MXPA00012789A - Protein c derivatives - Google Patents

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

DERIVATIVES OF PROTEIN C DESCRIPTION OF THE INVENTION This application claims the priority of Provisional Application No. 5 of Series 60 / 131,801 filed on April 30, 1999. This invention relates to novel polynucleotides, polypeptides encoded by them and with the use of such polynucleotides and polypeptides. More specifically, the invention relates to protein C derivatives resistant to serine inactivation, to their production, and to pharmaceutical compositions comprising those protein C derivatives. Protein C is a serine protease and is a protein. natural coagulant that plays a role in the regulation of homeostasis by the inactivation of factors Va and VIIIa in the coagulation cascade. Human protein C is produced in vi v as a single polypeptide of 461 amino acids. This polypeptide undergoes multiple post-translational modifications including 1) excision of a signal sequence of 42 amino acids; 2) cleavage of the lysine and arginine residues (positions 156 and 157) to produce an inactive precursor by 2-chain zymogen (a residual 155 amino acid light chain linked via a disulphide bridge to a Ref: 125916 _i »aaM_flt¡r¿_.3fc'¿ & & amp; & amp; amp; the heavy chain of 262 residual amino acids); 3) vitamin K-dependent carboxylation of nine glutamic acid residues of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate binding of four sites (one in the light chain and three in the heavy chain). Finally, the zymogen of 2 chains can be activated by the removal of a dodecapeptide in the N terminal dt? to heavy chain, producing active protein C (aPC) that has greater enzymatic activity than the zymogen of 2 chains. In conjunction with other polypeptides, aPC functions as an important anticoagulant in protection against thrombosis, has anti-inflammatory effects through its inhibition of cytokine generation (for example, TNF and IL-1), and exerts profibrinolytic properties that facilitate lysis of the clot Thus, aPC provides a mechanism for anticoagulation, anti-inflammation and fibrinolysis. The critical role of aPC in the control of haemostasis is exemplified by the increase in the speed of thrombosis in heterozygous deficiency, protein C resistance (eg, due to the mutation of Leiden from common Factor V) and the fatal result of untreated homozygous protein C deficiency. PCa derived from plasma and produced ecombinantly has been shown to be an effective and safe antithrombotic agent in a variety of animal models and 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) [Fourier, 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 Surq. 158: 491-494, 1989], burns [Lo, et al., Burns 20: 186-187 (1994)], syndrome of adult respiratory distension (ARDS) [Hasegawa, et al., Chest 105 (1): 268-277, 1994], and transplants [Gordon, et al., Bone Marrow Trans. 11: 61-65 [1993]]. In addition, there are numerous diseases with abnormalities or thrombotic complications in which aPC can be useful in the treatment, such as: heparin-induced thrombocytopenia (HIT) [Phillips, et al., Annals of Pharmacotherapy 28: 43-45, 1994 ], sickle-cell disease or thalassemia [Karayalcin, et al., The American Journal of Pediatric Hemetology / Oncology 11 (3): 320-323, 1989], fever or viral hemorrhagic [Lacy, et al., Advances in Pediatric Infectious Diseases 12: 21-53, 1997], thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) [Moake, Semmars in Hematology 34 (2): 83-89, 1997]. In addition, the aPC in combination with the protein that increases the permeability * -. "1 * ar J_j3Ss-5 &_ Bactericidal (BPI) may be useful in the treatment of sepsis [Fisher, et al., Crit. Care Med. 22 (4): 553-558, 1994]. Platelet inhibition is effective both in the prevention and treatment of thrombotic disease, however, the use of antiplatelet agents, such as aspirin, increases the risk of bleeding, which limits the dose of the agent and the duration of treatment. The combination of aPC and antiplatelet agents results in a synergy that allows dose reduction of both aPC and antiplatelet agents.The reduction of agent doses in combination therapy in turn results in the reduction of side effects such as increased bleeding often observed in combination with anticoagulant / antiplatelet therapy Several methods to obtain plasma C protein and produce protein C, aPC and protein C / aPC polypeptides through recombinant DNA technology are known in the art and have been described. See, for example, U.S. Patent Nos. 4,775,624 and 5,358,932. Despite improvements in the methods to produce aPC through recombinant DNA technology, aPC and polypeptides thereof have difficulties and are expensive to produce and have had a relatively short half life in vivo. x »t * w * a? ti¡t? One reason for the short half-life is that aPC levels in blood are regulated by molecules known as serpins (Serine Protease Inhibitors), which are covalently bound to aPC forming an inactive serpin / aPC complex. Serpin / aPC complexes are formed when the aPC binds and proteolytically cleaves a loop from the reactive site within the serpin; After the excision, the serpin undergoes a conformational change that irreversibly inactivates the aPC. The serpina / aPC complex is then removed from the blood stream via hepatic receptors for the serpina / aPC complex. As a result, aPC has a relatively short half-life compared to the zymogen; approximately 20 minutes for the aPC against approximately 10 hours of the human protein C zymogen (Okajima et al., Thromb Haemost 63 (l): 48-53, 1990). It has been shown that changes in the amino acid sequences of serine protease in residues that interact directly with the substrate (generally in or near the active site) can alter the specificity of the serine protease, potentially providing greater specific activity towards coagulation factors appropriate, as well as greater resistance the serpins (Rezaie, J Biol Chem 271 (39): 23807-23814, 1996; Rezaie and Esmon, Eur. J. Biochem 242: 477-484, 1996). Therefore, an aPC polypeptide exhibits greater resistance to inactivation by serpin, and which maintains both the desirable biological activities of the aPC (eg, anticoagulant, fibronolytic, and anti-inflammatory activities), provides a compound that has a longer plasma half-life, and therefore, is effectively more potent than the original compound , requiring substantially reduced dose levels or less frequent administrations for therapeutic applications. Potential advantages are especially important in disease states in which serpin levels are elevated. Physiologically, the two serpins that serve as primary inactivators of aPC are the protein C inhibitor (PCI) and the cyti-antritipsin (cti-AT) [Heeb, et al. , J Biol Chem 263 (24): 11613-6, 1988]. Both PCI and ax-AT have been shown to be primary physiological inactivators of aPC in disease states such as disseminated intravascular coagulation (Scully, et al., Thromb Haemost 69 (5): 48-53, 1993), and observed elevated levels of a? AT 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). Elevated serine levels inactivate aPC resulting in increased susceptibility to coagulopathies associated with decreased protein C levels. Attempts have been made to increase the plasma half-life of aPC by increasing resistance to serpins by modifying the human protein C molecule (e.g., U.S. Patent No. 5,358,932). Frequently, the increase in immunogenicity is observed when a natural protein is significantly modified and then administered to a patient. Through experiments and scientific analysis, we have identified the binding sites of serpin and protein C essential for the formation of serpin / aPC complexes. We modified those residual amino acids directed in the aPC molecule and we found surprisingly that we could inhibit the formation of the serpin / aPC complex (the complex which irreversibly inactivates the aPC) and at the same time retain the specificity of the polypeptide for the substrates of the aPC (for example, factors Va and Villa). In particular, three recognition sites within the active site of the aPC show distinctive differences between the substrate recognition sequences and the inhibitor recognition sequences: S2, S3 'and S4'. We found the inhibition of aPC serpin / 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 amino acids selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gly, and Gln, provided 195 is not replaced 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 native protein C (SEQ ID NC: 7) and have substantially longer half-lives in human blood. Therefore, these compounds provide several advantages, for example less frequent administration and / or smaller doses and in this way, a reduction in the total cost of the production of the therapy. Additionally, those compounds exhibit an advantage in disease states with significantly a? -AT levels and evades such as sepsis. Importantly, increases in the half-lives of protein C derived from plasma can be achieved via substitutions of u. amino acid loci, which are less likely to be immunogenic compared to molecules that contain multiple amino acid substitutions (U.S. Patent No. 5,358,932; Holly, et al., Biochemistry 3 • 1176-1880, 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 the 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 Gln, provided that amino acid 195 is not Ala and amino acid 154 is not Thr. The invention further provides the activated form of the protein C derivatives identified above. The present invention also provides recombinant DNA molecules that code for the protein C derivatives of the previous paragraph, in particular, those comprising SEQ ID NOS: 8, 9, and 10. Another aspect of the present invention provides protein sequences of those same protein derivatives C, particularly those comprising SEQ ID NOS: 3, 4, and 5 and the activated forms of those protein derivatives C. The present invention comprises methods for treating vascular occlusive disorders and hypercoagulable states including: sepsis, disseminated vascular coagulation, fulminating purpura, major trauma, major surgery, burns, adult respiratory distress syndrome, transplants, deep vein thrombosis, induced thrombocytopenia by heparin, sickle-cell disease, thalassemia, viral haemorrhagic fever, thrombotic thrombocytopenic purpura, and haemolytic uremic syndrome. The invention further provides for the treatment of those same diseases and conditions using the activated form of the protein C derivatives identified above. Another embodiment of the present invention is a method for treating sepsis, comprising administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative of this invention in combination with protein that increases bacterial permeability. The present invention comprises methods for treating acute coronary syndromes such as myocardial infarction and unstable angina. The present invention also comprises methods for treating thrombotic disorders. Such disorders include, but are not limited to, stroke, abrupt closure after angioplasty or placement of a stent device, 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. The human protein C derivatives for the indications and pharmaceutical compositions mentioned previously they are preferably selected from L194S, L194S: T254S, and L194A: T254S. The methods and aspects for producing the novel isolated protein polypeptides are the aspect of this invention. Finally, one aspect of the invention comprises treating diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibition to sequences of S2, S3 'and S4' recognition sequences from serpins, PCI and a_-AT . This final aspect of the invention contemplates any and all modifications to any aPC molecule that result in inhibition of binding to PCI and oti-AT serine inhibitor recognition sequences. The inhibition of binding to the specific inhibitor recognition sequences of serpins (S2, S3 'and S4') is an important contribution of this aspect of the invention. Figure 1. Inactivation of polypeptides of human aPC during incubation with normal human plasma. The remaining activity was measured as amidolytic activity normalized to the activity at the beginning of the experiment (time = 0); the error bars indicate the standard error of the experiments in triplicate. The data shown are for natural protein C (WT, circles), T254S (squares), L194S (triangles), and A195G (diamonds). Figure 2. Inactivation of polypeptides of human aPC during incubation with normal human plasma. The remaining activity was measured as amidolytic activity normalized to the activity at the beginning of the experiment (time = 0); the error bars indicate the standard error of the experiments in triplicate. The data shown are for natural protein C (WT, circles), L194 / T254S (squares), L194S / T254S (triangles). Figure 3. Inactivation of aPC polypeptides by normal human plasma containing Heparin (10 U / mL). The remaining activity was measured as amidolytic activity normalized to the activity at the beginning of the experiment (time = 0); the error bars indicate the standard error of the experiments in triplicate. The data shown are for natural protein C (WT, circles), T254S (squares), L194S (triangles), and A195G (diamonds). Figure 4. Inactivation of aPC polypeptides by purified ai-antitrypsin plasma. The remaining activity was measured as amidolytic activity normalized to the activity at the beginning of the experiment (time = 0); the error bars indicate the standard error of the experiments in triplicate. The data shown are for natural protein C (WT, i «'. circles), T254S (squares), L194S (triangles), and A195G (diamonds). Figure 5. Plasma aPC levels after an IV bolus dose of 0.1 mg / kg in normal, conscious rabbits (N = 3). Activated protein C levels were determined using the 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 standard samples. The data shown are for the natural protein (WT, circles), T254S (squares), L194S (triangles), and L194S / T254S (diamonds).

The plotted values are the mean the standard error for the three animals. For the purposes of the present invention, as described and claimed herein, the following terms are as defined below. Antiplatelet agent - one or more agents alone or in combination, which reduce 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, Ninth Edition, Vol II. Pages 924-25, Mack Publishing Co., incorporated herein by reference. Such agents include but are not limited to aspirin (ASA), clopidrogel, ReoPro (abciximab), dipyridamole, ticlopidine and Ilb / IIIa antagonists. The aPC or active protein C refers to recombinant aPC. The aPC includes and is preferably recombinant human PC, although the aPC may also include other protein C species having proteolytic, amidolytic, stereic and biological (anticoagulant, anti-inflammatory, or profibronolytic) activities. "Protein C derivatives" refers to the recombinantly produced polypeptides of this invention that differ from natural human protein C but when activated retain the essential properties, ie, proteolytic, amidolytic, stereolytic and biological (anti: coagulant, anti-inflammatory activities) and profibrinolítica). The definition of protein C derivatives as used herein also includes the activated form of the protein derivatives C identified above. Tratapuerto - describes the management and care of a patient for the purpose of combating a disease, condition, or disorder to either eliminate the disease, condition, or disorder or prophylactically to prevent the onset of symptoms or complications of the disease, condition or disorder.

Continuous infusion - substantially continuous and uninterrupted introduction of a solution or suspension into a vein for a specific period of time. Bolus injection - the injection of a drug in a defined amount (called a bolus) for 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. 10 Unit dosage form - refers to physiologically discrete units suitable as unit doses for human subjects, each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a pharmaceutically suitable excipient. Hypercoagulable states - excessive coagulability associated with disseminated intramuscular coagulation, pre-thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of coagulation factors such as aPC. Zymogen - protein C zymogen, as used herein, refers to inactive secreted forms, either a chain or two chains, of protein C. Pharmaceutically effective amount - a therapeutically effective amount of a pharmaceutical compound. The áí ^^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^? ?? ? A? ¡£: '. -. _. "_ ^ L_ ^ itil ^ fc__É_B_Si ^^ _ ^ fe? AiBe ^^ K ^ ü & ' 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 that is being treated, patient characteristics and similar considerations. Acute coronary syndromes - clinical manifestations of coronary arteriosclerosis complicated by rupture of the coronary plaque, coronary thrombosis superimposed, and dangerous coronary blood flow that results in coronary ischemia and / or myocardial infarction. The spectrum of acute coronary syndromes includes unstable angina, non-Q-wave myocardial infarction (ie, non-ST segment elevation), myocardial infarction Q wave (ie, ST segment elevation). Thrombotic disorders - a disorder related to or affected by the formation or presence of a blood clot within a blood vessel. Such disorders include, but are not limited to, stroke, abrupt closure after angioplasty or placement in a stent device, and thrombosis as a result of peripheral vascular surgery. Fulminating purpura - ecchymotic skin lesions, fever, hypotension associated with bacterial sepsis, Mm¿ ^^ t? ^^^^^^^^ viral, bacterial or protozoal infections. Disseminated intravascular coagulation is usually present. Serpine - any of a group of structurally related proteins that are typically serine protease inhibitors whose inhibitory activity is conferred by an active site in a loop or cycle of a highly variable and mobile peptide including, but not limited to, the inhibitor of the protein C (PCI) and cti-antitrypsin (ai- AT). Inhibitor sequence S2: 2nd N-terminal residue to the cleavage site PCI or cti-AT. S3 'inhibitor recognition sequence; the 3rd C-terminal residue to the ICP cleavage site by cti-AT. Recognition sequence of inhibitor S4 '; the 4t0 C-terminal residue to the PCI cleavage site by a_-AT. Natural protein C - type of protein C that predominates in the natural human population in contrast to natural or laboratory protein C polypeptides. Protein that increases bactericidal permeability - includes the protein that increases the bactericidal permeability (BPI) produced naturally and recombinantly; natural, synthetic and recombinant biologically active polypeptide fragments of the BPI protein; biologically active polypeptide variants of the BPI protein or fragments thereof, including proteins or fusion and dimers; analogous biologically active variants of the DPI protein or fragments or variants thereof, including analogs substituted with cysteine, and peptides derived from DPI. The complete amino acid sequence of the DPI, as well as the nucleotide sequence of the DNA encoding the DPI have been determined by Gray, et al., 1989, J. Biol. Chem 264: 9505. Recombinant genes coding for and methods for the expression of BPI proteins, including holoprotein DPI and fragments of DPI are described in U.S. Patent No. 5,198,541, incorporated herein by reference. Abbreviations of amino acids 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 the aPC or polypeptides of isolated human aPC can be produced by activating the zymogen of recombinant human protein C or zymogen derived from recombinant protein C in vi tro or by the direct secretion of the activated form of protein C. The media by the which activation occurs are not critical and the process aspects of this invention include any and all activation means. Protein C derivatives can be produced in eukaryotic cells, transgenic animals or transgenic plants, including, for example, secretion of human kidney 293 cells as purified zymogen and then activated by techniques known to those skilled in the art. The present invention provides protein C derivatives, including the activated forms thereof, which have higher resistances to serpins, and consequently result in prolonged plasma half-lives. Specific protein C derivatives include L194S, L194S: T254S, and L194A: T254S and activated forms thereof. The C-protein derivative L194S preferably contains a serine residue at position 194 instead of a leucine residue normally found in this position. One skilled in the art would understand that all amino acid substitutions at residue 194 in addition to Ser can impart greater resistance to serines in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gln. The activated form derived from protein C L194S demonstrates a prolonged life in plasma (Figure 1) and increased resistance to serpins, for example cti-antitrypsin (ai-AT), Figure. Protein derivative L194S: T254S preferably contains serine residue at position 194 instead of a leucine residue normally found in this position and a serine residue at position 254 instead of the threonine residue normally found in this position. It is evident to one skilled in the art that other amino acid substitutions at residue 194 and 254 in addition to the Ser containing more resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gln, and Gly, provided that amino acid 254 is not substituted with Thr. The form of the human protein C derivative L194S: T254S demonstrates a prolonged half-life in normal human plasma as compared to the native protein C, Figure 2. The protein derivative L194A: T254S preferably contains alanine residue at position 194 instead of a leucine residue normally found in this position and serine residue at position 254 instead of residue r? - eonine normally found in this position. It is evident to one skilled in the art that other substitutions of ar_noacid in residues 194 and 254 in addition to the Ser-trusting one, more resistance to serpins in the resulting polypeptide molecule. Examples of such innocent ar substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, ln, and Gly, provided that amino acid 254 is not substituted co-Thr. The form of the human protein C derivative L194A: T254S demonstrates a prolonged half-life in normal human plasma compared to the native protein C, Figure 2. The additional embodiments of the present invention include the C: L194T protein derivatives, L194A, A195G, L228Q, T254S, F316N, Y249E, and Y302Q, and activated forms thereof, which have higher resistance to serpins. The protein derivatives C L194T or L194A Preferably they contain a threonine residue or an alanine residue at position 194, instead of a leucine residue normally found in this position. One skilled in the art would understand that other amino acid substitutions in residues 194 in addition to Ser may impart greater resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, His, Lys, Arg, Asn, Asp, Glu, and Gln. The protein derivative C195G preferably contains a glycine residue at position 195 instead of an alanine residue normally found in this position. One skilled in the art would understand that other amino acid substitutions at residue 195 in addition to Gly can impart greater 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 Gln. The activated form of the protein derivative C A195G demonstrates a prolonged half-life in plasma (Figure 1) and increased resistance to serpins, eg, cti-Antitrypsin (oii-AT), Figure 4. The protein derivative C L228Q preferably contains a glutamine residue at position 228 instead of a leucine residue normally found in this position. One skilled in the art would understand that other amino acid substitutions at residue 228 in addition to Gln can impart higher resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gln, and Gly. The C-T254S protein derivative preferably contains a serine residue at position 254 instead of a threonine residue normally found in that position. It is evident to one skilled in the art that other amino acid substitutions at residue 254 in addition to Ser can impart greater resistance to serpins to the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gln, and Gly, provided that amino acid 254 is not substituted with Thr. The activated form of the protein derivative C T254S demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, eg, cti-antitrypsin (ai-AT), Figure 4. The protein derivative C F316N preferably contains a Asparagine residue at position 316 instead of a phenylalanine residue normally found in this position. One skilled in the art would understand that the amino acid substitutions at residue 316 in addition to Asn can impart greater resistance in the serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gln, and Gly. The protein derivative C Y249E preferably contains a glutamic acid residue at position 249 instead of a tyrosine residue normally found in this position. An additional polypeptide contains an Asp at position 249 instead of a tyrosine residue. One skilled in the art would understand that other amino acid substitutions at residue 249 in addition to Glu and Asp can impart greater resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asn, Gln, and Gly.

The protein derivative C Y302Q preferably contains a glutamine residue at position 302 instead of a tyrosine residue normally found in this position. An additional polypeptide contains a Glu at position 302 instead of a tyrosine residue. One skilled in the art would understand that other amino acid substitutions at residue 302 in addition to Glu or Gln can impart greater resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include the Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, and Gly. In addition, protein C derivatives can include proteins that represent functionally equivalent gene products. Such a protein C derivative can contain deletions, additions or substitutions of residual amino acids within the amino acid sequence encoded by the protein C polypeptide genetic sequences described above, but which result in a silent change, thereby producing a genetic product derived from functionally equivalent protein C. The amino acid substitutions can 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 an identity of at least 90% with the corresponding fragment of SEQ ID NOS: 3, 4, or 5. Preferably, all 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 that is, those that substitute a residue with another of similar characteristics. Typical substitutions are found between the Ala, Val, Leu, and Lie; between the Self and Thr; between the acid residues Asp and Glu; between Asn and Gln; and between the basic waste Lys and Arg; or the 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 the production of protein C derivatives. Those DNA compounds comprise the sequence encoding the light chain of the zymogen of human protein C or zymogen derived from protein C placed immediately adjacent to, downstream of of, and in the framework of translational reading with the prepropeptide sequence of the zymogen of human protein C or zymogen derived from protein C. *. ~. . x ^ __ *, ^ * ^^ _ fl? fc »« toii »s6¡d8Mh ---. A * _B_»., DNA sequences preferably code for the dipeptide Lys-Arg which is processed during the maturation of the protein C molecule, activation peptide and heavy chain of 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, all teachings of which, are incorporated herein by reference, describes the natural form of the human protein C molecule. The person skilled in the art could easily determine which changes in the DNA sequence could code for the exact polypeptides as described herein. * The invention is not limited to the specific DNA sequences described. Accordingly, the construction described below and the accompanying examples for the preferred DNA compounds are illustrative only and do not limit the scope of the invention. All of the DNA compounds of the present invention were prepared during the use of site-directed mutagenesis to change particular positions within the zymogen or human protein C. The methods used for the identification of the residues that form Critical contacts at those particular positions are described in Example 1. Protein C derivatives can be produced by methods well known in the art using eukaryotic cell lines, transgenic animals or transgenic plants. Those skilled in the art will readily understand that appropriate host eukaryotic cell lines include but are not limited to HepG2, LLC-MK2, CH0-K1, 293, or AV12 cells, examples of which are described in U.S. Patent No. 5,681,932 , incorporated here as a reference. In addition, examples of transgenic production of recombinant proteins are described in U.S. Patent Nos. 5,589,604 or 5,650,503, incorporated herein by reference. Those skilled in the art 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). Preferred vectors of the present invention is pIG3 as described in Example 2. To be fully active and operate under the methods of the present, protein C derivatives produced by any of these methods must undergo posttranslational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of an erythro-beta-hydroxy-Asp (beta-hydroxylation), the addition of four oligosaccharides bound by Asn (glycosylation) and, the removal of the leader sequence (42 residual amino acids). Without such posttranslational modifications, the C protein polypeptides are not fully functional or functional. The methods for activating the zymogenic forms of human protein C and derivatives of protein C to activated human protein C and activated protein C derivatives are old and well known in the art. Human protein C can be activated by thrombin alone, or a thrombin / thrombomodulm complex, by RVV-X, a Russell snake venom protease, 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, transplants, vein thrombosis deep, thrombocytopenia produced by heparin, sickle-cell disease, thalassemia, fever ¿^ - p - ?? t ^ á Jm? Ktxii *. ^^ »^ < BW < Mt > __ - _ J __-_ »_ ^ .. a < Viral hemorrhagic, thrombotic trophocytopenic purpura, and haemolytic uremic syndrome. In another embodiment, the recombinant protein C derivatives of the present invention are useful for the treatment of sepsis in combination with the protein that increases bacterial permeability. In a further aspect of this invention, the activated protein C derivatives of the present invention are combined with antiplatelet agents 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, unstable angina with human protein C derivatives with resistance to serine inactivations compared to natural 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 after angioplasty or placement of a stent device, 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 the active agent and an aPC polypeptide and a pharmaceutically acceptable carrier or solid. For example, a desired formulation would be one that is a stable high purity lyophilized product comprising an & amp; additive agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and an activated protein C derivative. A stable, preferred lyophilized formulation comprises: 2.5 mg / ml of activated protein C polypeptide, 15 mg / ml of sucrose, 20 mg / ml of NaCl and citrate buffer, the formulation having a pH of 6.0. A further stable lyophilized formulation comprises: 5.0 mg / ml of activated protein C polypeptide, 30 mg / ml of sucrose, 38 mg / ml of NaCl and citrate buffer, the formulation having a pH of 6.0. Preferably, the human aPC polypeptides will be administered parenterally to ensure release into the bloodstream in an effective manner by injecting the appropriate dose with a continuous infusion for 1 to 240 hours. Most preferably, 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. Even more preferably, aPC polypeptides will be administered as a continuous infusion of 1 to 96 hours.

The amount of 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 from about 0.1 to about 25 μg / kg / hr. Even more preferably, the amount of human aPC polypeptide administered will be from about 1 μg / kg / hr to about 15 μg / kg / hr. The most preferable amounts of human aPC polypeptide administered will be from about 5 μg / kg / hr to 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 for a time from about 5 minutes to about 120 minutes, followed by the continuous infusion of the appropriate dose for up to about 240 hours. In another alternative, derivatives of human aPC will be administered by injecting a dose of 0.01 mg / kg / day to approximately 1.0 mg / kg / day, B.I.D. (2 times a day), for one to ten days. Preferably, the human aPC derivatives will be administered B.I.D. during three days. In yet another alternative, the human aPC polypeptides will be administered subcutaneously to ensure a Ug ^ ss ^^ - i.

Slower release into the bloodstream. The formulation for the subcutaneous preparations will be made using known methods to prepare such pharmaceutical compositions. A further aspect of the present invention comprises treating the diseases and conditions caused or resulting from the protein C deficiency as defined herein, by inhibiting the binding to the recognition sequences of the S2, 3 ', and S4' inhibitor of the serpins, PCI and cti-AT, as described in Example 1. This final aspect of the invention contemplates - any and all modifications to any molecule. of aPC that result in the inhibition of binding to such recognition sequences for the inhibition of PCI and cti-AT serpins. The polypeptides of human aPC described in this invention have essentially the same type of biological activity as natural human aPC, with substantially longer half-lives in human blood. Therefore, those who use DS will require less frequent administration and / or lower doses. In addition, these compounds will exhibit a vente. in disease states with significantly elevated levels of cti-AT such as sepsis. Finally, higher increases in the plasma half-lives of the human aPC polypeptide can be achieved via a _ • &, or two amino acid substitutions, which are probably less immunogenic compared to a greater number of substitutions. The following Examples are provided solely to better illustrate the present invention. The scope of the invention should not be considered only as consistent with the following Examples.

Example 1 Targeted site-directed mutagenesis The use of site-directed mutagenesis to change particular positions within a human protein C molecule that decrease inactivation by serpins is described, and as a result result in prolonged plasma half-lives. The recognition sequences in the two inhibitors of primary aPC oii-AT and PCI reveal some differences that can be exploited by altering the residues in the aPC that interact with those sequences. Table I describes the sequences recognized by the aPC. The cleavage site occurs between the two residues shown in italics. The residues that occupy the specific sites SI, S3 ', and S4', are underlined. In general, the sites recognized in the factor Va are different from the sites in either the Villa factor or the inhibitors, therefore, it is possible to design the active site of an aPC to preferably cleave the most critical coagulant factor Va, and at the same time decrease the probability that aPC is inhibited by serpins.

Table I.

Coagulation factors S2 S3'S4 ' Factor Va 300--313 N C 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 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 d N Q I M S D Serpinas PCI G T I F T F R S S R L N S Q In particular, three recognition sites within the active site show distinct differences between the substrate recognition sequences and the inhibitor recognition sequences: S2 (the 2nd N-terminal residue at the cleavage site, S3 'site, and S4) The S2 site is occupied mainly by polar residues in the factor Va sequences, unlike the PCI and ai-AT, which have hydrophobic residues in this position.The S3 'site occupied by polar side chains in all sequences of the substrate, but remarkably, a hydrophobic side chain in the oti-AT sequence The S4 'site is occupied by charged residues in the three sequences of the factor Va, but is occupied by hydrophobic residues in the Villa factor and inhibitor sequences Based on the crystalline structures of aPC inhibited by PPACK (Mather, et al., EMBO J. 15 (24) -6822-6831, 1996) and thromboma 3-mib? da by Hirulog (Qiu, et al., Biochemistry 31 (47): 11689-97, 1992), they created two aPC substrate model structures and energy was minimized using a CHARMm protocol: (1) The sequence representing the R506 cleavage sequence of factor Va. (2) The oci-AT recognition sequence, with the Met substituted with Arg (corresponding to an ax-AT polypeptide exhibiting an extremely high affinity for aPC).

. «- Those models allowed the identification of waste which forms critical contacts in those three specific sites. A summary of the residues that form specific contacts within the active site, and replacements that are expected to provide greater specificity and / or activity are summarized in Table II. In general, mutations of contact residues within specific subsites of the active site were designed to reflect changes in the environment to direct the specificity of human aPC polypeptides from the recognition of the two primary physiological inhibitors, and potentially increase proteolytic activity of the human aPC polypeptide.

Table II. Mutations constructed for alterations of specificity APC Residual Site Substitutions Contact of the constructed substrate S2 Thr254 Be aliphatic part of the side chain S3 'Tyr302 Glu, Gln End of the side chain S4 'Leul94 Ser, Thr, Aliphatic part of the wing side chain Table II. Mutations constructed for alterations of specificity (continued) APC Residual Site Substitutions Contact of the constructed substrate S4 'Alal95 Gly Aliphatic part of the side chain S4 'Leu228 Gln End of the side chain S4 'Phe316 Asn Aliphatic part of the side chain Example 2 Construction and Production of Protein C Polypeptide Protein C derivatives were constructed using the polymerase chain reaction (PCR) following standard methods. The source of the natural coding sequence of the plasmid pLPC (Bio / Technology 5: 1189-1192, 1987). The universal PCR primers used included: PCOOlb; 5'- GCGATGTCTAGAccaccATGTGGCAGCTACACAAGCCTCCTGC -3 ', which codes for a restriction site Xbal (underlined) used for subcloning, a consensus sequence of Kozak (lower case) (Kozak, J Cell Biol 108 (2): 229-41, 1989 ) Y the 5 'end of the coding region for protein C: PC002E; 5'- CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3 ', which codes 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 antisense directions, respectively), include: PC194SF, 5'-CCTCAAAGAAGAAGTCCGCCmrCGGGGCAGTGC-3 'and PC194SR, 5'-GCACTGCCCCGCAGGCGGACTTCTTCTTTGAG-3' which encode the mutation from Leu (CTG) to Ser (TCC) ) at position 194 (of the bold type); PCA195GF, 5'-GAAGAAGCTGGGGTGCGGGGCAGTGC-3 ', and PCA195GR, 5'-GCACTGCCCCGCACCCCAGCTTCTTC-3, which encode for the mutation of Ala (GCC) to Gly (GGG); PCT254SF, 5'-GCAAGCGCACCAGCGACAATGAC-ATCGC-3 'and PCT254SR, 5'-GCGATGTCATTGTCGCTGGTGCTCTTGC-3', which code for the mutation of THR (£ CC) to Ser (AGC) at position 254 (in bold). The first round of the PCR was used to amplify two fragments of the protein C gene; the 5 'fragment was generated using PCOOlb and the antigen gone 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 product of the PCR was digested with Xbal and Bcll and subcloned into expression vector similarly digested pIG3. A natural construct was also generated by PCR using the two universal primers and the plasmid pLPC as a standard, followed by subcloning in pIG3. The mutations were confirmed by DNA sequencing of both coding and non-coding strands. The full-extraction plasmids were designated pIG3-HPC (natural protein C), pGH41 (T245S), pGH51 (A195G), and pGH94 (L194S). The pIG3 vector was generated by the insertion of a gene from the "internal ribosomal 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) in the expression vector of mammal pGTD (Gerlitz, et al, Biochem J 295 (Pt 1): 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) _5485-6, 1992) directs the expression of a bicistronic mRNA (5'-cDNA-IRES-GFP-3 '). Efficient translation of the first cistron is initiated by the classical assembly of the ribosomal subunits in the methylated cap structure at the 5 'position of the mRNA, while the normally inefficient translation of a second cistron is overcome by the IRES sequence, which allows the montage and »i ^ kshimi ¿aa? _i ____ a¡ _? _ á internal ribosomal on the mRNA. The coupling of the cDNA and the reporter on a single mRNA, translated as separate proteins, makes it possible to select clones with higher production based on the intensity of the fluorescence. The expression vector also contains a cassette of ampicillin resistance for the maintenance of the plasmid in E. coli, and a murine DHFR gene with appropriate expression sequences for purposes of selection and amplification in expression in mammalian tissue. The Syrian hamster AV12-664 cell line transformed with adenovirus 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 μg / mL of vitamin Kl. One day before transfection, the cells were cultured at a density of approximately 10 5 cells / 24 cm 2. Plasmids linearized with FspI were transfected using 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 mM methotrexate for selection. The methotrexate-resistant colonies were pooled 2-3 weeks after the application of the í & J! .and selection of the drug and be expanded. The collected fractions were subjected to cell separation activated by fluorescence based on the intensity of the GFP fluorescence (Cormack, 1996), with the cells with a more intense fluorescence with 5% being retained and expanded. To obtain material for purification, recombinant cells were grown in a modified mixture of Dulbecco's modified Eagle's medium and leaving F12 (1: 3) containing 1 μg / mL of human insulin, ul μg / mL of human transferrin and 10 μg. / mL of vitamin Kl. The conditioned media were collected, adjusted to a final concentration of 5 mM and 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). The purified protein was desalted / concentrated in 30.00 NMWL ultrafiltration units (Millipore) using buffer A (150 mM NaCl, 20 mM Tris-HCl, pH 7.4), and quantified by Pierce BCA assay using bovine serum albumin (BSA) as the standard.

Example 3 Activation of Recombinant Protein C Complete activation of zymogenic forms of protein C and polypeptides was achieved by incubation with thrombin sepharose. Thrombin sepharose was washed extensively with buffer A. 200 μl of packed thrombin sepharose were mixed with 250 μg of protein C in 1 mL of the same buffer and incubated at 37 ° C for 4 hours with agitation on a rotating platform. During the course of the incubation, the degree of activation of protein C was verified by briefly sedimenting the thrombin-sepharose, and assaying a small aliquot of supernatant to determine the activity of the aPC using the chromogenic substrate S-2366 (DiaPharma). After completing the activation, the thrombin-sepharose was pelleted, and the supernatant collected. The concentration of aPC was verified by the Pierce BCA assay, and the aPC was then assayed directly, or frozen in aliquots at -80 ° C. All polypeptides were analyzed by SDS-PAGE with Coomassie blue staining or Western electroblot analysis to confirm complete activation (Laemmli, Natura 227: 680-685, 1970).

Example 4 Functional Characterization The amidolytic activity of recombinant human aPC polypeptides was determined by hydrolysis of the tripeptide substrates S-2366 (Glu-Pro-Arg-p-nitroanilide), s-2238 (Pip-Pro-Arg-p-nitroanilide ), and S-2288 (lie-Pro-Arg-p-nitroanilide), Table III. The anticoagulant activity was demonstrated as coagulation time measured in an aPTT at 500 ng mL "1 of aPC The amidolytic activities were measured using chromogenic substrate S-2366. The analyzes were carried out at 25 ° C, in Shock absorber A containing 1 mL "1 BSA, 3 mM CaCl 2, t aPC 0.5 nM. The reactions (200 μL / well) were carried out in a 96-well microtiter plate, and the amidolytic activity was measured as the change in absorbance units / min at 405 nm as verified in a ThermoMax kinetic micrometric plate reader . The kinetic constants were derived by adjusting the velocity data at various substrate concentrations (16 μm to 2 nM) to the 5 Michaelis-Menten equation. The changes in the A4os were converted to mmol of product using an optical pitch length of 0. 53 cm (Molecular Devices Technical Applications Bulletin 4-1), and an extension coefficient for the liberated p-nitroanilide of 9620 M "1 (Pfleiderer, Methods Enzymol 19: 514-521, 1970.) The anticoagulant activity was evaluated by measuring the prolongation of the coagulation time and the coagulation assay of activated partial thromboplatin time (Helena Laboratories). The coagulation reactions were verified by a kinetic microtiter plate reader ThermoMax, measuring the time for Vmax in the change in turbidity.

Table III. Functional characterization of protein C polypeptides.

Example 5 Inactivation of aPC Polypeptide The inactivation rates of aPC polypeptides were determined by incubating normal human plasma (Helena Labs) with 20 nM aPC (or any polypeptide) at 37 ° C (Figure 1). The plasma concentration was 90% (v / v) in the final reactant buffer containing 150 mM NaCl, 20 mM Tris, pH 7., and 1 mL mL of BSA. selected times, and the activity was measured as Amidolytic activity used S-2366 at a final concentration of lmM. The measured half-lives are summarized in Table IV. To assess the impact of inactivation of protein C polypeptide activated by PCI, heparin (10 U mL "1) was added, which is known to cause a stimulation of approximately 100 times inactivation of aPC by PCI (Heeb, et al., J Biol Chem 263 (24): 11613-11616, 1988, Spain, et al., Thronb Res 55 (3): 369-84, 1989, Aznar, et al., Tromb Haemost 76 (6): 983-988, 1996), to a similar reaction (Figure 3) Inactivation by cti-antitrypsin (a_-AT) was determined by incubating the aPC or derivatives at 20 nM with oti-At 40 nM (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 1 mM.

Table IV. Average lives for activation of activated protein C polypeptides in normal human plasma Table IV. Average lives for Inactivation of activated protein C polypeptides in normal human plasma (CONTINUED) EXAMPLE 6 Jn vivo pharmacokinetics In vivo pharmacokinetic experiments were performed in normal rabbits to verify the effects observed in the middle life as a result of the mutations. A marginal vein and a central artery of the ear were cannulated in the conscious rabbit. Protein C polypeptides activated in buffer A (300 μg / ml) were used to administer a dose of 100 μg / Kg or a bolus of 0.1 mg / kg through the marginal vein catheter of the ear. Blood was sampled (0.45 ml) in a syringe containing 0.05 ml of 3.8% citrate containing benzamidine - adjustments were made to compensate for the empty space of the syringe / needle to produce the final concentration of citrate / benzamidine: 9 parts of blood. Samples were collected at 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 minutes after the treatment, centrifuged as soon as convenient after harvesting, and aliquots of 200 μl of plasma were made into 96 well plates. The level of activated protein C polypeptides was determined using an enzyme capture assay (ECA), as described above (Gruber, et al., Blood 79 (9): 2340-2348, 1992), which compared to standards range from 1 to 250 ng / ml diluted in pooled rabbit plasma. The results of the natural and the Leul94Ser are shown in Figure 5. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. js £ - ti.j.

Claims (37)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. A protein C derivative, characterized in that it comprises 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 by an amino acid selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gly, and Gln, provided that amino acid 195 is not substituted with Ala and amino acid 254 is not substituted with Thr.
2. 2. The protein C derivative according to claim 1, characterized in that the amino acid substitutions result in greater resistance to inactivation by serpins when compared to the natural activated human protein C.
3. 3. The protein C derivative according to claim 1, characterized in that the protein C derivative is in its activated form.
4. The protein C derivative according to claim 1, characterized in that the Leu at position 194 is replaced with Ser (SEQ ID NO: 3).
5. 5. The protein derivative C according to claim 1, characterized in that the Leu at position 194 is replaced with Ser and liPThr at position 254 is replaced with Ser (SEQ ID NO: 4).
6. 6. The protein derivative C according to claim 1, characterized in that the Leu at position 194 is replaced with Ala and the Thr at position 254 is replaced with Ser (SEQ ID NO: 5).
7. 7. A recombinant DNA molecule encoding the protein C derivative according to claim 4, characterized in that the DNA molecule is SEQ ID NO: 8.
8. 8. A recombinant DNA molecule encoding the protein derivative C according to claim 5, characterized in that the DNA molecule is SEQ ID NO: 9.
9. 9. The recombinant DNA molecule that codes for the protein C derivative according to claim 6, characterized in that the DNA molecule is SEQ ID NO:
10. 10. A method for treating vascular occlusive disorders and hypercoagulable states, characterized in that it comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with greater resistance to the selected serpins of the group consisting of L194S, L194S: T254S, and L194A: T254S.
11. 11. Occlusive ocular disorders and hypercoagulable states according to claim 10, characterized in that it comprises: sepsis, disseminated intravascular coagulation, fulminating purpura, major trauma, major surgery, burns, adult respiratory distress syndrome, transplants, deep vein thrombosis, thrombocytopenia induced by heparin, sickle cell disease, thalassemia, viral haemorrhagic fever, thrombotic t mbocytopenic purpura, and haemolytic uraemic syndrome.
12. 12. A method for treating thrombotic disorders and disease states that predispose to thrombosis, characterized in that it comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with higher resistance to serpins of a selected group consisting of L194S, L194S: T254S, and L194A: T254S.
13. 13. Thrombotic states and disease states as claimed in claim 12, characterized in that it comprises; myocardial infarction, unstable angina, and apoplen.
14. 14. A method for treating sepsis, characterized in that it comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative with increased resistance to serpins selected from a group consisting of L194S, L194S: T254S, and L194A: T254S in combination with protein that increases bacterial permeability.
15. 15. A pharmaceutical composition, characterized in that it comprises: a protein C derivative with greater resistance to serpins selected from the group consisting of L194S, L194S: T254S, and L194A: T254S, in a pharmaceutically acceptable diluent.
16. 16. The pharmaceutical composition according to claim 15, characterized in that the protein C derivative is activated.
17. 17. A vector, characterized in that it comprises a nucleic acid according to claim 7.
18. 18. A host cell, characterized in that it is transformed by the vector according to claim 17.
19. 19. A vector, characterized in that it comprises a nucleic acid of according to claim 8.
20. 20. A host cell, characterized in that it is transformed by the vector according to claim 19.
21. 21. A vector, "face" because it comprises a nucleic acid according to claim 9.
22. 22. A host cell, characterized in that it is transformed by the vector according to claim 21.
23. 23. An isolated nucleic acid, characterized in that it comprises: a polynucleotide of Human protein C coding for at least 90% of the amino acids of a protein sequence selected from SEQ ID NO: 3, 4, and 5.
24. 24. The isolated nucleic acid according to claim 22, characterized in that it also comprises , at least one substitution selected from the group consisting of 194T, or 194A, of SEQ ID NO: 3.
25. 25. A protein C derivative, characterized in that it comprises at least 90% of the amino acids of a protein sequence selected from the SEQ ID NO: 3, 4 and 5.
26. 26. The protein derivative C according to claim 25, characterized in that it also comprises at least one substitution selected from 194T and 1. 94A of SEQ ID NO: 3.
27. 27. The protein C derivative according to claim 25, characterized in that the protein C derivative is activated.
28. 28. A method for treating thrombotic disorders, characterized in that it comprises: administering to a patient in need thereof a pharmaceutically effective amount of a protein C derivative, with greater resistance 5 to serpins selected from a group consisting of L194S, L194S: T254S, and L194A: T254S, in combination with an antiplatelet agent.
29. 29. A method for treating thrombotic disorders or vascular occlusive disorders and hypercoagulable conditions, Characterized in that it comprises: inhibiting the binding of active protein C polypeptides to PCI and al-AT serotonin inhibitor recognition sequences.
30. 30. The method according to claim 29, characterized in that the recognition positions of the 15 inhibitor are selected from S2, S3 'and S4'.
31. 31. An article of manufacture for human pharmaceutical use, characterized in that it comprises: packaging material and a bottle comprising lyophilized human protein C derivative with inactivation resistance by 20 serpins when compared to the natural protein C.
32. 32. The article of manufacture for human pharmaceutical use according to claim 31, characterized in that the packaging material comprises a label, which indicates that the protein derivative C is .jBfcia_. administered in a dose of approximately 0.01 μg / kg / hr up to 50 μg / kg / hr.
33. 33. A human protein C derivative, with resistance to inactivation by serpins compared to the natural protein C produced by the process, characterized in that it comprises: (a) transforming a host cell with a vector containing nucleic acid encoding a derivative of human C protein. (b) culturing the host cell in a medium suitable for the expression of the human protein C derivative; (c) isolating the human protein derivative from the culture medium; and (d) activating the human protein C derivative.
34. 34. The vector according to claim 33, characterized in that the nucleic acid codes for a human protein C derivative consisting of L194S, L194S: T254S, and L194A: T254S.
35. 35. The host cell according to claim 33, characterized in that the host cell is selected from the group consisting of 293 cells and AVI2 cells.
36. 36. A method for treating acute coronary syndromes and disease states that predispose to thrombosis, which comprises: administering to a patient in need thereof a therapeutically effective amount of a human protein C derivative with resistance to inactivation by serpins compared to the natural protein C, the derivative is selected from the group consisting of L194S, L194S: T254S, and L194A: T254S.
37. 37. The method according to the claim 36, characterized in that the acute coronary syndrome and the disease states that predispose to thrombosis are selected from the group consisting of myocardial infarction and unstable angina. SUMMARY OF THE INVENTION Novel protein C derivatives are disclosed. These polypeptides retain the biological activity of natural human protein C with substantially longer lifetimes in human blood. These polypeptides will require a less frequent administration and / or lower dose than the protein C r ural in the treatment of vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states that predispose to thrombosis. SEQUENCE LIST < 110 > Gerlitz, Bruce E. Jones, Bryan E. < i20 > ACTGVATED PROTEIN c POLYPEPTIDES < 130 > X-11901 < 140 > X-11901 < 141 > 2000-04-28 < 150 > P-1190 < 151 > 1999-04-30 < 160 > 10 < 170 > Patentln Ver. Í.O < 210 > 1 < 211 > 419 < 212 > PRT < 213 > Homo sapiens < 400 > 1 Wing Asn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu 1 5 10 15 Cye He Glu Glu Lie Cys Asp Phe Glu Glu Wing Lys Glu He Phe Gln 20 25 30 Asn Val Asp Aep Thr Leu Wing Phe Trp Ser Lyß His Val Asp Gly Asp 35 40 45 G n Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala Ser Leu Cys Cys 50 55 60 Gly His Gly Thr Cyü He Asp Gly He Gly Ser Phe Ser Cys Asp Cyß 70 75 80 65 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu 100 105 110 Val Gly Trp Arg ftrg Cye Ser Cys Wing Pro Gly Tyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys Hiß Pro Wing Vafel-ys Phe Pro Cys Gly Arg Pro 130 135 '14 Trp Lye Arg Met Glu Lys Lys Arg Ser His .Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln Glu? Sp Gln Val Asp Pro Arg Leu He Asp Gly Lys Met Thr 165 170 175 Arg Arg Gly Asp Ser Pro Trp Val Val Leu Leu Asp Ser Lys Lys 180 185 190 Lys Leu Ala Cys Gly Ala Val Leu He His Pro Ser Trp Val Leu Thr 195 200 205 Wing Wing Hie Cye Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp He 225 230 235 «240 Lye Glu Val Phe Val H e Pro Asn Tyr Ser Lys Ser Thr Thr Aep Asn 245 250 255 Asp He Wing Leu Leu Leu His Leu Wing Gln Pro? Thr Leu Being sln Thr 260 265 270 He Val Pro He Cys Leu Pro? Sp Ser Gly Leu? The Glu Arg Glu Leu 275 280 285 Asn Gln Wing Gly? »Ln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser 290 295 300 Ser Arg Glu Iys Glu? The Lys Arg Asn Arg Thr Phe Val Leu? Sn Phe 305 310 315 320 He Lys He Pro Val val Pro His Asn Glu Cys Ser Glu Val Met Ser 3? 330 335? Sn Met Val Ser Glu Asn Met Leu Cys Wing Gly He Leu Gly? ßp? Rg 340 345 350 Gln? S Wing Cys Giu Gly Asp Ser Gly Gly Pro Met Val? The Ser Phe 355 360 365 His Gly Thr Trp Pfte Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His? Sn Tyr Gly Val Tyr Thr Lys Val Ser? Xg Tyr Leu 385 390 395 400? Sp Trp He His Gly His He Arg Asp Lys Glu? La Pro Gln Lys Ser 405 41 415 Trp? Pro < 210 > 2 < 211 > 461 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Trp Gln Leu Thi Ser Leu Leu Leu Phe Val? Thr Trp Gly He 1 5 10 15 Ser Gly Thr Pro? Pro Leu? Sp Ser Val Pbe Ser Ser Glu? Rg 20 25 30? The His Gln Val Leu ? rg Zle Arg Lys? rg Ala? sn Ser Phe Leu Glu 35 40 45 Glu Leu? rg His Ser Ser Leu Glu? rg Glu Cys He Glu Glu He Cys 50 55 60 Aßp Phe Glu Glu? the Lys Glu He Phe Gln Asn Val? Sp Asp Thr Leu 65 70 75 80 Wing Phe Trp Ser Lys His Val Asp Gly Asp Gln Cys Leu Val Leu Pro 85 90 95 Leu Glu His Pro Cye Wing Ser Leu Cys Cys Gly Hie Gly Thr Cys He 100 105 110 Asp Gly He Gly Ser Phe Ser Cys Asp Cys Arg Ser Gly Trp Glu Gly 115 120 125? Rg Phe Cye Gln? Rg Glu Val Ser Phe Leu? Sn Cys Ser Leu? Sp? Sn 130 135 140 Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Val Gly Trp Arg? Rg Cys 145 150 155 160 Ser Cys? Pro Gly Tyr Lys Leu Gly Asp? Sp Leu Leu Gln Cys Hi? ** O¡á &JBkM & 165 170 175 Pro Ala Val Lye Phe Pro Cys Gly? Rg Pro Trp Lys? Rg Met Glu Ly? 180 185 190 Lys Arg Ser His Leu Lys? Rg? Sp Thr Glu? Sp Gln Glu? Sp sln Val 195 200 205? Sp Pro? Rg Leu He? S Gly Lys Met Thr? G? Rg Gly? Sp Ser Pro 210 215 220 Trp Gln Val Val Leu Leu? Sp Sea: Lys Lys Ly? Leu? The Cys Gly? 225 230 235 240 Val Leu He His Pro Ser Trp Val Leu Thr? La? His Cys Met? Sp 245 250 255 Glu Ser Lys Lys Leu Leu Val? Rg Leu Gly Gl? Tyr? Sp Leu? Rg? Rg 260 265 270 Trp Glu Lys Trp Glu Leu? Sp Leu? Sp He Lys Glu Val Phe Val His 275 280 285 Pro? Sn Tyr Ser Lys Ser Thr Thr? Ep? Sn? Sp He? Leu Leu His 290 295 300 Leu? The Gln Pro? The Thr Leu Ser Gln Thr He Val Pro He Cys Leu 305 310 315 320 Pro? ßp Ser Gly Leu? The Glu? Rg Glu Leu? Sn Gln? The Gly Gln ßlu 325 330 335 Thr Leu Val Thr Gly Trp Gly Tyr His Ser Ser? Rg Glu Ly? Glu? 340 345 350 Lys? Rg? Sn? Rg Thr Phe Val Leu? Sn Pbe He Lys He Pro Val Val 355 360 365 Pro His? Sn Glu Cys Ser Glu Val Met Ser? Sn Met Val Ser Gl? ? sn 370 375 380 Met Leu Cys? the Gly He Leu Gly? sp? rg Gln? sp? the Cy? Glu Gly 385 390 395 400? sp Ser Gly Gly Pro Met Val? the Ser Phe His Gly Thr Trp Phe Leu 405 410 415 Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gly Leu Leu Hiß? Sn Tyr Pt * titfttftMt 420 425 430 Glv Val Tyr Thr Lys Val Ser Arg Tyr Leu Asp Trp He His Gly His 44CF 445 435 He Arg Asp Lys Glu? The Pro Gln Lys Ser Trp? The Pro 450 455 460 < 210 > 3 «211 > 419 < 212 > PRT < 213 > Homo sapiens < 400 »3? La? Sn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu 1 5 10 15 Cys He Glu Glu He Cys? Sp Phe Glu Gl? Ala Lys Glu He Phe Gln 20 25 30 Asn Val Asp Asp Thr Leu? The Phe Trp Ser Lys His Val? Sp Gly? Sp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys? The Ser Leu Cys Cys 50 55 60 Gly Hie Gly Thr Cys He? Sp Gly He Gly Ser Phe Ser Cys? Sp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly? Rg Phe Cys Gln? Rg Glu Val Ser Phe Leu 85 90 95 Aen Cys Ser Leu? Sp? Sn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu 100 105 110 Val Gly Trp? Rg? Rg Cys Ser Cys Wing Pro Gly Tyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys His Pro? The Val Lys Phe Pro Cys Gly? Rg Pro 130 135 140 Trp Lye? Rg Met Glu Lys Lys? Rg Ser Hie Leu Lye? Rg? Sp Thr Glu 150 155, 160 145 Asp Gln Glu Asp Gln Val? Sp Pro Arg Leu He Asp Gly Lys Met Thr 165 170 175 • * »• * & & amp; É_fc__í ___ á_S _._ A__í_jl? Rg? Rg Gly? Sp Ser Pro Trp Gln Val Val Leu Leu? Sp Ser Lys Lys 180 185 190 Lyß Ser Ala Cys Gly? Val Leu Ilj? His Pro Ser Trp Val Leu Thr 195 200 205? ? His Cys Met? sp Glu Ser Lys Lys Leu l-eu Val? rg Leu Gly 210 215 220 Glu Tyr Asp Leu? rg? rg Trp Glu Lys Trp Glu Leu? sp Leu? sp He 225 230 235 240 Lys Glu Val Phe Val His Pro? Sn Tyr Ser Lys Ser Thr Thr? Sp? Sn 245 250 25S? Sp He? The Leu Leu His Leu? The Gis Pro? The Thr Leu Ser Gln Thr 260 265 270 He Val Pro He Cye Leu Pro? Sp Ser Gly Leu Wing Glu Arg Glu Leu 275 280 285? Sn Gln? The Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser 290 295 300 Ser? Rg Glu Lys Glu? The Lys? Rg Aen Arg Thr Phe Val Leu Asn Phe 305 310 315 320 He Lys He Pro Val Val Pro His Asn Glu Cys Ser Glu Val Met Ser 325 330 335? Sn Met Val Ser Gl? Aen Met Leu Cys Wing Gly Xle Leu Gly Asp Arg 340 345 350 Gln Aep Wing Cye Glu Gly Asp Ser Gly Gly Pro Met Val? The Ser Phe 355 360 365 His Gly Thr Trp Ph «Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu Hie? Sn? _? R Gly Val Tyr Thr Lys Val Ser? Rg Tyr Leu 385 ^ 90 395 400? Ep Trp He His Gl / His He? Rg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp Ala Pro ____! ___! ____ < 210 > 4 < 211 > 419 < 212 > PRT < 213 > Homo sapiens < 400 > 4? La? Sn Ser Phe Leu Glu Glu Leu? Rg His Ser Ser Leu Glu? Rg Glu 1 5 10 15 Cys He Glu Glu He Cys? Sp Phe Glu Glu? The Lys Glu He Phe Gln 20 25 30? In Val? Sp? Sp Thr Leu? The Phe Trp Ser Lys His Val? Sp Gly? Sp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys? The Ser Leu Cys Cys 50 55 60 Gly His Gly Thr Cye He? Sp Gly He Gly Ser Phe Ser Cys? Sp Cys 65 70 75 80? Rg Ser Gly Trp Glu Gly? Rg Phe Cys Gln ? rg Gl? Val Ser Phe Leu 85 90 95? Sn Cys Ser Leu? Sp? Sn Gly Gly Cys Thr His Tyr Cyß Leu Glu Glu 100 105 110 Val Gly Trp Arg Cyg Ser Cys Ala Pro Gly Tyr Lyß Leu Gly? Sp 115 120 125 Asp Leu Leu Gln Cys His Pro Wing Val Lys Phe Pro Cys Gly? Rg Pro 130 135 140 Txp Lys Arg Met Glu Lye Lys Arg Ser His Leu Lyß Arg Asp Thr Glu 145 150 155 '160 Asp Gln Glu Asp Gln Val Asp Pro Arg Leu He Asp Gly Lyß Met Thr 165 170 175 Arg Arg Gly Asp Ser Pro Trp Val Val Leu Leu? Sp Ser Lyß Lyß 180 185 190 Lys Ser? La Cys Gly Ala Val Leu He His Pro Ser Trp Val Leu Thr 195 200 205 Ala Wing Cys Met Asp Glu Ser Lye Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr? Sp Leu? Rg? Rg Trp Glu Lys Trp Glu Leu Asp Leu? Sp Xle 225 230 235 240 Lys Glu Val Phe Val His Pro? Sn Tyr Ser Lys Ser Thr Ser? Sp Asn 245 250 255? Sp He? The Leu Leu His Leu? The Gln Pro? The Thr Leu Ser Gln Thr 260 265 270 He Val Pro He Cye Leu Pro? Sp Ser Gly Leu? La Glu? Rg Glu Leu 275 280 285? Sn Gln? The Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser 290 295 300 Ser? Rg Glu Lys Glu? The Lys? Rg? Sn ? rg Thr Phe Val Leu? sn Phe 305 310 315 320 Xle Lys He Pro Val Val Pro His? Sn Glu Cys Ser "Glu Val Met Ser 325 330 335? Sn Met Val Ser Glu? Sn Met Leu Cys? The Gly He Leu ßly? Sp? Rg 340 345 350 Gln? Sp? La Cys Glu Gly? A Ser Gly Gly Pro Met Val? The Ser Phe 355 360 365 His Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly ßlu Gly Cyß 370 375 380 Gly Leu Leu His? Sn Tyr Gly Val Tyr Thr Lys Val Ser? Rg Tyr Leu 385 390 395 400? ßp Trp He His Gly His Zle? Rg? Sp Lys Glu? Pro Gln Ly? Ser 405 410 415 Trp? Pro < 210 > 5 < 211 > 419 < 212 > PRT < 213 > Homo sapiens < 400 > 5? La? Sn Ser Phe Leu Glu Glu Leu? Rg His Ser Ser Leu Glu? Rg Glu 10 15 Cys He Glu Glu He Cys? Sp Phe Glu Glu? The Lys Glu Xle Phe 01 »20? Sn Val? Sp? Sp Thr Leu? The Pbe Trp Ser Lys His Val? Sp Gly? Sp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys? The Ser Leu Cys Cyß 50 55 60 ? rg Ser Gly Trp Glu Gly? rg Phe Cys Gln? rg Glu Val Ser Phe Leu 85 90 95? sn Cys Ser Leu? sp? sn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu or 11n0 100 105 Val Gly Trp? rg ? rg Cys Ser Cyß? the Pro Gly Tyr Lys Leu Gly? sp 115 120 125 Asp Leu Leu Gln Cys His Pro Wing Val Lys Phe Pro Cyß Gly Arg Pro 140 130 «s Trp Lys Arg Met Glu Lyß Lyß Arg Ser Hiß Leu Lys? Rg? ßp Thr Olu 145 150 1SS '? Sp Gln Glu? Sp Gln Val Pro? rg Leu Zle? sp Gly Lyß Met Thr 165 170 175? rg? rg Gly? sp Ser Pro Trp Gln Val Val Leu Leu? sp Ser Lys Ly? 180 185 190 Lys Wing Wing Cys Gly Wing Val Leu Zle His Pro Ser Trp Val Leu Thr 195 200 205 Wing? His Cye Met? Sp Glu Ser Lys Lys Leu Leu Val? Rg Leu Gly 210 215 220 Gl? Tyr? Sp Leu? Rg? Rg Trp Glu Lys Trp Glu Leu? Sp Leu? Sp He 225 230 235 240 Lys Glu Val Phe Val Hie Pro? ßn Tyr Ser Lys Ser Thr Ser? Ep? Sn 245 250 255 Asp He? The Leu Leu His Leu? The Gln Pro? The Thr Leu Ser Gln Thr 260 265 270 Xle Val Pro Xle Cys Leu Pro? Sp Ser Gly Leu? The Glu? Rg Glu Leu 275 280 285? Sn Gln? The Gly Gln Glu Thr Leu val Thr Gly Trp Gly Tyr His Ser 290 295 300 Ser? Rg Glu Lys Gl? ? the Lys? rg? sn? rg Thr Phe Val Leu? sn Phe 305 310 315 320 He Lys He Pro Val Val Pro His? Sn Glu Cys Ser Glu Val Met Ser 325 330 335? Sn Met Val Ser Glu? Sn Met Leu Cys? The Gly Xle Leu Gly? Sp? Rg 340 345 350 Gln? Ep? The Cys Glu Gly? S Gly Ser Gly Pro Met Val? S Ser Pbe 355 360 365 Hie Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys 370 375 380, Gly Leu Leu His Aen Tyr Gly Val Tyr Thr Lyß Val. Ser? Rg Tyr Leu 385 390 395. 400? Sp Trp He Hiß Gly His Xle? Rg? Sp Lyß Glu? The Pro Gln Lyß Ser 405 410 415 Trp Wing Pro < 210 > 6 < 211 > 1260 < 212 > DNA < 213 > Homo sapiens < 400 > 6 gccaactcct tcctggagga gctccgtcac agcagcctgg agcgggagtg catagaggag 60 atctgtgact tcgaggaggc caaggaaact ttccaaaatg tggatgacac actggccttc 120 tggtccaagc acgtcgacgg egaccagtgc ttggtcttgc ccttggagca cccgtgcgcc 180 agcctgtgct gcgggcacgg cacgegcatc gacggcatcg gcagctecag ctgcgactgc 240 cgcagcggct gggagggccg cttctgccag cgcgaggtga gcttcctcaa ttgctcgctg 300 gacaacggcg gctgcacgca ttactgccta gaggaggtgg gctggcggcg ctgtagctgt 360 gcgcctggct acaagctggg ggacgacctc ctgcagtgtc accccgcagt gaagttccct 420 tgtgggaggc cctggaagcg gatggagaag aagcgcagtc acctgaaacg agacacagaa 480 gaccaagaag accaagtaga tccgcggctc attgatggga agatgaccag gcggggagac 540 agcccctggc aggtggtcct gctggactca aagaagaagc tggcctgcgg ggcagtgctc 600 atccacccct cctgggtgct gacagcggcc cactgcatgg atgagtccaa gaagctcctt 660 gtcaggcttg gagagtatga cctgcggcgc tgggagaagt gggagctgga cctggacatc 720 aaggaggtct tcgtccaccc caactacagc aagagcacca ccgacaatga catcgcactg 780 ctgcacctgg cccagcccgc caccctctcg cagaccatag tgcccatctg cctcccggac 840 agcggccttg cagagcgcga gctcaatcag gccggccagg agaccctcgt gacgggctgg 900 gcagccgaga ggctaccaca gaaggaggcc aagagaaacc gcaccttcgt cctcaacttc 960 atcaagattc ccgtggtccc gcacaatgag tgcagcgagg tcatgagcaa catggtgtct 1020 gagaacatgc tgtgtgcggg catcctcggg gaccggcagg atgcctgcga gggcgacagt 1080 ggggggccca tggtcgcctc cttccacggc acctggttcc tggtgggcct ggtgagctgg 1140 ggtgagggct gtgggctcct tcacaactac ggcgtttaca ccaaagtcag ccgctacctc 1200 gactggatcc atgggcacat cagagacaag gaagcccccc agaagagctg ggcaccttag 1260 < 210 > 7 < 211 > 1386 c212 > DNA < 213 > Homo sapiens < 400 > 7 _ atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240 gccttctggt ccaagcacgt cgacggrtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gcggctggga gactgccgca gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480 agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 aagaagacca acagaagacc agtagatccg cggctcattg atgggaagat gaccaggcgg 660 ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagctggc ctgcggggca 720 gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ggcttggaga ctccttgtca gtatgacctg cggcgctggg agaagtggga gctggacctg 840 gacatcaagg agg tcttcgt ccaccccaac tacagcaaga gcaccaccga caatgacatc 900 gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag. 1386 < 210 > 8 < 211 > 1386 < 212 > DNA < 213 > Homo sapi ns tKlKA j_fe_-C._ s__ ^^ _-; Jew. ^^^ MaB & < 400 > 8 atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240 gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gcggctggga gactgccgca gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480 agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 aagaagacca acagaagacc agtagatccg cggctcattg atgggaagat gaccaggcgg 660 ggagacagcc CCTGG AGGT ggtcctgctg gactcaaaga agaagtccgc ctgcggggca 720 gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ggcttggaga ctccttgtca gtatgacctg cggcgctggg agaagtggga gctggacctg 840 gacatcaagg aggtr ct cgc ccaccccaac tacagcaaga gcaccaccga caatgacatc 900 gcactgctgc acctggc .. '. < gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcg gcctt? -AGA gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtg agggct TGG gctccttcac aactacggcg tttacaccaa agtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag 1386 < 210 > 9 < 211 > 1386 < 212 > DNA < 213 > Homo ßap enc, < 400 > 9 atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgcca actcctzoct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240 gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcc tgtgctj -75 gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gcggctggya gactgccgca gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tcgctggaca acggrggc -.g cacgcattac tgcctagagg aggtgggctg gcggcgctgt agctgtgcgc tggct R0? Caa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 aagaas acagaagacc _. '. Ca 660 agtagatccg cggctcattg atgggaagat gaccaggcgg ggagacagcc cctggcasct ggtcctgctg gactcaaaga agaagtccgc ctgcggggca 720 gtgctcatcc CACS ctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ctccttgtca gtatgacctg cggcgctggg agaagtggga gctggacctg 840 gacatcaagg acgtcttcgt ccaccccaac gcaccagcga tacagcaaga caatgacatc 900 gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagae cctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtgggg ggcc catggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtg agggctgtgg gctccttcac aactacggcg tttaca. »A agtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag 1386 <; 210 > 10 < 211 > 1386 < 212 > DNA < 213 > Homo sapiens < 400 > 10 atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttg actcagtgtc ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240 gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gcggctggga gactgccgca gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480 agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 aagaagacca acagaagacc agtagatccg cggctcattg atgggaagat gaccaggcgg 660 ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaaggccgc ctgcggggca 720 gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ctccttgtca ggcttggagt gtatgacctg cggcgctggg agaagtggga gctggacctg 840 gac atcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccagcga caatgacatc 900 gcactgctgc acctggr.cca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag 1386