MXPA00011807A - Human protein c polypeptide - Google Patents

Abstract

An isolated human protein C polypeptide with a truncated heavy chain is described. This isolated polypeptide retains the biological activity of the wild-type human protein C. This polypeptide will be useful in the treatment of vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states predisposing to thrombosis.

Description

PO IPEPTIDO OF PROTEINA C HUMANA Background of the Invention The present invention is found in the field of human medicine. More specifically, the invention relates to an isolated human protein C polypeptide having a truncated heavy chain, to methods for using this human protein C polypeptide, and to pharmaceutical compositions of this human protein C polypeptide.

Description of the Invention Protein C is a serine-dependent vitamin K protease and a naturally occurring anticoagulant that plays a role in the regulation of vascular homeostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human protein C is made mainly in the liver as a single polypeptide of 461 amino acids. This precursor molecule undergoes various post-translational modifications including 1) cleavage of a signal sequence of 42 amino acids; 2) proteolytic removal of the zymogen REF. 124918 single-stranded lysine residue at position 156 and the arginine residue at position 157 to make the double-stranded form of the molecule, (ie, a light chain of 155 amino acid residues bound by a bisulfide bridge to the heavy chain that contains the protease serine of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues grouped in the first 42 amino acids of the light chain, resulting in 9 gamma-carboxyglutamic acid residues; and 4) binding of a carbohydrate at 4 sites (one in the light chain and 3 in the heavy chain). Finally, the circulating double-stranded zymogen is activated by the action of the thrombin / thrombomodulin complex that cleaves the activation peptide (residues 158 to 169) of the activated protein C (aPC) producing the circulating zymogen. In conjunction with other proteins, protein C functions as perhaps the most important regulator regulator of blood coagulation factors that promotes thrombosis. Therefore, the protein C enzyme system represents a major physiological mechanism of anticoagulation.

The critical role of protein C in controlling hemostasis is exemplified by the increased speed in thrombosis in a heterozygous deficiency, 'resistance to protein C (for example, due to the common mutation of factor V Leiden) and the fatal outcome of the homozygous deficiency of protein C without treatment. Activated human protein C, both derived from plasma and recombinant, has been shown to be an effective and safe antithrombotic agent in a variety of animal models, both for venous and arterial thromboses. It has been shown in recent clinical studies that protein C is effective for human thrombotic diseases including the treatment of protein C deficiencies and microvascular thrombosis, such as disseminated intravascular coagulation associated with sepsis. Unfortunately, during the activation of protein C, the C-terminus of the heavy chain is cleaved which has the potential to change the structure of the protein, which in turn can lead to a less elegant pharmaceutical preparation. Applicants have discovered that this truncated form of aPC is biologically active. The present invention, therefore, provides an isolated aPC polypeptide with a truncated heavy chain, a method for preferentially preparing this polypeptide, and its use as a medicament. The present invention provides a human protein C isolated polypeptide comprising: a light chain and a truncated heavy chain wherein this polypeptide is SEQ ID NO: 1. The present invention further provides a recombinant DNA molecule encoding the isolated polypeptide of human protein C with a truncated heavy chain, wherein this DNA molecule is SEQ ID NO: 2. The present invention further provides a method for treating a thrombotic disease in a patient in need thereof, which comprises, administering to this patient a pharmaceutically effective amount of a polypeptide isolated from human protein C with a truncated heavy chain.

Methods and aspects for producing the isolated human protein C polypeptide with a truncated heavy chain are also an aspect of this invention. For the purposes of the present invention, as described and claimed herein, the following terms are as defined below.

The term aPC or activated protein C, either recombinant or derived from the plasma-aPC includes and preferably is human protein C, although aPC can also include other species or derivatives having biological, sterolitic, idolytic, proteolytic activities (anticoagulants or profibrinolytic) of protein C. Examples of protein C derivatives are described in U.S. Patent No. 5,453,373 and U.S. Patent No. 5,516,650, the complete teachings of which is incorporated herein by reference. APTT - activated partial thromboplastin time. HPC - human protein C zymogen. r-hPC - Recombinant zymogen of human protein C, produced in prokaryotic cells, eukaryotic cells or transgenic animals. r-aPC - recombinant human activated protein C produced by activating r-hPC in vi tro by direct secretion of the activated form of protein C from prokaryotic cells, eukaryotic cells, or transgenic animals [WO97 / 20043] including, for example, the secretion of human kidney 293 cells in the manner of a zymogen which is then purified and activated by methods that are well known to the skilled artisan and which are demonstrated in U.S. Patent No. 4,981,952, and, which the complete teachings are incorporated into the present for your reference. Zymogen - refers to the secreted, inactive forms of protein C either single chain or two chain. Truncated heavy chain - refers to the heavy chain of protein C that has cleaved its 4 amino acids C-term. For activated human protein C, the truncated heavy chain contains residues 170-415 of amino acids as indicated in SEQ ID NO: 1. Light chain - refers to the light chain of protein C. For human activated protein C, the light chain contains residues 1-155 of amino acids or polypeptides having one or more amino acids that are deleted from the C-terminus. Thrombotic disorder - a disorder that is related to, or that is affected by the formation or presence of a clot in the blood within a blood vessel. Thrombotic disorders include, but are not limited to, stroke, myocardial infarction, unstable angina, abrupt closure followed by angioplasty or placement of a plunger and thrombosis as a result of peripheral vascular surgery. Vascular occlusive disorders and hypercoagulable states are disorders that include but are not limited to sepsis, disseminated intravascular coagulation, fulminating purpura, major trauma, major surgery, burns, respiratory agony syndrome in adults, transplants, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, hemorrhagic viral fever, thrombotic thrombocytopenic purpura, and haemolytic uraemic syndrome. Pharmaceutical formulation - a formulation or solution that is suitable for administration as a therapeutic agent. The term "pharmaceutically effective amount" as used herein, represents an amount of a compound of the invention that is capable of inhibiting a thrombotic disorder in mammals. The particular dose of the compound administered according to this invention is, of course, determined by the particular circumstances surrounding the case, including the compound administered, the particular condition to be treated, and similar considerations. The structure of HPC is rather complex due to the number of post-translational modifications. The HPC structure consists of a light chain (residues 1-155) and a heavy chain (residues 158-419). The HPC molecule is originally expressed as a polypeptide of 419 amino acids, but before the secretion of the cell, most of the protein is converted to its heterodimeric form by removing the dipeptide Lys-Arg at positions 156-157 . Human recombinant protein C (r-hPC) is 'analogous to the HPC in its structure and complexity. During the conversion of r-hPC, in r-aPC, thrombin selectively cleaves the activation dodecapeptide (residues 158-169). However, applicants have discovered conditions where a tetrapeptide (residues 416-419) can also be excised from its C-terminus of the heavy chain resulting in the formation of des polypeptide 416-419 aPC. Applicants have further discovered that this form of aPC is biologically active (see Example 1, Table 1), which entails its use as a therapeutic agent alone or in combination with a natural aPC. The present invention therefore provides a des (416-419) aPC, isolated, a method for preferentially preparing des (416-419) aPC, and its use as a medicament. The invention also provides DNA compounds for use in the preparation of protein C having a truncated heavy chain. These compounds of DNA comprises the coding sequence for the light chain of human protein C placed immediately adjacent to, 3 'of, and in a transducer reading frame with the wild-type zymogen prepropeptide sequence of protein C. DNA sequences they also code for the dipeptide Lys-Arg that is processed during the maturation of the protein C molecule, the activation peptide and the truncated heavy chain of the protein C molecule. Those skilled in the art recognize that, due to the ease of degeneracy of the genetic code, a variety of DNA compounds can encode the activated protein C polypeptide described above. U.S. Patent No. 4,775,624, of which its full teachings are incorporated herein by reference, discloses and claims the DNA sequence encoding the wild-type form of the human protein C molecule. The right-handed artisan can easily determining which changes in the DNA sequences can be used to structure the other DNA sequences encoding the exact polypeptide as described herein, the invention however is not limited to specific DNA sequences.

Consequently, the structure described below for the preferred compound of DNA, vectors and transformants of the invention are merely illustrative and do not limit the scope of the invention. The DNA compound of the present invention can be prepared by sequence-directed mutagenesis of the human protein C gene. The cultures are obtained and the plasmids are isolated using conventional techniques, and can then be directly transfected into eukaryotic host cells for the production of protein C with a truncated heavy chain. It is preferable to transfect the plasmids in the host cells they express for the immediately preceding gene product of the ElA adenoviruses, in which the BK enhancer found in the GBMT transcription control unit functions to efficiently enhance expression in the presence of from ElA. The GBMT transcription control unit is described more fully in U.S. Patent No. 5,573,938 and Serial European Patent Application No. 91301451.0, of which the full teachings are incorporated herein by reference. Skilled technicians realize that a variety of host cells are expressing, or can cause them to express, a product of an immediate previous gene of a large DNA virus. The most preferred cell line for the expression of the human protein C derivatives of the present invention is the human kidney cell line 293 which is described in U.S. Patent No. 4,992,373, of which the full teachings are incorporated in the present for your reference. . After expression in the cell line, the derivatives are purified from the supernatant of the cell culture using the procedure in the Patent of E. U. A. No. 4, 981,952, of which his complete teachings are incorporated herein for reference. The DNA sequence of the invention can be synthesized chemically, or by combining restriction fragments, or by a combination of procedures that are known in the art. Instruments for synthesizing DNA are available and can be used to structure the DNA compounds of the present invention. Illustrative vectors of the invention comprise the GBMT transcription unit placed to stimulate the transcription of the coding sequences by the adenovirus late promoter. Those skilled in the art recognize that a large number of promoters, enhancers, and eukaryotic expression vectors are known in the art and can be used to express the DNA sequences to produce the derivatives of protein C of the present invention. Those skilled in the art also recognize that a eukaryotic expression vector can function without an enhancer element. The key aspect of the present invention resides in the novel DNA sequences and the corresponding aPD with a truncated heavy chain that is made from those sequences. Alternatively, the activated protein C polypeptide described herein can be prepared by reacting the activated protein C with a thrombin to cleave the tetrapeptide (residues 416-419) of the C-terminus of the heavy chain. Additional cleavage is obtained by exposing aPC to thrombin for an extended period of time, generally for 10 minutes or for 3 to 5 hours under conditions that are appreciated in the art. The aPC polypeptides that are prepared by treating a r-aPC with thrombin or by direct expression of eukaryotic cells having similar activity as aPC. Therefore, aPC having a truncated heavy chain is effective for the treatment of thrombotic diseases in humans that include replacement therapy in the treatment of protein C deficiencies, vascular occlusive disorders and hypercoagulable states including: sepsis, disseminated intravascular coagulation, fulminating purpura, major trauma, major surgery, burns, adult respiratory distress syndrome, transplants, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral haemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uraemic syndrome, as well as thrombotic disorders and in disease states that predispose to thrombosis, such as myocardial infarction and stroke, by administering a polypeptide isolated from protein C of 'human having a truncated heavy chain. Another embodiment of the present invention is a method for treating thrombotic disorders comprising: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C isolated polypeptide having a truncated heavy chain in combination with an antiplatelet agent . Another embodiment of the present invention is a method of treating sepsis comprising administering to a patient in need thereof a pharmaceutically effective amount of a human protein C isolated polypeptide having a truncated heavy chain in combination with an enhancer protein. of bacterial permeability. A human protein C isolated polypeptide having a truncated heavy chain can be formulated in a manner analogous to aPC with a pharmaceutically acceptable diluent. Preferably, it includes a sugar such as sucrose, salt and a citrate buffer. Preferably, the aPC derivatives are prepared at a pH of 5.5 to 6.5. Generally, the pharmaceutical doses of the aPC derivatives described herein are analogous to those of natural aPC, preferably 0.01 mg / kg / hr to 0.05 mg / kg / hr. The following preparations and examples are for illustrative purposes only. Someone skilled in the art realizes that there are additional methods to prepare and activate recombinant protein C.

Preparation 1 Preparation of Human Protein C Recombinant human protein C (r-HPC) is produced in Human Kidney 293 cells by techniques known to the skilled artisan, such as those set forth in US Patent No. 4,981,952 , of which the complete teachings are incorporated into the present for your reference. The gene encoding human protein C is described and claimed in the patent of E. U. A. No. 4,775,624, of which the full teachings are incorporated herein for reference. The plasmid that is used to express human C protein in 293 cells is a pLPC plasmid which is described in U.S. Patent No. 4,992,373 and U.S. Patent No. 5,661,002, of which the full teachings are incorporated in the present for your reference. The structuring of the pLPC plasmid is also described in European Patent Publication No. 0 445 939, and in Grinnell, et al. , 1987, Bi o / Technolgy 5: 1189-1192, of which his teachings are incorporated into the present for his reference. In brief, the plasmid is transfected into 293 cells, then the stable transformants are identified, subcultured and grown in a serum-free medium. After fermentation, the cell-free medium is obtained by microfiltration. Human protein C is separated from the culture fluid by an adaptation of the techniques in U.S. Patent No. 4,981,952, of which the full teachings are incorporated herein by reference. The purified medium is made 4 mM in EDTA before it is absorbed in an anion exchange resin (Fast-Flow Q, Pharmacia). After washing with 4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumes of 20 mM Tris, 150 mM NaCl, pH 7.4, the agglutinated zymogen of recombinant human protein C is eluted with 20 mM Tris , 150 mM NaCl, 10 mM CaCl 2, pH 7.4. The eluted protein is more than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis. Further purification of the protein is achieved by making the 3 M protein in NaCl followed by an adsorption of a hydrophobic interaction resin (Toyopearl Phenyl 650M, TodoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl 2, pH 7.4. After washing with two column volumes of the equilibrium buffer without CaCl 2, the recombinant human protein C is eluted with 20 mM Tris, pH 7.4. The eluted protein is prepared for activation by removing residual calcium. The recombinant human protein C is passed through an affinity metal column (Chelex-100, Bio-Rad) to remove the calcium and again agglutinate the anion exchanger (Fast Flow Q, Pharmacia). Both of these columns are serially configured and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4. After loading the protein, the Chelex-100 column is washed with a column volume of the same buffer before disconnecting it from the series. The ion exchange column is washed with 3 column volumes of the equilibrium buffer before eluting the protein with 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. The protein concentrations of the solutions of recombinant human protein C and recombinant activated protein C are quantified by UV 280 nm extinction EO .1% = 1.81 or 1.85, respectively, Preparation 2 Activation of Recombinant Human Protein C Bovine thrombin is coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4 ° C. The coupling reaction is performed on resin already packed in a column using approximately 5,000 thrombin units / ml resin. The thrombin solution is circulated through the column for approximately 3 hours before adding MEA at a concentration of 0.6 ml / l of the circulating solution. The solution containing MEA is circulated for an additional 10-12 hours to ensure complete blockage of the unreacted amines on the resin. Then, from blocking, the resin coupled with the thrombin is washed with 10 column volumes of 1M NaCl, 20 mM Tris, pH 6.5 to remove all the non-specifically bound protein, and is used in the activation reactions after equilibrating in the activation damper.

The purified r-HPC is made in 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg / ml with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material is passed through a thrombin column equilibrated at 37 ° C with 50 mM NaCl either with 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate is adjusted to allow a duration of approximately 20 minutes of contact time between the r-HPC and the thrombin resin. The effluent is collected and immediately evaluated for its amidolytic activity. If the material does not have a specific activity (amidolytic) comparable to an established aPC standard, it is recycled on the thrombin column to activate the r-HPC until its completion. This is followed by a 1: 1 dilution of the material with 20 mM buffer as above, with a pH of either 7.4 or 6.5 to maintain the aPC at lower concentrations while waiting for the next process step. The removal of the leached thrombin from the aPC material is carried out by agglutinating the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in an activation buffer (either in 20 mM Tris, pH 7.4 or 20 mM Tris -acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact with the anion exchange resin under these conditions, but passes "through the column in the effluent of the sample application." Once the aPC is loaded onto the column, a wash with 2-6 volumes of column with 20 mM of the equilibration buffer is made before eluting the agglutinated aPC with an elution step using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. The anticoagulant antiviral of active protein C is determined by quantifying the prolongation of the clotting time in the clotting titration of activated partial thromboplastin time (APTT). in a dilution buffer (1 mg / mL bovine serum albumin for radioimmunoassay quality [BSA], 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) that covers the protein concentration 1 C from 125-1000 ng / mL, while the samples are prepared in various dilutions at this concentration range. For each sample cuvette, 50 μL cold equine plasma and 50 μL of the part-time activated thromboplastin reconstituted reagent (APTT Reagent, Sigma) are added and incubated at 37 ° C for 5 minutes. After incubation, 50 μL of the appropriate samples or standards are added to each cuvette. The dilution buffer is used in place of the sample or standard to determine the baseline coagulation time. The timer of the fibrometer (CoA Hemostasis Detection Analyzer, American Labor) begins immediately after the addition of 50 μL 37 ° C 30 mM CaCl2 to each sample or standard. The concentration of activated protein C in the samples is calculated from the linear regression equation of the standard curve. The coagulation times reported here are the average of a minimum of three replicates, including samples of the standard curve.

Example 1 Preparation of Activated Pro-Ein Des 416-419 The aPC is used as the raw material for preparing des 416-419 aPC. The immobilized resin of thrombin (10 mg thrombin / ml CH-Sepharose 4B resin) is used. N-glycosidase F is purchased from Boehringer Mannheim. Equine plasma is a product of Animal Technologies, Inc. (Tyler, TX). Activated CH Sepharose ® 4B is purchased from Pharmacia Biotech. All other chemicals are reactive ACS grade and are commercially available. An amount of 6 mL of immobilized thrombin resin is placed in a 0.2 micron filter. The resin is washed with approximately 5x20 mL of 40 mM Tris buffer, pH 7.02. The immobilized washed thrombin resin is transferred to a 50 mL polypropylene bottle, an aliquot of 12 mL of a 2.67 mg / mL aPC solution is added (120 mg aPC in 45 mL of 40 mM Tris buffer, pH 7.02 ) to the bottle and the final volume of the suspension is adjusted by approximately 21 L with the Tris buffer. The suspension is incubated at room temperature with constant gentle agitation. After the incubation times of 10, 25, 50, 100, 160 and 240 minutes, 3 mL of aliquots of the suspension are removed from the bottle. These aliquots are centrifuged at 2,000 RPM (ICE Centrifuge CRU-5000) for 1 minute and the supernatants are transferred to various 1.5 mL polypropylene bottles. These jars are placed immediately in a dry ice bath to freeze the solution. A control sample is prepared at the same time using deactivated CH-Sepharose 4B resin that does not contain the immobilized thrombin. Protein Content Assessment. The aliquots (150 mcL) of the sample solution are diluted with 450 mcL of 40 mM Tris buffer, pH 7.02 or reactive water. The sample cell is rinsed twice with the sample solution and the UV absorbance (at? = 280 nm) of the solution is quantified. The Tris buffer or reactive water are used as the model for this quantification. LC / MS Assessment for Protein Polypeptide Distribution. The aliquots of approximately 600 mcL of the sample solution are mixed with 240 mg of urea, 88 mcL of 3 M Tris buffer (pH = 8.0) and 15 mcL of 50 mg / mL of a solution of dithiothreitol and the mixture incubate at 37 ° C for 30 minutes. The sample is rented by adding 50 mcL of 50 mg / mL of iodoacetamide solution and incubated at room temperature in the dark for 30 minutes. The samples are then desalted on a disposable gel filtration column, deglycosylated with N-glycosidase F and analyzed by LC / MS. RP-HPLC evaluation. From 300 to 400 microliters of aliquots of the solution of the thawed sample are mixed with a sufficient volume of a 0.1% TFA solution to obtain a solution of approximately 1 mg / mL. This solution is used as the high concentration sample. The low concentration sample is prepared by mixing 50 mcL of aliquots of the high concentration sample with 450 mcL of a 0.1% TFA solution. One hundred microliters of aliquots of each sample of high concentration and. low are injected into the HPLC system. APTT rating. The sample is evaluated in an Automated Activated Thromboplastin Part Time Analyzer (APTT) CoaLab. All samples are diluted using manual pipettes to concentrations between 410 ng and 420 ng of aPC / mL. A reference standard for aPC that has an assigned power of 303 U / mg is used for this assessment. Des (416-419) aPC that is generated as described above has a biological activity similar to that of natural aPC that is quantified by APTT titration. The relationship between the anticoagulant activity of APTT and the percentage of Des 416-419 aPC is shown in Table 1. The percentage of Des 416-419 aPC can be as high as 68% and still maintains essentially the same anticoagulant activity as the aPC natural. In general, aPC which is made by the methods described herein contain from about 1% to about 25% Des 416-419 aPC.

Table 1 It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (8)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A polypeptide isolated from human protein C characterized in that it comprises: a light chain and a truncated heavy chain.

2. The polypeptide according to claim 1, characterized in that this polypeptide is SEQ ID NO: 1.

3. A recombinant DNA molecule encoding the human protein C polypeptide according to claim 1.

4. The recombinant DNA molecule according to claim 3, characterized in that this DNA molecule is SEQ ID NO: 2.

5. The polypeptide isolated from human protein C according to claim 1, characterized in that this human protein C polypeptide is activated.

6. A method for treating thrombotic disorders, vascular occlusive disorders and hypercoagulable states in a patient in need thereof, comprising: administering to this patient a pharmaceutically effective amount of an isolated active protein C polypeptide with a truncated heavy chain in accordance with claim 1.

7. A vector, characterized in that it comprises a nucleic acid according to claim 2.

8. A host cell characterized in that it comprises an isolated nucleic acid according to claim 2. SUMMARY OF THE INVENTION A polypeptide isolated from human protein C with a truncated heavy chain is described. This isolated polypeptide retains the biological activity of the wild-type human protein C. This polypeptide is useful for the treatment of vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states that predispose to thrombosis. LIST OF SEQUENCES < 110 > Eli Lilly and Company < i20 > Poiipépti or Protein C < X30 > heavy chain truncated protein C «X40 > X12279 < 141 > 1999-06-02 < 160 > 2 < 170 patßntln Ver. 2.0 < 210 > X < 211 > 1244 < 212 > DNA < 213 > Artificial Sequence < 220 > 223 > Description of Artificial Sequence: recombinant human protein C truncated in the C-term < 400 > i gecaactect tcctggagga gctccg cac agcagcctgg agcgggagtg catagaggag 60 atctgtgact tcgaggaggc caaggaaatt ttceaaaatg tggatgacac actggccttc 120 tggtccaagc acg cgacgg tgaccagtgc ttggtcttgc ccttggagca cccgtgcgcc ISSO agcctgtgct gcgggcacgg cacgtgcatc gacggcatcg gcagcttcag ctgcgactgc 240 cgcagcggct gggagggccg cttctgscag cgcgaggtga gcttcctcaa ttgctcgctg 300 gacaacggcg gctgcacgca ttactgccta gaggaggtgg gctggcggcg ctgtagctgt 360 gcgcetggct acaagctggg ggacgacctc ctgcagtgtc accccgcagt gaagttccct 420 tg gggaggc cctggaagcg gatggagaag aagcgcagtc acctgaaacg agacacagaa 480g & ccaagaag accaagaga tecgcggetc attgatggga agatgaceag gcggggagac 540 agcccctggc aggggtcct gctggactca aagaagaagc tggcctgcgg ggcagtgctc OSs atccacocct cctggggct gacagcggcc cactgcatgg atgagtccaa gaagctcctt 660 g caggcttg gagagatga cctgcggcgc tgggagaagt gggagctgga cctggacatc 720 aaggaggtct tcgtccacce aagagcacca caactacagc ccgacaatga catcgcactg 780 ctgcacctgg cccagcccgc caccctctcg cagaccatag tgcccatctg cctcccggac 840 agcggccttg cagagc gcga gctcaatcag gecggccagg agaccctcgt gacgggctgg 900 ggctaccaca gcagccgaga gaaggaggcc aagagaaacc gcaccttcgt cctcaacttc 960 Alcaagattc ccgtggtccc gcacaatgag tgcagcgagg tcatgagcaa catggtgtct 1020 gagaacatgc tgtgtgcggg catcctcggg gaccggcagg atgcctgega gggcgacagt 1080 ggggggccca tggccgeetc cttccacggc acctggttcc tggtgggcct ggtgagctgg 1140 ggtgagggct gtgggctcct tcacaactac ggcgtttaca ccaaagtcag ccgctacctc 1200 gactggatcc atgggcacat cagagacaag gaagcccccc agaag 1245 < 210 > 2 211 > 415 < 212 PH < 2i3 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence; amino acid sequence of recombinant human protein C chorus truncation at the C-term < 400 > 2 Wing Asn Ser Phe Leu Glu Glu Leu Arg His Being be Leu Glu Arg Glu 1 5? O 15 Cys He Glu Glu He Cys Asp Phe Glu Glu Wing Lys Glu lie Phe Gln 20 25 30 As Asp Asp Thr Iißu Wing Phe Trp Ser Lys His Val Asp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu Hi? Pro Cys Ala Ser Leu Cys Cys 50 55 60 Gly His Gly Thr Cys He Asp Gly lie Gly Ser Phe Ser Cys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu 85 90 95 Aßn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu 100 105 lio Val Gly Trp Arg Cyg Ser Cyß Wing Pro Gly Tyr Lys Leu Gly Asp 115 120 125? ßp Leu Leu Gln Cys His Pro Wing Val Lys Phe Pro Cys ßly Arg Pro 130 135 140 Trp Lys Arg Met Glu Lyß Lys Arg Ser His Leu Lys Arg Asp Thr Qlu 145 150 155 160 Asp Gln Glu Asp Gln Val? Sp Pro? Rg Leu Xle Asp Gly Lys Met Thr 165 170 175 Arg Arg Gly? Sp Ser Pro Trp Gln Val Val Leu Leu? Sp Ser Lys Lys 180 185 190 Lys Leu Ala Cys Gly? Val Leu He His Pro Ser Trp Val Leu Thr 195 200 205? The? His Cys Met? Sp Glu Ser Lys Lys Leu Leu Val? Rg Leu Gly 210 215 220 Glu Tyr? Sp Leu? Rg ? rg Trp Glu Lys Trp Glu Leu? sp Leu? sp He 225 230 235 240 Lys Glu Val Phe Val Hxs Pro? Sn Tyr Ser Lys Ser Thr Thr? Sp? Sn 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 Cys Leu Pro? Sp Ser Gly Leu? La ßlu? Rg Glu Leu 275 280 285? Sn Gln Ala 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 He Lys Xle Pro Val Val Pro His? Sn Glu Cys Ser Glu Val Met Ser 325 330 335 Asn Met Val Ser Glu? Sn Met Leu Cys? La ßly He Leu Gly? Sp? Rg 340 345 350 Gln? Sp? The Cys Glu Gly? Sp 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 Glu Gly Cys 370 375 '380 .Gly Leu Leu His? S "nTyr Gly Val Tyr Thr Lys Val Ser? Rg Tyr Leu 385 390 395 400 Asp Trp Xle His Gly His Xle? Rg? Sp Lys Glu? The Pro G n Lys 405 410 415