WO2000050612A2 - Recombinant protein c activator and uses therefor - Google Patents

Abstract

A highly glycosylated form of Protein C Activator (PCA), produced recombinantly in yeast, has properties which distinguish it from PCAs obtained by prior art methods. Isolated nucleotide sequences encoding poylpeptides comprising natural PCA proteins or biosynthetic variants thereof, and the production of synthetic DNA molecules which encode a PCA protein of the snake Agkistrodon contortrix contortrix (ACC) or biosynthetic variants thereof are provided. Substantially pure protein preparations including polypeptides comprising a PCA protein, for methods of producing these preparations and blood coagulation diagnostic kits including these preparations are also provided.

Description

WO 00/50612 PCTtUSOO/04406

RECOMBINANT PROTEIN C ACTIVATOR AND USES THEREFOR

Field of the Invention

The present invention relates to recombinantly produced proteins which have activity affecting the blood coagulation system, and to their use in diagnostic kits. In particular, the invention relates to recombinant Protein C Activators that are useful in diagnostic kits for the evaluation of the level or activity of blood coagulation factors. Background of the Invention

Protein C is an enzyme in the blood coagulation pathway that is normally activated by a complex series of events to become Activated Protein C (APC). APC has an anticoagulant effect by inactivating Factors Va and Villa and, thereby, slowing the conversion of fibrinogen to fibrin and clot formation. Certain snake venoms contain very potent Protein C Activator (PCA) proteins which promote the conversion of Protein C to APC, thereby inhibiting clotting and allowing the venom to reach the systemic circulation. Snake venom PCA preparations are used in certain diagnostic assays in which it is desirable to activate Protein C.

The Protein C pathway has gained increasing clinical and research attention as inherited disorders have been discovered involving Protein C, its cofactor Protein S, and APC resistant forms of Factor V. In particular, congenital or acquired deficiencies of Protein C (PC) and Protein S (PS), and genetic mutation of Factor V constitute major factors affecting thrombotic risk. Physiologically, PC is activated by thrombin bound to an epithelial cell surface molecule, thrombomodulin. Activation of PC results in a remarkable prolongation of plasma clot time, a property that is useful in the development of assays for PC and PS. In the presence of normal levels of PS the prolongation of the plasma clot time is directly proportional to the PC concentration. Similarly, in the presence of normal levels of activated PC the prolongation of the plasma clot time is directly proportional to the PS concentration. Therefore, PC assays can be developed by careful optimization of the PS concentration and vice versa. Current commercial tests for PC and/or PS achieve activation of endogenous PC via a purified fraction from Agkistrodon contortrix contortrix (ACC) venom, commercialized under the trade name of Protac® (Pentapharm AG, Basel, Switzerland) (see also Martinoli and Stocker 1986). A "Protein-C-Activator Activity" (PCAA) was first reported in extracts from ACC venom in 1985 (Exner et al. 1985). Since then, five groups have reported on various preparations of PCAA from ACC, purified by different methods (Klein and Walker 1986; Stocker, et al. 1987; Kisiel, et al. 1987a and 1987b; Orthner, et al. 1988; Exner and Vaasjoki 1988), and two groups have obtained patents (U.S. Pat. No. 4,849,403 to Stocker and Svendsen, and U.S. Pat. No 4,908,314 to Orthner). Only one of the purified ACC-PCAAs, "ACC-C", has been subjected to amino acid sequencing (McMullen et al. 1989). The McMullen sequence showed that ACC PCA is a typical serine protease, bearing homology with other snake venom serine proteases as well as with thrombin. It also has the conserved amino acid residues that are involved in charge relays found in the active centers of other serine proteases.

Interestingly, the properties published by each group studying their respective ACC-PCA preparations are contradictory. For example, although it has been reported that Protac® is not sensitive to serine protease inhibitors (Stocker, et al. 1987), other reports clearly claim a serine protease activity for ACC-PCA preparations (Orthner et al. 1988; McMullen et al. 1989; Exner and Vaasjoki 1988). On the other hand, another paper (Stϋrzbecher et al. 1991) appears to claim that Protac® contains serine protease activity. The relatively high level of impurities in preparations obtained from snake venom may contribute to the variability and inconsistency within in vitro diagnostics assays.

Optimization of PC and PS tests on any instrument system requires that assay characteristics and specifications be met. Important specifications include a relatively constant activation range, a significant difference between 0 and 100% activity, a maximum clot time at 100% activity, and a maximum reportable clot time at 120% activity. Manufacturing of PC and PS kits conforming to rigid specifications is difficult due largely to the lack of batch-to-batch consistency in commercially available ACC-PCAA preparations. Titration of these preparations to achieve these specifications has been particularly fastidious. This imposes severe restrictions on instrument systems since the performance of these assays is compromised below or above the critical concentration. Extensive and costly software modification of current systems would be required to allow use of such preparations outside their limited optimal concentration range. Therefore, there remains a need in the art for a consistent, reliable, and easily produced Protein C Activator protein preparation. Summary of the Invention The present invention is based, in part, on the surprising discovery that a highly glycosylated form of Protein C Activator (PCA), produced recombinantly in yeast, has unexpected and beneficial properties which distinguish it from PCAs obtained by prior art methods.

In one series of embodiments, the present invention provides isolated nucleic acids including nucleotide sequences encoding polypeptides comprising natural PCA proteins or biosynthetic variants thereof. In particular embodiments, the present invention provides for the production of synthetic DNA molecules which encode a PCA protein of the snake Agkistrodon contortrix contortrix (ACC) or biosynthetic variants thereof. In preferred embodiments, the PCA nucleic acids reflect the yeast codon bias.

In another series of embodiments, the present invention provides for recombinant nucleic acid constructs in which the PCA sequences are operably joined to exogenous sequences to form cloning vectors, expression vectors, fusion vectors and the like. Thus, in accordance with another embodiment of the invention, a recombinant vector for transforming a host cell to express PCA in the cells is provided.

In another series of embodiments, the present invention provides for host cells which have been transformed with one of the nucleic acids of the invention.

In another series of embodiments, the present invention provides for substantially pure protein preparations including polypeptides comprising a PCA protein.

In another series of embodiments, the present invention provides for methods of producing substantially pure protein preparations including polypeptides comprising a PCA protein.

In another series of embodiments, the present invention provides for blood coagulation diagnostic kits which include the substantially pure PCA protein preparations of the invention.

Detailed Description of the Invention The present invention is based, in part, upon the surprising discovery that a highly glycosylated form of Protein C Activator (PCA) protein, produced recombinantly in yeast, has unexpected and beneficial properties which distinguish it from PCA proteins obtained by prior art methods. Relative to a commercially available PCA preparation purified from ACC venom (i.e., Protac® from Pentapharm AG, Basel, Switzerland), the recombinant PCA protein preparations of the invention appear to have a greater specificity for PC as a substrate, do not directly affect the biological activities of Factor VIII/NIIIa, and have more reproducible Protein C Activator activity. These and other advantages and features of the invention disclosed will be made more apparent from the following description. I. Definitions In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms which are used in the following description and the claims appended hereto.

Protein C Activator protein. As used without further modification herein, the terms "Protein C Activator protein" and "PCA protein" refer to the Protein C Activator found in snake venom, including, but not limited to, the Protein C Activator of the snake Agkistrodon contortrix contortrix (ACC), as well as artificial and synthetic variants which have Protein C Activator activity and which have at least 60% amino acid sequence similarity to SEQ ID ΝO:l. In addition, the terms "Protein C Activator protein" or "PCA protein" are specifically intended to include the proteins in glycosylated, partially glycosylated, or unglycosylated forms. PCA fusion protein. As used herein, the term "PCA fusion protein" refers to a protein comprising a PCA protein sequence and a second polypeptide sequence, and in which the PCA portion of the fusion protein retains Protein C Activator activity. The second polypeptide sequence may be N-terminal or C-terminal to the PCA portion of the PCA fusion protein.

Capable of activating Protein C. As used herein, the phrase "capable of activating Protein C" refers to the ability to convert normal human Protein C to Activated Protein C (APC) in a standard in vitro blood coagulation assay.

Protein C Activator activity. As used herein, the phrase "Protein C Activator activity" refers to the ability to directly activate Protein C as determined by measuring the increase of the activated partial thromboplastin time (APTT) of normal plasma, or Protein C deficient plasma which is supplemented with purified PC. For example, 50 μl of sample of normal plasma, or PC- deficient plasma supplemented with purified PC to contain from 100-300%) of normal PC activity, may be mixed with 50 μl of an APTT clotting reagent, and incubated at 37°C for 5 minutes. 50 μl CaCl₂. is then added to initiate clotting, and clotting time is measured (using, for example, an Automatic Coagulation Lab (ACL), Instrumentation Laboratory, Lexington, MA). Protein C Activator activity is expressed as Units/ml (units are defined as the amount of PCA activity that activates Protein C in 1 ml of normal plasma.) Substantially pure. As used herein with respect to PCA protein preparations, the term "substantially pure" means a preparation which contains at least 60%> (by dry weight) the protein of interest, exclusive of the weight of other intentionally included compounds. Preferably the preparation is at least 75%, more preferably at least 90%>, and most preferably at least 99% by dry weight the protein of interest, exclusive of the weight of other intentionally included compounds. Purity can be measured by any appropriate method, e.g., column chromatography, gel electrophoresis, or HPLC analysis. If a preparation intentionally includes two or more different proteins of the invention, a "substantially pure" preparation means a preparation in which the total dry weight of the proteins of the invention is at least 60%> of the total dry weight, exclusive of the weight of other intentionally included compounds. Preferably, for such preparations containing two or more proteins of the invention, the total weight of the proteins of the invention be at least 75%, more preferably at least 90%, and most preferably at least 99% of the total dry weight of the preparation, exclusive of the weight of other intentionally included compounds. Thus, if the proteins of the invention are mixed with one or more other proteins (e.g., serum albumin) or other compounds (e.g., diluents, detergents, excipients, salts, polysaccharides, sugars, lipids) for purposes of administration, stability, storage, and the like, the weight of such other proteins or compounds is ignored in the calculation of the purity of the preparation.

Molecular Weight. As used herein, the term "molecular weight" refers to the actual molecular weight or molecular mass as determined by a standard empirical method such as electrophoresis or chromatography.

Km. As used herein, the abbreviation "Km" means the Michaelis-Menten constant of a given enzyme. As is known in the art, the Km is a parameter that describes the affinity of an enzyme for its substrate and equals the substrate concentration that yields the half-maximal reaction rate. The Km values disclosed herein were determined by the method as described in Segel (1975), "Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems," John Wiley & Sons, Inc., New York.

Directly Affect Factor VIH/VIIIa. As used herein, the phrase "directly affect Factor Vlll/VIIIa" refers to direct interaction with Factor VIII/NIIIa at a molecular level resulting in inhibition or activation of the biological activity characteristic of Factor VIH/VIIIa, for example, by proteolytic cleavage or by steric hindrance of the active site or binding sites of Factor VIH/VIIIa. Such a direct interaction may be measured by reacting 0.01-0.20 mM of a PCA protein for 1 minute at 37°C in either an APTT-based FVIII assay or a chromogenic-based FVIII assay, where substantially pure FVIII is present at a concentration equivalent to approximately 100% of the activity of normal plasma. A PCA protein does not directly affect Factor VIII/NIIIa if the presence or absence of the PCA protein has no significant effect on Factor VIII activity in such an assay. Similarity. As used herein with respect to amino acid sequences, the "similarity" between two sequences means the percentage of amino acid residue positions, after aligning the sequences according to standard techniques, at which the two sequences have identical or similar residues. "Similar" residues include those which are regarded in the art as "conservative substitutions" (see, e.g., Dayhoff et al. (1978), Atlas of Protein Sequence and Structure Vol. 5 (Suppl. 3), pp. 354 352, Νatl. Biomed. Res. Found., Washington, D.C.); which fall within the groups (a) methionine, leucine, isoleucine and valine, (b) phenylalanine, tyrosine and tryptophan, (c) lysine, arginine and histidine, (d) alanine and glycine, (e) serine and threonine, (f) glutamine and asparagine, and (g) glutamate and aspartate; or which are otherwise shown to have no substantial effect on the biological activity of the protein. Isolated nucleic acid. As used herein, an "isolated nucleic acid" is a ribonucleic acid, deoxyribonucleic acid, or nucleic acid analog comprising a polynucleotide sequence that has been synthesized apart from, or which has been isolated or separated from, sequences that are immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DΝA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDΝA or a genomic DΝA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DΝA which is part of a hybrid gene encoding additional polypeptide sequences and/or including exogenous expression control sequences.

Expression Control Sequences. As used herein, the term "expression control sequences" refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are operably joined or to target the encoded protein to different locations inside or outside the cell. The nature of such control sequences differs depending upon the host organism: in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequences; in eukaryotes, such control sequences generally include promoter and transcription termination sequences. Operably joined. As used herein, the term "operably joined" refers to coding sequences and expression control sequences that are covalently linked in such a way as to place the expression or transcription of the coding sequences under the influence or control of the expression control sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of promoter function results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression control sequences to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, expression control sequences would be operably joined to a coding sequence if the expression control sequences were capable of effecting transcription of the coding sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

Yeast Codon Bias. As used herein, the term "yeast codon bias" means the preferred codons used in strongly expressed genes in yeast as described in Bennetzen and Hall (1982) J. Biol. Chem. 257:3026-3031.

Transformed cell. As used herein, a "transformed cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid molecule of interest. The nucleic acid of interest will typically encode a peptide or protein. The transformed cell may express the sequence of interest, or may be used only to propagate the sequence. The term "transformed" may be used herein to embrace any method of introducing exogenous nucleic acids into cells including, but not limited to, transformation, transfection, electroporation, micro injection, viral-mediated transfection, and the like.

Stringent hybridization conditions. Stringent hybridization conditions is a term of art understood by those of ordinary skill in the art. For any given nucleic acid sequence, stringent hybridization conditions are those conditions of temperature, chaotrophic acids, buffer, and ionic strength which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences. The exact conditions which constitute "stringent" conditions depend upon the nature of the nucleic acid sequence, the length of the sequence, and the frequency of occurrence of subsets of that sequence within other non-identical sequences. By varying hybridization conditions from a level of stringency at which non-specific hybridization occurs to a level at which only specific hybridization is observed, one of ordinary skill in the art can, without undue experimentation, determine conditions which will allow a given sequence to hybridize only with complementary sequences. Suitable ranges of such stringency conditions are described in Krause and Aaronson (1991), Methods Enzymol. 200:546- 56. Hybridization conditions, depending upon the length and commonality of a sequence, may include temperatures of 20°C-65°C and ionic strengths from 5x to 0.1 x SSC. Stringent hybridization conditions may include temperatures as low as 40-42°C (when denaturants such as formamide are included) or up to 60-65°C in ionic strengths as low as O.lx SSC. These ranges, however, are only illustrative and, depending upon the nature of the target sequence, and possible future technological developments, may be more stringent than necessary. Less than stringent conditions are employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence. II. Isolated Nucleic Acids

In one aspect, the present invention provides isolated nucleic acids encoding the PCA proteins of the invention. As described more fully below, the PCA proteins of the invention include a PCA protein found in ACC snake venom (SEQ ID NO: 1), as well as biosynthetic variants and fusion proteins based upon or derived from that sequence. In addition, the present invention provides such PCA protein-encoding nucleic acids operably joined to exogenous sequences, such as expression control sequences or sequences encoding other polypeptides, in cloning, expression or fusion vectors. In a preferred series of embodiments in which the PCA proteins of the invention are produced by transformed yeast cells, isolated nucleic acid sequences are provided which reflect the yeast codon bias. That is, at least 50% of the codons of a nucleic acid encoding a PCA protein are chosen from the 25 codons which are favored in yeast (see Bennetzen and Hall (1982), J. Biol. Chem. 257:3026-3031). Preferably, at least 75% or, more preferably, at least 90%) of the codons are selected from the 25 codons which are favored in yeast. In a most preferred embodiment, all of the codons are selected from the 25 codons which are favored in yeast.

In one preferred embodiment, a PCA protein-encoding nucleic acid sequence is provided comprising the nucleotide sequence of SEQ ID NO:2. This sequence reflects the yeast codon bias and encodes a biosynthetic variant of a PCA protein from ACC venom. In other preferred embodiments, nucleic acids are provided which have at least 70%, preferably 80%, and more preferably 90% sequence identity with the nucleotide sequence of SEQ ID NO:2, and which encode a PCA protein that is capable of activating Protein C. In another preferred embodiment, nucleic acids are provided which hybridize with the complement of the nucleotide sequence of SEQ ID NO:2 under stringent hybridization conditions, and which encode a PCA protein that is capable of activating Protein C. In another series of embodiments, the present invention provides nucleic acids in which the PCA protein-encoding sequences described above are operably joined to endogenous or exogenous 5' and/or 3' expression control sequences. Exogenous expression control sequences may be operably joined to the PCA protein-encoding sequences in order to drive expression of an mRNA transcript encoding the PCA protein. Appropriate 5' expression control sequences typically include promoter elements but may also include additional elements, such as secretory sequences, operator or enhancer sequences, ribosome binding sequences, RNA capping sequences and the like. Depending upon the nature of the host cells to be transformed, the expression control sequences may be selected from those that control the expression of genes of prokaryotic cells, eukaryotic cells, viruses, and combinations thereof. Preferably, the expression control sequences are derived from yeast genes and the nucleic acids are used to transform yeast cells. Expression control sequences are preferably chosen which allow for high yield production of recombinant PCA protein in the chosen host cells. For example, where the host cell is yeast, the regulatory elements may include an inducible 5' AOX1 (alcohol oxidase) promoter. The 3' AOX1 sequences may also be included 3' to the PCA protein-encoding sequences to promote targeted integration of the recombinant construct into the yeast genome.

In another series of embodiments, the present invention provides for isolated nucleic acids encoding a PCA protein in the form of a fusion protein. Useful exogenous sequences include, short sequence "tags" such as poly-His tags or c-myc epitope tags which may be used to aid in the identification and/or purification of the resultant fusion protein. Alternatively, the non- PCA sequences may encode a large protein or protein fragment, such as an enzyme or binding protein which also may assist in the identification and purification of the protein, or which may be useful in an assay, such as an blood coagulation assay. In addition, an α-factor secretion signal or other leader sequence which promotes secretion of the PCA protein is preferably encoded by the 5' end of the coding sequence. Finally, the isolated nucleic acids of the present invention include any of the above described sequences when included in vectors. Appropriate vectors include cloning vectors and expression vectors of all types, including plasmids, phagemids, cosmids, episomes, and the like, as well as integration vectors. The vectors may also include various marker nucleic acid sequences which are useful in identifying transformed host cells (e.g., by antibiotic resistance or susceptibility). In addition, the vectors may include expression control sequences to which the nucleic acids of the invention are operably joined, and/or may also include coding regions such that the nucleic acids of the invention, when appropriately ligated into the vector, are expressed as fusion proteins. The vectors may be chosen to be useful for prokaryotic, eukaryotic, or viral expression systems, as needed or desired for the particular application.

As described below, the pPICZαA vector (Invitrogen, San Diego, CA) has been successfully used to cause high level expression of recombinant PCA protein in transformed cells of the yeast Pichia pastoris. The vector construct may include, for example, an inducible AOXl promoter operably joined to the nucleic acid sequence of SEQ ID NO:2 (which encodes an α- factor secretion signal, followed by a PCA protein sequence, followed by a C-terminal poly-His tag and a c-myc epitope).

Vectors may be introduced into the recipient or "host" cells by various methods well known in the art including, but not limited to, electroporation, calcium phosphate transfection, strontium phosphate transfection, DEAE dextran transfection, lipofection, microinjection, ballistic insertion on micro-beads, protoplast fusion or, for viral or phage vectors, by infection with the recombinant virus or phage. III. Transformed Host Cells In another aspect, the present invention provides for cells or cell lines, both prokaryotic and eukaryotic, which have been transformed with the nucleic acids of the present invention so as to cause clonal propagation of those nucleic acids and/or expression of the proteins or peptides encoded thereby. Such cells or cell lines will have utility in the propagation and production of the nucleic acids and proteins of the present invention. As used herein, the term "transformed cell" is intended to embrace any cell, or the descendant of any cell, into which has been introduced any of the nucleic acids of the invention by any means.

Prokaryotic cells useful for producing the transformed cells of the invention include members of the bacterial genera Escherichia (e.g., E. coli), Pseudomonas (e.g., P. aeruginosa), and Bacillus (e.g., B. subtillus, B. stearothermophilus), as well as many others well known and frequently used in the art. Prokaryotic cells are particularly useful for replicating and propagating the nucleic acids of the invention. Bacterial cells (e.g., E. coli) may be used with a variety of expression vector systems including, for example, plasmids with the T7 RNA polymerase/promoter system, bacteriophage λ regulatory sequences, or Ml 3 Phage mGPI-2. All of these, as well as many other prokaryotic expression systems, are well known in the art and widely available commercially. However, because prokaryotic cells do not produce proteins with the glycosylation patterns typical of eukaryotic proteins, prokaryotic cells are not preferred host cells for producing the PCA proteins of the invention.

In preferred embodiments the PCA proteins of the invention are produced in eukaryotic cells which cause a high degree of glycosylation of the protein. Eukaryotic cells and cell lines useful for producing the transformed cells of the invention include mammalian cells and cell lines, insect cells lines, yeast, and fungi. Particularly preferred are yeast cells of the species Pichia pastoris. As described below, a functionally active recombinant PCA protein, encoded by the nucleic acids of SEQ ID NO:2, has been expressed at high levels in the yeast Pichia pastoris. IV. Substantially Pure Protein Preparations

In another aspect of the invention, substantially pure preparations of PCA proteins are provided. The PCA proteins of the invention may comprise the complete amino acid sequence of a naturally occurring PCA protein of the snake Agkistrodon contortrix contortrix (ACC) (SEQ ID NO: 1), or may comprise only a minimal active fragment of that protein. For example, small N- terminal or C-terminal deletions of the amino acid sequence (e.g., 1-10, preferably 1-5 residues) are not expected to cause a loss of activity and, therefore, such N-terminally or C-terminally truncated biosynthetic variants may be employed. In addition, biosynthetic variants of this PCA protein are provided in which amino acid substitutions, preferably conservative amino acid substitutions, and/or small insertions or deletions, are made to the amino acid sequence of SEQ ID NO: 1. Such biosynthetic variants comprise an amino acid sequence having at least 60%) or 70% similarity, preferably at least 80%) similarity, and most preferably at least 90% amino acid sequence similarity to the PCA protein of SEQ ID NO: 1. In all cases, however, the PCA proteins of the invention must be capable of activating Protein C and, therefore, biosynthetic variants which lack Protein C Activator activity are excluded. One example of a biosynthetic variant PCA protein is provided in SEQ ID NO:3. The PCA protein disclosed in SEQ ID NO:l is a trypsin-type serine protease with a high degree of homology to other snake venom proteases (e.g., batroxobin, flavoxobin, Russell's viper venom Factor V activator). The catalytic site is believed to be formed, in part, by residues His- 40, Asp-85 and Ser-177 of SEQ ID NO:l, and there are three N-linked glycosylation sites at residues Asn-21, Asn-78 and Asn-129. Therefore, in producing biosynthetic variants, these residues should not be substituted or deleted, and insertions or deletions close to these residues are preferably avoided or minimized. In addition, residues which are highly conserved amongst the snake venom proteases (see, for example, McMullen et al. (1989), Biochem. 28:674-679) are also preferably conserved in biosynthetic variants, whereas residues which vary widely amongst these proteins, but which are not implicated in the active site or glycosylation sites, may be more freely altered by substitution, deletion or insertion by standard methods known in the art.

The predicted molecular weight of the PCA protein of SEQ ID NO: 1 , based solely on the molecular weights of its constituent amino acid residues, is approximately, 25 kDa. Depending upon the nature of any amino acid substitutions, insertions, or deletions relative to SEQ ID NO: 1, the weights of biosynthetic variant PCA proteins are generally expected to have predicted molecular weights of about 22-30 kDa, preferably about 23-29 kDa, and most preferably about 24-28 kDa. In preferred embodiments, however, the PCA proteins of the invention are produced in yeast cells and are highly glycosylated. Therefore, the PCA proteins of the invention have an actual molecular weight of at least 40 kDa, in which the fraction of the molecular weight due to glycosylation is at least 15%. In preferred embodiments, the PCA proteins of the invention have an actual molecular weight at least 50 kDa, in which the fraction of the molecular weight due to glycosylation is at least 40%), or an actual molecular weight at least 60 kDa, in which the fraction of the molecular weight due to glycosylation is at least 50%. In a most preferred embodiment, the PCA proteins of the invention have an actual molecular weight about 68 kDa, in which the fraction of the molecular weight due to glycosylation is about 58%. If desired, the amount of glycosylation may be enzymatically reduced after isolating the substantially pure proteins from the fermentation medium.

In preferred embodiments, relative to commercially available PCA protein preparations from ACC venom (e.g., Protac® from Pentapharm AG, Basel, Switzerland), the PCA proteins of the invention have a reduced degree of affinity, and therefore a relatively higher Km, for synthetic substrates used in the measurement of Protein C Activator activity. Thus for example, the PCA proteins of the invention have a Km of at least 2 mM, preferably at least 3 mM, and most preferably about 3.21 mM for the synthetic substrate Tos-Gly-Pro-Arg-4-NA (Chromozym® TH, Pentapharm AG, Basel Switzerland), and a Km of at least 0.9 mM, preferably at least 1.3 mM, more preferably at least 1.5 mM, and most preferably about 1.8 mM for the synthetic substrate H-D-Phe-Pip-Arg-pNA (S-2238, Chromogenix AB, Molndal, Sweden). In evaluating the Km of a PCA protein of the invention, such synthetic substrates may be employed in a standard assay procedure, such as that set forth in Segel (1975), "Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems," John Wiley & Sons, Inc., New York. The values reported herein were obtained by first reacting 0.15 μg/ml PCA protein and 0.08 μM purified human Protein C, in 20 mM HEPES buffer, pH 7.2, at 37^°C, for 30, 60, and 120 seconds to assess the time course of the reaction. The Km values for the different substrates were then obtained by standard methods using an Automatic Coagulation Lab (ACL) from Instrumentation Laboratory, Lexington, MA).

In preferred embodiments, the PCA proteins of the present invention have no direct affect on Factor VIII/NIIIa activity under specific conditions. That is, the PCA proteins of the invention do not directly interact with Factor VIII or Factor Villa at a molecular level in a way which alters their activity. This is in contrast to a commercially available PCA protein preparation from ACC venom (i.e., Protac® from Pentapharm AG, Basel, Switzerland). This is advantageous because any direct effect of a PCA protein preparation with Factor VIH/VIIIa activity will obscure the effect of APC on Factor VIII/NIIIa activity and interfere with diagnostic assays which seek to measure, for example, APC activity or APC resistance.

The PCA protein preparations of the invention may be substantially purified using standard protein purification procedures including, but not limited to, gel filtration chromatography, ion-exchange chromatography, high-performance liquid chromatography (RP- HPLC, ion-exchange HPLC, size-exclusion HPLC, high-performance chromatofocusing chromatography, immunoprecipitation, or immunoaffmity purification. Gel electrophoresis (e.g., PAGE, SDS-PAGE) can also be used to isolate a protein or peptide bases on its molecular weight, charge properties and hydrophobicity. For example, as shown below, recombinant PCA has been substantially purified to greater than 75%) purity from fermentation media using Hydrophobic Interaction Chromatography (HIC) and Cation Exchange Chromatography (i.e., SP- Sepharose Chromatography).

In some embodiments, a PCA protein of the invention may be conveniently purified by creating a fusion protein including the desired PCA protein sequence joined to another polypeptide sequence, such as an antigenic determinant, poly-His tag, or a relatively large protein (e.g., fusion to lacZ, trpE, maltose-binding protein, or glutathione-S-transferase). Fusion proteins in which the PCA protein sequences are fused to a functional polypeptide from another blood coagulation pathway protein may be particularly useful. The fusion protein may be expressed and recovered from transformed prokaryotic or eukaryotic host cells by any standard method WO 00/50612 PCTtUSOO/04406

- 14 - which exploit the non-PC A protein portion of the fusion protein. Thus, for example, fusion proteins including non-PCA protein antigenic determinants (e.g., the c-myc epitope) may be purified by affinity chromatography using antibodies to the antigenic determinant. Similarly, fusion proteins employing a poly-His tag can be affinity purified by using a Ni+ affinity column. Methods of preparing and using such fusion constructs for the purification of proteins are well known in the art.

V. Blood Coagulation Assay Kits

The substantially pure protein preparations of the invention are useful as components of functional assay kits for Protein C, Protein S, or other components of the blood coagulation system. For example, sensitive functional assays for both Protein C (Martinoli et al. (1986), Thromb. Res. 43:253-264; Frances et al. (1987), Am. J. Clin. Pathol. 87:619-625; McCall et al. (1987), Thromb. Res. 45:681-685) and Protein S (Suzuki et al. (1988), Thromb. Res. 49:241- 251) have been developed which utilize, as one component, and activator of Protein C. Other components which may be included in such kits include chromogenic substrates for Protein C or Factors V/Na or VIII/NIIIa, APTT reagents, normal control plasmas, deficient plasmas, and the like. For example, the recombinant Protein C of the present invention may be used in the IL Test™ Proclot assay and/or IL Test™ Pro-Chrom assay.

Examples Practice of the invention will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.

A synthetic gene was constructed based on the polypeptide sequence of an Agkistrodon contortrix contortrix Protein C Activator published by McMullen et al. (1989). This DΝA encodes a polypeptide with identical amino acid sequence except for two amino acid substitutions (T76S; A158G), which were purposely introduced at the ligation junctions among the three PCR amplified fragments. A recombinant Pichia expression vector, pIL-m4, was constructed using the nucleic acid disclosed as SEQ ID ΝO:2. The pPICα vector (Invitrogen, San Diego, CA) uses the AXO1 promoter, AOXl 3' sequences for genomic integration, and C- terminal fusion of a myc epitope and a poly-histidine tail for purification. Pichia pastoris strain X33 (Mut⁺, His⁺) was used to generate the recombinant strains that contained these expression control sequences. PCR analysis revealed that 9 out of 10 transformed strains had genomic integrations of the recombinant PCA DNA sequence. The recombinant strains were screened in shaker flask for the expression of recombinant Protein C Activator. After 53 hours of induction in methanol, both conditioned cultured broth and cell extracts were assayed directly for their ability to prolong the coagulation time of human plasma. The fermentation of a selected recombinant strain reached a production of up to -230 mg/L of recombinant PCA with an activity (by the chromogenic method) of 74 U/ml in its culture broth. A two step purification scheme was devised to purify recombinant PCA to ~75% purity. The recombinant PCA produced was glycosylated and had an apparent molecular weight of 68 kDa. The apparent molecular weight dropped to 28 kDa when the protein was deglycosylated. Steady-state kinetic analysis revealed that recombinant PCA activated purified human Protein C with a K_m of 77 nM and k_cat of 0.39 sec^"1. In clotting assays, recombinant PCA prolonged the coagulation time of human plasma. This effect had a dose dependent relationship with both Protein C and Protein S. The recombinant PCA protein preparation can be produced reliably and in large quantities under controlled manufacture conditions. It has enzymatic and coagulation properties suitable for using it as a reagent for Protein C activation, in assays measuring Protein C or Protein S, as well as other assays utilizing similar principles, and as an alternative to the native venom preparations.

Materials

Plasmids pPICZαA and pHIL, Pichia pastoris Strain X33, Zeocin, and anti-myc monoclonal antibodies were obtained from Invitrogen (San Diego, CA). Enzymes for DNA manipulations and EndoH were obtained from New England Biolabs (Beverly, MA). Oligo nucleotides were obtained from Stratagene (La Jolla, CA). Zymolyase was obtained from Seikagaku Corporation (Chuo-ku, Tokyo). AmpliTaq DNA polymerase was obtained from Perkin-Elmer (Branchberg, NJ). Protac® was obtained from Pentapharm AG (Basel, Switzerland). Chromozym® TH was obtained from Boehringer Mannheim (Indianapolis, IN). S-2366 peptide substrate was obtained from Dia Pharma Group (Franklin, OH). Purified human Protein C was obtained from Enzyme Research Laboratories (South Bend, IN). Soybean trypsin inhibitor was obtained from Sigma. Anti recombinant PCA antiserum was custom produced by Biosynthesis (Lewisville, TX). Alkaline phosphatase (AP) conjugated goat-anti-mouse and goat- anti-rabbit antibodies and AP conjugate substrates were obtained from BioRad (Hercules, CA). Mazu DF 60 P was obtained from PPG Industries (Gurnee, IL). All chemicals used in the cell culture and fermentations were obtained from Sigma, Fisher, Chempure, Difco, or BIO 101 and had ACS or higher grades. Nitro phenyl guanidine benzoate (NPBG) was obtained from Sigma (St. Louis, MO). Resins, columns, and FPCL system used for chromatography were obtained from Amersham-Pharmacia (Uppsala, Sweden). BCA protein reagent was obtained from Pierce Chemical Company (Rockford, IL). Dialysis tubing with a 6,000 to 8,000 molecular weight cut off was obtained from Spectrapor (Laguna Hills, CA). Cotton-fiber filters were obtained from Whatman (Hillsboro, OR).

Yeast Extract Peptone Dextrose Media (YPD), Buffered Glycerol Complex Media (BMGY), Buffered Methanol Complex Media (BMMY) were prepared according to Invitrogen's Pichia pastoris Instruction Manual. Fermentation basal media and PTM4 trace salts solution were prepared according to Brierley et al. (1990).

PC Deficient Plasma, ProClot Diluent, Working Diluent, Activated Partial Thromboplastin Time Reagent (APTT), Calibration Plasma, PC Abnormal Control Plasma, PC Normal Control Plasma, PS Deficient Plasma, Bovine Thromboplastin Reagent, Calibration Plasma, PS Abnormal Control Plasma, PS Normal Control Plasma, FV Deficient Plasma, Factor Diluent, Prothrombin Time Reagent (PT), FVIII Deficient Plasma, Normal Control Plasma,

Factor Reagent (Factor IXa + Factor X + Thrombin + CaCl₂ + phospholipid), S-2765 and 1-2581, Buffer Stock Solution (BSS), Fresh Frozen Plasma (FFP) were obtained from Instrumentation Laboratory, Lexington, MA.

To produce Fresh Frozen Plasma, nine parts freshly drawn venous human blood was added to one part 3.8 g/dL trisodium citrate anticoagulant. Blood was collected into siliconized glass tubes, mixed with anticoagulant and centrifuged at 2500Xg for 15 min. The supernatant human plasma was removed and stored at or below -70°C. This plasma is referred to as fresh frozen plasma (FFP) Protein C Assay: Assays for Protein C activity were performed as follows:

1. A Calibration plasma, PC Normal Control plasma, PC Abnormal Control plasma, or FFP plasma were diluted 10-fold with Working Diluent comprising 4.3 parts ProClot Diluent and 1.0 part a PCA preparation (either Protac® or the recombinant PCA of the invention).

2. A zero calibration point was prepared by diluting PC Deficient Plasma 10-fold with Working Diluent. 3. Diluted FFP plasma, Calibration plasma or Control plasma (32 μL each) were mixed with PC Deficient Plasma (32 μL) and APTT Reagent (64 μL). The reaction was incubated for 5 minutes at 37°C.

4. CaCl₂ (64 μL of 0.025M) was added to each incubation, mixed and the time to clot was measured.

5. The procedure was automated on the ACL System. A run was accepted only if the calibration curve correlation coefficient was >0.980. The system automatically outputs the PC percent activity for Control plasma and FFP plasma.

Protein S Assay: Assays for Protein C activity were performed as follows:

1. A Calibration plasma, PS Normal Control plasma, PS Abnormal Control plasma, or FFP plasma (4 μL) was mixed with Activated PS Deficient Plasma (76 μL) comprising 1.0 part of PCA protein preparation (either Protac® or recombinant PCA) and 2.0 parts PS Deficient Plasma. 2. A zero calibration point was prepared using Activated PS Deficient Plasma (80 μL).

3. Bovine Thromboplastin Reagent (80 μL) was added to each sample.

4. Reaction mixtures were mixed and the time to clot was measured.

5. The procedure was automated on the ACL System (Instrumentation Laboratory, Lexington, MA). A run was accepted only if the calibration curve correlation coefficient was >0.980. The system automatically outputs the PS percent activity for Control plasma and FFP plasma.

Factor V Assay (PT-based):

Assays for Factor V activity were performed as follows:

1. A Calibration plasma, Normal Control plasma, or FFP plasma was diluted 5 -fold with Factor

Diluent. 2. Test conditions were designed so that results are within the 25-150% calibrated range.

3. Diluted samples (40 μL) were mixed with FV Deficient Plasma (40 μL).

4. Prothrombin Time (PT) Reagent (80 μL) was added to each sample.

5. Reaction mixtures were mixed and the time to clot was measured.

6. The procedure was automated on the ACL System. A run was accepted only if the calibration curve correlation coefficient was >0.980. The system automatically outputs the FV percent activity for Control plasma and FFP plasma. Factor VIII Assay (APTT-based): Assays for Factor V activity were performed as follows:

1. A Calibration plasma, Normal Control plasma, or FFP plasma was diluted 5-fold with Factor Diluent.

2. Test conditions were designed so that results are within the 25-150% calibrated range. 3. Diluted samples (40 μL) were mixed with Factor VIII Deficient Plasma (40 μL) and an APTT Reagent (40 μL). The reaction was incubated for 5 minutes at 37°C.

4. CaCl₂ (40 μL of 0.025 M) was added to each incubation, mixed and the time to clot was measured.

5. The procedure was automated on the ACL System. A run was accepted only if the calibration curve correlation coefficient was >0.980. The system automatically outputs the FVIII percent activity for Control plasma and FFP plasma. Factor VIII Assay (Chromogenic-based):

Assays for Factor VIII activity were performed as follows:

1. A solution of rFVIII was prepared at 2.78 U/mL in a Buffer Working Solution (a 10-fold dilution of Buffer Stock Solution).

2. A Factor Reagent (50 μL comprising Factor IXa + Factor X + Thrombin + CaCl₂ + phospholipid) was mixed with 30 μL of various concentrations of a PCA protein preparation (either Protac® or recombinant PCA). The reaction was incubated for 1 minute at 37°C.

3. rFVIII (20 μL) was added to each reaction and further incubated for 1 or 10 minutes. 4. 50 μL of a substrate (S-2765 + 1-2581) was added to each incubation, mixed and the change in optical density was measured at 405 nm for 2 minutes.

5. The change in optical density for each reaction represents its relative FVIII activity. IL Test™ PROCLOT

1. Calibration Plasma, Normal Control, Abnormal Control or FFP is diluted 10-fold with Working Diluent.

2. A zero calibration point is prepared by diluting PC Deficient Plasma 10-fold with Working Diluent.

3. Diluted FFP, Calibrators or Controls (32 μL each) are mixed with PC Deficient Plasma (32 μL) and APTT Reagent (64 μL). Reaction is incubated for 5 minutes at 37°C. 4. CaC12 (64 μL) is added to each incubation, mixed and the time to clot is measured. 5. The procedure is automated on the ACL™ System. A run is accepted only if the calibration curve correlation coefficient is >0.980. The system automatically outputs the PC percent activity for Controls and FFP. IL Test™ PRO-CHROM 1. FFP, Calibrators or Controls (14 μL each) are mixed with Diluent (42 μL) Protein C Activator (56 μL of ≡0.8 U/ml). Reaction is incubated for 4 minutes at 37°C. 2. Add Substrate (56 μL) and measure Absorbance at 405 nm.

The procedure is automated on the ACL™ System. A run is accepted only if the calibration curve correlation coefficient is >0.980. The system automatically outputs the PC percent activity for Controls and FFP. IL Test™ PROTEIN S

1. Calibration Plasma, Normal Control, Abnormal Control or FFP (4 μL) is mixed with Activated PS Deficient Plasma (76 μL).

2. A zero calibration point is prepared using Activated PS Deficient Plasma (80 μL). 3. Bovine Thromboplastin Reagent (80 μL) is added to each sample.

4. Reaction mixtures are mixed and the time to clot is measured.

5. The procedure is automated on the ACL™ System. A run is accepted only if the calibration curve correlation coefficient is >0.980. The system automatically outputs the PS percent activity for Controls and FFP. Effect of PC Activator on PC activity:

FFP plasma and zero calibrator were mixed with Working Diluent containing increasing doses of PC Activator and tested for PC activity. Effect of PC Activator on PS activity:

FFP plasma and zero calibrator were mixed with Activated PS Deficient Plasma containing increasing doses of PC Activator and tested for PS activity. Effect of PC Activator on FV activity:

FFP plasma was reacted with increasing doses of PC Activator and assayed for FV activity either immediately or after a 5 minute pre-incubation. Effect of PC Activator on FVIII activity (APTT-based): FFP plasma was reacted with increasing doses of PC Activator for 5 minutes and assayed for FVIII activity. Effect of PC Activator on rFVIII activity (Chromogenic-based): Purified rFVIII was reacted with increasing doses of Protein C Activator in Factor Reagent for 1 or 10 minutes and assayed for FVIII activity Effect of PC.

PC-deficient plasma was spiked with purified PC to contain from 100%) to 300%) PC activity. Each sample was activated with either 0.5 or 1.0 U/ml recombinant PCA. The activated samples were assayed for APTT clotting time, in the following fashion: Sample (50 μl) was mixed with APTT reagent (50 μl) and incubated for 5 min. at 37°C, then CaCl₂ (50 μl) was added to each incubation and the clot time was measured automatically on an ACL. Effect of PS. PS-deficient plasma was spiked with purified PS to contain from 5% to 30% PS activity.

Each sample was activated with 1.0 U/ml recombinant PCA. The activated samples were assayed for PT clotting time in the following fashion: Sample (50 μl) was mixed with PT reagent (100 μl) and the clot time was measured immediately in an ACL. Human Plasma: Nine parts freshly drawn venous blood added to one part 3.8 g/dL trisodium citrate anticoagulant. Blood was collected into siliconized glass tubes, mixed with anticoagulant and centrifuged at 2500Xg for 15 min. The supernatant plasma was removed and stored at or below - 70°C. This plasma is referred to as fresh frozen plasma (FFP). Vector Construct. Polypeptide sequence encoding a ACC Protein C Activator (McMullen et al., 1989) was reverse-translated into DNA sequence using S. cerevisiae codon usage preference. Twelve overlapping oligonucleotides were synthesized according to this sequence to cover the entire polypeptide length, with additional nucleotide sequences at their 5' and 3' ends that contain engineered restriction sites. Three DNA fragments, FI, F2, and F3, were synthesized by an asymmetric PCR method using groups of 4 primers, namely, FI was synthesized using IL-acc-1 (SEQ ID NO:4), IL-acc-2 (SEQ ID NO:5), IL-acc-3 (SEQ ID NO:6), and IL-acc-4 (SEQ ID NO:7); F2 was synthesized using IL-acc-5 (SEQ ID NO: 8), IL-acc-6 (SEQ ID NO:9), IL-acc-7 (SEQ ID NO: 10), and IL-acc-8 (SEQ ID NO:l 1); and F3 was synthesized using IL-acc-9 (SEQ ID NO:12), IL-acc-10 (SEQ ID NO:13), IL-acc-11 (SEQ ID NO: 14), and IL-acc-12 (SEQ ID NO:15) (Sandhu et al., 1992). After gel cleaning, FI was digested with EcoR I and Xba I, F2 with Xba I and NgoM IV, and F3 with NgoM IV and Not I. These DNA fragments were ligated using T4 DNA ligase and then PCR amplified using SEQ ID NO:4 and SEQ ID NO: 15 as primers to give a full length Protein C Activator (SEQ ID NO:2). To express this gene in Pichia pastoris an expression vector was constructed. The nucleic acid encoded by SEQ ID NO:2 was inserted into EcoR I-Not I sites of pPICZαA to give plasmid pIL-m4. Amino acid Substitutions PCA encoding sequence of the construct was based on the amino acidic sequence for the

PCA protein isolated by Kisiel, et. al. and sequenced by McMullen et al., using the codon bias of Saccharomyces. The yeast codon bias used in this synthetic gene may have contributed to the high level of recombinant PCA production achieved, since such sequences are in general compatible with yeast cells' translational apparatus and tRNA pool composition. The amino acid sequence encoded by this synthetic cDNA has two amino acid substitution mutations compared to the published peptide sequence (T76S; A158G). The positions of these mutations were placed at regions that are away from amino acid residues His-40, Asp-85, and Ser-177, putative catalytic residues by an analogy to other homologous serine proteases. Transformation of Pichia pastoris strain. Pichia pastoris strain X33 cells were made competent and transformed by electroporation following the manufacture's recommendations (Invitrogen, La Jolla ,CA). Plasmid DNA pIL-m4 used for transformation was prepared using a plasmid midi prep kit (Qiagene), linearized by Sac I, and Ethanol precipitated. Roughly 5 μg of the linearized DNA and 80 μl of competent X33 cells were used for each transformation. Transformants were plated onto YPD plates containing 100 μg/ml or 1000 μg/ml Zeocin and incubated at 30°C for 3 - 4 days. The Zeocin resistant colonies were purified on plates containing the same Zeocin concentrations. Genomic DNA analysis.

To verify the recombinant gene integration the genomic DNA of a number of recombinant Pichia pastoris strains were analyzed directly by a PCR method (Linder, 1996). Single colonies were isolated from YPD plates and suspended in 15 μl H2O. Then 3 μl of 5 mg/ml Zymolyase were added to each cell suspension and the mixtures incubated at 30 °C for 10 min. These cell suspensions were then frozen and thawed before they were used as templates for PCR amplification. PCR amplifications were carried out using primers that are complementary to AOXl 5' and 3' regions of pPICZα. Thirty cycles of amplifications were carried out at 95°C for 1 min., 54°C for 1 min., and 72°C for 1 min. with hot start. Shaker flask expression studies. Single colonies were grown in 5 ml BMGY at 30 °C in 50 ml tubes to OD₆oo of 2 - 6. Cells were spun down and then resuspended in 10 ml BMMY and grown in 100 ml tissue culture flasks. 1 ml of 5% methanol was added to each flask at 24 hours and 48 hours. The cultures were stopped at 53 hours. Conditioned culture broth was separated from yeast cells, and concentrated 85 times by Filtron and Centricon of 30 kDa cut off. Cell pellets were suspended in breaking buffer (50 mM PIPES, 500 mM NaCl, 0.76 % Emulphogen) at a density of 4 OD units per μl breaking buffer, and the cell suspensions were mixed with equal volume of glass beads and vortexed eight times for 30 sec. each time , samples were chilled on ice for at least 30 seconds between vortexing. Both the concentrated culture broth and the cell extract samples were analyzed directly for their abilities to prolong the coagulation time of human plasma. Screening Recombinant Strains

There are wide variations among the recombinant strains derived from the same Pichia pastoris strain. This is because the genomic integration event in each strain is independent, both copy number and site of integration can affect the efficiency of the recombinant protein expression. The screening of these independent strains is, therefore, an important step in identifying a strain that is best suitable for the recombinant PCA production. To screen the recombinant strains we used an assay where a sample's ability to prolong the APTT time of normal human plasma was tested. Activated Protein C subsequently inhibits Factor Va and Factor Villa and slows down both intrinsic and extrinsic coagulation pathways. As a result, longer APTT time is correlated with higher PCA activity. Other methods that can be used in such a screening include immunological methods that detects the amount of recombinant protein, and DNA methods that determines the gene copy numbers. Copy number typically dictates the level of recombinant protein expression but there have been instances where copy numbers had inverse relationship to the levels of recombinant protein expression. For the purpose of screening recombinant strains the APTT time of human plasma in the absence and presence of testing samples was measured directly. Samples of indicated volumes were mixed with 110 μl of ProClot Diluent and 165 μl of Normal plasma in a total volumes of 300 μl, and the APTT times were measured on an Automatic Coagulation Lab (ACL) using APTT reagents (Instrumentation Lab, Lexington, MA). For the analysis of samples from fermentation and purification, Protein C Activator activity was assayed by a chromogenic method that measures the hydrolysis of Chromozym® TH catalyzed by the Protein C Activator. Appropriate dilutions of the samples were reacted with 0.475 mM Chromozym® TH in ProClot Buffer at 37°C for 10 min., and the absorbance at 405 nm was recorded on an ACL. A standard curve using 0 - 125 mU/ml Protac® was measured for each assay; and used to determine the recombinant PCA activity in the sample. SDS-PAGE and Western Blot Analysis. Crude and purified recombinant PCA were treated with EndoH following manufacture's recommendations. Proteins were separated on 4 -12 %> polyacrylamide gels under reducing conditions. The gels were either stained by Coomassie blue or subject to western blot analysis. For western blot a rabbit polyclonal antiserum (BSYN282) against a 15 amino acid recombinant PCA polypeptide (amino acids 73-87 of SEQ ID NO:3) was used at 1 :1000 dilution, and an anti- myc monoclonal antibody was used at 1 :2000 dilution. The western blot protocol was essentially as that recommended by Invitrogen. QS30 densitometry software (pdi, Huntington Station, NY), was used to assess the molecular weight and density of the bands on the gels. Silver stained gels were carried out as followed: Sample (1 μg) was subjected to reducing SDS-PAGE in a 12.5% phastgel (PhastSystem, Pharmacia, Uppsala, Sweden) and stained with silver according to manufacturer's recommendations with the exception that the development step was conducted outside of the PhastSystem development chamber. Large Scale Fermentation.

Fermentation growth of the recombinant strain yIL-m4-102 was carried out using a 5 liter BioFlo III bench-top fermentor (New Brunswick Scientific Co. Inc., Edison, NJ), using 4^°C, 50% polyethylene glycol solution for chilling, and compressed air for culture aeration. The fermentation media contained 3 L basal media (4% glycerol), 6 ml PTM4 trace salt solution, and 9 ml 20%) Mazu DF 60P as defoamer. A seed culture was grown in 200 ml BMGY in a baffled flask at 30°C and 250 rpm to OD₆₀o of 7 - 14, and was used to inoculated the fermentor to a OD₆oo of 0.5. The initial settings for the fermentor were: dissolved O2 (dO ) at 100%), temperature at 30^°C, agitation at 900 rpm, pH at 5.0, feeding rate at 0%>. The fermentation was carried out in two phases, a batch phase of approximately 20 hours, followed by a fed batch phase that lasted 145 - 160 hours. The entire process was monitored and controlled by a program, written in ABS-Biocommand (New Brunswick Scientific Co. Inc., Edison, NJ). At the end of the batch phase when dO2 has reached a minimum and subsequently increased to above 35 %, the control program turned on the media-feeding which was set at a rate of 0.23 ml/min. (0.08 ml/min/L) initially. The fed-batch feeding media contained 25% glycerol (w/v), 40% methanol (w/v), and 12 ml/L PTM4 trace metal solution. The program increased the feeding rate WO 00/50612 PCTtUSOO/04406

- 24 - automatically by multiply the rate by a factor of 1.0058 once every half minute. The maximum feeding rate was capped to 0.62 ml/min (0.21 ml/min/L) which took about 4 hours to reach. Additionally, throughout the fed-batch phase, the program automatically turns on and off the feeding depending on the dO2 level: If dO2 is greater than 35% the feeding is on, if the dO2 is less than 35% the feeding is off. Samples were taken from the fermentor at different time points and their OD and Protein C Activator activity were measured. At the end of fermentation the culture broth was separated from yeast cells by centrifugation, and was frozen to -70^°C.

The cell density continued to increase after the start of the fed-batch phase until it reached a saturation density of approximately 400 OD unit after about 50 hours of feeding. At the same time the production of recombinant PCA activity in the culture broth starts to increase, though very slowly at the beginning. The PCA activity continues to increase as the fed-batch culture continued, and this increase began to slow down after the induction time had reached 120 hours. The culture broth at the final time point (169 hours) contained about 74 U/ml PCA activity as assayed by the chromogenic activity. At 145 hours the total feeding volume was approximately 3 L, the same as the starting volume of the culture. This fermentation protocol was reproducible and has been scaled up to 50 L fermentation. The culture broth from different fermentation time points was analyzed by electrophoresis and by the chromogenic activity assay. Recombinant PCA purification.

Recombinant PCA was purified from fermented media by hydrophobic interaction chromatography (HIC) followed by cation exchange chromatography under control of the FPLC. After addition of ammonium sulfate to 1.5 M, fermented media was clarified by high speed centrifugation and filtration. Recombinant PCA was bound to Phenyl-Sepharose High Substitution resin at pH 5.2 in 1.5 M ammonium sulfate and was eluted in 20 mM sodium acetate, pH 5.2. Residual salt was removed by dialysis before binding the HIC-pool to SP-Sepharose. Recombinant PCA was eluted from the cation exchange column in 20 mM sodium acetate, 0.5 M NaCl, pH 5.2. Final product was buffer exchanged into 20 mM Hepes, 50 mM NaCl, pH 7.2, before storage at -70°C. Total protein concentration was determined using the BCA assay with bovine serum albumin as standard.

The yield information from a typical purification is summarized in Table 1. Final product was estimated to be at least 75% recombinant PCA by evaluation of silver stained protein profiles after deglycosylation by EndoH. The western blot of the purified recombinant PCA reacted with anti-recombinant PCA showed a diffuse band at -68 kDa. The final product represented less than 1% of the starting protein and contained 33%) of the starting amidolytic Units of activity, resulting in a 43 fold increase in the specific activity from the starting medium.

TABLE I

Physical characteristics of recombinant PCA glycosylation

Recombinant PCA produced from Pichia pastoris is a glycoprotein of 68 kDa apparent molecular weight. After the removal of the carbohydrates, its polypeptide backbone has an apparent molecular weight of 28-29 kDa. Recombinant PCA, like the venom ACC-C isolated by Kisiel et al., (1987) has three N-glycosylation consensus sites (Asn-21, Asn-78, Asn-129). These are presumably the sites of glycosylation in recombinant PCA. N-linked oligosaccharides produced in Pichia pastoris have the high-mannose-type and are 10 to 14 mannose in length (Montesino et al. (1998). Recently, O-glycosylation has also been observed in proteins produced in Pichia pastoris. Recombinant PCA has 15 serine and 13 proline residues; glycosylation on some of these sites may contribute to the large amount of total glycosylation observed. Deglycosylated recombinant PCA has similar activity as the native PCA, so glycosylation does not have apparent effect on the recombinant PCA produced. The transformation vector used has an ORF that encodes a polypeptide of about 38 kDa (signal sequence to the C-terminal fusion sequences, glycosylation not included). The signal sequence, if properly recognized, should be removed in the endoplasmic reticulum (ER) by Kex2 and Ste 13 proteases following the nascent polypeptide synthesis. As a result the processed polypeptide should have 260 amino acids or 28 kDa molecular weight. Since deglycosylated recombinant PCA has similar apparent molecular weight, it might possess most of the coded sequence except the C-terminal c-myc epitope that was truncated. This truncation was likely to have taken place extracellularly after the secretion event, since small amount of intracellular recombinant PCA detected had intact c-myc epitope. PC Activator Activity of recombinant PCA.

The effect of activated purified PC on the APTT clotting time was tested. PC was activated by recombinant PCA within a plasma milieu containing normal levels of PS. Activated PC was observed to bring about a dose-dependent prolongation of the APTT. The dose- dependent effect of purified PS on the PT clotting time was also tested. Normal level of endogenous PC was activated by recombinant PCA and was observed to bring about a prolongation of the PT. Steady state kinetic studies were carried out using an ACL (Instrumentation Laboratory,

Lexington, MA) which carries out multi-channel sample additions, incubations, and data recordings automatically. First 0.15 μg/ml recombinant PCA and 0.08 μM purified human Protein C were reacted, in 20 mM HEPES pH 7.2 at 37^°C, for 30, 60, and 120 seconds to assess the time course of the reaction. At the end of these and the other similar reactions described below soybean trypsin inhibitor and S-2366 (in a total volume of 50 μl) were added to final concentrations of 4 mg/ml and 1.5 mM respectively, and the absorbance at 405 nm (ABS405) were recorded immediately once every 0.45 seconds for a period of 15 minutes. To study the kinetics of the reaction between recombinant PCA and human Protein C, 0.15 μg/ml purified recombinant PCA was incubated with 0.00 μg/ml, 0.05μg/ml, 0.08μg/ml, 0.12μg/ml, and 0.25 μM purified human Protein C in 20 mM HEPES, pH 7.2, in a total volume of 100 μl for 30 seconds. Since the ABS405 vs. time plots were linear, their slopes were used to calculate the initial rates of these reactions. In separate reactions 0.125 μM activated Protein C was reacted with O.OOμg/ml, 0.42μg/ml, 0.83μg/ml, 0.167μg/ml, and 0.250 mM of S-2366 under the same conditions as above, and the ABS405 was recorded similarly. The initial rates (delta ABS sec^'1) of these reactions were calculated by linear regression of the ABS405 vs time data recorded during the first 30 seconds of the reactions, and converted to the unit of mM sec^"1 using an extinction coefficient of 9.75 mM^"1 cm^"1 for p-Nitro aniline. These reaction rates were plotted against S-2366 concentrations (Michaelis-Menten plots), and the Km and V for this reaction was determined from their double reciprocal plots to be 0.30 + 0.06 mM and 0.34 + 0.08 μM sec^" ' respectively. The kcat was 2.7 sec^" as determined by dividing Vm by the activated Protein C concentration. The reaction rates between recombinant PCA and Protein C were calculated by the equation

(delta ABS/884 sec) (0.3 mM+1.5 mM) 10⁶ nM/sec r = x x

0.5 cm x 9.75 mM^"1. cm^" 1.5 mM x 2.7 sec^"1 30 sec The reaction rates were plotted against the Protein C concentrations (Michaelis-Menten plots) and the Km and Vm were determined from their double reciprocal plots. The active site concentration of recombinant PCA was measured by titration with NPGB (Chase and Shaw, 1969), using a standard curve made with p-Nitrophenol under the same conditions. The kcat for the Protein C reactions was calculated by dividing Vm by the active site concentration. Neither recombinant PCA nor purified human Protein C alone catalyzed S-2366 hydrolysis.

Recombinant PCA activates purified human Protein C with classic first order kinetics, with a K_m of 77 nM, and a k_cat of 0.39 sec^"1. Since its affinity for Protein C is in the same range of the Protein C concentration in human plasma (65 nM), it is possible to use recombinant PCA as a reagent for activating Protein C in the human plasma. Furthermore, the kinetic properties of recombinant PCA is also similar to other snake venom PCAA (K_m of 80 nM and k_cat of 0.27 S^"1 in 50 mM Tris-HCl pH 7.5 at 30° C) as characterized by Orthner et al.(1988). In coagulation assays recombinant PCA behaved as a PC activator causing a prolongation of both APTT and PT clotting times. Consistent with theory, this effect correlated with PC concentration in the presence of normal levels of PS. Likewise the effect correlated with PS concentration in the presence of normal levels of PC.

Purified recombinant PCA was reacted with human Protein C at 37 C in Hepes, pH 7.2, for 0, 30, 60, and 120 sec; under the experimental conditions the reaction was essentially linear up to 30 sec, a time period subsequently chosen to carry out the kinetic studies for the reactions between recombinant PCA and Protein C. The rate of these reactions, calculated using the rate of S-2366 hydrolysis, its Km and kcat, and an extinction coefficient of 9.75 mM sec^"1, were plotted against Protein C concentrations. The double reciprocal plot of these reactions was linear (R2 > 0.99 in all four experiments). From these plots it was determined that Km = 77 + 2 nM; Vm = 1.75 +0.02 nM sec^"1 (mean + standard error, n=4). The titration of recombinant PCA with NPGB gave a molar concentration that is 75% of the estimated recombinant PCA protein concentration. Using the measured active sites concentration, the kcat was calculated to be 0.39 sec^"1.

Comparison of recombinant PCA and Protac® - Steady State Kinetics

Both recombinant PCA and Protac® activate Protein C. In these reactions both a consensus peptide sequence and the overall confirmation of Protein C are recognized and they both contribute to the reaction kinetics. In addition to Protein C, certain chromogenic substrates can also be cleaved by recombinant PCA or Protac® because their peptide sequence resemble the recognized peptide sequence in the natural substrate Protein C. Chromozym® TH (Tos-Gly-Pro- Arg-pNA) that is a tripeptide substrate for serine proteases, especially thrombin, has the identical PI and P2 amino acids but a different P3 amino acid from Protein C (P3=Asp, P2=Pro, Pl=Arg). S-2238 (H-D-Phe-Pip- Arg-pNA), another peptide substrate for thrombin, has the identical PI amino acid but has different P2 and P3 amino acids from Protein C (P3=Asp, P2=Pro, Pl=Arg).

As seen in Table II, recombinant PCA and Protac® have very similar affinities for human Protein C. These affinity values are also in the same range of the Protein C concentration in human plasma (0.065 μM), making it possible to use these proteins as reagents for activating Protein C in the human plasma. The turn-over number of recombinant PCA is slightly lower than that of Protac® (0.4 sec^"1 vs 0.6 sec^"1), but these are essentially in the same range considering the possible errors in the assessment of the protein concentrations of both recombinant PCA and Protac® used.

Table II: Summary of steady-kinetic parameters for recombinant PCA and Protac®

Although recombinant PCA and Protac® behaved very similarly toward human Protein C, their reactions with Chromozym® TH are more different. The affinity of recombinant PCA for Chromozym® TH is five times lower than that of Protac®, and consequently, the overall catalytic effⁱciency of recombinant PCA is also three times lower than Protac®. The difference WO 00/50612 PCTtUSOO/04406

- 29 - between recombinant PCA and Protac® was even more pronounced in their reactions with S-2388. Recombinant PCA has three times lower affinity, and six times lower turnover number for S-2388 than Protac®. Consequently its overall catalytic efficiency is 19 times lower. Chromozym® TH and S-2238 are both synthetic peptide substrates for thrombin and other serine proteases. They are not components of human Plasma samples or any coagulation assays, so the fact that recombinant PCA has lower reactivity with these synthetic substrates has no influence on its potential performance in these assays. On the contrary, recombinant PCA has a more stringent requirement for the three consensus amino acids that are recognized in its substrates, it is therefore, at least theoretically, less likely to react with potential contaminants and/or interfering substances in the samples or reagents. In other words, these data suggest that recombinant PCA is a much more specific activator of Protein C than Protac®. Comparison of recombinant PCA and Protac® - Serine Protease Inhibitor Sensitivity

Recombinant PCA and Protac® are both sensitive to the serine-protease inhibitors tested (soybean trypsin inhibitor, PMSF, TLCK, p-AminoBenzomidine, and NPGB), confirming that both are serine proteases. Compared to Protac®, recombinant PCA is slightly less sensitive to three out of four inhibitors for which a dose response have been determined. The inhibitions of recombinant PCA and Protac® by five serine protease inhibitors were studied. Recombinant PCA and Protac®, were incubated in the absence or presence of various concentrations of soybean trypsin inhibitor, PMSF, TLCK, p-AminoBenzomidine, or NPGB at 37°C for 60 min (except for soybean trypsin inhibitor, in which case the incubation time was 15 min). The remaining activities of recombinant PCA and Protac® were measured by reacting with 1.5 mM of Chromozym® TH on an ACL and recording the ABS 405nm. The initial rate of each reaction was calculated using linear regression of ABS data recorded for the first 20 seconds of the reactions. Comparison of recombinant PCA and Protac® - Optimal Concentration Range

Consistent manufacturing of in vitro diagnostics kits based on Protac® has traditionally been problematic. Particularly troublesome is the optimization of Protac® activity for each distinct assay. This task becomes more difficult since its activity can only be adjusted over a narrow concentration range. Outside this critical range unwanted interferences are often observed that compromise assay specificity and performance. The lack of batch-to-batch homogeneity between lots of Protac® has also been discouraging. A well defined and characterized recombinant PCA may avoid these shortcomings. Preliminary testing surprisingly shows that activation of PC (within a Protein S assay) by recombinant PCA is optimal over a much broader concentration range as compared to Protac®. It was observed that the clot time at 100%) PS is practically constant from 1.0-2.5 U/mL recombinant PCA, while clot time was constant only between 1.5-2.0 U/mL Protac®. In addition, the maximum clot time at 100% PS was always within specifications with recombinant PCA (<150 seconds) but not with Protac®.

A constant plasma clot time at 100% PS between 1.0-2.5 U/mL recombinant PCA was observed. The clot time at 100% PS was constant only between a narrow 1.5-2.0 U/mL Protac® range. Furthermore, the maximum clot time at 100% PS was always <150 seconds with recombinant PCA but not with Protac®. Protein C activation was similar for Protac® and recombinant PCA at the concentrations of activator tested. Possible Secondary Activities of Protac® - FVIII

Until recently, Protac® was thought to be a specific activator of PC. However, secondary Protac® activities were reported on coagulation Factor V (FV) and Factor VIII (FVIII) (Gable et al. 1997). These secondary activities could potentially introduce strict limitations on product performance since a high degree of assay specificity is desirable. Studies therefore examined whether recombinant PCA exhibits similar secondary activities on FV and/or FVIII. Testing confirmed the activation of FV by Protac® and observed a similar activity with recombinant PCA. Results with FVIII were unexpected and differed from that reported above. Testing was performed with both APTT-based and chromogenic-based assays. The APTT-based method showed activation of FVIII by both activators, although to a lesser extent by recombinant PCA. The FVIII chromogenic assay system provides a well-defined and characterized system using purified components of the blood coagulation pathway. This assay system, unlike the plasma system used above, excludes other possible side reactions that can obscure the interpretation of experimental results. Surprisingly, it was found that both Protac® and recombinant PCA inhibited FVIII activity in a dose-dependent manner at 10 minutes incubation. On the other hand, recombinant PCA has no direct effect on FVIII activity, while Protac® at equivalent concentrations inhibits FVIII activity at 1 minute incubation. The observed FVIII inhibition by Protac® was the opposite effect as described in the literature, and may reflect the different assay systems used to perform each investigation. The different activities displayed on short incubation with FVIII suggest that recombinant PCA and Protac® are different molecules. The broader recombinant PCA optimal concentration in the PS assay will allow an easier, more consistent and cost effective way to manufacture PS kits that meet the desired specifications and customer expectations. Furthermore, recombinant PCA is a better characterized activator that will allow improvements in the performance and design of PC and PS assays.

Similar dose-dependent activation of plasma FV by Protac® and recombinant PCA within a PT-based assay system has been demonstrated. Plasma FVIII activation by both Protac® and recombinant PCA was demonstrated within an APTT-based assay. FVIII activation, however, was less with recombinant PCA than with Protac®.

A dose-dependent inactivation of rFVIII pre-incubated for 1 minute with Protac® was observed using by a chromogenic-based assay. Recombinant PCA had no effect under the same conditions. Pre-incubation of rFVIII for 10 minutes with Protac® or recombinant PCA resulted in each case in a dose-dependent inactivation. Comparison of recombinant PCA and Protac® - Summary & Conclusions

Serine-protease-specific inhibitors affect both recombinant PCA, and Protac®, demonstrating that both are serine-proteases. Steady state kinetic studies with purified human Protein C, Chromozym® TH, and S-2238 showed that when reacting with purified human Protein C, the catalytic efficiencies of recombinant PCA and Protac® differ only slightly from each other (less than two fold difference). But when reacting with Chromozym® TH, recombinant PCA has five times lower affinity and three times lower catalytic efficiency as compared to Protac®, and when reacting with S-2238, recombinant PCA has three times lower affinity and 19 times lower catalytic efficiency as compared to Protac®. These data suggest that recombinant PCA reacts more specifically with Protein C than Protac® does, therefore it is a better reagent for coagulation assays. These data and the different activities displayed on short incubation with FVIII suggest that recombinant PCA and Protac® are different molecules. The broader recombinant PCA optimal concentration in the PS assay will allow an easier, more consistent and cost effective way to manufacture PS kits that meet the desired specifications and customer expectations. Furthermore, recombinant PCA is a better characterized activator that will allow improvements in the performance and design of PC and PS assays.

Equivalents/Other Embodiments The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

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Venom of Agkistrodon contortrix contortrix, can Activate Factor V and Factor VIII," Thromb. Res. 86:79-84.

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Claims

CLAIMS What is claimed is: 1. A substantially pure PCA protein preparation, comprising a PCA protein having at least 60% sequence similarity with the amino acid sequence of SEQ ID NO: l, and wherein said PCA protein is capable of activating Protein C.

2. A substantially pure PCA protein preparation, comprising a PCA protein having at least 70%> sequence similarity with the amino acid sequence of SEQ ID NO: l, and wherein said PCA protein is capable of activating Protein C.

3. A substantially pure PCA protein preparation, comprising a PCA protein having at least 80%> sequence similarity with the amino acid sequence of SEQ ID NO: 1, and wherein said PCA protein is capable of activating Protein C.

4. A substantially pure PCA protein preparation, comprising a PCA protein having at least 90%> sequence similarity with the amino acid sequence of SEQ ID NO: l, and wherein said PCA protein is capable of activating Protein C.

5. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein comprises the amino acid sequence of SEQ ID NO:3.

6. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 40 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 25%.

7. A substantially pure PCA protein preparation as in claim 1, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 50 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 40%.

8. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 60 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 50%.

9. A substantially pure PCA protein preparation as in claim 1, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of about 68 kDa and wherein the fraction of said molecular weight due to glycosylation is about 58%.

10. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of about 3.21 mM for the synthetic substrate Tos-Gly-Pro-Arg-4-NA.

11. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of about at least 2 mM for the synthetic substrate Tos-Gly-Pro- Arg-4-NA.

12. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of about at least 3 mM for the synthetic substrate Tos-Gly-Pro-Arg-4-NA.

13. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of about 1.8 mM for the synthetic substrate H-D-Phe-Pip- Arg-pNA.

14. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of at least 0.9 mM for the synthetic substrate H-D-Phe-Pip- Arg-pNA.

15. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of at least 1.3 mM for the synthetic substrate H-D-Phe-Pip- Arg-pNA.

16. A substantially pure PCA protein preparation as in claim 1 , wherein said PCA protein has a Km of at least 1.5 mM for the synthetic substrate H-D-Phe-Pip-Arg-pNA.

17. A substantially pure PCA protein preparation as in claim 1, wherein said PCA protein has no direct affect on Factor VIII/NIIIa activity.

18. A kit for assaying activity of a component for the blood coagulation system, comprising a container containing a substantially pure PCA protein preparation as in any one of claims 1-17, and instructions for performing diagnostic assays employing said PCA protein preparation.

19. A method of producing a PCA protein preparation comprising: (a) transforming a host cell with a nucleic acid encoding a PCA protein as in any one of claims 1-17; (b) maintaining said host cell under conditions whereby said nucleic acid is expressed to produce said PCA protein; and (c) recovering said PCA protein.

20. A method as in claim 19 wherein said host cell is a yeast cell.

21. A method as in claim 20 wherein said yeast is Pichia pastoris.

22. A method as in any one of claims 20-21 wherein said nucleic acid comprises a coding sequence having a yeast codon bias.

23. A method as in claim 19, further comprising the step of purifying said PCA protein preparation.

24. A method as in claim 23, wherein said step of purifying said PCA protein preparation comprises hydrophobic interaction chromatography.

25. A method as in claim 23, wherein said step of purifying said PCA protein preparation comprises cation exchange chromatography.

26. A method as in any one of claims 19-25, further comprising the step of lyophilizing said PCA protein preparation.

27. A method as in claim 19, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 40 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 25%.

28. A method as in claim 19, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 50 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 40%.

29. A method as in claim 19, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of at least 60 kDa and wherein the fraction of said molecular weight due to glycosylation is at least 50%.

30. A method as in claim 19, wherein said PCA protein is a glycoprotein having a glycosylated molecular weight of about 68 kDa and wherein the fraction of said molecular weight due to glycosylation is about 58%.

31. A method as in claim 19, wherein said PCA protein has a Km of about 2 for the synthetic substrate Tos-Gly-Pro-Arg-4-NA.

32. A method as in claim 19, wherein said PCA protein has a Km of about 3 for the synthetic substrate Tos-Gly-Pro-Arg-4-NA.

33. A method as in claim 19, wherein said PCA protein has a Km of about 3.21 mM for the synthetic substrate Tos-Gly-Pro- Arg-4-NA.

34. A method as in claim 19, wherein said PCA protein has a Km of 1.8 mM for the synthetic substrate H-D-Phe-Pip-Arg-pNA.

35. A method as in claim 19, wherein said PCA protein has a Km of 1.3 mM for the synthetic substrate H-D-Phe-Pip- Arg-pNA.

36. A method as in claim 19, wherein said PCA protein does not directly affect Factor VIII/NIIIa.

37. An isolated nucleic acid which encodes a PCA protein of any one of claims 1-17.

38. An isolated nucleic acid as in claim 37 comprising the nucleotide sequence of SEQ ID NO:2.

39. An isolated nucleic acid as in claim 37 having at least 60% sequence identity with the nucleotide sequence of SEQ ID NO:2.

40. An isolated nucleic acid as in claim 37 having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:2.

41. An isolated nucleic acid as in claim 37 having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:2.

42. An isolated nucleic acid as in claim 37 having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:2.

43. An isolated nucleic acid as in claim 37 that hybridizes under stringent hybridization ccoonnddiittiioonnss ttoo aa nnuucclleeiicc aacciidd hhaavviinngg aa sseeqquueennccee ccoommpplleemmeennttaarryy ttoo tthhee nnuucclleeoottiiddee sseeqquueennccee ooff SEQ T IDD NNΠO:2?.

44. A vector comprising a nucleic acid as in any one of claims 37-43.

45 A vector comprising a nucleic acid as in any one of claims 37-43 operably joined to an expressi ^'on control sequence.

46. The vector as in claim 45, wherein said expression control sequence comprises an AOXl pprroommootteerr..

47. A vector comprising a nucleic acid as in any one of claims 37-43 encoding a PCA fusion protein.

48. The vector as in claim 47, wherein said PCA fusion protein comprises a polypeptide selected from the group consisting of a poly-histidine tag, a c-myc epitope, and the secretion signal from the alpha-mating factor of S. cerevisiae.

49. A host cell which has been transformed with a nucleic acid as in any one of claims 37-43 and which expresses PCA protein.

50. A host cell as in claim 49, wherein said host cell is a yeast.

51. A host cell as in claim 49, wherein said yeast is Pichia pastoris.