PROTEIN C DERIVATIVES

Abstract: Novel protein C derivatives are described. These polypeptides retain the biological activity of the wild-type human protein C with substantially longer half-lives in human blood. These polypeptides will require either less frequent administration and/or smaller dosage than wild-type human protein C in the treatment of vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states predisposing to thrombosis.

Description: Protein C Derivatives This application claims priority of Provisional Application Serial No. 60/131,801 filed April 30, 1999. This invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides. More specifically, the invention relates to protein C derivatives resistant to serpin inactivation, to their production, and to pharmaceutical compositions comprising these protein C derivatives . Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V a and Villa in the coagulation cascade. Human protein C is made in vivo as a single polypeptide of 461 amino acids. This polypeptide undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive precursor or zymogen (a 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain) ; 3) vitamin K-dependenr carboxylation of nine glutamic acid residues of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain) . Finally, the 2-chain zymogen may be activated by removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing greater enzymatic activity than the 2-chain zymogen. In conjunction with other polypeptides, aPC functions as an anti-coagulant important in protecting against thrombosis, has anti-inflammatory effects through its inhibition of cytokine generation (e.g., TNF and IL-1), and exerts profibrinolytic properties that facilitate clot lysis. Thus, aPC provides a mechanism for anti-coagulation, anti-inflammation, and fibrinolysis . The critical role of aPC in controlling hemostasis is exemplified by the increased rate of thrombosis in heterozygous deficiency, protein C resistance (e.g., due to the common Factor V Leiden mutation) and the fatal outcome of untreated homozygous protein C deficiency. Plasma- derived and recombinantly produced aPC have been shown to be effective and safe antithrombotic agents in a variety of animal models of both venous and arterial thrombosis. Protein C levels have also been shown to be abnormally low in the following diseases and conditions : disseminated intravascular coagulation (DIC) [Fourrier, et al . , Chest 101:816-823, 1992], sepsis [Gerson, et al . , Pediatrics 91:418-422, 1993], major trauma/major surgery [Thomas, et al., Am J Surg. 158:491-494, 1989], burns [Lo, et al . , Burns 20:186-187 (1994)], adult respiratory distress syndrome (A DS) [Hasegawa, et al . , Chest 105 (1) : 268-277 , 1994], and transplantations [Gordon, et al . , Bone Marrow Trans . 11:61- 65 (1993)] . In addition, there are numerous diseases with thrombotic abnormalities or complications that aPC may be useful in treating, such as: heparin-induced thrombocytopenia (HIT) [Phillips, et al . , Annals of Pharmacotherapy 28: 43-45, 1994], sickle cell disease or t alassemia [Karayalcin, et al . , The American Journal of Pediatric Hematology/Oncology 11 (3) : 320-323 , 1989], viral hemorrhagic fever [Lacy, et al . , Advances in Pediatric Infectious Diseases 12:21-53, 1997], thrombotic thrombocytopenic purpura (TTP)and hemolytic uremic syndrome (HUS) [Moake, Seminars in Hematology 34(2):83-89, 1997]. In addition, aPC in combination with Bactericidal Permeability Increasing Protein (BPI) may be useful in the treatment of sepsis [Fisher, et al . , Crit . Care Med . 22 (4) : 553-558 , 1994] . Finally, platelet inhibition is efficacious in both prevention and treatment of thrombotic disease. However, the use of antiplatelet agents, such as aspirin, increase the risk of bleeding, which limits the dose of the agent and duration of treatment. The combination of aPC and antiplatelet agents results in a synergy that allows the reduction of the dosages of both aPC and the antiplatelet agent (s) . The reduction of the dosages of the agents in combination therapy in turn results in reduced side effects such as increased bleeding often observed in combination anti- coagulant/anti-platelet therapy. Various methods of obtaining protein C from plasma and producing protein C, aPC and protein C/aPC polypeptides through recombinant DNA technology are known in the art and have been described. See e.g., U.S. Patent Nos . 4,775,624 and 5,358,932. Despite improvements in methods to produce aPC through recombinant DNA technology, aPC and polypeptides thereof are difficult and costly to produce and have a relatively short half-life in vivo . A reason for the short half-life is that blood levels of aPC are regulated by molecules known as serpins (Serine Protease Inhibitors) , which covalently bind to aPC forming an inactive serpin/aPC complex. The serpin/aPC complexes are formed when aPC binds and proteolytically cleaves a reactive site loop within the serpin; upon cleavage, the serpin undergoes a conformational change irreversibly inactivating aPC. The serpin/aPC complex is then eliminated from the bloodstream via hepatic receptors for the serpin/aPC complex. As a result, aPC has a relatively short half-life compared to the zymogen; approximately 20 minutes for aPC versus approximately 10 hours for human protein C zymogen (Okajima, et al . , Thromb Haemost 63(l):48-53, 1990). It has been shown that changes to serine protease amino acid sequences at residues which interact directly with the substrate (generally within or near the active site) can alter the specificity of the serine protease, potentially providing increased specific activity towards appropriate coagulation factors, as well as increased resistance to serpins (Rezaie, J Biol Che 271 (39) : 23807-23814 , 1996; Rezaie and Esmon, Eur. J. Biochem 242:477-484, 1996). Therefore, an aPC polypeptide exhibiting increased resistance to serpin inactivation, while maintaining the desirable biological activities of aPC (e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities), provides a compound that has an increased plasma half-life and, therefore, is effectively more potent than the parent compound, requiring substantially reduced dosage levels or less frequent administrations for therapeutic applications. The potency advantages are especially important in disease states in which serpin levels are elevated. Physiologically, the two serpins that serve as the primary inactivators of aPC are protein C inhibitor (PCI) and α^-antitrypsin (α^-AT) [Heeb, et al . , J Biol Chem 263 (24) :11613-6, 1988). Both PCI and α^-AT have been demonstrated to be the primary physiological inactivators of aPC in disease states such as disseminated intravascular coagulation (Scully, et al . , Thromb Haemost 69 (5) : 448-53 , 1993) , and elevated levels of α^-AT have been observed in a number of disease states involving an inflammatory response (Somayajulu, et al . , J Pathol Microbiol 39(4):271-5, 1996; Morgan, et al . , Int J Biochem Cell Biol 29 (12) : 1501-11, 1997) . The elevated serpin levels inactivate aPC resulting in an increased susceptibility of coagulapathies associated with decreased protein C levels. Attempts have been made to increase the plasma half-life of aPC by increasing the resistance to serpins by modifying the human protein C molecule (e.g., U.S. Patent No. 5,358,932). An increase in immunogenicity is often observed when a natural protein is significantly modified and then administered to a patient. Through scientific experiment and analysis, we identified serpin and protein C binding sites essential to formation of serpin/aPC complexes. We modified targeted amino acid residues in the aPC molecule and surprisingly found that we were able to inhibit formation of the serpin/aPC complex (the complex which irreversibly inactivates aPC) while at the same time retaining the specificity of the aPC polypeptide for aPC's natural substrates (e.g. factor Va and Villa) . In particular, three sites of recognition within the aPC active site show distinctive differences between substrate recognition sequences and inhibitor recognition sequences: S2 , S3', and S4' . We found inhibition of serpin/human aPC polypeptide binding by substituting one or more of the following amino acids: 194 (Leu), 195 (Ala), 228 (Leu), 249 (Tyr) , 254 (Thr) , 302 (Tyr) , and 316 (Phe) of SEQ ID NO: 7 with an amino acid(s) selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gly, and Gin, provided that 195 is not substituted with Ala and 254 is not substituted with Thr. Accordingly, the present invention describes novel protein C derivatives. These protein C derivatives retain the important biological activity of the wild-type protein C (SEQ ID NO: 7) and have substantially longer half-lives in human blood. Therefore, these compounds provide various advantages, eg. less frequent administration and/or smaller dosages and thus a reduction in the overall cost of production of the therapy. Additionally, these compounds exhibit an advantage in disease states with significantly elevated α^-AT levels such as sepsis. Importantly, the increases in protein C derivative plasma half-lives may be achieved via single amino acid substitutions, which are less likely to be immunogenic in comparison to molecules which contain multiple amino acid substitutions (U.S. Patent No. 5,358,932; Holly, et al . , Biochemistry 33:1876-1880, 1994). The present invention provides a protein C derivative comprising SEQ ID NO: 1 and the corresponding amino acids in SEQ ID NO: 2, wherein one or more of amino acids 194, 195, 228, 249, 254, 302, or 316 is substituted with an amino acid selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gly, and Gin, provided that amino acid 195 is not Ala and amino acid 254 is not Thr. The invention further provides the activated form of the above-identified protein C derivatives . The present invention also provides recombinant DNA molecules encoding the protein C derivatives in the preceding paragraph, in particular those comprising SEQ ID NOS: 8, 9, and 10. Another aspect of the present invention provides protein sequences of these same protein C derivatives, particularly those comprising SEQ ID NOS: 3, 4, and 5 and the activated forms of these protein C derivatives. The present invention comprises methods of treating vascular occlusive disorders and hypercoagulable states including: sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin- induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome. The invention further provides treating these same diseases and conditions employing the activated form of the above- identified protein C derivatives. Another embodiment of the present invention is a method of treating sepsis comprising the administration to a patient in need thereof a pharmaceutically effective amount of a protein C derivative of this invention in combination with bacterial permeability increasing protein. The present invention comprises methods of treating acute coronary syndromes such as myocardial infarction and unstable angina. The present invention further comprises methods of treating thrombotic disorders. Such disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery. The present invention also provides a pharmaceutical composition comprising a protein C derivative of this invention. Human protein C derivatives for the above-mentioned indications and pharmaceutical compositions are preferably selected from L194S, L194S:T254S, and L194A:T254S. Methods and aspects of producing the novel isolated human protein polypeptides are also an aspect of this invention. Finally, an aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and α^-AT. This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and αη_-AT. The inhibition of binding to the specific inhibitor recognition sequences of the serpins (S2, S3', and S4 ' ) being an important contribution to this aspect of the invention. Figure 1. Inactivation of human aPC polypeptides during incubation with normal human plasma. Remaining activity is measured as amidolytic activity normalized to activity at the start of the experiment (time=0) ; error bars indicate the standard error of triplicate experiments. Data are shown for the wild-type protein C (WT, circles) , T254S (squares) , L194S (triangles) , and A195G (diamonds) . Figure 2. Inactivation of human aPC polypeptides during incubation with normal human plasma. Remaining activity is measured as amidolytic activity normalized to activity at the start of the experiment (time=0) ; error bars indicate the standard error of triplicate experiments. Data are shown for the wild-type protein C (WT, circles) , L194A/T254S (squares) , and L194S/T254S (triangles) . Figure 3. Inactivation of human aPC polypeptides by normal human plasma containing Heparin (10 U/mL) . Remaining activity is measured as amidolytic activity normalized to activity at the start of the experiment (time=0) ; error bars indicate the standard error of triplicate experiments. Data are shown for the wild-type protein C(WT, circles), T254S (squares) , L19 S (triangles) , and A195G (diamonds) . Figure 4. Inactivation of human aPC polypeptides by purified α^-antitrypsin. Remaining activity is measured as amidolytic activity normalized to activity at the start of the experiment (time=0) ; error bars indicate the standard error of triplicate experiments. Data are shown for the wild-type protein (WT, circles) , T254S (squares) , L194S (triangles) , and A195G (diamonds) . Figure 5. Plasma aPC levels following a bolus IV dose of 0.1 g/kg in normal, conscious rabbits (N=3) . Activated protein C levels were determined using immunocapture assay, and compared to a standard curve generated from dilutions of the purified protein in rabbit plasma; the standard curve ranged from 1 to 250 ng/mL, with the calculated values within 10% of the standard samples. Data are shown for the wild-type protein (WT, circles) , T254S (squares) , L194S (triangles) , and L194S/T254S (diamonds) . The values plotted are the mean and standard error for the three animals. For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. Antiplatelet agent - one or more agents alone or in combination which reduces the ability of platelets to aggregate. Agents understood and appreciated in the art include those cited in, for example, Remington, The Science and Practice of Pharmacy, Nineteenth Edition, Vol II, pages 924-25, Mack Publishing Co., herein incorporated by reference. Such agents include but are not limited to aspirin (ASA) , clopidogrel, ReoPro® (abciximab) , dipyridamole, ticlopidine and Ilb/lIIa antagonists. aPC or activated protein C refers to recombinant aPC . aPC includes and is preferably recombinant human aPC although aPC may also include other species having protein C proteolytic, amidolytic, esterolytic, and biological (anti- coagulant, anti-inflammatory, or pro-fibrinolytic) activities . Protein C derivative (s) refers to the recombinantly produced polypeptides of this invention that differ from wild-type human protein C but when activated retain the essential properties i.e., proteolytic, amidolytic, esterolytic, and biological (anti-coagulant, anti- inflammatory, pro-fibrinolytic activities) . The definition of protein C derivatives as used herein also includes the activated form of the above identified protein C derivatives . Treating - describes the management and care of a patient for the purpose of combating a disease, condition, or disorder whether to eliminate the disease, condition, or disorder, or prophylactically to prevent the onset of the symptoms or complications of the disease, condition, or disorder. Continuous infusion - continuing substantially uninterrupted the introduction of a solution or suspension into a vein for a specified period of time. Bolus injection - the injection of a drug in a defined quantity (called a bolus) over a period of time up to about 120 minutes. Suitable for administration - a lyophilized formulation or solution that is appropriate to be given as a therapeutic agent . Unit dosage form - refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient . Hypercoagulable states - excessive coagulability associated with disseminated intravascular coagulation, pre- thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors such as aPC. Zymogen - protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chains, of protein C . Pharmaceutically effective amount - a therapeutically efficacious amount of a pharmaceutical compound. The particular dose of the compound administered according to this invention will, of course, be determined by the attending physician evaluating the particular circumstances surrounding the case, including the compound administered, the particular condition being treated, the patient characteristics and similar considerations. Acute coronary syndromes - clinical manifestations of coronary atherosclerosis complicated by coronary plaque rupture, superimposed coronary thrombosis, and jeopardized coronary blood flow resulting in coronary ischemia and/or myocardial infarction. The spectrum of acute coronary syndromes includes unstable angina, non-Q-wave (i.e., non- ST-segment elevation) myocardial infarction, and Q-wave (i.e., ST-segment elevation) myocardial infarction. Thrombotic disorders - a disorder relating to, or affected with the formation or presence of a blood clot within a blood vessel. Such disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery. Purpura fulminans - ecchymotic skin lesions, fever, hypotension associated with bacterial sepsis, viral, bacterial or protozoan infections. Disseminated intravascular coagulation is usually present. Serpin - any of a group of structurally related proteins that typically are serine protease inhibitors whose inhibiting activity is conferred by an active site in a highly variable and mobile peptide loop and that include but are not limited to protein C inhibitor (PCI) and α^- antitrypsin (α]_-AT) . Inhibitor recognition sequence S2 : the 2 nd residue N-terminal to the cleavage site of PCI or α^-AT. Inhibitor recognition sequence S3': the 3 rd residue C-terminal to the cleavage site of PCI or α^-AT. Inhibitor recognition sequence S4 ' : the 4 th residue C-terminal to the cleavage site of PCI or α^-AT. Wild-type protein C - the type of protein C that predominates in a natural population of humans in contrast to that of natural or laboratory mutant or polypeptide forms of protein C. Bactericidal permeability increasing protein - includes naturally and recombinantly produced bactericidal permeability increasing (BPI) protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active variant analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides. The complete amino acid sequence of human BPI, as well as the nucleotide sequence of DNA encoding BPI have been elucidated by Gray, et al . , 1989, J. Biol. Chem 264:9505. Recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI are disclosed in U.S. Patent No. 5,198,541, herein incorporated by reference . The amino acid abbreviations are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. 1.822 (d) (1) (1998) . The activated form of aPC or isolated human aPC polypeptides may be produced by activating recombinant human protein C zymogen or recombinant protein C derivative zymogen in vitro or by direct secretion of the activated form of protein C. The means by which the activation occurs is not critical and the process aspects of this invention include any and all means of activation. Protein C derivatives may be produced in eukaryotic cells, transgenic animals, or transgenic plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan. The present invention provides protein C derivatives, including activated forms thereof, which have increased resistance to serpins, and consequently result in extended plasma half-lives. Specific protein C derivatives include L194S, L194S:T254S, and L194A:T254S and activated forms thereof . Protein C derivative L194S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position. One with skill in the art would realize that other amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin. The activated form of protein C derivative L194S demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, for example, αι_-antitrypsin (α^-AT) , Figure 4. Protein C derivative L194S:T254S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr. The activated form of human protein C derivative L194S:T254S demonstrates a prolonged half-life in normal human plasma compared to wild- type protein C, Figure 2. Protein C derivative L194A:T254S preferably contains an alanine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr. The activated form of human protein C derivative L194A:T254S demonstrates a prolonged half-life in normal human plasma compared to wild-type protein C, Figure 2. Further embodiments of the present invention include protein C derivatives: L194T, L194A, A195G, L228Q, T254S, F316N, Y249E, and Y302Q, and activated forms thereof which have increased resistance to serpins. Protein C derivatives L194T or L194A preferably contain a threonine residue or an alanine residue at position 194 rather than a leucine residue normally found at this position. One with skill in the art would realize that other amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, His, Lys, Arg, Asn, Asp, Glu, and Gin. Protein C derivative A195G preferably contains a glycine residue at position 195 rather than an alanine residue normally found at this position. One with skill in the art would realize that other amino acid substitutions at residue 195 in addition to Gly may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin. The activated form of protein C derivative A195G demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, for example, a ] _-antitrypsin (α^-AT) , Figure 4. Protein C derivative L228Q preferably contains a glutamine residue at position 228 rather than a leucine residue normally found at this position. One with skill in the art would realize that other amino acid substitutions at residue 228 in addition to Gin may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gly. Protein C derivative T254S preferably contains a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residue 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr. The activated form of protein C derivative T254S demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, for example, α^-antitrypsin (αχ-AT) , Figure 4. Protein C derivative F316N preferably contains an asparagine residue at position 316 rather than a p enylalanine residue normally found at this position. One with skill in the art would realize that other amino acid substitutions at residue 316 in addition to Asn may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gin, and Gly. Protein C derivative Y249E preferably contains a glutamic acid residue at position 249 rather than a tyrosine residue normally found at this position. An additional polypeptide contains an Asp at position 249 rather than a tyrosine residue. One with skill in the art would realize that other amino acid substitutions at residue 249 in addition to Glu and Asp may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Gin, and Gly. Protein C derivative Y302Q preferably contains a glutamine residue at position 302 rather than a tyrosine residue normally found at this position. An additional polypeptide contains a Glu at position 302 rather than a tyrosine residue. One with skill in the art would realize that other amino acid substitutions at residue 302 in addition to Glu or Gin may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, and Gly. In addition, protein C derivatives may include proteins that represent functionally equivalent gene products. Such an equivalent protein C derivative may contain deletions, additions, or substitutions of amino acid residues within the amino acid sequence encoded by the protein C polypeptide gene sequences described above, but which result in a silent change, thus producing a functionally equivalent protein C derivative gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Thus, the polypeptides of the present invention include polypeptides having an amino acid sequence at least identical to that of SEQ ID NOS: 3, 4, or 5, or fragments thereof with at least 90% identity to the corresponding fragment of SEQ ID NOS: 3, 4, or 5. Preferably, all of these polypeptides retain the biological activity of human aPC. Preferred polypeptides are those that vary from SEQ ID NOS: 3, 4, or 5 by conservative substitutions i.e., those that substitute a residue with another of like characteristics. Typical substitutions are among Ala, Val , Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are polypeptides in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. The invention also provides DNA compounds for use in making the protein C derivatives. These DNA compounds comprise the coding sequence for the light chain of human protein C zymogen or protein C derivative zymogen positioned immediately adjacent to, downstream of, and in translational reading frame with the prepropeptide sequence of human protein C zymogen or protein C derivative zymogen. The DNA sequences preferably encode the Lys-Arg dipeptide which is processed during maturation of the protein C molecule, the activation peptide and the heavy chain of the protein C derivative . Those skilled in the art will recognize that, due to the degeneracy of the genetic code, a variety of DNA compounds can encode the polypeptides described above. U.S. Patent No. 4,775,624, the entire teaching of which is herein incorporated by reference, discloses the wild-type form of the human protein C molecule. The skilled artisan could readily determine which changes in the DNA sequences which could encode the exact polypeptides as disclosed herein. The invention is not limited to the specific DNA sequences disclosed. Consequently, the construction described below and in the accompanying Examples for the preferred DNA compounds are merely illustrative and do not limit the scope of the invention. All of the DNA compounds of the present invention were prepared by the use of site-directed mutagenesis to change particular positions within human protein C zymogen. The methods used for the identification of residues which form critical contacts in these particular positions are described in Example 1. The protein C derivatives can be made by techniques well known in the art utilizing eukaryotic cell lines, transgenic animals, or transgenic plants. Skilled artisans will readily understand that appropriate host eukaryotic cell lines include but are not limited to HepG2 , LLC-MK 2 , CHO-K1, 293, or AV12 cells, examples of which are described in U.S. Patent No. 5,681,932, herein incorporated by reference. Furthermore, examples of transgenic production of recombinant proteins are described in U.S. Patent Nos . 5,589,604 and 5,650,503, herein incorporated by reference. Skilled artisans recognize that a variety of vectors are useful in the expression of a DNA sequence of interest in a eukaryotic host cell . Vectors that are suitable for expression in mammalian cells include, but are not limited to: pGT-h, pGT-d; pCDNA 3.0, pCDNA 3.1, pCDNA 3.1+Zeo, and pCDNA 3.1+Hygro (Invitrogen) ; and, pIRES/Hygro, and pIRES/neo (Clonetech) . The preferred vector of the present invention is pIG3 as described in Example 2. To be fully active and operable under the present methods, the protein C derivatives made by any of these methods must undergo post-translational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta- hydroxylation) , the addition of four Asn-linked oligosaccharides (glycosylation) and, the removal of the leader sequence (42 amino acid residues) . Without such post-translational modifications, the protein C polypeptides are not fully functional or are non-functional . Methods for the activation of zymogen forms of human protein C and protein C derivatives to activated human protein C and activated protein C derivatives are old and well known in the art. Human protein C may be activated by thrombin alone, by a thrombin/thrombomodulin complex, by RW-X, a protease from Russell's Viper venom, by pancreatic trypsin or by other proteolytic enzymes. The recombinant protein C derivatives of the present invention are useful for the treatment of vascular occlusive disorders or hypercoagulable states associated with sepsis, disseminated intravascular coagulation, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin- induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome. In another embodiment, the recombinant protein C derivatives of the present invention are useful for the treatment of sepsis in combination with bacterial permeability increasing protein. In yet another aspect of this invention the activated protein C derivatives of the present invention are combined with an antiplatelet agent (s) to treat or prevent various disorders, such as, thrombotic disease . The present invention further provides for the treatment of acute coronary syndromes comprising myocardial infarction, and unstable angina with human protein C derivatives with resistance to serpin inactivation as compared to wild-type aPC. The recombinant human protein C derivatives of the present invention are also useful for the treatment of thrombotic disorders such as stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery. The protein C derivatives can be formulated according to known methods to prepare a pharmaceutical composition comprising as the active agent an aPC polypeptide and a pharmaceutically acceptable solid or carrier. For example, a desired formulation would be one that is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and an activated protein C derivative. A preferred stable lyophilized formulation comprises: 2.5 mg/ml activated protein C polypeptide, 15 mg/ml sucrose, 20 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0. An additional stable lyophilized formulation comprises: 5.0 mg/ml activated protein C polypeptide, 30 mg/ml sucrose, 38 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0. Preferably, the human aPC polypeptides will be administered parenterally to ensure delivery into the bloodstream in an effective form by injecting the appropriate dose as a continuous infusion for 1 to 240 hours. More preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 192 hours. Even more preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 144 hours. Yet even more preferably, the aPC polypeptides will be administered as a continuous infusion for 1 to 96 hours. The amount of human aPC polypeptide administered will be from about 0.01 μg/kg/hr to about 50 μg/kg/hr. More preferably, the amount of human aPC polypeptide administered will be about 0.1 μg/kg/hr to about 25 μg/kg/hr. Even more preferably the amount of human aPC polypeptide administered will be about 1 μg/kg/hr to about 15 μg/kg/hr. The most preferable amounts of human aPC polypeptide administered will be about 5 μg/kg/hr or about 10 μg/kg/hr. Alternatively, the human aPC polypeptide will be administered by injecting a portion (1/3 to 1/2) of the appropriate dose per hour as a bolus injection over a time from about 5 minutes to about 120 minutes, followed by continuous infusion of the appropriate dose for up to 240 hours . In another alternative the human aPC derivatives will be administered by injecting a dose of 0.01 mg/kg/day to about 1.0 mg/kg/day, B.I.D. (2 times a day), for one to ten days. More preferably, the human aPC derivatives will be administered B.I.D. for three days. In yet another alternative, the human aPC polypeptides will be administered subcutaneously to ensure a slower release into the bloodstream. Formulation for subcutaneous preparations will be done using known methods to prepare such pharmaceutical compositions. An additional aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and α_-AT, as described in Example 1. This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and α^-AT. The human aPC polypeptides described in this invention have essentially the same type of biological activity as the wild-type human aPC, with substantially longer half-lives in human blood. Therefore, these compounds will require either less frequent administration and/or smaller dosage. Additionally, these compounds will exhibit an advantage in disease states with significantly elevated α^-AT levels such as sepsis. Finally, superior increases in human aPC polypeptide plasma half-lives may be achieved via one or two amino acid substitutions, which are less likely to be immunogenic compared to greater numbers of substitutions. The following Examples are provided merely to further illustrate the present invention. The scope of the invention shall not be construed as merely consisting of the following Examples. Example 1 Site-Directed Mutagenesis The use of site-directed mutagenesis to change particular positions within human protein C molecule that decrease inactivation by serpins, and consequently result in extended plasma half-lives is described. The recognition sequences in the two primary aPC inhibitors α x -AT and PCI reveal some differences that can be exploited by altering the residues in aPC that interact with these sequences. Table I depicts the sequences recognized by aPC. The cleavage site occurs between the two residues shown in italics. Residues occupying the specific subsites, S2, S3', and S4 ' , are underlined. In general, the recognized sites in factor Va are different from the sites in either factor Villa or the inhibitors, therefore, it is possible to engineer the active site of aPC to preferentially cleave the more critical coagulant factor Va, while at the same time decrease aPC's likelihood of being inhibited by serpins. Table I Coagulation Factors S2 S3 / S4' Factor Va 300- -313 N C P K K T R N L K K I T R Factor Va 500- -513 S R S L D R R G I Q R A A A Factor Va 673- -685 S T V M A T R K M H D R L E Factor Villa 330- -341 P E E P Q L R M K N N E E A Factor Villa 560- -571 K E S V D Q R G N Q I M S D Serpins PCI G T I F T F R S A R L N S Q cq-AT F L E A I P M S I P P E V K In particular, three sites of recognition within the active site show distinctive differences between substrate recognition sequences and inhibitor recognition sequences: S2 (the 2 nd residue N-terminal to the cleavage site) , S3' site, and S4' . The S2 site is primarily occupied by polar residues in the factor Va sequences; unlike PCI and α^-AT, which have hydrophobic residues at this position. The S3' site occupied by polar side chains in all of the substrate sequences, but notably, a hydrophobic side chain in the α^- AT sequence. The S4' site is occupied by charged residues in all three factor Va sequences, but is occupied by hydrophobic residues in the factor Villa and inhibitor sequences . Based upon the crystal structures of the PPACK- inhibited aPC (Mather, et al . , EMBO J. 15 (24) : 6822-6831, 1996) and Hirulog 3-inhibited thrombin (Qiu, et al . , Biochemistry 31 (47) : 11689-97 , 1992), two aPC-substrate model structures were created and energy minimized using a CHARMm protocol : (1) The sequence representing the factor Va R506 cleavage sequence . (2) The recognition sequence of o^-AT, with the Met substituted with Arg (corresponding to a polypeptide of α^_- AT which exhibits extremely high affinity for aPC) . These models allowed for the identification of residues which form critical contacts in these three specific sites. A summary of residues which may form specific contacts within the active site, and replacements that are expected to provide enhanced specificity and/or activity are summarized in Table II. In general, mutations of residues that form contacts within the specific subsites of the active site are designed to reflect changes in the environment to drive the specificity of human aPC polypeptides away from the recognition of the two primary physiological inhibitors, and potentially enhance human aPC polypeptide' s proteolytic activity. Table II. Mutations constructed for alteration of specificity Site aPC Constructed Substrate Contact Residue replacements S2 Thr254 Ser Aliphatic part of sidechain S3' Tyr302 I Glu, Gin End of sidechain S4' Leul94 Ser, Thr, Ala , Aliphatic part of sidechain S4' Alal95 ■ Gly Aliphatic part of sidechain S4' Leu228 Gin End of sidechain S4' Phe316 Asn Aliphatic part of sidechain Example 2 Protein C Polypeptide Construction and Production. Protein C derivatives were constructed using the polymerase chain reaction (PCR) following standard methods. The source of the wild-type coding sequence was plasmid pLPC (Bio/Technology 5:1189-1192, 1987). The universal PCR primers used include: PCOOlb; 5'- GCGATGTCTAGAccaccATGTGGCAGCTCACAAGCCTCCTGC -3', which encodes for an Xbal restriction site (underlined) used for subcloning, a Kozak consensus sequence (lowercase) (Kozak, J Cell Biol 108 (2) .229-41, 1989), and the 5' end of the coding region for protein C: PC002E; 5'- CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3 ' , which encodes for the 3' end of the coding region for human protein C, and includes a Bell restriction site (underlined) for subcloning. Mutagenic PCR primers (sense and anti-sense directions, respectively) include: PC194SF, 5'- CTCAAAGAAGAAGTCCGCCTGCGGGGCAGTGC-3 ' and PCI94SR, 5 ' - GCACTGCCCCGCAGGCGGACTTCTTCTTTGAG-3' which encode for a Leu (CTG) to Ser (TCC) mutation at position 194 (boldfaced type) ; PCA195GF , 5 ' -GAAGAAGCTGGGGTGCGGGGCAGTGC-3 ' , and PCA195GR, 5' -GCACTGCCCCGCACCCCAGCTTCTTC-3 ' , which encode for a Ala(GCC) to Gly(GGG) mutation; PCT254SF, 5'- GCAAGAGCACCAGCGACAATGAC-ATCGC-3' and PCT254SR, 5'- GCGATGTCATTGTCGCTGGTGCTCTTGC-3' , which encode for a Thr (ACC) to Ser (AGC) mutation at position 254 (boldfaced type) . The first round of PCR was used to amplify two fragments of the protein C gene; the 5' fragment was generated using PCOOlb and the antisense mutagenic primer, and the 3' fragment was generated using PC002e and the sense mutagenic primer. The resulting amplified products were purified by standard procedures. These fragments were combined and then used as a template for a second round of PCR using primers PCOOlb and PC002e. The final PCR product was digested with Xbal and Bell and subcloned into similarly digested expression vector pIG3. A wild-type construct was similarly generated by PCR using the two universal primers and the plasmid pLPC as the template, followed by subcloning into pIG3. The mutations were confirmed by DNA sequencing of both the coding and non-coding strands. The completed expression plasmids were designated pIG3-HPC (wild-type protein C) , pGH41 (T254S) , pGH51 (A195G) , and pGH94 (L194S) . The pIG3 vector was generated by the insertion of an "internal ribosome entry site" (IRES) (Jackson, et al . , Trends Biochem Sci 15 (12) : 447-83 , 1990) and green fluorescent protein (GFP) (Cormack, et al . , Gene 173:33-38, 1996) gene into the mammalian expression vector pGTD (Gerlitz,et al . , Biochem J 295(Pt l):131-40, 1993). When a cDNA of interest is cloned into the multiple cloning site of pIG3 , the GBMT promoter (Berg, Nucleic Acids Res 20(20) : 5485-6, 1992) drives expression of a bicistronic mRNA (5'- cDNA - IRES - GFP - 3'). Efficient translation of the first cistron is initiated by classical assembly of ribosome subunits on the 5' -methylated cap structure of the mRNA; while the normally inefficient translation of a second cistron is overcome by the IRES sequence which allows for internal ribosome assembly on the mRNA. The coupling of the cDNA and reporter on a single mRNA, translated as separate proteins, allows one to screen for the highest-producing clones on the basis of fluorescence intensity. The expression vector also contains an ampicillin resistance cassette for maintenance of the plasmid in E. coli , and a murine DHFR gene with appropriate expression sequences for selection and amplification purposes in mammalian tissue expression. The adenovirus-transformed Syrian hamster AV12-664 cell line was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 μg/mL gentamicin, 200 μg /mL Geneticin (G418) , and 10 μg /mL vitamin Kl . One day prior to transfection, cells were plated at a density of about 10 5 cells/ 25 cm 2 . Fspl- linearized plasmids were transfected using either the calcium phosphate method (ProFection, Gibco BRL-Life Technologies) or FuGene-6 (Boehringer Mannheim) , following the manufacturer's instructions. Approximately 48 hours after transfection, the medium was replaced with medium containing 250 nM methotrexate for selection. Colonies resistant to methotrexate were pooled 2-3 weeks after applying drug selection and expanded. The pools were subjected to fluorescence activated cell sorting based upon GFP fluorescence intensity (Cormack, 1996) , with the most intense 5% of fluorescent cells being retained and expanded. To obtain material for purification, recombinant cells were grown in a modified mixture of Dulbecco's modified Eagle's and Ham's F-12 media (1:3) containing 1 μg/mL human insulin, 1 μg/mL human transferrin, and 10 μg/mL vitamin Kl . Conditioned media were collected, adjusted to a final concentration of 5 mM benzamidine and 5 mM EDTA, pH 8.0, and protein C was purified via anion-exchange chromatography as described (Yan, et al . , Bio/Technology 8:655-661, 1990). Purified protein was desalted/concentrated in Ultrafree-CL 30,000 NMWL filtration units (Millipore) using Buffer A (150 mM NaCl, 20 mM Tris-HCl, pH 7.4), and quantitated by Pierce BCA assay using bovine serum albumin (BSA) as the standard. Example 3 Activation of Recombinant Protein C Complete activation of the zymogen forms of protein C and polypeptides was accomplished by incubation with thrombin-sepharose . Thrombin-sepharose was washed extensively with Buffer A. 200 μL of packed thrombin- sepharose was mixed with 250 μg of protein C in 1 mL of the same buffer and incubated at 37 °C for 4 hours with gentle shaking on a rotating platform. During the course of the incubation, the degree of protein C activation was monitored by briefly pelleting the thrombin-sepharose, and assaying a small aliquot of the supernatant for aPC activity using the chromogenic substrate S-2366 (DiaPharma) . Following complete activation, the thrombin-sepharose was pelleted, and the supernatant collected. aPC concentration was verified by Pierce BCA assay, and the aPC was either assayed directly, or frozen in aliquots at -80°C. All polypeptides were analyzed by SDS-PAGE with either Coomassie-blue staining or Western Blot analysis to confirm complete activation (Lae mli, Nature 227:680-685, 1970). Example 4 Functional Characterization The amidolytic activity of recombinant human aPC polypeptides were determined by hydrolysis of the tri- peptide substrates S-2366 (Glu-Pro-Arg-p-nitroanilide) , S- 2238 (Pip-Pro-Arg-p-nitroanilide) , and S-2288 (Ile-Pro-Arg- p-nitroanilide) , Table III. The anticoagulant activity is shown as measured clotting time in an aPTT at 500 ng mL "1 aPC. Amidolytic activites were measured using the chromogenic substrate S-2366. Assays were performed at 25 °C, in Buffer A containing 1 mg mL "1 BSA, 3 mM CaCl 2 , and 0.5 nM aPC . Reactions (200 μL/well) were performed in a 96 -well microtiter plate, and amidolytic activity was measured as the change in absorbance units/min at 405 nm as monitored in a ThermoMax kinetic micrometer plate reader. Kinetic constants were derived by fitting velocity data at varying substrate concentrations (16 μM to 2 mM) to the Michaelis-Menten equation. Changes in A405 were converted to mmol product using a path length of 0.53 cm (Molecular Devices Technical Applications Bulletin 4-1) , and an extinction coefficient for the released p-nitroanilide of 9620 M "1 cm "1 (Pfleiderer, Methods Enzymol 19:514-521, 1970). Anti-coagulant activity was assessed by measuring the prolongation of clotting time in the activated partial thromboplastin time clotting assay (Helena Laboratories) . Clotting reactions were monitored in a ThermoMax kinetic microtiter plate reader, measuring the time to V max in the change in turbidity. Table III. Functional characterization of protein C polypeptides Protein Anticoagulant activity Amidolytic APTT Clotting Time activity JTcat/Km (mM s _1 ) Control 36 seconds N/A WT-aPC 114 seconds : 98 Leul94S : 108 seconds : 84 Alal95G 120 seconds 66 Thr254S 108 seconds 63 Example 5 Inactivation of aPC Polypeptides The rates of inactivation of aPC polypeptides were determined by incubating normal human plasma (Helena Labs) with 20 nM aPC (or either polypeptide) at 37°C (Figure 1) . Plasma concentration was 90% (v/v) in the final reaction buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM. The measured half-lives are summarized in Table IV. To assess the impact of activated protein C polypeptide inactivation by PCI, heparin (10 U mL " 1 ) , which is known to cause about 100- fold stimulation in the inactivation of aPC by PCI (Heeb, et al . , J Biol Chem 263(24) .11613-11616, 1988; Espana, et al . , Thromb Res 55 (3) :369-84, 1989; Aznar, et al . , Thromb Haemost 76 (6) :983-988, 1996), was added to a similar reaction (Figure 3) . Inactivation by α^-antitrypsin (α^-AT) was determined by incubation of aPC or derivatives at 20 nM with 40 mM α^-AT (Sigma) in a reaction buffer consisting of 3 mM CaCl 2 , 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM. Table IV. Half-lives for inactivation of activated protein C polypeptides in normal human plasma. Protein tl/2 -Fold increase (min) relative to wild-type Wild-Type 28 1 Leul94Ser 180 1 6.5 Leul94Ala 88 , 3.1 Example 6 In vivo Pharmacokinetics In vivo pharmacokinetic experiments were performed in normal rabbits to verify the observed in vi tro effects in half-life as a result of the mutations. A marginal ear vein and a central ear artery was cannulated in the conscious rabbit. Activated protein C polypeptides in buffer A (300 μg/ml) were used to administer a dose of 100 μg/Kg or 0.1 mg/kg bolus through the marginal ear vein catheter. Blood was sampled (0.45 ml) into a syringe containing 0.05 ml of 3.8% citrate containing benzamidine - adjustments were made to compensate for the syringe/needle dead space to yield the final concentration of 1 part citrate/benzamidine : 9 parts blood. Samples were collected 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 minutes post treatment, spun as soon as convenient after collection, and 200 μl of plasma was aliquoted into 96-well plates. The level of activated protein C polypeptides were determined using an enzyme capture assay (ECA) , as described previously (Gruber, et al., Blood 79 (9) :2340-2348, 1992), compared to standards ranging from 1 to 250 ng/mL diluted in pooled rabbit plasma. The results for wild-type and Leul94Ser are shown in Figure 5.

Claims: WE CLAIM : 1. A protein C derivative comprising SEQ ID NO: 1 and the corresponding amino acids in SEQ ID NO : 2, wherein one or more amino acids at position 194, 195, 228, 249, 254, 302, or 316 is substituted with an amino acid selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gly, and Gin, provided that amino acid 195 is not substituted with Ala and amino acid 254 is not substituted with Thr. 2. The protein c derivative of Claim 1 wherein said amino acid substitutions result in increased resistance to inactivation by serpins when compared to wild-type activated human protein C . 3. The protein C derivative of Claim 1, wherein said protein C derivative is in its activated form. 4. The protein C derivative of Claim 1 wherein the Leu at position 194 is replaced with Ser (SEQ ID NO: 3) . 5. The protein C derivative of Claim 1 wherein the Leu at position 194 is replaced with Ser and the Thr at position 254 is replaced with Ser (SEQ ID NO: 4) . 6. The protein C derivative of Claim 1 wherein the Leu at position 194 is replaced with Ala and the Thr at position 254 is replaced with Ser (SEQ ID NO: 5) . 7. A recombinant DNA molecule encoding the protein C derivative of Claim 4, wherein said DNA molecule is SEQ ID NO: 8. 8. A recombinant DNA molecule encoding the protein C derivative of Claim 5, wherein said DNA molecule is SEQ ID NO: 9. 9. A recombinant DNA molecule encoding the protein C derivative of Claim 6, wherein said DNA molecule is SEQ ID NO: 10. 10. A method of treating vascular occlusive disorders and hypercoagulable states which comprises : administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with increased resistance to serpins selected from the group consisting of L194S, L194S:T254S, and L194A:T254S. 11. The vascular occlusive disorders and hypercoagulable states of Claim 10 comprising: sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome. 12. A method of treating thrombotic disorders and disease states predisposing to thrombosis which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with increased resistance to serpins selected from the group consisting of L194S, L194S:T254S, and L194A:T254S. 13. The thrombotic disorders and disease states of Claim 12 comprising: myocardial infarction, unstable angina and stroke. 14. A method of treating sepsis which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with increased resistance to serpins selected from the group consisting of L194S, L194S:T254S, and L194A:T254S in combination with bacterial permeability increasing protein. 15. A pharmaceutical composition comprising: a protein C derivative with increased resistance to serpins selected from the group consisting of L194S, L194S:T254S, and L194A:T254S, in a pharmaceutically acceptable diluent. 16. The pharmaceutical composition of Claim 15 wherein said protein C derivative is activated. 17. A vector, comprising a nucleic acid according to Claim 7. 18. A host cell transformed by the vector according to Claim 17. 19. A vector, comprising a nucleic acid according to Claim 8. 20. A host cell transformed by the vector according to Claim 19. 21. A vector, comprising a nucleic acid according to Claim 9. 22. A host cell transformed by the vector according to Claim 21. 23. An isolated nucleic acid, comprising: a human protein C polynucleotide encoding at least 90% of the amino acids of a protein sequence selected from SEQ ID NOS: 3, 4, and 5. 24. An isolated nucleic acid of Claim 22, further comprising at least one substitution selected from the group consisting of 194T or 194A of SEQ ID NO : 3. 25. A protein C derivative comprising at least 90% of the amino acids of a protein sequence selected from SEQ ID NOS: 3, 4, and 5. 26. A protein C derivative of Claim 25, further comprising at least one substitution selected from 194T or 194A of SEQ ID NO: 3. 27. The protein C derivative of Claim 25 wherein said protein C derivative is activated. 28. A method of treating thrombotic disorders which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with increased resistance to serpins selected from the group consisting of L194S, L194S:T254S, and L194A:T254S, in combination with an anti-platelet agent. 29. A method of treating thrombotic disorders or vascular occlusive disorders and hypercoaguable states, comprising: inhibiting binding of activated protein C polypeptides to inhibitor recognition sequences of the serpins, PCI and α^-AT. 30. The method of Claim 29, wherein the inhibitor recognition positions are selected from S2 , S3', and S4 ' . 31. An article of manufacture for human pharmaceutical use comprising: packaging material and a vial comprising lyophilized human protein C derivative with resistance to inactivation by serpins when compared to wild-type human protein C. 32. The article of manufacture for human pharmaceutical use of Claim 31, wherein said packaging material comprises a label which indicates that said protein C derivative be administered at a dosage of about 0.01 μg/kg/hr to about 50 μg/kg/hr. 33. A human protein C derivative with resistance to inactivation by serpins compared to wild-type protein C produced by the process comprising: (a) transforming a host cell with a vector containing nucleic acid encoding a human protein C derivative; (b) culturing said host cell in a medium appropriate for expression of said human protein C derivative; (c) isolating said human protein C derivative from the culture medium; and (d) activating said human protein C derivative. 34. The vector according to Claim 33 wherein the nucleic acid encodes a human protein C derivative selected from the group consisting of L194S, L194S:T254S, and L194A:T254S. 35. The host cell according to Claim 33 wherein said host cell is selected from the group consisting of 293 cells and AV12 cells. 36. A method of treating acute coronary syndromes and disease states predisposing to thrombosis which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C derivative with resistance to inactivation by serpins compared to wild-type protein C, said derivative is selected from the group consisting of L194S, L194S:T254S, and L194A:T254S. 37. The method of Claim 36 wherein acute coronary syndromes and disease states predisposing to thrombosis are selected from the group consisting of myocardial infarction and unstable angina. Novel protein C derivatives are described. These polypeptides retain the biological activity of the wild-type human protein C with substantially longer half-lives in human blood. These polypeptides will require either less frequent administration and/or smaller dosage than wild-type human protein C in the treatment of vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states predisposing to thrombosis.

Inventors: GERLITZ BRUCE EDWARD, JONES BRYAN EDWARD, GERLITZ, BRUCE, EDWARD, JONES, BRYAN, EDWARD
Applicants: GERLITZ BRUCE EDWARD, JONES BRYAN EDWARD, LILLY CO ELI, ELI LILLY AND COMPANY, GERLITZ, BRUCE, EDWARD, JONES, BRYAN, EDWARD
Application year: 2000
Main classification: C12N15
IPC codes: A61K38, A61P7, A61P9, A61P11, A61P13, A61P17, A61P31, A61P37, C12N1, C12N5, C12N9, C12N15