RECOMBINANT PROTEIN C AND PROTEIN S VARIANTS

Abstract: The present invention is concerned with a variant blood coagulation component, which is substantially homologous in amino acid sequence to wild-type blood coagulation component capable of expressing anticoagulant activity in the protein C-anticoagulant system of blood and selected from protein C (PC), activated protein C (APC) and protein S (PS), said variant component being capable of expressing an anticoagulant activity, which is enhanced in comparison with the anticoagulant activity expressed by the corresponding wild-type blood coagulation component, and said variant component differing from the respective wild-type component, in that it contains in comparison with the said wild-type component at least one amino acid residue modification in its amino acid residue sequence. The present invention is also concerned with methods to produce such variants based on DNA technology, DNA segments intended for use in the said methods, and the use of the said variants for therapeutic and diagnostic purposes.

Description: Recombinant protein C and protein S variants The present invention is directed to functional variants of recombinant protein C and protein S, which express enhanced anticoagulant activity, and to the use of such variants for therapeutic or diagnostic purposes. Protein C and protein S are vitamin K-dependent proteins of major physiological importance which participate in an anticoagulant system of the blood, which is commonly designated the protein C-anticoagulant system. In this system protein C, in concert with other proteins including the cofactors protein S and intact Factor V (FV), which act as synergistic cofactors to protein C in its activated form (APC, Activated Protein C), functions as a down-regulator of blood coagulation, thereby preventing excess coagulation of blood and, thus, inhibiting thrombosis. This anticoagulant activity expressed by the activated form of protein C emanates from its capa- * city to inhibit the reactions of blood coagulation by specifically cleaving and degrading activated Factor VIII (F Villa) and activated Factor V (FVa), these being other cofactors of the blood coagulation system. As a result thereof, activation of components necessary for blood coagulation, viz. Factor X (FX) and prothrombin, is inhibited and the activity of the coagulation system is dampered. Both protein C and protein S are, thus, of major physiological importance for a properly functioning blood coagulation system. The importance of protein C can be deduced from clinical observations. For in- stance, severe thromboembolism affects individuals with homozygous protein C deficiency and affected individuals develop thrombosis already in their neonatal life. The resulting clinical condition, purpura fulminans, is usually fatal unless the condition is treated with protein C. On the other hand, heterozygous protein C deficiency is associated with a less severe thromboembolic phenotype, constituting only a relatively mild risk factor for venous thrombosis. It has been estimated that carriers of this genetic trait have a 5- to 10-fold higher risk of thrombosis in comparison with individuals with normal protein C levels. More importantly, however, the most common genetic defect associated with thrombosis is also affecting the protein C system. This condition is usually referred to as APC resistance and is most frequently caused by a single point mutation in the FV-gene, which mutation leads to replacement of the amino acid residue Arg506 with a Gin residue in the FV amino acid sequence. Arg506 constitutes one of three cleavage sites in activated FV (FVa), which are sensitive for cleavage action by APC, and such mutated FVa is less efficiently degraded by APC than normal FVa (Dahlback, J. Clin. Invest. 1994, 94: 923-927). Protein S is also an anticoagulant protein of major physiological importance as is illustrated by the association between protein S deficiency and thromboembolic disorders. Homozygous deficiency, which is extremely rare, gives a neonatal fatal disease, whereas heterozygous deficiency is a risk factor for venous thrombosis in adult life. Indeed, protein C deficiency or protein S deficiency is found in approximately 5-10% of all individuals exhibiting venous thrombosis. The physiological importance of protein C, activated protein C (APC) and protein S as anticoagulant components in the blood coagulation system indicate potential use of these substances for therapeutic purposes. Indeed, protein C and its activated form APC have already been used to some ex- tent for therapeutic purposes (Verstraete and Zoldholyi, Drugs 1995, 49: 856-884; Esmon et al, Dev. Biol. Stand. 1987, 67: 51-57; Okajima et al, Am. J. Hematol. 1990, 33: 277-278; Dreyfys et al, N. Engl. J. Med. 1991, 325: 1565-1568). More specifically, protein C purified from human plasma has been used as replacement therapy in homozygous protein C deficiency (Marlar and Neumann, Semin. Thromb. Haemostas. 1990, 16: 299-309) and has also been used successfully in cases with severe disseminated intravascular coagulation due to meningococcemia (Rivard et al, J. Pediatr. 1995, 126: 646-652). Moreover, in a baboon model of septicaemia (using E. coli), APC was shown to have a protective effect, which was particularly pronounced when the APC was given prior to the E. coli infusion (Taylor et al, J. Clin. Invest. 1987, 79: 918-925). In any event, the results obtained to date suggest that protein C may become a useful drug, not only for treatment of the above conditions but also for many other conditions, in which the coagulation system is activated, e.g. for the prevention and treatment of venous thrombosis, vascular occlusion after recanalization of coronary vessel after myocardial infarction (MI) and after angioplasty. As regards the therapeutic potential of protein S, Schwarz et al have described use of plasma-derived protein S for in vivo treatment of thrombosis and thromboembolic complications in US-A-5 143 901. Therapeutic use of specifically designed, recombinant protein S variants has also been suggested, as will be described in more detail below. It is envisaged that therapeutic treatment of various conditions related to blood coagulation disturbancies could be improved if variants of protein C and protein S having enhanced anticoagulant properties were available. Moreover, such variants would be useful as reagents to improve various biologial assays for other components of the protein C system in order to obtain assays having improved performance. The development of recombinant DNA technology in the past decades has had a tremendous impact on the possibilities to produce desired biological substances efficiently and/or to create biological substances having desired and optionally specifically designed properties. Indeed, functional variants of recombinant protein C and protein S have been disclosed in prior art. In US-A-4 775 624 (Bang et al) recombinant production of human protein C deri- vatives is disclosed. However, only production of protein C polypeptides having functional activities essentially corresponding to human wild-type protein C is disclosed. Use of protein C prepared by recombinant technique has also been disclosed in Berg et al, Biotechnique, 1993, 14: 972-978; Hoyer et al, Vox Sang. 1994, 67: Suppl. 3: 217-220). Moreover, functional variants of protein C obtained by mutagenesis directed to the activation peptide region, which includes residues 158-169, may have enhanced sensitivity to thrombin, such variants being activated by thrombin faster than wild-type protein C (Erlich et al, Embo. J. 1990, 9: 2367-2373; Richardson et al, Nature 1992, 360:261-264). In one of these studies, a number of mutations were introduced around the activation site leading to a mutant protein C, which was relatively easily activated by thrombin alone. Mo- re specifically, this mutant protein C was activated by thrombin, formed during coagulation of blood, even in absence of thrombomodulin, a membrane protein, which is usually required for efficient activation of protein C by thrombin (Richardson et al, Nature 1992, 360: 261-264). In Grinnell et al., J. Biol. Chem., 1991, 9778-9785 the role of glycosylation in the function of human protein C is examined, site-directed mutagenesis being used to singly eliminate each of the four potential N-linked glycosylation sites. In the Protein C variants disclosed therein Gin is substituted for Asn at positions 97, 248 and 313, resp., and it is shown, that the Protein C mutants having this substitution mutation at position 248 and 313 expressed a 2- to 3-fold enhanced anticoagulant activity in addition to other modified pro- perties. Protein C variants having enhanced interaction with thrombin are disclosed in Richardson et al., Nature, 1992, 360:261-264. These variants comprise mutations in the acti- vation peptide region, two putative inhibitory acidic residues near the thrombin cleavage site being altered. One protein C variant comprising the said altered residues in the activation peptide region and also the Asn 313 Gin mutation disclosed by Grinnell et al. (supra) has recently been shown to function well as an anticoagulant in experiments perfomed in vivo (Kurz et al., Blood, 1997, 89: 534-540). However, in this protein C variant the enhanced anticoagulant activity is due to the Asn 313 Gin mutation. In J. Biol. Chem. 1993, 268: 19943-19948 Rezaie et al. disclose a protein C mutant comprising a Glu 357 Gin mutation (i.e. Glu 192 Gin if chymotypsin numbering is used). Although this mutant inactivates FVa at an about 2- to 3-fold enhanced rate in a pure system, in plasma the anticoagulant activity is not enhanced in comparison to wild-type protein C since the mutant is rapidly inhibited by protease inhibitors such as alpha-1 antit- rypsin and antithrombin III. A protein C variant lacking the Gla-domain of native protein C and comprising a Thr 254 Tyr (i.e. Thr 99 Tyr based on the chymotrypsin numbering) is disclosed in J. Biol. Chem., 1996, 271: 23807-23814. This variant protein C has a 2-fold enhanced activity towards pure FVa, i.e. soluble FVa in absence of phospholipids, but is lacking anticoagulant activity in plasma by virtue of the missing Gla-domain. Functional variants of recombinant protein S have also been disclosed in prior art. Thus, Chang et al, Blood 78: 1099a (1991) report a functional variant of recombinant pro- tein S having retained anticoagulant activity but reduced binding affinity for the C4b binding protein (C4BP). This variant protein S differs from native protein S in that the carboxy terminal amino acid residues corresponding to residues 583-635 of native protein S have been deleted. In US-A-5 405 946 (Griffin et al), recombinant mutants of protein S are disclosed, which are highly homologous to wild-type protein S and exhibit reduced affinity for C4BP. These mutants contain one or more point mutations in the region between residues 425 and 432 of protein S and optionally also a mutation in the thrombin sensitive loop region of wild-type mature human protein S defined by residues 45-72 of protein S, the mutation in the latter region giving rise to enhanced resistance to cleavage by thrombin. In Chang et al., Circulation, 86:3241a (1992), it is reported that the anticoagulant activity of Protein S can be diminished or lost by cleavage at arginine residues within d e thrombin sensitive loop comprising residues 46-75. Functional variants, e.g. recombinant mutants, of protein S having enhanced anticoagulant activity have not been disclosed before. As regards protein C, even though protein C variants having enhanced anticoagulant activity have been disclosed by Grinnell et al. (supra), these variants comprise an alte- red glycosylation site. Obviously, variants of protein C and protein S having enhanced anticoagulant activity would be useful of therapeutic as well as diagnostic purposes. Thus, the present invention is concerned with functional variants of protein C and protein S, which express enhanced anticoagulant activity, the protein C variants having the same glycosylation sites as native protein C. As regards d e variants of protein C, diis enhanced activity is essentially expressed by APC, which is the active form of the protein C zymogen, said zymogen being virtually inactive. Accordingly, die present invention is also concerned with variants of APC having enhanced anticoagulant activity. The said enhanced activity of protein S essentially emanates from an enhanced activity of protein S as a cofac- tor to APC in the above mentioned protein C anticoagulant system, although APC- independent anticoagulant effects of protein S have been reported. Furthermore, the present invention is also concerned with methods to produce such variants based on DNA technology, DNA segments intended for use in the said methods, and the use of the said variants for therapeutic and diagnostic purposes. In the following, the present invention is disclosed in more detail with reference to the drawings, wherein: Fig. 1 illustrates schematically the protein C molecule. Fig. 2 illustrates schematically the protein S molecule. Fig. 3 illustrates die amidolytic activity of human and bovine wild-type APC and of APC mutants. Human APC (O), human APC-SP (•), bovine APC (D), bovine APC-SP (■)• Fig. 4A-C illustrate the effect of various APCs on the activated partial tiirombo- plastin times in human and bovine plasma. A) In human plasma: human APC (O), human APC-SP (•), bovine APC (D), bovine APC-SP (M). B) In human plasma supplemented wim bovine protein S (final concentration of 5 μg/ml): human APC (O), human APC-SP (•), bovine APC (D), bovine APC-SP (■). C) In bovine plasma: human APC (O), human APC-SP (•), bovine (D), bovine APC-SP (■). Fig. 5A-C illustrate me effect of various APCs on the inactivation of human factor Villa. Different concentrations of various APCs were preincubated with factor Villa, factor IXa, phospholipids and Ca 2+ mixture for 5 min in the presence of bovine factor V and human or bovine protein S. Factor X was activated by this solution and die rate of factor Xa formation was measured with a synmetic substrate. The absorbance was linearly related to die factor Villa activity, and results were expressed as percentage of respective control. A) Inactivation of factor Villa by high concentrations of APCs (final concentrations are indicated) in die presence of human protein S and bovine factor V: human APC (O), human APC-SP (•), bovine APC (G), bovine APC-SP (■). B) Inactivation of factor Wla by low concentrations of APCs (final concentrations are indicated) in the presence of human protein S och bovine factor V: human APC (O), human APC-SP (•), bovine APC (D), bovine APC-SP (■). C) Inactivation of factor Villa by APCs (final concentrations are indicated) in the presence of bovine protein S and bovine factor V: human APC (O), human APC-SP (•), bovine APC (D), bovine APC-SP (■). Fig. 6 A and B illustrate the effect of various APCs on the prothrombin times in human and bovine plasma. A) In human plasma: human APC (O), huma APC-SP (•), bovine APC (D), bovine APC-SP (■). B) In bovine plasma: human APC (O), human APC- SP (•), bovine APC (D), bovine APC-SP (■). Fig. 7 illustrates the inactivation of various APCs, viz. human APC (O), human APC-SP (•), bovine APC (D) and bovine APC-SP (■), by human plasma. Fig. 8 A and B illustrate d e APC-cofactor activity of protein S mutants. Increasing concentrations of protein S mutants (final concentrations of 0-10 μg/ml) were included to- gether with human APC (at a final concentration of 0.3 μg/ml) in an APTT-based assay using protein S-deficient human plasma, whereafter the respective clotting time was measu- red. The values are the means of triplicate measurements. A. Molecular arrangement of Protein C and Protein S As indicated above, protein C and protein S are both members of the vitamin Independent protein family, which means that they both contain a specific protein module in which the glutamic acid residues are modified to γ-carboxy glutamic acid residues (Gla). The Gla-modules provide the proteins with the ability to bind calcium and to bind negatively charged procoagulant phospholipids. More specifically, the protein C molecule is composed of four different types of ~ modules. As shown in Fig. 1, in me direction of amino terminus to carboxy terminus, these modules consist of a Gla-module, two EGF-like modules, i.e. Epidermal Grow Factor homologous modules, and finally a typical serine protease (SP) module. In plasma, most of die circulating protein C consists of the mature two-chain, disulfide-linked protein C zymogen arisen from a single-chain precursor by limited proteolysis. These two chains are die 20 kDa light chain, which contains the Gla- and EGF-modules and the 40 kDa heavy chain, which constitutes the SP-module. During activation by d rombin bound to thrombomodulin, a peptide bond Arg-Leu (residues 169 and 170) is cleaved in die N-terminal part of the hea- vy chain and an activation peptide comprising twelve amino acid residues (residues 158- 169) is released. The modular molecular arrangement of protein C is shown in Fig. 1. The thrombin-cleavage site is indicated w * ith an arrow and die numbers denote the C-terminal amino acid residues of d e light (155) chain and die heavy (419) chain. In connection with the present invention, die numbering of residues in me amino acid sequence of protein C and variants thereof is based on mature protein C. As is shown in Fig. 2, protein S has a modular molecular arrangement similar to that of protein C. Thus, protein S is a multi-modular molecule composed of a Gla-module, a thrombin sensitive disulfide loop, four EGF-like modules comprising high affinity calcium binding sites, and a C-terminal domain or module homologous to die Sex Hormone Binding Globulin (SHBG), die said SHBG-module containing three glycosylation sites. The amino acid sequences of protein C and protein S have been deduced from die corresponding cDNA-nucleotide sequences and have been reported in the literature. As regards protein S, Lundwall, A. et al., Proc. Natl. Acad. Sci. USA, vol. 83, p. 6716-6720 (human protein S) and Dahlback, B. et al., Proc. Natl. Acad. Sci. USA, vol. 83, p. 4199- 4203 (bovine protein S) have disclosed d e cDNA and the amino acid sequence of protein S. For protein C, the cDNA-nucleotide sequences and die corresonding amino acid sequences are available from the EMBL Gene database (Heidelberg, Germany) under die accession number X02750 for human protein C, which is designated HSPROTC, and d e accession number KO 2435 for bovine protein C, which is designated BTPBC. In connection with the present invention, die usual 1 -letter or 3-letter symbols are used as abbreviations for amino acids as is shown in d e following table of correspondence: TABLE OF CORRESPONDENCE SYMBOL 1 -Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I He isoleucine L Leu leucine T Thr direonine V Val valine P Pro proline K Lys lysine H His histidine Q Gin glutamine E Glu glutamic acid Z Glx Glu and/or Gin w Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine J Xaa Unknown or other B. Variants of Protein C and Protein S As stated above, die present invention is concerned widi functional variants of recombinant protein C and recombinant protein S having enhanced anticoagulant activity. These variants differ from wild-type recombinant protein C and wild-type recombinant protein S as regards one or more amino acid residues, said residues being inserted, deleted or substituted in die corresponding wild-type sequence, diereby giving rise to the present variants of protein C and protein S. Widi respect to die present functional variants of protein C, the said difference(s) is (are) maintained after activation to APC. Accordingly, the present invention is also concerned widi APC variants having enhanced anticoagulant activity. At present, such variants are conveniently obtained by mutagenesis, especially site- directed mutagenesis including use of oligonucleotide primers. However, the present invention is concerned with die functional variants per se irrespective of the mode of obtaining diese variants. In connection with the present invention, die expression "variant" means a modifi- ed wild-type molecule, such as a mutant molecule, which generally has a high degree of homology in comparison with die wild-type molecule. Suitably, die present invention is concerned with such variants of protein C, APC and protein S, which express enhanced anticoagulant activity and comprise one or more mutations contained in regions of d e amino acid sequence of the wild-type substance, which are previously known or have turned out to be essential for the functional anticoagulant activity of the wild-type substance. It is preferred diat such mutations encompass only a few amino acid residues, and possibly only one amino acid residue, in order to preserve substantial homology widi respect to d e wild-type substance. This is of particular importance in connection with use of the present variants for treatment in vivo to avoid, or at least re- duce, a possible immune response to the variant used for treatment. In connection with the diagnostic embodiments of die present invention, a high degree of homology is of course of less importance, the main requirement being mat die functional variant expresses die desired activity at an enhanced level. In accordance widi die present invention it has, d us, been discovered diat protein C (PC), the active state of PC designated APC, and protein S (PS) can be modified to express enhanced anticoagulant activity by introduction of one or more alterations, e.g. mutations, in one or more regions of their amino acid residue sequences, which regions may correspond to or may be odier than those previously known to be essential for the anticoagulant activity of the respective wild-type substance. In view of the close relationship between PC and APC, frequendy, no clear distinction is made between PC and APC in connection widi e present invention, but die designation PC/ APC is used and die context will reveal if one or bodi of diese substances- are considered. A suitable embodiment of the present invention is concerned wid functional variants of PC/ APC. In expeiimental work preceding die present invention and concerned widi the PC molecule, and more specifically the relationship between its structure and function, different mutations were introduced in d e cDNA for protein C and die mutated cDNA's were expressed in suitable host cells derived from eukaryotic cell lines. The resulting mutated protein C variants were isolated, activated and functionally characterized. During diis work it was found diat one of these recombinant protein C molecules expressed in its acti- vated form (APC) considerably enhanced activity, both against syndietic substrates and against die natural substrate of PC/ APC, i.e. Factor Villa, and probably also against Factor Va, although d is has not been experimentally verified yet. This enhanced proteolytic capa- city of the mutated PC/ APC means that the anticoagulant potential of die mutated PC/ APC is enhanced. In die experimental part of the present disclosure, d is is demonstrated bodi in plasma clotting systems (APTT system) and in a test concerned with degradation of purified Factor Villa. Accordingly, a suitable embodiment of the present invention is related to functional variants of PC/ APC, which have a high degree, suitably at least 95%, of amino acid sequence identity with wild-type mature PC/ APC and express enhanced proteolytic activity, said activity resulting in enhanced anticoagulant activity, which variants differ from me said wild-type PC/ APC with respect to at least one amino acid residue. It has quite unexpectedly been found that variants of PC/APC which have these improved properties can be obtained by introduction of one or more mutations in the SP- module of die amino acid residue sequence of die wild-type PC/APC. Except for Grinned et al. (supra), which is related to die role of glycosylation in the function of human PC, mere are no reports in the prior art literature, which indicate diat one or more mutations in diis module, i.e. me serine protease (SP) module, of die PC/APC molecule would lead to enhanced proteolytic and anticoagulant activity. How-ever, it was previously known d at, on one hand, human APC is inhibited by several serpins, i.e. snake venom proteins, by the protein C inhibitor (PCI) and by alpha 1-anti-trypsin (αlAT), whereas, on die other hand, bovine APC is not inhibited by αlAT. In an effort to understand diis phenomenon, Holl and Foster et al (Biochemistry, 1994, 33:1876-1880) constructed hybride molecules between human and bovine protein C and were able to demonstrate that the molecular background for this difference resides somewhere in the SP-module of protein C. However, it is not suggested in or obvious from diis report that mutations in die SP- module could lead to enhanced proteolytic and anticoagulant activities. The present Inventor has studied die SP-module in more detail in an attempt to locate closely the site in the SP-module, which is responsible for die different reactivities of human and bovine APC with αlAT. In connection with these studies, it was quite unexpectedly found d at an amino acid sequence between residues numbers 300 and 314 in human wild-type protein C is essential for proteolytic and amidolytic activities and, thus, for the anticoagulant activity of PC/APC and diat introduction of mutation(s) in this amino acid stretch could give rise to functional variants of PC/APC expressing the said enhanced activities. Thus, a suitable embodiment of die present invention is directed to functional variants of PC/APC, which express enhanced proteolytic and anticoagulant activities, which variants differ from die wild-type PC/APC in diat tiiey contain one or more mutations in their SP-module. In accordance with a specific embodiment, the present invention contemplates variants of PC/APC, wherein the mutation(s) in the SP-module is (are) located witiiin an amino acid stretch between the residues number 300 and 314 of wild-type human protein C. In human APC, the above mentioned sequence No. 300-314 consists of WGYHSSREKEAKRNR (1), the one letter code for amino acids being used. One preferred embodiment of the present invention is directed to a human PC/APC variant having an amino acid sequence identical with d at of the wild-type PC/APC molecule except for mutations contained in die said amino acid sequence (1), die mutated sequence being comprised of WGYRDETKRNR (2). The locations in die wild-type molecule of die mutations are obvious from the following representation of die mutated sequence: WGY...RD.ETKRNR (3), wherein the points illustrate deleted amino acids and substitutions are underlined. As is obvious from sequence (3), the PC/APC variant of this specific embodiment contains an amino acid stretch in die SP module which is shortened with four amino acid residues and contains two substitutions in comparison widi the wild-type PC/APC molecule. As stated above, the modified, i.e. variant or mutant, PC/APC or PS, of the pre- ~ sent invention has enhanced anticoagulant activity. Such anticoagulant activity can be determined i.a. as d e ability of the present variants to increase clotting time in standard in vitro coagulation assays. The enhanced anticoagulant activity is measured in comparison to wild-type PC/APC or PS which may be derived from plasma or obtained by recombinant DNA technique. Thus, to be useful in accordance with the present invention, die PC/APC or PS variants should express an anticoagulant activity which is higher tiian the anticoagulant activity of the wild-type substance. Suitably, the present variants express an anticoagulant activity which is enhanced at least about 50%, and suitably at least about 100%. For suitable PC/APC variants an enhancement of about 400% has been achieved. Suitable PS variants express an anticoagulant activity which is at least 200 % , suitably at least 500 % and preferably about 1000 % of die anticoagulant activity of wild-type PS. Moreover, the present variants preferably are substantially homologous to d e corresponding wild-type substance. Thus, die present variants preferably only contain point mutations, i.e. one or a few single amino acid residue substitutions, deletions and/or insertions. In accordance wid suitable embodiments of die present invention, variants of PC/APC contain one or more mutations, suitably point mutations, in the SP module e.g. between amino acid residue numbers 290 and 320 and preferably in die amino acid residue sequence within die region of PC/APC between amino acid residues 300 and 314 of wild- type mature PC/APC. Thus, a specific embodiment of die present invention is concerned widi PC/APC variants containing deletion and substitution mutations in the SP-module and suitably between amino acid residues 300 and 314, preferably die amino acid 303, 304, 305 and 308 being deleted and amino acids 307 and 310 being substituted (E307D/A310T), d e above mentioned PC/APC variant comprising die mutated sequence (2) being obtained. Accordingly, preferred variants containing mutations witiiin die said sequence contain a mutated sequence represented by die sequence formula (2) instead of die wild-type sequence represented by die sequence formula (1). Other PC/APC variants of die present invention could contain insertion mutations in the SP-module. In principal, any amino acid residue witiiin die amino acid sequence of PC/APC, and suitably within the SP-module, could be modified in accordance widi d e present invention, provided diat d e PC/APC variants thereby produced ex- presses me desired enhanced anticoagulant activity and, preferably, also enhanced proteolytic activity. This is also true for die present PS variants except diat die PS variants have an enhanced cof actor activity but no proteolytic activity. As regards PS variants of the present invention, suitable variants contain one or more mutations in d e amino acid residue sequence within the dirombin sensitive loop and/or die first two EGF-like modules. More specifically, suitable PS variants contain mutations in the dirombin sensitive (TS) loop and in die first EGF-like (EGFl) module, i.e. contain mutations between the amino acid residue number 46 (Val) and 116 (Asp). Suitably one or more of the following ami- no acid residues are mutated: 49, 52, 53, 61, 77, 81, 90, 92, 97, 99, 103, and 106. Suitable mutations are substitution mutations and, preferably, human PS variants are mutated in such a way that the PS variant contains at modified positions, d e corresponding bovine amino acid residue as replacement for the wild-type amino acid residue. Preferred PS variants contain at least d e mutations R49G and Q52R in die TS loop and P106S in d e EGFl module. Suitably, the PS variants also contain the mutation T53A and/or Q61L in die TS loop. In additions to the said mutations in die TS loop, suitable variants also contain some of the following mutations in the EGFl module: S81N, S92T, K97Q, S99T, T103I and P106S. A preferred PS variant contains all die above mutations in the TS loop and in the EGFl module. Preferred embodiments of me present invention are concerned widi human PC/APC and PS variants. However, the present invention is also concerned with PC/APC and PS variants of mammalian origin, e.g. bovine and murine, such as mouse and rat, origin, having enhanced anticoagulant activity. C. DNA segments and preparation thereof The present invention is also concerned with die deoxyribonucleic acid (DNA) segments or sequences related to die PC/APC and PS variants, e.g. the structural genes coding for tiiese variants, mutagenizing primers comprising the coding sequence for die modified amino acid stretch, etc.. In diis connection, die well-known redundancy of die genetic code must be taken into account. That is, for most of the amino acids used to make proteins, more than one coding nucleotide triplet (codon) can code for or define a particular amino acid residue. Therefore, a number of different nucleotide sequences may code for a particular amino acid residue sequence. However, such nucleotide sequences are considered as functionally equivalent since they can result in die production of d e same amino acid residue sequence. Moreover, occasionally, a methylation variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence, but such methylations do not effect the co- ding relationship in any way. Thus, such funtionally equivalent sequences, which may or may not comprise methylation variants, are also encompassed by the present invention. A suitable DNA segment of the present invention comprises a DNA sequence, diat encodes die modified (variant or mutant) PS/ APC and PS of d e present invention, that is, the DNA segment comprises the structural gene encoding the modified PC/APC or PS. However, a DNA segment of the present invention may consist of a relatively short sequence comprising nucleotide triplets coding for a few up to about 15 amino acid residues inclusive of the modified amino acid stretch, e.g. for use as mutagenizing primers. A structural gene of the present invention is preferably free of introns, i.e. the gene consists of an uninterrupted sequence of codons, each codon coding for an amino acid resi- due present in d e said modified PC/APC or PS. One suitable DNA segment of the present invention encodes an amino acid residue sequence d at defines a PC/APC variant that corresponds in sequence to die wild-type human PC/APC except for at least one amino acid modification (insertion, deletion, substitution), suitably in the amino acid sequence corresponding to d e SP-module of die wild-type protein. Otiier suitable DNA segments encode PC/APC variants, wherein said modifications) are contained in the amino acid residue sequence corresponding to amino acid residues 300-315 of the wild-type protein, die modified sequence preferably being comprised of WGYRDETKRNR (2). A further suitable DNA segment of die present invention encodes an amino acid residue sequence diat defines a PS variant d at corresponds in sequence to wild-type human PS except for at least one amino acid substitution in an amino acid sequence corresponding to die dirombin sensitive loop and/or die first and/or d e second EGF like module. Suitably one or more of the above mentioned substitutions disclosed for PS is(are) encoded by said DNA segment. In addition, the present invention is related to homologous and analogous DNA sequences d at encode die present PC/APC or PS variants, and to RNA sequences complementary tiiereto. The present DNA segments can be used to produce the PC/ APS or PS variants, - suitably in a conventional expression vector/host cell system as will be explained further below (Section D). As regards die DNA segments per se, tiiese can be obtained in accordance widi well-known technique. For instance, once the nucleotide sequence has been determined using conventional sequencing methods, such as die dideoxy chain termination sequencing method (Sanger et al., 1977), the said segments can be chemically syntiiesized, suitably in accordance with automated syndiesis methods, especially if large DNA segments are to be prepared. Large DNA segments can also be prepared by synthesis of several small oligo- nucleotides diat constitute the present DNA segments followed by hybridization and ligation of die oligonucleotides to form the large DNA segments, well-known methods being used. If chemical med ods are used to synthesize die present DNA segments, it is of course easy to modify d e DNA sequence coding for the wild-type PC/APC or PS by replacement, insertion and/or deletion of d e appropriate bases encoding one or more amino acid residues in die wild-type molecule. Suitably, recombinant DNA technique is used to prepare the present DNA segments comprising a modified structural gene. Thus, starting with recombinant DNA molecules comprising a gene, i.e. cDNA encoding wild-type PC/APC or PS, a DNA segment of me present invention comprising a structural gene encoding a modified PC/APC or PS can be obtained by modification of the said recombinant DNA molecule to introduce desired amino acid residue changes, such as substitutions, deletions and/or insertions, after expression of said modified recombinant DNA molecule. One convenient method for achieving tiiese changes is by site-directed mutagenesis, e.g. performed widi PCR-technology. PCR is an abbreviation for Polymerase Chain Reaction first reported by Mullis and Faloona (1987). Site-specific primer-directed mutagenesis is now standard in die art and is conducted using a synthetic oligonucleotide primer which primer is complementary to a single- stranded phage DNA comprising die DNA to be mutagenized, except for limited mismatching representing the desired mutation. Briefly, die synthetic oligonucleotide is used as a primer to direct syndiesis of a strand complementary to the phage DNA inclusive of the heterologous DNA and d e resulting double-stranded DNA is transformed into a phage- supporting host bacterium. Cultures of .the transformed bacteria are plated in top agar, permitting plaque formation from single cells that harbour die phage. In diis method, the DNA which is mutated must be available in single-stranded form which can be obtained after cloning in M13 phages. Site-directed mutagenesis can also be accomplished by "gapped duplex" method (Vandeyar et al., 1988; Raleigh and Wilson, 1986). In accordance wid a suitable embodiment of d e present invention, site-directed mutagenesis is performed with standard PCR-technology (Mullis and Faloona, 1987). Ex- amplary PCR based mutagenizing methods are described in d e experimental part of the present disclosure. In this example, the replication of the mutant DNA-segment is accomplished in vitro, no cells, neither prokaryotic nor eukaryotic, being used. Obviously, site-directed mutagenesis can be used as a convenient tool for construc- tion of die present DNA segments that encode PC/APC or PS variants as described herein, by starting, e.g. with a vector containing the cDNA sequence or structural gene that codes and expresses wild-type PC/APC or PS, said vector at least being capable of DNA replication, and mutating selected nucleotides as described herein, to form one or more of the present DNA segments coding for a variant of diis invention. Replication of said vector contai- ning mutated DNA may be obtained after transformation of host cells, usually prokaryotic cells, with the said vector. Illustrative methods of mutagenesis, replication, expression and screening are described in the experimental part of the present disclosure. D. Preparation of PC/APC or PS variants Such DNA segments, which comprise the complete cDNA sequence or structural gene encoding a PC/APC or PS variant, can be used to produce d e encoded variant by expression of die said cDNA in a suitable host cell, preferably a eukaryotic cell. Generally, such preparation of variants of the present invention comprises the steps of providing a DNA segment that codes a variant of this invention; introduction of the provided DNA segment into an expression vector; introduction of the vector into a compatible host cell; culturing die host cell under conditions required for expression of the said variant; and harvesting the expressed variant from the host cell. For each of the above mentioned steps suitable methods are described in the experimental part of the present disclosure. Vectors, which can be used in accordance widi die present invention comprise DNA replication vectors, which vectors can be operatively linked to a DNA segment of the present invention so as to bring about replication of this DNA segment by virtue of its capacity of autonomous replication, usually in a suitable host cell. To achieve not only DNA replication but also production of d e variant encoded by a DNA segment of die present invention, die said DNA segment is operatively linked to an expression vector, i.e. a vector capable of directing the expression of a DNA segment introduced ti erein. Replication and expression of DNA can be achieved from the same or diffe- rent vectors. The present invention is also directed to recombinant DNA molecules, which contain a DNA segment of die present invention operatively linked to a DNA replication and/or expression vector. It is well known that die choise of a vector, to which a DNA segment of die present invention can be operatively linked, depends directly on die functional properties desired for die recombinant DNA molecule, e.g. as regards protein expression, and die host cell to be transformed. A variety of vectors commercially available and/or disclosed in prior art literature can be used in connection with the present DNA segments, provided d at such vectors are capable of directing the replication of d e said DNA segment. In case of a DNA segment containing a strucmral gene for a PC/APC or PS variant, preferably, the vector is also capable of expressing d e structural gene when the vector is operatively linked to said DNA segment or gene. A suitable embodiment of me present invention is concerned widi eukaryotic cell expression systems, suitably vertebrate, e.g. mammalian, cell expression systems. Expres- sion vectors, which can be used in eukaryotic cells are well known in me art and are available from several commercial sources. Generally, such vectors contain convenient restriction sites for insertion of die desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPVl/pML2d (International Biotechnologies, Inc.), pXTl available from Stratagene (La Jolla, California), pJ5Eω available from The American Type Culture Collection (ATCC; Rockwille, MD) as accession number ATCC 37722, pTDTl (ATCC 31255) and die like eukaryotic expression vectors. In the experimental part of die present disclosure, pRc/CMV (available from Invitrogen, California, USA) and pGT-h (obtained from Lilly Research Laboratories, USA) have been used to obtain expression plasmids for use in adenovirus-transfected human kidney cells. Suitable eukaryotic cell expression vectors used to construct die recombinant DNA molecules of the present invention include a selection marker d at is effective in eukaryotic cells, preferably a drug resistance selection marker. A suitable drug resistance marker is me gene whose expression results in neomycin resistance, i.e. the neomycin phosphotransferase (neo) gene, Southern et al., J. Mol. Appl. Genet., 1:327-341 (1982). A further suitable drug resistance marker is a marker giving rise to resistance to Geneticin (G418). Alternatively, the selectable marker can be present on a separate plasmid, in which case die two vectors will be introduced by co-transfection of die host cell and selection is achieved by culturing in the appropriate drug for the selectable marker. Eukaryotic cells, which can be used as host cells to be transformed with a recombinant DNA molecule of die present invention, are not limited in any way provided diat a cell line is used, which is compatible with cell culmre methods, mediods for propagation of the expression vector and mediods for expression of the contemplated gene product. Suitable host cells include yeast and animal cells, vertebrate cells and especially mammalian cells being preferred, e.g. monkey, murine, hamster or human cell lines. Suitable eukaryotic host cells include Chinese hamster ovary (CHO) cells available from me ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL1658, baby hamster kidney cells (BHK) and the like eukaryotic tissue culture cell lines. In the experimental part of the disclosure, an adenovirus-transfected human kidney cell line 293 (available from The American Type Culmre Collection, Rockville, MD, USA) has been used. To obtain an expression system in accordance widi the present invention, a suitable host cell, such as a eukaryotic, preferably mammalian, host cell, is transformed with the present recombinant DNA molecule, known methods being used, e.g. such methods as disclosed in Graham et al., Virol., 52:456 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979). Thus, to express the DNA segment of die present invention in a eukaryotic host cell, generally, a recombinant DNA molecule of the present invention is used d at contains functional sequences for controlling gene expression, such as an origin of replication, a promoter which is to be located upstream of the DNA segment of the present invention, a ribosome-binding site, a polyadenylation site and a transcription termination sequence. Such functional sequences to be used for expressing die DNA segment of die present invention in a eukaryotic cell my be obtained from a virus or viral substance, or may be inherently con- tained in the present DNA segment, e.g. when said segment comprises a complete strucmral gene. A promoter which can be used in a eukaryotic expression system may, tiius, be ~ obtained from a virus, such as adeno-virus 2, polyoma virus, simian virus 40 (SV40) and die like. Expecially, the major late promoter of adenovirus 2 and d e early promoter and late promoter of S V40 are preferred. A suitable origin of replication may also be derived from a virus such as adenovirus, polyoma virus, SV40, vesicular stomatitis virus (VSV) and bovine papilloma virus (BPV). Alternatively, if a vector, that can be integrated into a host chromosome, is used as an expression vector, the origin of replication of die host chromosome may be utilized. Even if eukaryotic expression systems are preferred, prokaryotic expression sys- tems can also be used in connection with die present invention. Moreover, prokaryotic systems can advantageously be used to accomplish replication or amplification of me DNA-segment of the present invention, subsequently the DNA segments produced in said prokaryotic system being used for expression of the encoded product, e.g. in a eukaryotic expression system. Thus, a prokaryotic vector of d e present invention includes a prokaryotic replicon, i.e. a DNA sequence having the ability to direct autonomous replication and maintenance of e recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed tiierewith. Such replicons are well known in the art. In addition, tiiose embodiments that include a prokaryotic replicon also include a gene, whose expression confers drug resistance to a bacterial host transformed tiierewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline. If a prokaryotic system is used, not only for DNA replication but also as an expression system, these vectors that include a prokaryotic replicon also include a prokaryotic promoter capable of directing die expression, i.e. transcription and translation, of die pre- sent DNA segment containing a strucmral gene, in a bacterial host cell, such as E. coli, transformed tiierewith. A promoter is an expression control element formed by a DNA sequence diat permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pUC18, pBR322 and pBR329 available from BioRad Laboratories, Richmond, California, and pPL and pKK223 available from Pharmacia. Accordingly, to obtain a prokaryotic expression system which can express the gene product of die present invention appropriate prokaryotic host cells are transformed widi a recombinant DNA molecule of the present invention in accordance with well known methods diat typically depend on d e type of vector used, e.g. as diclosed in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). It is of course necessary that successfully transformed prokaryotic or eukaryotic cells can be distinguished and separated from non-transformed cells. For diis purpose, a variety of methods are known and have been described in prior art literature. In accordance with one such method, die presence of recombinant DNA is assayed for by examining die DNA content of monoclonal colonies derived from cells which have been subjected to a transformation procedure. Such methods have been disclosed by Southern, J. Mol. Biol. 98:503 (1975) and Berent et al., Biotech., 3:208 (1985). Successful transformation can also be confirmed by well known immunological methods, e.g. using monoclonal or polyclonal antibodies specific for the expressed gene product, or by the detection of the biological activity of the expressed gene product. Thus, cells successfully transformed widi an expression vector can be identified by me antigenicity or biological activity that is displayed. For this purpose, samples of cells suspected of being transformed are harvested and assayed for either the said biological acti- vity or antigenicity. Such selected, successfully transformed cells are used to produce the desired PC/APC or PS variants as disclosed above. E. Assays for biological activity Suitable methods for assaying the biological activity of the PC/APC or PS variants of the present invention are based on clotting systems, such as an APTT system, and on tests related to degradation of purified Factor Villa. Such methods are disclosed in more detail in die experimental part of the present disclosure. F. Discussion In accordance with a suitable embodiment of die present invention, which is related to PC/APC variants, the present variants comprise a mutated shortened amino acid sequence (2) instead of d e sequence (1) of die wild-type substance. This shortened amino acid sequence (2) is identical with die corresponding amino "" acid sequence of the bovine SP module. Since a comparison between me human, bovine, rat, and mouse sequences of die SP module revealed that the rat and mouse PC/APC moel- cules were more similar to human PC/APC man was the case with bovine PC/APC, mutants were prepared and investigated, which mutants comprised deletion and substitution mutations in human PC/APC making the 300-314 amino acid sequence identical widi die corresponding sequence of bovine PC/APC, and, vice versa, insertion and substitution mutations were introduced in bovine PC/APC to extend die bovine sequence corresponding to amino acid numbers 300-314 of human PC/APC and to make mat sequence identical with the hu- man amino acid sequence No. 300-314. In die experimental part of die present disclosure, isolation and characterization of mutants of human PC/APC and bovine PC/APC are described. Using standard PCR technology (Mullis and Faloona (1987), Meth. Enzymol. 155, 335-350), the above deletion, substitution and insertion mutations were made in d e cDNA's of human PC/APC and bovine PC/APC. Thus, after expression of tiiese mutated cDNA's in a eukaryotic system, a mutated human PC/APC molecule comprising sequence (2) and a mutated bovine PC/APC molecule comprising sequence (1) were produced and purified to homogeneity. In addition, d e cDNA's of wild-type human and bovine protein C/APC were expressed in d is eukaryotic system and the expression products were purified to homogeneity. To characterize the purified protein C/APC wild-type molecules and variants thereof obtained in these procedures, these molecules were activated by dirombin and the dirombin activation products were separated by S-Sepharose chromatography. The functional properties of the isolated PC/APC molecules were men characterized. The different PC/APC contracts, obtained by expression of die above mentioned cDNA's and a subsequent purification procedure, are referred to as follows: wt-hPC/APC, the wild-type human PC/APC; Δ- hPC/APC, human protein C comprising the shortened sequence corresponding to sequence (2); wt-bPC/APC, wild-type bovine PC/APC; ins-bPC/APC, bovine PC/APC comprising an extended sequence corresponding to sequence (1). These mutatnts, Δ-hPC/APC and ins- bPC/APC, are also designated human APC-SP and bovine APC-SP, resp., the latter designations being used mainly in me following Exemple 1 and the Figures referred to in this example. As is obvious from Example 1 , below, on standard SDS-polyacrylamide gel elect- rophoresis, these recombinant PC/APC constructs had die expected molecular weights when run under both reducing and non-reducing conditions. The amidolytic activity, i.e. die prό " - teolytic activity against a low molecular weights substrate, such as S-2238 (Chromogenix AB, Molndal, Sweden) was characterized and it was observed that d e mutated human PC/APC (Δ-hPC/APC) had much higher activity against the substrate than wild-type human PC/APC. The bovine mutation (ins-bPC/APC) on the otiier hand, had much lower activity against die syntiietic substrate, which suggested that the deletion insertion mutations affected the catalytic site of die PC/APC, even though die mutations were positioned at some distance from die active site. Thus, in accordance widi die present invention, it has unexpectedly been found, diat mutations in the SP-module of PC/APC, which mutations are positioned at some distance from the active site of PC/APC may give rise to PC/APC variants having enhanced anticoagulant activity due to enhanced proteolytic, and more specifically enhanced amidolytic, activity. The conclusion that the present mutations are not located within or adjacent to this active site is based on a published hypothetical molecule model of APC and a re- centiy elucidated model of die three-dimensional structure of the SP-module of APC, which is disclosed in EMBO Journal, 1996, 15: 6810-6821 (Mather et al.). From these models, it appears that die mutations contained in die above constructs of die present invention are located in loop 5 of the SP-module, which loop is not directly in contact widi the active site region. Furthermore, in die experimental part of the present disclosure, me kinetics of die synthetic substrate cleavages of the above recombinant PC/APC molecules have been characterized, i.e. d e values of Km, Vmax and kcat were determined by changing the substrate concentration. These parameters are elucidated in more detail in Example 1, below. In summary, it was found diat die value of Km was decreased, suggesting diat die affinities of die various APC-molecules for the substrate were higher. Moreover, die values of Vmax were very different, Δ-hPC/APC having at least a 7-fold higher value of Vmax than the wt- hPC/APC; ins-bPC/APC on the other hand, had a distinctiy lower value of Vmax whereas the value of Km was virtually unaffected. These results suggest diat die increased activity of Δ-hPC/APC was due to a combination of increased catalytic activity and increased affinity for the substrate caused by die mutations. In addition, in Example 1, below, die anticoagulant activities of the above mentioned mutated PC/APC molecules were measured in a plasma clotting systems based on die APTT (activated partial thrombopiastin time) reaction (activation by intrinsic padiway). Iτ was observed in tiiese tests, that when added to human plasma die Δ-hPC/APC had enhanced anticoagulant response as compared to wt-hPC/APC. In the absence of added bovine protein S, both wt-bPC/APC and ins-bPC/APC had very poor anticoagulant response, whe- reas both these bovine recombinant compounds expressed distinct anticoagulant activity when bovine protein S was also included in die reaction mixture. The above results indicate that the deletion-mutation in me human APC led to enhanced activity against the natural substrates present in human plasma (FVa and FVHIa) whereas the insertion-mutation in bovine APC did not significantly affect the reactivity against the natural substrates even though the activity against the synthetic substrates was impaired. To confirm that die deletion mutation in human APC indeed led to increased proteolytic activity against me natural substrate FVffla, the effect of the recombinant APCs in a FVIIIa degradation system using purified components (previously described system, Shen and Dahlback, J. Biol. Chem. 1994, 269:18735-18738) was investigated. The system inclu- ded FIXa, FVIIIa, phospholipid vesicles and calcium, the activity of FVHIa was measured by d e addition of FX and, after a short incubation time, addition of a synthetic substrate against FXa. The effect of the various APC molecules was tested by the addition of APC together with its synergistic cofactors protein S (of the same species as die APC) and bovine FV. In this system it was obvious that the Δ-hPC/APC had higher activity than wt- hPC/APC, whereas the two bovine PC/ APCs were relatively similar to each other. As regards the degradation of purified FVa, the various APCs have not been tested yet but is is expected diat Δ-hPC/APC will have higher activity than wt-hPC/APC. It is of course possible that die introduced changes in die human and bovine APCs might influence the rate of inhibition. To elucidate this possibility, the rate of inhibition of the mutated APC molecules was tested in human plasma. Thus, APC was added to plasma and at various intervals, the remaining amidolytic activity was measured. It was found that the mutated human molecule had d e same half-life as the wild-type human APC suggesting that die mutation did not affect the rate of inhibition by serpins. To test diis further, the rate of inhibition of mutated and wild-type APC by purified PCI and αlAT was tested and found to be essentially identi- cal. Bovine APC and die mutated bovine APC on the other hand were not inhibited by αlAT, which demonstrates mat the hypoti esis about the mutated region being involved in determining die rate of inhibition was not correct i.e. that the explanation for the different inhibition pattern of human and bovine APC was not caused by die identified sequence difference but by another sequence difference yet to be defined. In conclusion, the results obtained in Example 1 demonstrate that d e deletion- mutation in hAPC led to a molecule which had higher catalytic activity against die natural substrates FVIIIa and FVa as well as against low molecular substrates, whereas d e mutation did not affect the rate of inhibition by serpins. G. Potential use of the present PC/APC variants As emphasized above, it is obvious that a recombinant protein C molecule which after its activation to APC expresses enhanced amidolytic and anticoagulant activity has great potential use both as a possible therapeutic compound and as a reagent to be used in various biological assays for other components of the protein C system. In accordance with the present invention it has been shown for the first time that mutations in the SP module of the protein C molecule which do not affect the glycosylation sites of protein C can lead to enhanced proteolytic efficiency against both syntiietic and natural substrates. Thus, it can be expected diat a systematic search for such mutations may produce other protein C molecules with even better properties. For instance, it could become possible to design APC molecules with highly specific functions, e.g. molecules which mainly work against FVUla or those mainly cleaving FVa; optionally it may be possible to produce an APC which works well against the mutated FV which is present in me blood coagulation disorder APC-resistance. Accordingly, the PC/APC variants prepared in Example 1 only illustrate the invention without limitation thereof. Thus, the present invention is directed to all mutated protein C molecules widi enhanced amidolytic and anticoagulant activity, e.g. also such APC variants, which may be found and which are not inhibited by die naturally occurring serpins and, tiius, may by beneficial for specific therapeutic and diagnostic purposes. It is envisaged that d e present protein C and protein S variants expressing enhanced anticoagulant activity will be useful in all situations where undesired blood coagulation is to be inhibited. Thus, me present variants could be used for prevention or treatment of thrombosis and otiier thromboembolic conditions. Illustrative of such conditions are disseminated intravascular coagulation (DIC), arterioschlerosis, myocardial infarction, various hypercoagulable states and thromboembolism. The present variants could also be used for thrombosis prophylaxis, e.g. after thrombolytic dierapy in connection widi myocardial infarction and in connection with surgery. A combination of the present protein C and protein S variants could be useful, which combination also could include Factor V expressing activity as a cofactor to APC. As regards diagnostic use of die present PC/APC variants, there is a great need for improved functional assays for protein S and also for die anticoagulant activity of factor V. It is likely that a mutated APC with enhanced amidolytic and anticoagulant activity will be very useful in such assays because such APCs will give stronger signal and this will lead to increased signal to noise ratios in different assays. This is confirmed by the initial in vitro characterization of the mutated APC molecules which has been made in Example 1 and which shows that die amidolytic activity is much higher for the mutant hAPC than for nor- mal APC and also tiiat the anticoagulant effect is higher for the mutant hAPC than for normal APC. The interaction of the mutated molecule with its cofactors protein S and intact FV appeared unaffected by die mutations in me SP-module which suggests diat die concept of using the mutated hAPC (Δ-hAPC) in in vitro tests is correct. It might be possible to combine mutations in the APC-module with mutations in otiier parts of protein C to produce protein C with very unique properties. The scientist at Ely Lilly (Ehrlich et al, Embo. J. 1990, 9:2367-2373; Richardson et al, Nature 1992,360:261-264) and also other groups have already shown that mutations around die activation peptide region yielded protein C which was easily activated even in the absence of TM. Similarly, another set of mutations in the activation peptide region led to a protein C molecule which was secreted in active form from the synthesizing cells (Ehrlich et al, J. Biol, Chem. 1989,264:14298-14304). In a future perspective is may become interesting to combine mutations affecting the activation process with mutations in the SP-module which affect the catalytic activity and also with future mutations which may even enhance the interactions between APC and its cofactors. The present invention is of course directed to die protein C and protein S variants defined herein irrespective of die mode of production thereof. In die previous sections, e.g. in section D, some suitable methods are disclosed. However, other methods such as methods concerned with transgenic animals are foreseen to be useful. For instance, it is referred to Velander et al., "Transgenic Livestock as Drug Factories" in Scientific American, Jan. 1997, wherein a transgenic pig producing human protein C in her milk is disclosed. Thus, it seems likely that transgenic animals producing die present protein C or S variants could be obtained. EXPERIMENTAL PART In the following examples suitable embodiments are disclosed to illustrate die present invention. However, these examples should not be construed as limiting me invention . EXAMPLE 1. Variants of protein C In diis example the following materials were used. Human αl- antitrypsin (αlAT) and Protein C inhibitor (PCI) were kind gifts from Drs. Carl-B. Laurell and Margareta Kjellberg, respectively (Dept. of Clinical Chemistry, University Hospital, Malmo, Sweden). HPC 4 immunoaffinity columns were obtained from Dr. Charles T. Esmon (Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, USA). Fast Flow Q-Sepharose (FFQ) and Octonative M (as source of factor Vm) were purchased from Pharmacia, Sweden. Lipofectin and Geneticin (G418) are available from Life Technologies AB, Sweden, and Dulbecco's Eagle's modified medium (DMEM) is available from Gibco Corp.. Purified bovine factor IXa, factor X, phospholipid vesicles and the chromogenic substrate S-2222 were generous gifts from Dr. Steffen Rosen at Chromogenix AB, Sweden. Hirudin was obtained from Sigma Chemical Co., USA, and D-Phe-Pro-Arg Chloromethyl Ketone (PPACK) from Calbiochem, USA. Bovine factor V, α-dirombin, and human protein S as well as bovine protein S were purified according to previously described methods (Dahlback, et al., 1990; Dahlback and Hildebrand, 1994). (a) Site directed mutagenesis A full-length human protein C cDNA clone, which was a generous gift from Dr. Johan Stenflo (Dept. of Clinical Chemistry, University Hospital, Malmo, Sweden), and a full-lengdi bovine protein C cDNA clone, kindly provided by Dr. Donald Foster (Zymo- Genetics, Inc., USA) were separately digested with die restriction enzymes HinάTfl and Xbal and die resultant restriction fragment comprising the complete PC coding region, ei- dier human or bovine, diat is full length protein C cDNA, was cloned into a Hindlll and Xbal digested expression vector pRc/CMV. The resultant expression vectors containing the coding sequences for wild-type human or bovine protein C were used for site-directed mutagenesis of the SP-module of protein C, wherein a PCR procedure for amplification of target DNA was performed as de- scribed below and as shown in die following reaction scheme (Scheme I). The nucleotide sequences of die primers used in this procedure are listed in Table I below. SCHEME I PCR reactions to mutate human protein C mutated region PCR 2 primer C primer D * PRC1 and 2 products were mixed and used in PCR 3 primer A PCR 3 primer D / \ SacII Apal PCR product 3 was cleaved widi SacII and Apal and the mutant fragment isolated and Iigated into pUC18 containing protein C cDNA fragments as defined in the text and as shown below 1311 Tht full length mutated protein C cDNA was isolated after Hindϋ-Xbal difeβtion and Iigated into Hindlϋ-Xbal cleaved pRc/CMV vector and used for tranafection of 293 cells. To obtain a mutagenized human protein C cDNA, a fragment of human Protein G cDNA containing the coding region from the 5' terminal amino acid up to position 313 was amplified widi die use of intact human protein C cDNA as a template and a pair of primers A and B, primer B being the mutagenic oligonucleotide (PCR1 of Scheme I). A second fragment of human Protein C cDNA containing remaining amino acids after position 303 and, thus, partly overlapping the first fragment, was amplified widi d e use of intact human protein C cDNA as a template and a pair of primers C and D, primer C being the mutagenic oligonucleotide (PCR2 of Scheme I). From the above PCR amplification procedures, two partly overlapping, doub- lestiranded cDNA fragments were obtained, which botii contain the mutagenized DNA sequence. These two cDNA fragments were used as templates together wid two primers A and D in a further PCR procedure to amplify a full length human protein C cDNA containing the desired mutated amino acids (PCR3 of Scheme I). The reagent mixture for each of the above PCR reactions was 100 μl containing 0.25 μg of template DNA, 200 μM each of the deoxyribonucleoside triphosphates (dNTP: dATP/dCTP/dGTP/dTTP), 0.5 μM of each primer and 2.5 U of Pwo-DNA polymerase (Boehringer Mannheim) in Tris-HCl buffer (10 mM Tris, 25 mM KC1, 5 mM (NH 4 ) 2 SO 4 , and 2 mM MgSO 4 , pH 8.85). The sample was subjected to 30 cycles of PCR comprised of a 2 min denaturation period at 94°C, a 2 min annealing period at 55°C and a 2 min elongation period at 72°C. After amplification, the DNA was subjected to electrophoresis on 0.8 % agarose gel in 40 mM Tris-acetate buffer containing 1 mM EDTA. All PCR amplification products were purified by using JET Plasmid Miniprep-Kit (Saveen Biotech AB, Sweden). The resultant human protein C cDNA containing the desired mutations was digested with SacII and Apal, and then the fragment from the SacII and Apal digestion (nu- cleotides 728-1311) was cloned into the vector pUC18 which contains intact human protein C fragments (Hindlll-SacII, 5' end-nucleotide 728; and Apal-Xbal, nucleotide 1311-3' end) to produce human protein C full length cDNA comprising the desired mutations, viz. coding for a human protein C mutant comprising the mutated sequence (2) instead of the human wild-type sequence (1). In addition, bovine protein C cDNA was mutagenized and d e mutated cDNA was amplified essentially as disclosed above, except diat different primers and templates were used. The PCR amplification product of bovine protein C cDNA containing die desired mutations was cleaved with Sail and Bglll, and the fragment from digestion with Sail and Bglll (nucleotides 600-1123) was cloned into a vector pUClδ containing intact bovine protein C fragments (Hindlll-Sall, 5' end-nucleotide 600bp; and Bglll-Xbal, nucleotide 1123-3' end) to produce mutated bovine protein C full length cDNA in the vector pUC18, whereaf- ter Hindlll and Xbal were used to cleave bovine protein C full length cDNA containing the desired mutations, viz. coding for a bovine protein C mutant comprising the mutated sequence (1) instead of the bovine wild-type sequence (2). Then, each of the above mutated human and bovine protein C cDNA's was digested with Hindlll and Xbal and the appropriate restriction fragment was cloned into the vector pRc/CMV, which had been digested with the same restriction enzymes. The vectors obtained were used for expression of mutated human or bovine protein C in eukaryotic cells. Before transfection of the appropriate host cells, all mutations were confirmed by DNA sequencing by the dideoxy chain termination method of Sanger et al. , supra. For the above site-directed mutagenesis procedure, the following oligonucleotide primers listed in Table I in the 5' to 3' direction were used. TABLE I Primer designation Nucleotide sequence A 5'-AAA TTA ATA CGA CTC ACT ATA GGG AGA CCC AAG CTT-3' B 5'-GTT TCT CTT GGT CTC GTC ACG GTA GCC CCA GCC CGT CAC GAG-3' C 5'-CGTGACGAG ACC AAG AGA AAC CGC ACC TTC GTC CTC-3' D 5'-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCC TCT AGA-3' E 5'-GGC CTC CTT CTC TCG GCT GCT GTG GTA GCC CCA GCC CGTCAC-3' F 5'-CAC AGC AGC CGA GAG AAG GAG GCC AAG AGA AAC CGC ACC TTC-3' . Primers A-D were used to mutagenize and amplify human protein C cDNA, as disclosed above. To mutagenize and amplify bovine protein C cDNA, likewise, two pair of primers were used, viz. primers A and E and primers F and D, primers E and F being mutagenic primers. The nucleotide sequences of these primers are related to parts of the vector nucleotide sequence or parts of the protein C cDNA nucleotide sequence as explained below. Primer A corresponds to nucleotides 860-895 in die vector pRc/CMV and provides a Hindlll restriction site between the pRc/CMV vector DNA and d e protein C cDNA. Primer B corresponds to a partial, modified antisense nucleotide sequence of human protein C cDNA, the modified sense sequence coding for: LVTGWGYRDETKRN. This amino acid residue sequence corresponds to a modified sequence of human protein C from amino acid residue number 296 to 313, inclusive, wherein the sequence of residues 303-310 contains mutations, i.e. the residues 303, 304, 305 and 308 are deleted and residues 307 and 310 are substimted, d e resulting sequence RDET being identical with d e corresponding part of bovine protein C (residue numbers 305-308). Primer C corresponds to a partial modified nucleotide sequence of human protein C cDNA coding for: RDETKRNRTFVL. This amino acid residue sequence corrsponds to a modified sequence of human protein C from amino acid residue number 303 to 318, inclusive, which contains die same mutations as disclosed for primer B above, i.e. the residue numbers 303-305 and 308 are deleted and residue numbers 307 and 310 are substimted. Thus, primer C encodes a shortened sequence RDET which is identical with the corresponding sequence of bovine protein C. Primer D corresponds to die antisense sequence to d e sequence of nucleotides 984-1019 in the vector pRc/CMV and provides a Xbal restriction site between the pRc/CMV vector DNA and the protein C cDNA. Primer E corresponds to a partial modified antisense nucleotide sequence of bovine protein C cDNA, die modified sense sequence coding for: VTGWGYHSSREKEA. This amino acid residue sequence corresponds to a modified sequence of bovine protein C from amino acid residue number 299 to 308, inclusive, wherein the sequence corresponding to residue numbers 305-308 (RDET) contains mutations, viz. four insertions and two substitutions, the mutated sequence being HSSREKEA which is identical with the cor- ^ responding part of human protein C (residues numbers 303-310). Primer F corresponds to a partial modified antisense nucleotide sequence of bovine protein C cDNA coding for: HSSREKEAKRNRTF. This amino acid residue sequence corresponds to a modified sequence of bovine protein C from amino acid residue number 305 to 314, inclusive, which contains the same mutations between positions 305 and 308 as stated for primer E above. Thus, primer F encodes an extended sequence HSSREKEA which is identical widi the corresponding sequence of human protein C. (b) Production of stable transformants producing variant or wild-type protein C. To produce stable transformants producing variant or wild-type protein C, adeno- virus-transfected human kidney cell line 293, was grown in DMEM medium containing 10% of fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin and 10 μg/ml vitamin K,, and transfected with an expression vector comprising wild-type or mutagenized protein C cDNA from step (a). The transfection was performed in accordance with the Lipofectin method as described earlier (Feigner et al., 1987). In brief, 2 μg of vector DNA which was diluted to 100 μl with DMEM containing 2 mM of L-glutamine was mixed with 10 μl Lipofectin (1 μg/μl) which was diluted to 100 μl with d e same buffer. The mixture was kept at room temperature for 10-15 min and was diluted to 1.8 ml with the medium, and then added to the cells (25-50% confluence in a 5-cm Petri dish) that had been washed twice with d e same medium. (c) Expression of variant or wild-type protein C. The transfected cells from (b) were incubated for 16 hours, whereafter the medium was replaced wid complete medium containing 10% calf serum and die cells were incu- bated for additional 48-72 hrs. The cells were tiien trypsinized and seeded into 10-cm dishes contaning selection medium (DMEM comprising 10% serum, 400 μg/ml G418, 2 mM L- glutamine, 100 U/ml penicillin, 100 U/ml streptomycin and 10 μg/ml vitamin K,) (Grinnell, et al. 1990). G418-resistant colonies were obtained after 3-5 weeks selection. From each DNA transfection procedure, 24 colonies were selected and grown until confluence. All colonies were screened by dot-blot assays using monoclonal antibody HPC 4 (for human protein C) or monoclonal antibody BPC 5 (for bovine protein C) to examine die protein C expression. High expression cell colonies were selected and grown until confluence in d e selection medium. Thereafter, these cells were grown in a condition medium (selection medium lacking serum) to iniate expression of protein C or a variant tiiereof, which medium, like the selection medium was replaced every 72 h. After a suitable time period, die condition medium containing me respective expression product was collected for purification of said product in section (d) below. (d) Purification of recombinant wild-type and mutated proteins (i) Bovine recombinant protein C and its mutant were purified by the method described previously (Yen et al., 1990). Five mM of EDTA and 0.2 μM of PPACK were added to d e condition medium collected in section (c). The medium was then applied to a Pharmacia FFQ anion-exchange column and eluted with a CaCl 2 gradient (starting solution, 20 mM Tris-HCl/150 mM NaCl, pH 7.4; limiting solution, 20 mM Tris-HCl/150 mM NaCl/30 mM CaCl 2 , pH 7.4) at room temperature. The CaCl 2 was removed by overnight dialysis (20 mM Tris-HCl, 150 mM NaCl, pH7.4) in combination with Chelex 100 treatment. The dialysate was men applied to a second FFQ column to readsorb protein C or its mutant to the column, whereafter protein was eluted with a NaCl gradient solution (starting solution 20 mM Tris-HCl/150 mM NaCl, pH 7.4; limiting solution, 20 mM Tris-HCl/500 mM NaCl, pH 7.4). (ii) Culmre medium obtained in section (c) from transformants producing human wild-type or mutant protein C was first subjected to column purification and, then, applied to an affinity column carrying monoclonal antibodies HPC 4 as described earlier (Rezaie and Esmon, 1994) except for slight modifications (He et al., 1994). The purified proteins obtained in (i) and (ii) were concentrated on YM 10 filters (Amicon), dialyzed against TBS buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.4) for 12 hrs and stored at - 80°C until use tiiereof. The purity and homogeneity of the above wild-type and mutant protein C's were established by SDS-PAGE. This electrophoresis procedure was run as a polyacrylamide (10- 15%) slab-gel electrophoresis in the presence of 0.1 % of SDS (sodium dodecyl sulphate) under reducing and non-reducing conditions wherein the said proteins were visualized by silver staining (Morrissey, 1981). The results from SDS-PAGE analysis using an acrylamide concentration gradient of 5-15% and run on the proteins purified above, indicated tiiat all recombinant protein C's obtained from the expression in Example 1(c) migrated as single bands with relative mo- lecular masses similar to those of the respective plasma-derived proteins under nonreducing conditions. Human protein C had an apparent molecular mass of 62 KDa, whereas the molecular mass of bovine protein C was somewhat smaller. In agreement with previous reports, it was found tiiat plasma-derived human protein C, recombinant wild-type protein C and mutant protein C exhibited two subforms corresponding to α and β protein C as glycosylation variants (Miletich and Broze, 1990). However, these two subforms were not obvious in bovine protein C. Under reducing conditions, die heavy chain from each recombinant protein C migrated as a double-band (Mr 41 KDa). A light chain (Mr 21 KDa) was also observed. This indicates mat the transformed cells from Example 1(b) produce recombinant wild-type and mutant protein C derivatives in a similar manner, (e) Characterization of protein C mutants (1) To characterize the protein C mutants obtained in the previous steps, mutant and wild-type protein C were activated and their activity measured in accordance with the following test methods. Activity inhibition tests, as disclosed below, were also performed. (1) (i) Activation of Protein C and Amidolytic Activity Assay Activation of protein C to activated form (activated protein C, APC) by thrombin was performed as described previously (Solymoss et al., 1988) except for slight modifica- tions. In brief, the protein C was incubated with α-thrombin (1:10, w/w) at 37°C for 2 hrs in TBS in the presence of 5 mM EDTA. After incubation, the mixture was passed d rough a sulfopropyl-Sepharose column to remove dirombin. It was confirmed by the mobility difference between reduced protein C and APC on SDS-PAGE, diat protein C was fully activated. The amidolytic activity of APC was measured by determination of die hydrolysis of a syntiietic substrate, S2238 (Chromogenix AB, Sweden), which process was monitored at 405 nm at room temperature in a Vmax kinetic microplate reader (Victor, Molecular De- vices Corp., USA). (1) (ii) Activated Partial Thromboplastin Time (APTT) Assay Quantitative determination of APC activity was based on d e prolongation of APT time. Coatest APC Resistance kit (Chromogenix AB, Molndal, Sweden) was used for APTT assay of APC. Fifty μl of human or bovine citrated normal plasma was incubated widi 50 μl of APTT reagent at 37°C for 200 sec, and then 100 μl of CaCl 2 (12.5 mM) containing APC (final concentrations (of 0-10 nM) were added. The clotting time was measured using an Amelung-Coagulometer KC 10 (Swedish Labex AB). All dilutions were made in TBS buffer in die presence of 0.1 % bovine serum albumin (BSA). (1) (iii) FVIIIa Inactivation Assay Different concentrations of human or bovine recombinant APCs (0-32 nM) were mixed widi protein S (20 nM) and factor V (20 nM) in microtiter plate wells (Linbro, Flow Laboratories) with a final volume of 25 μl in 50 mM Tris-HCl, 150 mM NaCl buffer containing 10.5 mM CaCl 2 , 0.1 % BSA, pH 7.4. Eighty μl of factor Villa reagent (containing bovine factor IXa, human factor Villa, CaCl 2 and phospholipids) was added to d e mixture. After 5 min of incubation at room temperature, bovine factor X was added. The amount of activated factor X subsequently formed was measured by addition of 50 μl of a syntiietic substrate S-2222 after 5 min of incubation. The reaction was stopped by adding 50 μl of 20% acetic acid after 5 min of incubation in dark at room temperature and die absorbance at 405 nm was monitored. The production of factor Xa is linearly correlated to the activity of factor Villa, which is expressed as percent of activity of respective control (Shen and Dahl- back, 1994). All reagent concentrations given above are final concentrations. (1) (iv) Prothrombin Time (PT) Assay The inactivation of factor V by APC was measured according to the PT assay. One hundred μl of human or bovine plasma (1: 3 dilution) was incubated at 37°C for 120 sec, whereafter clotting was initiated by adding 300 μl of a mixture of Neoplastin and APC (Neoplastin: APC, 2: 1, v/v). The final concentrations of APC were from 0 to 30 nM. The assay was performed on an Amelung-Coagulometer KC 10. (1) (v) Inactivation of Protein C and Protein C Mutants In Human Plasma APC derived from activation of protein C, either human or bovine wild-type or mutants tiiereof, were diluted to 70 nM with 300 μl of citrated human plasma at 37°C. Sam- pies (40 μl) were collected and diluted 5-fold in cold TBS at points of time in a range of 0 to 60 minutes. From each diluted sample, 60 μl were added to 50 μl of a synthetic substrate S-2238 (Chromogenix AB, Sweden) (1 mM) in wells on a microtiter plate. The rate of ami- dolysis of S-2238 by APC was recorded continuously for 0-10 min at 405 nm (Holly and Foster, 1994). (1) (vi) Inactivation of Protein C and Mutants thereof by αlAT Wild-type or mutated human APC or bovine APC (170 nM of each) were incubated separately with human αlAT (0-16 μM) in 80 μl TBS buffer containing 0.1 % BSA at 37°C overnight (Holly and Foster, 1994). Samples (20 μl) were collected and added to 100 μl of S-2238 (1 mM) in wells on a microtiter plate. The rate of hydrolysis of S-2238 was monitored at 405 nm at room temperature for 0-10 min in a Vmax kinetic plate reader. (1) (vii) Inactivation of Protein C and Protein C Mutants by PCI Various recombinant APCs (40 nM) were incubated widi 88 nM of PCI in 1 ml TBS buffer containing 0.1 % BSA at 37°C. After incubation, samples (50 μl) were collected and placed on ice at points of time ranging from 0 to 120 min, and tiien added to 50 μl S- 2238 (1 mM). The rate of hydrolysis of S-2238 was measured from 0-10 min at 405 nm at room temperature. (2) The results from activity tests performed as disclosed above are summarized below. (2) (i) After activation of the protein C's from Example 1(d), SDS-PAGE run on die activated protein C's, indicated that die molecular masses of all recombinant wild-type and mutant APCs were similar to the corresponding plasma-derived APC, but smaller than the respective inactive forms. No intact protein C bands were observed in d e APC samples, and d e purity of all these proteins were more than 90% on the gel. The amido-lytic activity of all APCs were measured with the syndietic substrate S-2238. For wild-type human and bovine APC the initial rate was essentially the same, whereas the initial rate for the mutant recombinant human activated protein C (designated human APC-SP) was approximately 5- fold higher than for wild-type APC. However, for the mutant recombinant bovine activated protein C (designated bovine APC-SP) the initial rate was only about 1/10 of wild-type APC. These results are shown in Fig. 3. (2) (ii) In the APTT assay, the anticoagulant activity of recombinant wild-type and mutant APCs was analyzed in human plasma, in human plasma supplemented with bovine protein S and in bovine plasma. As is obvious from Fig. 4A, in human plasma, human APC-SP expressed a higher anticoagulant activity than wild-type human APC, whereas neither wild-type APC nor bovine APC-SP expressed any substantial anticoagulant activity. On the other hand, all these APCs expressed anticoagulant function in bovine plasma and in human plasma supplemented wid bovine protein S. However, bovine APC and human APC-SP showed a higher anticoagulant activity than human APC and bovine APC-SP (Fig. 4B, 4C). (2) (iii) In the Factor Villa Inactivation Assay performed in the presence of human protein S and factor V, die activity of factor Villa was inactivated by all APCs from section (2)(i) above but high concentrations were needed. At low concentrations, neidier bovine wild-type APC nor bovine APC-SP could inactivate factor Villa. Human APC-SP expressed more potent anticoagulant activity than that of wild type human APC (Fig. 5A, 5B). Wild- type human and bovine APC as well as the mutants thereof were able to inhibit factor VHIa activity in the presence of bovine protein S and bovine factor V, but both wild-type bovine APC and bovine APC-SP worked more efficiently than wild-type human APC and human APC-SP (Fig. 5C). (2) (iv) In accordance with the PT assay of (l)(iv), inactivation of factor Va by die wild-type APCs and the mutants thereof was tested in human plasma and bovine plasma. Bod wild-type human APC and human APC-SP increased clotting times essentially in this PT assay. Moreover, human APC-SP was more active than wild-type human APC. Neither wild-type bovine APC nor bovine APC-SP had any effect in human plasma (Fig. 6A). As is obvious from Fig. 6B, wiid-type human APC and human APC-SP efficiently prolong clotting time in bovine plasma, whereas wild-type bovine APC and its mutant expressed only weak anticoagulant acitivity in bovine plasma (Fig. 6B). (2) (v) Results from APC inactivation tests. The above APC inactivation test (l)(v) showed that d e amidolytic activity of wild- type and mutant APCs declined with about 60 to 90% from 0 to 60 min (Fig. 7). Thus, tiiese APCs should be inactivated by some serine protease inhibitors, such as PCI, αlAT, O j -macroglobulin, etc.. Indeed, both wild-type human APC and human APC-SP were substantially inhibited by high concentrations of αlAT in test (l)(vi). However, wild-type bovine APC and bovine APC-SP were almost completely resistant to the inhibition. Test results obtained in accordance with (l)(vii) showed diat bovine wild-type APC was efficiently degraded by human PCI, whereas bovine APC-SP was less efficiently inhibited by human PCI. On the other hand, the amidolytic activity of human APC-SP declined much faster than for wild-type human APC but at a rate similar to the rate for wild-type bovine APC. EXAMPLE 2. Variants of protein S In this example the following materials were used. Kits for DNA sequencing and the T7-Gen In Vitro Mutagenesis were obtained from United State Biochemicals Corporation, (USA). Q-Sepharose Fast Flow was obtained from Pharmacia (Sweden) and Hygromycin B (Hyg) from Calbiochem. Saline of phosphate buffer (PBS) lacking calcium and carbonate, Lipofectin, Optimem medium and Dulbecco's Eagle's modified medium (DMEM) were available from Gibco Corp. The protein S-defici-ency plasma was prepared by incubating normal human plasma with immobilized polyclonal anti-protein S antibodies at 4°C overnight as described in Dahlback, B. (1986) J. Biol. Chem. 261, 12022-12027. Plasmas from different species were obtained from Dakopatts AB (Copenhagen, Denmark) or Department of Experimental Research, Lund University, Sweden. Protein S and activated protein C were prepared from human and bovine plasma using methods previously described ifi Dahlback et al., (1990) J. Biol. Chem. 265, 8127-8135. The monoclonal antibodies which react with human protein S have been previously characte- rized (Dahlback et al., (1990) loc. cit.). Full length human and bovine protein S cDNA clones were previously isolated and characterized (Dahlback et al., (1986), loc. cit., Malm et al., (1994) Biochem. J. 302, 845- 850). Since the original bovine protein S cDNA clone was missing one nucleotide, viz. an A in die second EGF-like module, to make expression of wild-type bovine protein S possible, site-directed mutagenesis as disclosed below was used to introduce the missing codon. In addition, BamHI sites were introduced at the 5'- as well as the 3'- end of the coding regions of full- length human and bovine cDNA clones as described previously in He, et al., (1995) Eur. J. Biochem. 277, 433-440. The numbering of me nucleotides of human and bovine protein S cDNA used herein is based on sequences available in the EMBL database having d e acces- sion number M 15036 and M 13044, respectively. (a) Preparation of cDNA coding for protein S variants. cDNA:s coding for human protein S variants or mutants containing mutations in the thrombin-sensitive region (TSR) and/or in the first EGF-like (EGFl) module were prepared as disclosed below. The protein S mutants encoded by these cDNA:s are listed in die following Table II, wherein the wild-type human protein S (wt-hPS) amino acid residue sequence from Asp38 to Cys 113 is shown. Mutated amino acid residues are underlined and the corresponding substitutions in the mutants are shown. As is obvious from the last line in mis table showing the wild-type bovine protein S (wt-bPS) amino acid sequence, the mutations made are such mat the wt-hPS amino acid residues are replaced with d e respective wt-bPS amino acid residue at the same position. Table II Protein S mutants TSR EGFl 50 60 70 80 ?0 100 110 Protein S mutants CLBSF4^GLFTAARQSTNAYPDLRSCVNAI£r_ C£PL^ no 1 -N- • F-T- -Q-T- I— S- 2 -G— RA— -L- 3 -G— RA— • L- -N- -P-T- -Q-T- -I— S- 4 -G— R ■ S- -N- -F-T- -Q- - -I— S- 5 -G— A— - — T- -Q-T- -I— S- 6 -G— RA— — N- — T- -Q — -I— S- 7 -G— A— — N- . — τ _ -Q-T- ■ I— s- 8 -G— A— ■ L- — T- --Q-T- -I— -s- 9 -G— RA-- -L- .__ N- — - -Q— -I— s- 10 -G— -RA— - • L- -— N- - — - -Q-T- -I — s- 11 -G— RA— - s- 12 -G— RA— -L- s- 13 -G — RA— - • L- N— -- T- Q-T- Bovine protein S -G— RA— - - -P-T- -Q-T- I— s- Thus, as is obvious from Table II, the mutants contained some of or all of the following substitutions R49G, Q52R, T53A and Q61L in the TS region and P77S, S81N, Y90F, S92T, K97Q, S99T, T103I and P106S in the EGFl module. In this table, listed mutants 1-3 are human/bovine protein S, chimeras in which the bovine TSR, EGFl or both modules have been introduced into human protein S, i.e. the bovine module(s) replace the corresponding human module(s). Mutant 1 containing the bovine EGFl module was prepared from the previously described chimera V (He, et al., (1995), loc. cit.). This chimera contains the Gla-, TS- and EGFl-modules of bovine origin with the rest being of human origin. A cleavage site for Hindi (position 454) located between TSR and EGFl in both human and bovine protein S cDNA was used to create the cDNA coding for mutant 1. BamHI-Xbal (Xbal cleaves at 1481) fragments of both human protein S cDNA and chimera V cDNA were cleaved with Hindi. The isolated larger HincII-Xbal fragment from chimera V cDNA was Iigated to the smaller BamHI-HincII fragment from human protein S cDNA and to the 3' Xbal-BamHI fragment to create full-length cDNA in BamHI cleaved pUC18. This full-length cDNA construct, coding for a chimeric protein S, which contains EGFl of bovine origin in a human protein S background, was transferred to the expression vector pGT-h as described below. The cDNA coding for mutant 3, which mutant contains bom TSR and EGFl of bovine origin, was constructed in a similar way utilizing an Neil cleavage site which was present in both human (position 372) and bovine (position 261) protein S cDNA between the Gla and TSR modules. In short, chimera V cDNA was cut with Neil and BamHI and the large fragment was isolated and Iigated to the small BamHI-Ncil fragment obtained from the human cDNA clone. The cDNA coding for mutant 2, which mutant contains only the TSR of bovine origin, was made from mutant 3 cDNA and human protein S cDNA. BamHI-Xbal fragments of both these cDNAs were cut with Hindi, and die Bam- HI-HincII fragment from mutant 3 cDNA was Iigated to the HincII-Xbal frag-ment from human protein S cDNA together with die 3' Xbal-BamHI fragment to create cDNA constructs coding for full-length molecules. Thus, a cDNA coding for a mutant, which mutant contains the TSR of bovine origin in a human protein S background, was obtained. To produce mutants 4-12, oligonucleotide-directed mutagenesis, wherein me oligo- nucleotides listed in Table III were used as primers, was performed to create cDNA coding for mutants containing mutations in the EGFl- and TS-regions of protein S. Suitably, mutants 1 and 2 were used to introduce mutation(s) in the TS- and EGFl -region, resp., to produce mutants having a bovine EGFl -region (from mutant 1) or a bovine TS-region (from mutant 2) and containing further mutations(s) in the other region of concern. All EGFl mutations were made using the BamHI-Xbal fragment, whereas a Bam- HI-Hindlll fragment (Hindlll cleaves at position 559) was used for the TSR mutations. These fragments of human protein S cDNA were subcloned into M13mpl8 and single-strand templates were prepared using standard methods, (Sambrook et al., (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY). Site-directed mutagenesis was accomplished with the T7-Gen In Vitro Mutagenesis kit from United State Biochemicals Corporation and performed in accordance with the "gapped duplex" method (Vandeyar et al., (1988) Gene 65 (1), 129-133; Raleigh et al., (1986) PNAS 83 (23), 9070-9074). The M13mpl8 clones carrying the protein S cDNA inserts were annealed with each specific oligonucleotide, subjected to die second strand synthesis with T7 DNA polymerase and ligation with T4 DNA ligase. The DNA was used to transfect competent Escherichia coli SDM (mcrA ' mcrB ' ) cells. From individual plaques, single stranded DNA was isolated and sequenced by the dideoxy chain termi- nation method using Ml 3 or protein S specific primers. (Sanger et al., (1977) PNAS 74 (12), 5463-5467). The mutated protein S cDNA inserts were isolated from double stranded phage DNA (HincII-Xbal for EGFl mutants and BamHI-HincII for TSR mutants) and the appropriate fragments of human protein S cDNA were used to construct full length human protein S cDNA in pUC18. With the help of the Hindi site between TSR and EGF 1 , combinations of the TSR and EGFl mutant cDNAs were prepared using standard restriction enzyme cleavage and fragment ligation procedures, as outlined above. Mutant cDNAs were isolated after BamHI digestion and subcloned into the Bell site of the expression plasmid pGT-h, which was a kind gift from Dr. B. W. Grinnell (Lilly Research Laboratories, Eli Lilly Company, Indianapolis, USA), (Berg et al., (1992) Nucl. Acids. Res. 20 (20), 5485-5486). The resultant protein S cDNA expression plasmids were prepared by CsCl gradient ultracentrifugation (Sambrook et al., loc. cit.) and used to transfect kidney cells, as disclosed below. TABLE HI Primers used in construction of mutations in thrombin-sensitive region and first EGF-like module of human protein S HS52 5'-GAA TAA CCC AGT TCG AAΛ AGA GCG AAG HS49/52 5*-AGCAGTGAATAACCC AGTTCG AAAAGA GCC AAG ACA AACTAA GTA TT-3* HS49/52/53 5'-AGC AGTGAA TAA CCC AGC TCG AAA AGA GCC AAG ACA AAC TAA GTA TT-3' HS61 5'-AGC ATTAGTTGA CAGACG TGC AGC AGT G-3' HS77 5'-GGA CTA CAC TGG TCC GAA ATG GCA TTG ACA CA-3' HS81 5'-GCA TGG CAG AGG ATT ACA CIO GTC TGG -3' HS92 5'-CCA TCTTTG CAG GTC ATA TATCCA CATTAC-3* HS97 5*-TAA AAG AAG CTT GTC CAT CITTGC AG-3' HS92/97 5'-TAA AAG AAG CTT GTC CATCTTTGC AGG TCA TATATC CATCTT C-3' HS92/97/99 5*-TGC AAGTAAAAG1AG CTTGTC CATCTTTGC AGG TCATATATC CATCTTC-3' HS 103/106 5'-CTC CTTGCC AAC CTG ATTTAC AAA TGC AAG TAA AAG-3' HS106 5'-CCTTGC CAACCTGATTTA CAA GTG C-3' The positions subjected to direcled-mutagenesis are given. The nucleotides giving rise to substitution are underlined. P33278Sl_.X.-b-lW8/GAG. LP (b) Cell Culture and Expression: The adenovirus-transfected human kidney cell line 293, was grown in DMEM medium supplemented with 10% fetal calf serum, 2 mM L-gluta-mine, 100 Units/ml of penicillin and streptamycin, 10 μg/ml vitamin K|. Transfection was performed using the Lipofectin method (Feigner et al., (1987) PNAS 84 (21). 7413- 7417). DNA (2-4 μg) was diluted to 0.1 ml with sterile water. Lipofectin was added (1 μg/μl) and samples were left at room temperature for 10-15 min. Cell monolayers (40-50% confluent in a 5 cm Petri-dish) were washed twice in serum-free Optimem medium (Gibco). The DNA/lipid mixture was diluted to 1 ml in Optimem medium, added to the cells and incubated overnight (16-20 hrs). The cells were fed with 2 ml complete medium containing 10% calf serum and left to recover for another 48-72 hrs. They were then trypsinized and seeded into 10 cm dishes with selection medium (DMEM containing 10% calf serum and 200 μg/ml Hygromycin B) at 1:5 (Grinned et al., (1990) Blood 76 (12), 2546-2554). Hygromy- cin-resistant colonies were obtained after 3-5 weeks of selection, pooled (30 colonies on average), grown to confluency and the media screened for protein S expression with an enzy- me-linked immunosorbent assay (ELISA). Conditioned media were collected in the presence of 10 μg/ml vitamin Ki. (c) Purification of recombinant wild-type and mutant protein S.: Tissue culture medium (approximately 2 liters) was filtered through glass wool and diluted with an equal volume of 10 mM Tris-HCl, pH 7.5, containing 10 mM benzamidine-HCl and incubated with 50 ml Q-Sepharose Fast Flow (Pharmacia) during gentle stirring at 4°C overnight. The gel was allowed to settle for 1 h and the supernatant discarded. The gel was packed into a column (3 cm x 50 cm) and washed with 10 mM Tris-HCl, pH 7.5, containing 10 mM benzami- dine-HCI, and then with the same buffer also containing 50 mM NaCl. Elution was performed with 1.0 M NaCl in the same buffer. The column was run at 4°C at a flow rate of 80 ml/h; 15 ml fractions were collected. Fractions containing protein S were identified by electroimmu- noassay or ELISA, pooled and dialyzed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, containing 5 mM benzamidine-HCl. After the addition of Ca 2+ to a final concentration of 2 mM and incubation for 2 h, the material was applied to an affinity column containing the Ca 2+ -dependent antibody HPS-21 (Dahlback et al., (1990), loc. cit.; Malm et al., (1990) Eur. J. Biochem. 187, 737-743.). The column was equilibrated with 50 mM Tris-HCl, 0.15 NaCI, pH 7.5, containing 5 mM benzamidine-HCl and 2 mM Ca 2+ . After application, the column was washed with the same buffer containing 1.0 M NaCl and protein S was then eluted with 50 mM Tris-HCl, 1.0 M NaCl, 10 mM EDTA, pH 7.5, containing 5 mM benzamidine-HCl. Fractions containing protein S were pooled and dialysed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5 overnight. After concentration by ultrafiltration using YM 10 filters (Amicon), the proteins were stored at -70°C. The purity and homogeneity of these proteins were established by SDS-PAGE. Poly- acrylamide (10-15%) slab gel electrophoresis (PAGE) was run in the presence of 0.1% sodium dodecyl sulphate (SDS) under reducing and nonreducing conditions using methods described previously (Dahlback et al., (1990) loc. cit.). Proteins were silver-stained on SDS-PAGE. (Morrissey, 1981). When subjected to SDS-PAGE under nonreducing conditions, the mutants migrated as single chain bands, like the wild-type human and bovine pro- tein S and the respective plasma-derived proteins. After reduction, the human and bovine wild-type proteins and some of the recombinants migrated as closely spaced doublets, just like the human and bovine plasma-derived protein S preparations. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the wild-type protein S and the protein S mutants, obtained above. Rabbit anti-protein S IgG (human or bo- vine) was used as catching antibody and biotinylated monoclonal antibody HPS-21 as detecting anti-body. Polyclonal antibodies were used at appropriate dilution in 50 mM Na 2 CO 3 , pH 8.5, to coat microtiter plates (Costar, Cambridge, MA, USA) overnight at 4°C. Plates were washed three times with 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 and incubated with 200 μl 1% BSA in the same buffer for 30 min. This was followed by three washes using 50 mM Tris-HCl, 150 mM NaCl. pH 7.5, containing 0.1% Tween 20. Samples containing protein S (diluted in 150 mM NaCl, 20 mM Tris-HCl, 0.1% BSA, pH 7.5, containing 2 mM CaCl 2 ) were incubated in the wells for 2 hrs at room temperature. After three washes, 100 μl of biotinylated HPS-21 (final concentration 1 μg/ml) was added and incubated for 1 hr at room temperature. After three washes, a mixture of streptavidin and biotinylated horseradish peroxidase (50 μl) was added and incubated for 30 min. After three washes, 50 μl of 0.04% 1,2- phenylenediamine dihydrochloride and 0.015% hydrogen peroxide in 0.1 M sodium phosphate was added. Hydrolysis of p-nitrophenylphosphate was stopped by die addition of 50 μl 2 N sulfuric acid and the absorbance measured at 492 nm using a Vmax plate reader (Victor, Molecular Devices Corporation, Menlo Park, CA, USA). The level of protein S expression was found to vary between 0.6 and 1.2 μg/10 6 cells, 24 hrs. After SDS-PAGE, performed as disclosed above proteins were transferred to Immo- bilon membranes, stained with Coomassie brilliant blue and sequenced in a gas phase sequencer as described by Matsudaira, P (1987) J. Biol. Chem. 262 (21), 10035-10038. The amino acid composition was determined after acid hydrolysis (6 M HC1, 24 h, 110°C in vacuum) using a Beckman 6300 amino acid analyzer. Gla was measured following alkaline hydrolysis as described in Fernlund et al., (1975) J. Biol. Chem. 250, 6125-6133. All mea-surements were performed at least in duplicate. Amino-terminal protein sequence analysis yielded the expected sequences for recombinant wild-type and mutant protein S. (d) Characterization of protein S mutants: The APC-cofactor activities of d e wild- types and mutants of protein S obtained above were determined in a accordance with plasma- based activated partial thromboplastin time (APTT) system. Human protein S-de- ficient plasma (100 μl) was incubated with 100 μl of APTT reagent (Chromogenix AB, Swe- den) for 5 min at 37°C, after which 100 μl 25 mM calcium chloride containing human or bovine APC (final concentration in assay of 0.3 μg/ml) and wild-type or mutant protein S samples (final concentration in assay of 0-10 μg/ml) were added and the clotting time measured in an Amelung-Coagulometer KC4A. This test was performed at seven different concentrations of protein S. All dilutions were made in Michaelis buffer (0.036 M sodiumacetate, 0.036 M sodium-barbitone, 0.14 M NaCl, pH 7.4). Each datapoint was the mean of three determinations; mutants 5, 6, 7 and 11 being measured on two occasions each including three individual measurements. In mis test, the APC cofactor activities of protein S mutants were tested in the presence of either human or bovine APC. In the absence of added protein S, both human and bovine APC yielded approximately 10 seconds prolongation of the clotting time. The addition of wild-type human or bovine protein S (or the plasma-derived proteins) yielded results in accordance with those on record, i.e. both human and bovine protein S potentiated the anticoagulant activity of human APC, whereas only bovine protein S functioned as cofactor to bovine APC. In this system, bovine protein S was found to be more potent than its human coun- terpart even in the presence of human APC. The plasma-derived human and bovine protein S gave results similar to those of their recombinant counterparts (results not shown). The results obtained in the above APTT performed in the presence of human APC are shown in Fig. 8 A and B, wherein clotting time (seconds) is plotted versus different protein S concentrations in presence of human APC. In absence of APC, the clotting time was 34 seconds. As is obvious from Fig. 8 A and B all mutants of the present invention expressed activity as a cofactor to human APC, which activity was better than that of wild-type human protein S. Mutants 1-3 are the different bovine/human TS/EGF1 chimeras. Mutant 3 compri- sing bovine TS and bovine EGFl functioned as a potent cofactor to human and bovine APC, whereas mutant 1 (comprising bovine EGFl) and mutant 2 (comprising bovine TS) only functioned well as cofactor to human APC. Some of the mutants 4-13 are distinctly more active as cofactor to human APC man the wild-type protein S. For instance, mutants 11 and 12 express an activity which is 200 % of the activity of wild-type human protein S. Mutants 5, 6, 7, 8 and 9 express enhanced cofactor activity to both human and bovine APC. One of the mutants prepared in Example 2, viz. mutant 10, has a much enhanced cofactor activity towards human as well as bovine APC. As is obvious from Fig. 8B, the activity of mutant 10 is 5-fold, or even 10-fold higher tiian the acti- vity of wild-type human protein S, as estimated from the (reduced) amounts of mutant 10 which produce the same clotting times as useful amounts of wild-type protein S.

Claims: PATENT CLAIMS 1. A variant blood coagulation component, which is substantially homologous in amino acid sequence to a wild-type blood coagulation component capable of expressing anticoagulant activity in the protein C-anticoaguIant system of blood and selected from protein C (PC), activated protein C (APC) and protein S (PS), said variant component being capable of expressing an anticoagulant activity, which is enhanced in comparison wid the anticoagulant activity expressed by the corresponding wild-type blood coagulation component, and said variant component differing from the respective wild-type component, in tiiat it contains in comparison with the said wild-type component at least one amino acid residue modification in its amino acid residue sequence. 2. The variant component of claim 1, which has at least 95% amino acid residue sequence identity with the corresponding wild-type component. 3. The variant component of * claim 1 or 2, wherein the said at least one amino acid residue modification is comprised of a substimted, deleted or inserted amino acid residue. 4. The variant component of any one of claims 1-3, wherein said component is a variant PC or a variant APC which expresses enhanced proteolytic, suitably amidolytic, activity in comparison with the wild-type component. 5. The variant component of claim 4 which contains essentially the same glycosylation sites as wild-type protein C. 6. The variant component of claim 4 or 5, said component being a variant PC or variant APC wherein the amino acid residue at position 97, 248 and/or 313 is(are) other tiian glutamine (Gin). 7. The variant component of claim 4, wherein the said variant component contains at least one amino acid residue modification in a region of its amino acid residue sequence, which corresponds to the serine-protease (SP) module of the wild-type component. 8. The variant component of claim 7, wherein the said at least one amino acid residue modification is contained in a region corresponding to an amino acid stretch between amino acid residue numbers 300 and 314 of die the wild-type component. 9. The variant component of claim 8, wherein the modified region, which corresponds to die wild-type amino acid residue numbers 300-314, contains the deletion Δ 303 . 304 . 305 . 308 and e substitution E307D/A310T and is represented by the formula WGYRDETKRNR (2). 10. The variant component of any one of claims 1-3, which is a variant PS containing at least one amino acid residue modification in a region of its amino acid residue sequence which corresponds to a region of the wild-type PS amino acid sequence selected from the thrombin sensitive loop and the first and the second epidermal growth factor ho- mologous module. 11. The variant component of claim 10, wherein die modification(s) is(are) contained in a region corresponding to an amino acid stretch between amino acid residue numbers 46 and 113, and, suitably, the modifications are selected from the group comprising at least R49G, Q52R and P106S. 12. The variant of claim 11, which contains the modifications R49G, Q52R, T53A, Q61L and P106S, and optionally at least one modification selected from S81N, S92T, K97Q, S99T and T103I. 13. The variant component of any one of claims 1-12, wherein the said wild-type blood coagulation component is of human origin. 14. A DNA segement comprising a nucleotide sequence coding for a variant blood coagulation component according to any one of claims 1-13. 15. A recombinant DNA molecule comprising a replicable vector, which suitably is an expression vector, and a DNA segment according to claim 14 inserted therein. 16. A host cell comprising a microorganism or an animal cell, suitably a cultured animal cell line, harbouring the recombinant DNA molecule of claim 15, which suitably is stably incorporated therein. 17. The host cell of claim 16, which is an adenovirus-transfected human kidney cell. 18. A method for producing a DNA segment of claim 14 coding for a variant blood coagulation component according to any one of claims 1-13, which comprises: (a) providing a DNA coding for the wild-type blood coagulation component; (b) introducing at least one nucleotide modification in said wild-type DNA to form a modified DNA segment coding for a variant blood coagulation component; and (c) replicating said modified DNA segment. 19. A method for producing a variant blood coagulation component according to any one of claims 1-13, which comprises: (a) providing a DNA-segment that codes for the said variant component; (b) introducing me said DNA segment provided in step (a) into an expression vector; (c) introducing the said vector, which contains the said DNA segment, into a compatible host cell; (d) culturing the host cell provided in step (c) under conditions required for expression of said variant component; and (e) isolating the expressed variant component from the cultured host cell. 20. A pharmaceutical composition comprising an effective amount of a variant blood coagulation component according to any one of claims 1-13 and a pharmaceutically acceptable carrier, diluent or excipient. 21. A diagnostic test system, suitably in kit form, for assaying components participating in the protein C-anticoagulant system of blood, said system comprising a variant blood coagulation component of any one of claims 1-13. 22. The diagnostic test system of claim 21 wherein die variant blood coagulation component is a variant APC and said test system is a system for assaying functional activity of protein S or intact anticoagulant Factor V. 23. A method for inhibiting coagulation in a patient comprising administering to said patient a physiologically tolerable composition comprising a coagulation-inhibiting amount of a variant blood coagulation component according to claim 1. 24. The method of claim 23, wherein thrombosis is inhibited. 25. The method of claim 22 or 23, wherein the variant blood coagulation component is a variant PC or a variant APC. 26. The method of claim 22 or 23, wherein the variant blood coagulation component is a variant PS. 27. Use of the pharmaceutical composition of claim 20 for treatment and prevention of coagulation disorders, such as thrombosis. 28. Use according to claim 27, wherein the pharmaceutical composition comprises a variant PC or a variant APC, optionally in combination with a variant PS.

Inventors: DAHLBAECK BJOERN, DAHLBAECK, BJOERN
Applicants: DAHLBAECK BJOERN, T A C THROMBOSIS AND COAGULATI, DAHLBAECK, BJOERN, T.A.C. THROMBOSIS AND COAGULATION AKTIEBOLAG
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, A61P7, A61P9, C07K14, C12N1, C12N5, C12N9, C12N15, C12P21, G01N33