 ° c . factor v ( 40 nm ) and factor viii ( 1 . 4 nm ) were added to tf , and thrombin generation was started by addition of an equal volume of zymogen - inhibitor mixture containing prothrombin ( 2 . 8 μm ) factors vii ( 20 nm ), viia ( 0 . 2 nm ), x ( 340 nm ) and ix ( 180 nm ), tfpi ( 5 nm ), and at - iii ( 6 . 8 μm ) prepared in hbs , 2 mm cacl 2 and preheated at 37 ° c . for 3 minutes . the final concentrations of the proteins in the reaction represent mean physiological values . final concentration of tf was 12 . 5 pm . final concentration of pcps was 200 μm . following initiation of the reaction , at selected time points , 10 μl aliquots were withdrawn from the reaction mixture and quenched in 20 mm edta in hbs ( ph 7 . 4 ) containing 0 . 2 mm spectrozyme th and assayed immediately for thrombin activity . the hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a molecular devices v max spectrophotometer . thrombin generation was calculated from a standard curve prepared by serial dilutions of α - thrombin . the total active thrombin generated was evaluated by integrating the area under thrombin vs time curve . the rest of experiments ( fig7 ) were conducted in the absence of factor viii ( hemophilia a situation ) and in the presence of factor viia at 0 . 1 nm ( physiological concentration ) and pcps at 200 μm (▾) or in the presence of factor viia at 10 nm and pcps at 3 μm (*), 8 mm (×), 14 μm (▴), 20 μm (▪), 50 μm (), 100 μm ( δ ), and 200 μm (□). parameters of thrombin generation presented in fig7 b , 7c , and 7 d are calculated from fig7 a data . the influence of 10 nm factor viia and 15 , μm pcps on thrombin generation in whole blood clotting in freshly drawn , non - anticoagulated whole blood was performed in 64 tubes ( four series per experiment — 16 tubes / series ). all tubes were loaded with corn trypsin inhibitor ( 100 μg / ml blood ), 10 pm relipidated tf ( pcps : tf = 2 , 000 ) in hbs with 2 mm cacl 2 ( all tubes in each series except phlebotomy control tube ), anti - factor ix antibody ( 50 μg / ml blood ) ( all tubes , three experiment series only ), 10 nm recombinant factor viia ( all tubes , one experiment series only ), 15 μm pcps ( all tubes , two experiment series only ), and equivalent volume of factor viia , pcps , and anti - factor ix antibody dilution buffer ( hbs , ph 7 . 4 , all tubes not loaded with corresponding adjuvants ). no more than 35 μl of reagents were loaded in each tube . the zero tube of each series was pretreated using 1 ml of 50 mm edta and 10 μl of 10 mm fprck ( diluted in 0 . 01 m hcl ). normal donor blood was drawn by venipuncture , delivered into the reagent - loaded tubes , and the tubes were periodically quenched with edta and fprck . the clotting time was observed visually by two observers . tubes were centrifuged and the supernatants were aliquoted for further tat analyses . the results are shown in fig8 wherein (♦) represents normal control ( no anti - factor ix antibody ); (▪) represents induced hemophilia b ( 50 μg / ml anti - factor ix antibody ); ( δ ) represents induced hemophilia b with 15 μm pcps addition ; (×) represents induced hemophilia b with 15 mm pcps and 10 nm factor viia addition ; and , () represents induced hemophilia b with 10 nm factor vi la addition . several models of blood coagulation were used to examine hemophilia a treatment with recombinant factor viia . in one of them , synthetic “ plasma ” mixtures were prepared with purified proteins involved in blood coagulation and its regulation . synthetic phospholipids ( pcps ; 75 % phosphatidylcholine and 25 % phosphatidylserine ) or washed platelets were also present . at selected time points samples of the reaction mixture were taken and analyzed for thrombin generation . in the second model , fresh whole blood was added to tubes containing corn trypsin inhibitor ( cti ; prevents contact pathway - initiated coagulation ) and tf . at selected time points blood in the tubes is quenched and samples are analyzed . in both these models , the procoagulant process may be divided into two phases : an initiation phase and a propagation phase ( fig1 ). the initiation phase is characterized by the appearance of nanomolar amounts of thrombin , femto - and picomolar amounts of other coagulation enzymes and the activation of pro - cofactors factors v and viii . the propagation phase is characterized by explosive , extensive prothrombin activation . in the whole blood model , when normal blood was used , clotting time corresponded to the beginning of the propagation phase [ fig2 arrow “ a ” and filled circles ; thrombin generation is evaluated as thrombin - antithrombin - iii complex ( tat ) formation ]. in hemophilia a blood , the clotting time ( initiation phase ) was slightly prolonged ( arrow “ c ”), however , the bolus of thrombin ( tat ) produced during the propagation phase did not occur ( open circles ). the addition of factor viii restored the clotting time ( arrow “ b ”) and the propagation phase of thrombin generation ( open squares ). when factor viia at a final concentration of 10 nm was added in vitro to cti - inhibited fresh blood of a hemophilia a patient whose factor viii level was & lt ; 1 % and blood coagulation was initiated by 12 . 5 pm tf , clotting occurred 3 : 20 min after initiation . in the control experiment ( without factor viia addition ) the clotting time of the same blood was 6 : 25 min . blood taken from a healthy volunteer under the same conditions clotted at 3 : 15 min after initiation . when 10 nm factor viia was added to the normal blood , the clotting time was shortened to 1 : 53 min ( fig3 a , arrows indicate clotting time ). thus , 10 nm recombinant factor viia was able to correct the visually observed clotting time of hemophilia a blood . however , the thrombin generation ( fig3 a ; diamonds ) analyses showed that thrombin generation was barely elevated by the addition of factor viia to the hemophilia a blood compared to blood without factor viia addition ( fig3 a ; circles ), i . e ., no propagation phase in both cases . tat levels in the blood of a normal person without factor viia addition , reach significantly higher levels ( fig3 a ; squares ) than in hemophilia a blood with factor viia addition . similarly , the addition of factor viia had no effect on the solid clot formation ( fig3 b ; compare middle and bottom panels ). however , the addition of 10 nm factor viia together with pcps to the hemophilia blood corrects thrombin generation at approximately 10 μm pcps ( fig3 c ; compare open squares and filled circle ). pcps alone ( without factor viia addition ) has almost no effect on thrombin generation ( fig3 c ; filled squares ). tf initiated clotting of cti inhibited factor viii deficient plasma showed that an increasing concentration of recombinant factor viia decreased clotting time to that of normal plasma ( fig4 ; dotted lines ). saturation was achieved at approximately 10 nnm factor viia in the presence of 50 μm pcps . thrombin generation in synthetic “ plasma ” initiated with 25 pm relipidated tf and in the presence of washed platelets at physiological concentration is shown in fig5 . squares represent a complete system with all procoagulants present at physiological concentrations . thrombin generation in the absence of factor viii ( circles ) was suppressed . factor viia at 10 nm slightly increased thrombin generation , however it was not able to restore normal thrombin levels or the duration of the initiation phase ( triangles ). a further increase in factor viia concentration to 40 nm did not increase thrombin generation ( diamonds ). in a similar synthetic plasma experiment , when platelets were replaced by 2 μm pcps ( this concentration is equivalent to the contribution of normal platelets when the latter are present at physiological concentration ), neither 10 nm nor 40 nm factor viia was able to restore normal thrombin generation ( fig6 ). in a synthetic plasma mixture initiated with 12 . 5 pm relipidated tf , thrombin generation was not detectable in the absence of factor viii and in the presence of 10 nm factor viia when pcps was present at 3 μm or below ( fig7 a ; stars ). increasing the pcps concentration increased the total thrombin generated ( integrated area under the thrombin generation curve ) ( fig7 b ) and the maximum thrombin generation rate ( fig7 c ) and decreased the duration of the initiation phase ( fig7 d ). in the absence of factor viii and in the presence of 10 nm factor viia , the normal levels of total thrombin generated and the duration of the initiation phase ( similar to those achieved in the presence of physiological concentrations of factors viii and viia ; open circles in fig7 a , filled triangles in fig7 b and 7d ) were achieved at ˜ 50 μm pcps . total thrombin generated in the presence of platelets in a complete system is indicated by the filled circle , and in the absence of factor viii by the open circle ( fig7 b ). human coagulation factors vii , x , ix , and prothrombin , were isolated from fresh frozen plasma using the general methods of bajaj et al . 3 and were purged of trace contaminants and traces of active enzymes as described 4 . human factor v and at - iii were isolated from freshly frozen plasma 5 , 6 . recombinant factor viii and recombinant tissue factor ( tf ) ( residues 1 - 242 ) were provided as gifts from drs . shu len liu and roger lundblad ( hyland division , baxter healthcare corp , duarte , calif .). recombinant human factor viia was purchased from novo pharmaceuticals ( copenhagen , denmark ). recombinant full - length tfpi produced in escherichia coli was provided as a gift by dr . kirk johnson ( chiron corp , emeryville , calif .). the tf / lipid reagent ( 2 nm / 10μm ) and corn trypsin inhibitor ( cti ) were prepared as described 7 . phosphatidylserine ( bovine brain ) ( ps ), phosphatidylcholine ( egg yolk ) ( pc ), and edta were purchased from sigma ( st louis , mo .). phospholipid vesicles ( pcps ) composed of 25 % ps and 75 % pc were prepared as described 15 . spectrozyme th was purchased from american diagnostica , inc ( greenwich , conn .). d - phe - pro - argch 2 cl ( fprck ) was synthesized in house . elisa thrombin - at - iii ( tat ) kit ( enzygnost tat ) was purchased from behring ( marburg , germany ). monoclonal anti - factor ix antibody ( α - fix - 91 ) was obtained from the biochemistry antibody core laboratory ( university of vermont ). fibrinogen , fibrinopeptides a ( fpa ) and b ( fpb ), and solid clot analyses were performed as described 2 . relipidated tf ( tf / pcps : 200 pm / 400 nm ) and varying concentrations of recombinant factor viia ( 0 - 600 pm ) were incubated at 37 ° c . in hbs , 2 mm cacl 2 , 0 . 1 % peg , ph 7 . 4 containing 10 μm pcps for 10 min . factor x at 170 nm concentration was added and 20 μl aliquots were removed every 30 s for 5 min and quenched into 160 μl hbs containing 20 mm edta . 20 μl of 2 mm spectrozyme xa was added and the rate of substrate hydrolysis was estimated . factor xa generation rate was evaluated from a standard curve using serial dilutions of purified factor xa . the concentration of active tf was estimated using a scatchard plot ( factor viia bound vs . bound / free ratio ) assuming 1 : 1 stoichiometry for tf and factor viia . three healthy donors were recruited , advised according to a protocol approved by the university of vermont human studies committee 16 , 7 and their consent to participate obtained . donors with history of thrombosis / hemorrhage , or regular aspirin or drug use were excluded . all individuals exhibited normal values for the pt ( 11 . 6 - 13 . 0 s ), inr ( 1 . 0 - 1 . 1 ), fibrinogen ( 2 . 4 - 3 . 4 mg / ml ) and blood coagulation protein levels , and platelet counts ( 1 . 8 - 2 . 7 × 10 8 / ml ). an in vitro equivalent of natural acquired hemophilia b was manufactured in fresh cti - inhibited normal whole blood by the addition of 50 μg / ml α - fix - 91 . at this concentration , the antibody prolonged the aptt of normal plasma from 38 to 115 s . the aptt of commercial factor ix - deficient plasma (& lt ; 1 % of factor ix ; george king ) was 112 s . the “ titer ” of the α - ix - 91 at 50 μg / ml was 27 bethesda units . purified plasma factor ix addition returned the aptt to 39 s . the procedure used is a modification of lawson et al . 17 and van &# 39 ; t veer et al 4 and involves mixing proteins , lipids , platelets and relipidated tf to produce the equivalent of a thrombin - generating “ synthetic plasma ”. relipidated tf ( 20 pm ) was incubated with 4 μm pcps ( when desired , additional amounts of pcps ( 6 - 400 μm ) were added ) in hbs , 2 mm cacl 2 for 10 min at 37 ° c . factor v ( 40 nm ) and factor viii ( 1 . 4 nm ) were added to the relipidated tf prior to the initiation . prothrombin ( 2 . 8 μm ), factors vii ( 20 nm ), viia ( 0 . 2 nm ), x ( 340 nm ) and ix ( 180 nm ), tfpi ( 5 ram ), and at - iii ( 6 . 8 μm ) were incubated in hbs , 2 mm cacl 2 on ice for 7 min and the preheated at 37 ° c . for 3 min . the reaction was started by mixing equal volumes of both solutions resulting in mean physiological concentrations of the proteins , with a final tf concentration of 10 pm . following initiation of the reaction , at selected time points , 10 μl aliquots were withdrawn from the reaction mixture and quenched in 20 mm edta in hbs ( ph7 . 4 ) containing 0 . 2 mm spectrozyme th and assayed immediately for thrombin activity . the hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a molecular devices v max spectrophotometer . thrombin generation was calculated from a standard curve prepared by serial dilutions of α - thrombin . the protocol used is a modification of rand et al . 16 . clotting in freshly drawn , nonanticoagulated whole blood was performed in tubes placed on a rocking table at 37 ° c . and loaded with cti ( 100 μg / ml blood ). experiments were performed in 4 series of tubes ( 16 tubes / series ; 2 blood draws ). 62 tubes ( 15 in each of 4 series and two supplementary ) were loaded with cti and 4 pm relipidated tf ( pcps : tf 5000 ) in hbs with 2 mm cacl 2 . four phlebotomy control tubes ( 1 tube / series ) contained no tf . 50 tubes ( 3 experimental series and supplementary ) were loaded with 50 μg / ml of α - fix - 91 . 16 of 50 tubes ( first experimental series ) had no additional reactants added , 16 ( second experimental series ) were loaded with 15 μm pcps ( final concentration after the addition of blood ) and other 16 tubes ( third experimental series ) were loaded with 15 μm pcps and 10 nm factor viia . two supplementary tubes were loaded with 10 nm factor viia ( no pcps ). no more than 35 μl of all reagents were loaded in each tube . the zero tube of each series was pretreated using 1 ml of 50 mm edta and 10 μl of 10 mm fprck ( diluted in 10 mm hcl ). normal blood was drawn by venipuncture , 1 ml aliquots were delivered into the reagent - loaded tubes , and the tubes were periodically quenched with edta and fprck . the clotting time was observed visually by two observers . two supplementary tubes were quenched 10 min after the clotting time . tubes were centrifuged and the supernatants were aliquoted for further analyses . elisa for tat was performed . fpa and fpb release was evaluated by hplc , fibrinogen depletion by western blots . solid clots were lyophilized , weighed , solubilized and analyzed by gel electrophoresis 2 . in these experiments all 41 tubes ( 10 tubes / series and phlebotomy control tube ) were loaded with cti and varying concentrations of pcps in duplicates ( 0 , 1 , 3 , 8 , and 25 μm ) ( no pcps in phlebotomy control tube ). in control experiment one series of tubes was loaded with 5 pm tf alone ( normal control ), another one with 5 pm tf and 50 μg / ml of α - fix - 91 ( hemophilia b control ). in a second experiment all tubes were loaded with 50 μg / ml of α - fix - 91 and 10 nm factor viia . in one series tf was present in another absent . fresh blood ( 1 ml ) was delivered into reagent - loaded tubes . tubes were quenched 10 min after the clotting time . elisa for tat was performed . in all experiments of this series , the concentration of tf was 10 pm . results are presented in fig9 a - c . fig9 a depicts thrombin generation profiles over time at varying pcps concentrations . maximum rates of thrombin generation and the initiation phase duration dependence on the pcps concentration ( fig9 b and c , respectively ) are calculated from the data of fig9 a . in the control experiment ( complete system ) with 200 μm pcps , thrombin was generated at a maximum rate of 320 nm / min after an initiation phase of ˜ 4 min (♦). the highest level of thrombin observed was 340 nm . in the absence of factor viii ( approximation of hemophilia a ) with 200 μm pcps , the initiation phase was prolonged to 15 min (; fig9 b ) and the maximum rate of thrombin generation was decreased to 6 . 2 nm / min (; fig9 c ). only 18 nm thrombin was detected after 20 min . the addition of 10 nm factor viia in the absence of factor viii at 200 μm pcps , decreased the initiation phase to 1 min (▪; fig9 b ) and increased the maximum thrombin generation rate to 140 nm / min (▪; fig9 c ). a decrease in pcps to 100 μm had little effect on the maximum rate of thrombin generation during the propagation phase ( 110 nm / min ) and maximum thrombin levels (˜ 250 nm at both pcps concentrations ), while the initiation phase was prolonged to 2 min (; fig9 b ). further reductions in pcps concentration caused prolongation of the initiation phase and decreased rates of prothrombin activation during the propagation phase . at 50 μm pcps ( δ ) the initiation phase duration was similar to that of the normal control , while the maximum rate of thrombin generation was only 60 nm / min with the maximum thrombin level of 160 nm . at 14 μm pcps the initiation phase was extended to 9 min , thrombin generation rate decreased to 13 nm / min (▴; fig9 c ) and the maximum thrombin level decreased to 70 nm . no thrombin generation was observed in the absence of pcps (∘; fig9 c ). cti - inhibited normal blood activated with 5 pm tf / 25 nm pcps clotted in 6 . 9 min ( table 1 ; fig1 a , ♦). the addition of 1 μm pcps decreased the clotting time to 5 . 5 min . further increases in pcps concentration ( to 25 μm ) had no additional effect on the clotting time . “ acquired ” hemophilia b blood in the presence of 5 pm tf / 25 nm pcps clotted in 11 . 8 min ( ). at 3 μm pcps the clotting time of this blood was decreased to 10 . 1 min . an increase in pcps to 8 μm decreased clotting time to 5 . 8 min . a further increase in pcps to 25 μm had no additional effect on clotting time . the addition of 10 nm factor viia at 5 pm tf / 25 ram pcps decreased the visually observed clotting time of “ acquired ” hemophilia b blood from 11 . 8 min ( without exogenous factor viia ; fig1 a ) to 4 . 3 min (* fig1 a ). increases in pcps concentration , decreased the clotting time gradually to 2 . 5 min at 25 μm phospholipid . when tf was omitted from the reaction , the “ acquired ” hemophilia b blood clotted in 35 min in the presence of 10 nm factor viia ( δ ; fig1 a ). in the absence of tf , the additions of pcps ( 1 - 25 μm ) had no procoagulant effect on the clotting time of the “ acquired ” hemophilia b blood ( clotting time varied from 33 to 41 min ). the maximum thrombin concentration ( evaluated as the plateau level of the tat generation ) achieved in normal blood activated with 5 pm tf / 25 nm pcps was 500 nm ( table 1 ). an increase in pcps to 3 μm increased the maximum thrombin observed to ˜ 700 nm . further increases in the pcps concentration ( up to 25 μm ) had no additional effect on the ultimate thrombin concentration . the maximum thrombin level observed in “ acquired ” hemophilia b blood at the same initiator concentration ( no additional pcps ) was ˜ 240 nm . the addition of pcps ( 25 μm ) increased the thrombin level to 570 nm . when 10 nm factor viia was added to “ acquired ” hemophilia blood at 5 pm tf and 3 μm pcps , an equivalent thrombin level ( 570 nm ) was achieved . no further increase in thrombin was observed when pcps was increased to 8 and 25 μm . in the absence of tf , the addition of 10 nm factor viia to “ acquired ” hemophilia b blood produced a maximum thrombin level of 56 nm after 45 min . in the absence of tf , the addition of pcps ( 1 - 25 μm ) with 10 nm factor viia to “ acquired ” hemophilia b blood did not increase the final thrombin concentration . thrombin generation rates presented in fig1 b were calculated dividing maximum thrombin levels generated ( based on tat formation ) by the clotting time . in normal blood activated with 5 pm tf ( no exogenous factor viia added ), thrombin generation rates varied from 60 nm / min without pcps addition to 135 nm / min at 25 μm pcps ( table 1 ; fig1 b ; ♦). in “ acquired ” hemophilia b blood activated with the same tf concentration in the absence of additional pcps , the thrombin generation rate was approximately ⅓ that of normal blood ( 21 nm / min ) (; fig1 b ). with increasing pcps this rate increased , reaching 97 nm / min at 25 μm phospholipid . the addition of 10 nm factor viia to “ acquired ” hemophilia b blood induced to clot with 5 pm tf increased the thrombin generation rate at all pcps concentrations tested (*; fig1 b ). at 3 μm pcps , the thrombin generation rate in “ acquired hemophilia b blood with 10 nm factor viia was similar to that observed in normal blood at the same pcps concentration in the absence of exogenous factor viia ( 134 and 132 nm / min , respectively ). in the absence of tf and pcps , the addition of 10 nm factor viia to “ acquired ” hemophilia b blood produced low levels of thrombin ( 56 nm ) at a low ( 1 . 6 nm / min ) rate (▴; fig1 b ). additions of pcps ( 1 - 25 μm ) did not increase thrombin generation rate . these data indicate that for normal blood in the presence of tf , saturation with pcps was achieved at 3 μm phospholipid . in “ acquired ” hemophilia b blood in both the absence and presence of 10 nm exogenous factor viia , the maximum thrombin generation rates were observed at 25 μm pcps . in the absence of tf and in the presence of 10 nm factor viia , thrombin generation in “ acquired ” hemophilia b blood was negligible and almost independent of pcps . in all experiments of this series blood was activated with 4 pm tf / 20 nm pcps . normal cti - inhibited blood clotted 5 . 5 min after the addition of tf ( table 2 ; fig1 ; ♦). thrombin generation occurred at a maximum rate of 68 nm / min and the reaction was complete ˜ 15 min after tf addition . the clotting time of “ acquired ” hemophilia b blood at the same tf concentration was extended to 7 . 8 min ( ) with a maximum thrombin generation rate of 5 . 7 nm / min and the maximum level of 36 nm ; values less than 10 % those observed in normal blood . the addition of 15 μm pcps to “ acquired ” hemophilia b blood (; fig1 a ) decreased the visually observed clotting time to 3 . 9 min , i . e . a clot was observed almost 2 min sooner than in normal blood , however , the inception of the propagation phase of thrombin generation occurred at the same time as in normal blood ( 6 min ). the maximum rate of thrombin generation was enhanced to 48 nm / min and the maximum level increased to 70 nm . the addition of 10 nm factor viia to “ acquired ” hemophilia b blood activated with 4 pm tf in the presence of 15 μm pcps (*) had almost no effect on the visually observed clotting time ( 3 . 7 min ), i . e . it was similar to that observed in the absence of added factor viia . the initiation phase of thrombin generation , however , was shortened to 3 min , and maximum levels increased to 200 nm . the addition of 10 nm factor viia alone , without pcps to “ acquired ” hemophilia b blood activated with 4 pm tf produced 60 nm thrombin over 15 min of the reaction (▴). platelet activation ( based upon platelet osteonectin release ) in normal blood induced to clot with 4 pm tf ( fig1 b ; ♦) was detected between 1 and 2 min after the initiation of the reaction and release occurred at a rate of 1 . 3 nm / min , approximately 10 % the maximum rate ( 12 . 9 nm / min ). in “ acquired ” hemophilia b blood ( ), detectable platelet activation occurred after similar interval with initial rates as in normal blood . however , the maximum rate of this process ( 6 . 9 nm / min ) was decreased by half and delayed , occurring between 7 and 9 min of the reaction . the addition of 15 μm pcps to “ acquired ” hemophilia b blood had little effect on platelet activation (). when both 10 nm factor viia and 15 μm pcps were added to this blood , early and rapid platelet activation occurred at a rate of 9 . 6 nm / min (*). in normal blood activated with 4 pm tf , approximately 70 % of total fpa was released at the clotting time at a maximum rate of 4 . 2 μm / min ( table 2 ; fig1 c ; ♦). in “ acquired ” hemophilia b blood , detectable fpa release was delayed by more than 1 min and the maximum rate decreased to 1 . 2 μm / min ( ). approximately 60 % of this peptide were released at the clotting time . the addition of 15 μm pcps to “ acquired ” hemophilia b blood , substantially shifted the inception of the fpa release to the left although maximum rate of release ( 1 . 3 μm / min ) was almost not affected (). less than 50 % of fpa were released at clotting time . when 10 nm factor viia together with 15 μm pcps were added to “ acquired ” hemophilia b blood , the maximum rate of the fpa release was increased to 2 . 9 μm / min (*). at the visually observed clotting time , 85 % of total available fpa was in solution . soluble fibrinogen depletion and insoluble clot formation in normal and induced hemophilia b blood are presented in fig1 and table 2 . in normal blood induced to clot with 4 pm tf ( fig1 , panel a ), soluble fibrinogen was almost completely removed from the solution at the visually observed clotting time . in “ acquired ” hemophilia b blood , fibrinogen depletion and solid clot formation were delayed and approximately 40 % of total fibrinogen was in solution at the clotting time ( fig1 , panel b ). only 35 % of total fibrinogen was depleted from the solution at the clotting time when 15 μm pcps were added to “ acquired ” hemophilia b blood ( fig1 , panel c ). when both 10 nm factor viia and 15 μm pcps were added to “ acquired ” hemophilia b blood ( fig1 , panel d ), the onset of the fibrinogen depletion started substantially earlier than in normal blood ( compare fig1 , panels a and d ). only 10 % of fibrinogen was in solution at the visually observed clotting time . the final clot weights in normal blood were 1 . 0 mg / ml of blood ( table 2 ; fig1 ). in “ acquired ” hemophilia b blood , final clot weights were decreased to 0 . 7 mg and not affected by the addition of 10 nm factor viia . when 15 μm pcps was added to “ acquired ” hemophilia b blood , final clot weights increased to an average of 0 . 9 mg , whereas addition of both 10 nm factor viia and 15 μm pcps increased clot weights to 1 . 1 mg . the dynamics of clot growth over time revealed a pronounced dependence upon the conditions of the reaction . in normal blood , initial clot weights were ˜ 60 % of those final ( on average 0 . 6 vs . 1 . 0 mg ). in “ acquired ” hemophilia b blood , initial weights were ˜ 50 % of final ( 0 . 33 vs . 0 . 7 mg ). in “ acquired ” hemophilia b blood with 15 μm pcps , although clots were formed relatively early ( 4 min after the initiation ), they were initially small ( 0 . 1 - 0 . 3 mg ) and stayed such for ˜ 5 min . in the presence of 10 nm exogenous factor viia and 15 μm pcps in “ acquired ” hemophilia b blood , clots of a normal weight ( 0 . 9 - 1 . 2 mg ) were formed early in the reaction ( 3 min after tf addition ). the data indicate that anionic phospholipids remarkably improve the hemostatic potential of factor viia in the tissue factor - induced coagulation of “ acquired ” hemophilia b blood in vitro . in the presence of factor viia at a pharmacological concentration ( 10 nm ) and phospholipids at 8 μm , all parameters of thrombin generation and clot formation in “ acquired ” hemophilia b blood are similar to those observed in normal blood . factor viia and pcps alone are able to correct the visually observed clotting time . in the absence of tf , only negligible amounts of thrombin are generated at a low rate when 10 nm factor viia is added to the hemophilia b blood , and they are not increased by the addition of phospholipids . these data are consistent with our previous conclusion that tf is essential to achieve normal hemostasis in hemophilia blood in the presence of factor viia at pharmacological concentrations ( see butenas s , brummel k e , branda r f , paradis s g , mann k g . “ mechanism of factor viia - dependent coagulation in hemophilia blood .” blood . 2002 ; 99 : 923 - 930 ). our results also show thrombin generation profiles are observed for the reactions accomplished either in the presence of platelets present at physiological concentrations or in the presence of 1 - 2 μm phospholipids ( see butenas s , branda r f , van &# 39 ; t veer c , cawthern k m , mann k g . “ platelets and phospholipids in tissue factor - initiatedtthrombin generation .” thromb haemost . 2001 ; 86 : 660 - 667 ). these concentrations of phospholipids , however , are much lower than those required to saturate the complex enzymes of blood coagulation . an addition of phospholipids to blood provides an extra surface for the reactions of blood coagulation to occur and , as a consequence , increases the potency of enzymatic complexes of blood coagulation . the proteolytic activity of the factor viia / tf complex is substantially affected due to the high saturating concentrations of phospholipids (& gt ; 100 μm ) required for the maximum efficiency of this enzymatic complex ( see butenas s , branda r f , van &# 39 ; t veer c , cawthern k m , mann k g . “ platelets and phospholipids in tissue factor - initiatedtthrombin generation .” thromb haemost . 2001 ; 86 : 660 - 667 ). at phospholipid addition , factor viia / tf is able to generate factor xa at a high rate . an addition of 10 nm factor viia to blood saturates tf present and , as a consequence , increases concentration of the factor viia / tf complex . however , neither phospholipids alone nor factor viia alone are able to increase factor xa concentration to the levels required for an efficient propagation phase of thrombin generation to occur . only a combined effect of both increased concentration and increased efficiency of the factor viia / tf complex produces factor xa levels substantial enough to drive thrombin generation over the threshold . in summary , phospholipids substantially improve the hemostatic potential of factor viia in the tissue factor induced coagulation of “ acquired ” hemophilia b blood . the in vivo efficacy of factor viia replacement therapy probably requires a simultaneous presence of tf and excess membrane phospholipids . this can lead to decreased both amounts of factor viia and duration of treatment required to achieve normal hemostasis in hemophilia blood . 1 . higgins d l , mann k g . the interaction of bovine factor v and factor v - derived peptides with phospholipid vesicles . j . biol chem . 1983 ; 258 : 6503 - 6508 . 2 . brummel k e , butenas s , mann k g . an integrated study of fibrinogen during blood coagulation . j . biol chem . 1999 ; 274 : 22862 - 22870 . 3 . bajaj s p , rapaport s i , prodanos c . a simplified procedure for purification of human prothrombin , factor ix and factor x . prep biochem . 1981 ; 11 : 397 - 412 . 4 . van &# 39 ; t veer c , mann k g . regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor ,. antithrombin - iii , and heparin cofactor - ii . j . biol chem . 1997 ; 272 : 4367 - 4377 . 5 . katzmann j a , nesheim m e , hibbard l s , mann k g . isolation of functional human coagulation factor v by using a hybridoma antibody . proc nat &# 39 ; l acad sci usa . 1981 ; 78 : 162 - 166 . 6 . griffith m j , noyes c m , church f c . reactive site peptide structural similarity between heparin cofactor ii and antithrombin iii . j . biol chem . 1985 ; 260 : 2218 - 2225 . 7 . cawthern k m , van &# 39 ; 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t veer c , gaffney d p , mann k g . blood clotting in minimally altered whole blood . blood . 1996 ; 88 : 3432 - 3445 . 17 . lawson j h , kalafatis m , stram s , mann k g . a model for the tissue factor pathway to thrombin . i . an empirical study . j . biol chem . 1994 ; 269 : 23357 - 23366 . 18 . santagostino e , morfini m , rocino a , baudo f , scaraggi f a , gringeri a . relationship between factor vii activity and clinical efficacy of recombinant factor viia given by continuous infusion to patients with factor viii inhibitors . thromb haemost . 2001 ; 86 : 954 - 958 .