MXPA00003916A - Modified vitamin k-dependent polypeptides - Google Patents

Abstract

The invention provides vitamin k-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.

Description

MODIFIED POLYPEPTIDES DEPENDENTS OF VITAMIN K BACKGROUND OF THE INVENTION Vitamin K dependent proteins contain 9 to 13 gamma-carboxyglutamic acid (Gla) residues at their amino terminal residues 45. Gla residues are produced by enzymes in the liver that use vitamin K to carboxylate the side chains of the glutamic acid residues in protein precursors. Vitamin K dependent proteins are involved in a variety of biological processes, of which the best described is blood coagulation (reviewed in Furie, B. and Furie, B.C., 1988, Cell, 53: 505-518). Vitamin K dependent proteins include Z protein, S protein, prothrombin, factor X, factor IX, protein C, factor XII and Gas6. The last protein works in the regulation of cell growth. Matsubara et al., 1996, Dev. Biol., 180: 499-510. Gla residues are necessary for proper calcium binding and membrane interaction by these proteins. It is thought that the membrane contact site of factor X resides within amino acid residues 137. Evans and Nelsestuen, 1996, Protein Science 5: suppl. 1, 163 Abs. Although the Gla containing regions of the plasma proteins show a high degree of sequence homology, they have at least a 1000-fold range in membrane affinity. McDonald, J.F. et al., 1997, Biochemistry.36: 5120-5137. The factor VII works in the initial stage of blood coagulation and can be a key element in the formation of blood clots. The inactive precursor, or zymogen, has a low enzymatic activity that increases enormously by proteolytic cleavage to form factor Vlla. This activation can be catalyzed by factor Xa, as well as by the Vlla-tissue factor, an integral membrane protein found in a variety of cell types. Fiore, M.M., et al., 1994, J. Biol. Chem., 269: 143-149. Vlla-tissue factor activation is referred to as autoactivation. It is involved in both the activation (factor Vlla factor V formation) and the subsequent factor Vlla activity. The most important route for in vivo activation is not known. The factor Vlla can activate factors IX and X of blood coagulation. The tissue factor is expressed at high levels on the surface of some tumor cells. A role for this tissue factor is possible, and for the factor Vlla, in tumor development and tissue invasion. Vrana, J.A. et al., Cancer Res., 56: 5063-5070. Cell expression and tissue factor action is also a major factor in the toxic response to endotoxic shock. Dackiw, A. A. et al., 1996, Arch. Surq., 131: 1273-1278.

Protein C is activated by thrombin in the presence of thrombomodulin, an integral membrane protein of endothelial cells. Esmon, N.L. et al., 1982, J. Biol. Chem. 257: 859-864. Activated protein C (APC) degrades the factors Va and Villa in combination with its cofactor, protein S. Resistance to APC is the most common form of hereditary thrombosis disease. Dahlback, B., 1995, Blood, 85-607-614. Vitamin K inhibitors are commonly administered as a prophylaxis for thrombosis disease. Vitamin K-dependent proteins are used to treat certain types of hemophilia. Hemophilia A is characterized by the absence of active factor VIII, factor Villa, or the presence of inhibitors for factor VIII. Hemophilia B is characterized by the absence of active factor IX, factor IXa. The factor VII deficiency, although rare, responds well to the administration of factor VII. Bauer, K.A., 1996, Haemostasis, 26: 155-158, suppl. 1. Factor VIII replacement therapy is limited due to the development of high titre inhibitory factor VIII antibodies in some patients. Alternatively, factor Vlla can be used in the treatment of hemophilia A and B. Factor IXa and factor Villa activate factor X. Factor Vlla eliminates the need for factors IX and VIII by activating factor X directly, and It can overcome the problems of factor IX and VIII deficiencies with few immunological consequences. Hedner et al., 1993, Transfus. I measured Rev., 7: 78-83; Nicolaisen, E.M. et al., 1996, Thromb. Haemost., 76: 200-204. Effective levels of factor Vlla administration are often high (45 to 90 μg / kg body weight) and administration needs to be repeated every few hours. Shumalv, S. et al., 1996, Thromb. Haemost..75: 432-436. It has been found that a soluble form of tissue factor (soluble tissue factor or sTF) that does not contain the membrane contact region is effective in the treatment of hemophilia when co-administered with factor Vlla. US Patent no. 5,504,064. In dogs, it was shown that sTF reduces the amount of Vlla factor needed to treat hemophilia. The membrane association by sTF-Vlla is completely dependent on the membrane contact site of factor VII. This contrasts with normal tissue-factor complex Vlla, which binds to the membrane through both tissue factor and Vll (a).

BRIEF DESCRIPTION OF THE INVENTION It has been discovered that modifications within the? -carboxyglutamic acid (GLA) domain of vitamin K-dependent polypeptides intensify their membrane binding affinities. Vitamin K-dependent polypeptides modified in such a manner have an enhanced activity and can be used as anti-coagulants, pro-coagulants or for other functions utilizing vitamin K-dependent proteins. For example, a molecule of factor Vi Improved treatment can provide several benefits by decreasing the dosage of V1 needed, the relative frequency of administration and / or by providing qualitative changes that allow a more effective treatment of deficiency states. The invention features vitamin K-dependent polypeptides that include a modified G LA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding peptide dependent natural vitamin K. The modified GLA domain is from about amino acid 1 to about amino acid 45 and includes at least one amino acid substitution. For example, the amino acid substitution may be in the amino acid 1 1, 1 2, 29, 33 or 34. Preferably, the substitution is in amino acid 1 1, 33 or 34. The modified GLA domain may include amino acid sequence, which, in the saturated state of calcium, forms a tary structure having a cationic nucleus with a halo of electronegative charge.

The vitamin K dependent peptide can be, for example, protein C, activated protein C, factor IX, factor IXa, factor VII, factor Vlla or factor Vlla modified from active site. The modified GLA domain of protein C or activated protein C can include a glutamic acid residue at amino acid 33 and a residue of aspartic acid at amino acid 34 (SEQ ID NO 19) The modified Gla domain of protein C or activated protein C may also include a glutamine or glutamic acid residue in the amino acid 11 (SEQ ID NO 20 and SEQ ID NO 21, respectively) Additionally, a glycine residue can be substituted at amino acid 12 in the Gla domain of activated protein C or protein C (SEQ ID NO 24 or SEQ ID NO. NO 35) The modified factor VII domain Gla, Vlla factor, and active site-modified factor Vlla, can contain a substitution at amino acid 11 and 33 For example, a glutamine residue at amino acid 11 can be substituted and a glutamic acid residue at amino acid 33 (SEQ ID NO 30) The invention also features an isolated nucleic acid encoding a vitamin K-dependent polypeptide. As used herein, the term "isolated" (purified) refers to a corresponding sequence. or part or all of the gene encoding a vitamin K-dependent polypeptide, but free of sequences that normally flank one or both sides of the gene in a mammalian genome. The vitamin K dependent peptide includes a modified GLA domain that enhances the affinity of membrane binding of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. Modified sunscreen includes at least one amino acid substitution. The invention also features a mammalian host cell that includes a nucleic acid vector encoding a vitamin K-dependent polypeptide. The vitamin K-dependent polypeptide includes a modified GLA domain that enhances the affinity of membrane binding of the polypeptide. in relation to a corresponding natural vitamin K dependent polypeptide. The modified G LA domain includes at least one amino acid substitution in, for example, the amino acid 1 1, 1 2, 29, 33 or 34. The vitamin K dependent polypeptide can be, for example, the Vi factor. Vl lae includes a substitution of amino acid in amino acid 1 1 and amino acid 33. For example, the amino acid substitution may include a glutamine residue at amino acid 1 1 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30). The invention also relates to a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of vitamin K-dependent polypeptide effective to inhibit the formation of clots in a mammal. The vitamin K-dependent polypeptide includes a modified Gla domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K na dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The vitamin K dependent polypeptide can be, for example, protein C, activated protein C or factor V1 the modified active site. Protein C or activated protein C may include, for example, a glutamic acid residue at amino acid 33 and a residue of aspartic acid at amino acid 34 (SEQ ID NO: 19). Protein C or activated protein C may further include a glutamine at amino acid 11 (SEQ ID NO: 20) or a glutamic acid at amino acid 11 (SEQ ID NO: 21). The amino acid substitution may also include a glycine at amino acid 12 (SEQ ID NO: 24 or SEQ ID NO: 35). The active site modified Vlla factor can include, for example, a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 33). The invention also features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of a vitamin K-dependent polypeptide effective to increase clot formation in a mammal. The vitamin K-dependent polypeptide includes a modified Gla domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified Gla domain includes at least one amino acid substitution. The vitamin K-dependent polypeptide can be, for example, the factor VII, factor Vlla, factor IX or factor IXa. The pharmaceutical composition may also include soluble tissue factor. Factor VII or Factor Vlla can include an amino acid substitution at amino acid 11 and an amino acid 33. For example, a glutamine residue can be substituted at amino acid 11 and a glutamic acid residue can be substituted at amino acid 33 (SEQ ID NO: 30).

A vitamin K-dependent polypeptide for use in treating a coagulation disorder is also disclosed. The vitamin K-dependent polypeptide includes a modified Gla domain, which enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified Gla domain includes at least one amino acid substitution. Useful vitamin K-dependent modified polypeptides include, for example, protein C, activated protein C, factor VII, factor Vlla, factor Vlla modified from active site, factor IX and factor IXa, as described above. The invention also characterizes the use of a vitamin K-dependent polypeptide in the manufacture of a medicament for the treatment of a coagulation disorder. The vitamin K-dependent polypeptide includes a modified GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. Useful vitamin K-dependent modified polypeptides include, for example, protein C, activated protein C, factor VII, factor Vlla, factor Vlla modified from active site, factor IX and factor IXa as described above. A method for decreasing clots in a mammal is also described. The method includes administering an amount of an effective vitamin K-dependent polypeptide to decrease clot formation in the mammal. The vitamin K-dependent polypeptide includes a modified GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The vitamin K-dependent polypeptide can be, for example, protein C, activated protein C or active site-modified factor Vlla. Activated protein C or protein C may include a glutamic acid residue at amino acid 33 and a residue of aspartic acid at amino acid 34 (SEQ ID NO: 19). A glutamine or glutamic acid can additionally be substituted on activated protein C or protein C (SEQ ID NO: 20 or SEQ ID NO: 21, respectively). A glycine can also be substituted in amino acid 12 (SEQ ID NO: 24 or NO: 35). The active site modified Vlla factor can include a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30). The invention also characterizes a method for increasing the formation of clots in a mammal. The method includes administering an amount of an effective vitamin K-dependent polypeptide to increase clot formation in the mammal. The vitamin K-dependent polypeptide includes a modified GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The vitamin K-dependent polypeptide can be, for example, Factor VII, Factor Vlla, Factor IX or Factor IXa. The factor VII or factor Vlla can include an amino acid substitution at amino acid 11 and at amino acid 33. For example, the amino acid substitution can include a glutamine residue at amino acid 11 and a glutamic acid residue in amino acid 33 (SEQ ID NO: 30). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS The amino acid sequences of the wild-type GLA domain Vlla and VIIQ11E33 were found in SEQ ID NO: 3 and SEQ ID NO: 33, respectively. The amino acid sequences of the GLA domain of bovine factor X, bovine protein C, human protein C and bovine protein C-H11 were found in SEQ ID NO: 18, SEQ ID NO: 2, SEQ ID NO: 1 and SEQ ID NO. : 23, respectively. The amino acid sequence of the GLA domain of protein C VQ33E.N34D is found in SEQ ID NO: 19. Figure 1 shows the union, with standard deviations, of Vlla natural type (empty circles), VIIQ11E33 (filled circles) and bovine factor X (filled triangles), to membranes. Figure 2 shows the self-activation of VIIQ11E33. The dotted line shows activity in the absence of phospholipid. Figure 3 shows the activation of factor X by the factor Vlla. The results for the Vlla factor of natural type (empty circles) and VllaQ11E33 (filled circles) are given for a concentration of 0.06 nM. Figure 4 shows the coagulation of human plasma by Vlla and VllaQ11E33 with soluble tissue factor. Figure 5 shows the coagulation of plasma by factor VII zymogens and normal tissue factor. Figure 6 shows the inhibition of clot formation by active site modified factor VllaQ11E33 (DEGR-VllaQ11 E33). Figure 7 shows the circulation time of factor VIIQ11E33 in rats. Figure 8 shows the membrane interaction by normal and modified proteins. Panel A shows the interaction of wild type bovine protein C (open circles) and bovine C-H11 protein (filled circles) with vesicles. Panel B shows the interaction of wild type human protein C (empty circles) and human C-P11 protein (filled circles) with membranes. In both cases, the dotted line indicates the result of wer all the added protein was bound to the membrane. Figure 9 shows the influence of activated protein c on coagulation times. In panel A, the average and standard deviation are shown for three determinations of coagulation times for bovine plasma, for bovine natural-type APC (empty circles) and for bAPC-H11 (filled circles). In panel B, the average and standard deviation of three human plasma coagulation replicates for human-type APC (open circles) and human APC-P11 (filled circles) are shown. Figure 10 shows the inactivation of factor Va by bovine and human APC. Panel A shows the inactivation of factor Va by wild type APC bovine (empty circles) and bovine APC-H11 (filled circles). Panel B shows the inactivation of human Va factor in protein S deficient plasma either by natural type APC (open circles) and human APC-H11 (filled circles). Figure 11 shows the electrostatic distribution of Z protein. Vertical lines denote electropositive regions and horizontal lines denote electronegative regions. Figure 12 shows the membrane binding and activity of several Cs proteins. Panel a shows membrane binding by wild-type C protein (empty circles), mutant P11H protein c (filled squares), mutant Q33E, N34D (filled circles) and bovine prothrombin (empty squares). Panel B shows the inhibition of blood coagulation by these mutants. Panel C shows the inactivation of factor Va. Figure 13 compares membrane binding and activity of human protein C mutants. The panel compares the binding of natural type membrane (empty circles), E33 (empty triangles) and E33D34 (filled circles). Panel B compares the coagulation times using natural type (empty triangles), E33 (empty circles) and E33D34 (filled circles). Figure 14 compares the membrane binding (Panel A) and the inhibition of coagulation (Panel B) with wild type (empty squares), H11 (filled circles), E33D34 (empty triangles) and the triple H11E33D34 mutant (open circles) of Bovine protein C. Figure 15 shows the membrane interaction properties of different vitamin K dependent proteins. Panel A compares the interaction of human factor X membrane (filled circles) and bovine (empty circles). Panel B shows the interaction of membrane by fragment 1 of normal bovine prothrombin (empty circles), fragment 1 modified with TNBS in the absence of calcium (filled circles) and fragment 1 modified with TNBS in the presence of 25 mM of calcium (filled squares). Panel C shows the proportion of Z protein binding to vesicles at pH 9 (filled circles) and 7.5 (empty circles).

DETAILED DESCRIPTION In one aspect, the invention features a vitamin K-dependent polypeptide that includes a modified GLA domain with enhanced membrane binding affinity relative to a corresponding natural vitamin K-dependent polypeptide. Vitamin K-dependent polypeptides are a group of proteins that utilize vitamin K in their biosynthetic pathway to carboxylate the side chains of glutamic acid residues in protein precursors. The GLA domain contains 9-1 3 residues of α-carboxyglutamic acid in the N-terminal region of the polypeptide, typically from amino acid 1 to about amino acid 45. Z protein, S protein, factor X, factor II (prothrombin) , factor IX, protein C, factor Vi and Gas6 are examples of vitamin K dependent polypeptides. The amino acid positions of the polypeptides discussed herein are numbered according to factor IX. The protein S, protein C, factor X, factor Vi l and human prothrombin all have a less amino acid (position 4) and should be adjusted accordingly. For example, the actual 1 0 position of bovine protein C is a proline, but it is numbered here as 1 1 amino acid for ease of comparison throughout. As used herein, the term "polypeptide" is any chain of amino acids, with respect to length or post-translational modi? Cation. In the present, amóacids have been designated by standard abbreviations of three letters and one letter. Modifications of the GLA domain include at least one substitution of amino acid. The substitutions may be conservative or non-conservative. Conservative amino acid substitutions replace an amino acid with an amino acid of the same class, while its non-conservative amino acid substitutions replace an amino acid with an amino acid of a different class. Non-conservative substitutions may result in a substantial change in the hydrophobicity of the polypeptide or in the volume of a residual side chain. In addition, non-conservative substitutions can make a substantial change in polypeptide loading, such as reducing electropositive charges or introducing electronegative charges. Examples of non-conservative substitutions include a basic amino acid by a non-polar amino acid, or a polar amino acid by an acidic amino acid. The amino acid substitution can be at amino acid 1 1, 12, 29, 33 or 34. Preferably, the amino acid substitution is at amino acid 1 1, 33 or 34. The modified GLA domain can include an amino acid sequence the which, in the calcium-saturated state, contributes to the formation of a tertiary structure having a cationic core with a halo of electronegative charge. Without being left to a particular theory, the enhanced membrane affinity may result from a particular electrostatic pattern consisting of an electropositive core completely surrounded by an electronegative surface. Many vitamin K-dependent polypeptides are substrates for membrane-bound enzymes. Since no vitamin K-dependent polypeptide shows the maximum potential membrane binding affinity of a G LA domain, all must contain amino acids whose purpose is to reduce the affinity of an ion. Consequently, many vitamin K dependent polypeptides contain amino acids that are not optimal from the point of view of maximum affinity. These residues effectively break the binding site to provide a faster change for an enzymatic reaction. The decreased membrane affinity can serve several purposes. The high affinity is accompanied by a slow exchange, which can limit the reaction rates. For example, when the prothrombinase enzyme is mounted on membranes with high substrate affinity, the exchange of proteins from the membrane, instead of enzymatic catalysis, is the limiting factor. Lu, Y. And Nelsestuen, G.L., 1996, Biochemistry, 35: 8201-8209. Alternatively, the adjustment of membrane affinity by substitution with non-optimal amino acids can balance the competing processes of procoagulation (factor X, IX, VII and prothrombin) and anticoagulation (protein C, S). Although membrane affinities of natural proteins may be optimal for normal states, membrane affinity enhancement can produce proteins that are useful for in vitro study as well as improved therapeutics for regulating blood coagulation under pathological conditions in vivo. Various examples of vitamin K-dependent polypeptides of modified GLA domain are described below. The vitamin K dependent polypeptide can be activated protein C or protein C (APC). The amino acid sequences of the GLA domain of human protein (hC) and bovine (bC), wild-type, are shown in Table 1. X is a residue of Gla or glu. In general, a protein with neutral (eg, Q) or anionic (eg, D, E) residues at positions 11, 33 and 34 will have higher membrane affinity.

TABLE 1 hC: ANS-FLXXLRH11 SSLXRXCIXX21 ICDFXXAKXI31 FQNVDDTLAF4? WSKH (SEQ ID N0: 1) bC: ANS-FLXXLRP11 GNVXRXCSXX21 VCXFXXARXI31 FQNTXDTMAF41 WSFY (SEQ ID N0: 2) The modified GLA domain of protein C or APC may include, for example, a glutamic acid residue at amino acid 33 and a residue of aspartic acid at amino acid 34 (SEQ ID NO: 19). The glutamic acid at position 33 can be further modified to α-carboxyglutamic acid in vivo. For optimal activity, the modified GLA domain can include an additional substitution at amino acid 11. For example, a glutamine residue can be substituted can be substituted at amino acid 11 (SEQ ID NO: 20) or alternatively, a glutamic acid or a residue of aspartic acid (SEQ ID NO: 21 and SEQ ID NO: 22, respectively). A histidine residue can be substituted in the amino acid 11 in bovine protein C (SEQ ID NO: 23). A further modification may include a substitution at amino acid 12 for a glycine residue for serine (SEQ ID NO: 24 and SEQ ID NO: 35). The replacement of amino acid 29 by phenylalanine, the amino acid found in prothrombin, is another useful modification (SEQ ID NO: 25). The modified C protein with membrane binding affinity can be used in place of other injectable anticoagulants, such as heparin. Heparin is usually used in most types of surgery, but suffers from a low efficacy / toxicity ratio. In addition, modified C protein with enhanced membrane affinity may be used in place of oral anticoagulants in the coumarin family, such as warfarin. These modifications can also be done with APC modified from active site. The active site of APC can be chemically inactivated, for example, by N-dansyl-glutamyl-glycylglycine-chloromethyl ketone (DEGR) or by site-directed mutagenesis of the active site. Sorensen, B.B. et al., 1997, J. Biol. Chem. 272: 11863-11868. The modified active site APC functions as an inhibitor of the prothrombinase complex. The enhanced membrane affinity of active site-modified APC can result in a therapeutically more effective polypeptide. The vitamin K-dependent polypeptide can be either factor VII or the active form of factor VII, factor Vlla. Natural or naturally occurring factor VII polypeptide has low affinity for membranes. The amino acid sequences of the GLA domain of human type factor VII (hVII) and bovine (bVII) are shown in Table 2.

TABLE 2 hVII: ANA-FLXXLRPn GSLXRXCKXX21 QCSFXXARXI31 FKDAXRTKLF41 WISY (SEQ ID NO: 3) bVII: ANG-FLXXLRP11 GSLXRXCRXX21 LCSFXXAHXI31 FRNXXRTRQF41 WVSY (SEQ ID NO: 4) The modified GLA domain of factor VII or factor Vlla may include, for example, a residue of glutamic acid or aspartic acid at amino acid 11 (SEQ ID NO: 26 and SEQ ID NO: 27, respectively), a residue of phenylalanine in amino acid 29 (SEQ ID NO: 28) or a residue of aspartic acid at amino acid 33 (SEQ ID NO: 29). Preferably, the GLA domain of factor VII or factor Vlla can include a glutamine residue in amino acid 1 1 and a residue of glutamic acid in amino acid 33 (SEQ ID NO: 30). The vitamin K-dependent polypeptide mofridized in this manner has a higher affinity for membranes than the natural or wild-type polypeptide. It also has a much higher activity in autoactivation, in the generation of factor Xa and in several blood coagulation tests. The activity is particularly enhanced under marginal coagulation conditions, such as, low levels of tissue factor and / or phospholipid. For example, the modified Vi l factor is approximately 4 times as effective as the natural V l at optimal thromboplastin levels, but it is approximately 20 times as effective at 1% optimal thromboplastin levels. The signs of marginal procoagulation are probably very predominant in vivo. Currently, the available coagulation assays that use optimal levels of thromboplastin can not detect the differences in coagulation times between normal plasma and that of hemophilia patients. Coagulation differences between such samples are only detected when non-optimal levels of thromboplastin or diluted thromboplastin are used in coagulation assays. Another example of a vitamin K-dependent polypeptide is a modified active site Vl factor. The active site of factor V1 can be modified chemically, for example, by DEGR or by site-directed mutagenesis of the active site. The Vi l factor modified by DEGR is an effective inhibitor of coagulation by several administration routes. Arnljots, b. et al. , 1 997, J. Vasc. Surq. , 25: 341-346. Modifications of the GLA domain can make the modified active site Vl factor more efficient due to a higher membrane affinity. The modified active site factor Vlla GLA domain can include, for example, a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30). The vitamin K dependent polypeptide can also be factor IX or the active form of factor IX, factor IXa. As with the active site modified Vlla factor, active site modified IXa and Xa may be coagulation inhibitors. The amino acid sequences of the GLA domain of human-like factor IX (hlX) and bovine (blX) are shown in Table 3. For example, a residue of aspartic acid or glutamic acid can be substituted at amino acid 11 (SEQ ID. NO: 31 and SEQ ID NO: 32, respectively), a phenylalanine residue at amino acid 29 (SEQ ID NO: 33) or a residue of aspartic acid at amino acid 34 (SEQ ID NO: 34).

TABLE 3 hVII: YNSGKLXXFVQn GNLXRXCMXX21 KCSFXXARXV31 FXNXXRTTXF41 WKQY (SEQ ID NO: 5) bVII: YNSGKLXXFVQ11 GNLXRXCMXX2? KCSFXXARXV31 FXNTXKRTTXF4? WKQY (SEQ ID NO: 6) In another aspect, the invention features a mammalian host cell that includes a vitamin K-dependent polypeptide having a modified GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution as discussed above. The mammalian host cell may include, for example, modified factor VII or factor VII to modified. The GLA domain of modified factor Vl or Vl of modified factor may contain an amino acid substitution in amino acid 1 1 and in amino acid 33. Preferably, the amino acid substitution includes a glutamine residue in the amino acid 1 1 and a glutamic acid residue in amino acid 33 (SEQ ID NO: 30). Suitable mammalian cell cultures are capable of modifying vitamin K-dependent polypeptide glutamate residues to α-caboxiglutamate. Kidney cells derived from kidney and liver are especially useful as host cells. The invention also features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of a vitamin K-dependent polypeptide effective to inhibit the formation of clots in a mammal. The vitamin K-dependent polypeptide includes a modified GLA domain with at least one amino acid substitution that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K-dependent polypeptide. Modified vitamin K-dependent modified polypeptides of the pharmaceutical compositions may include, without limitation, C-protein or APC, active site-modified APC, active site-modified factor Vl, active site-modified factor IXa and active site-modified factor Xa, as discussed earlier. The concentration of an effective vitamin K-dependent polypeptide to inhibit the formation of clots in a mammal can vary, depending on a variety of factors, including the preferred dosage of the compound to be admired, the chemical characteristics of the compounds used, the formulation of the excipients of the compound and the route of administration. The optimal dosage of a pharmaceutical composition to be administered may also depend on variables such as the general health status of the particular patient and the relative biological efficacy of the selected compound. These pharmaceutical compositions can be used to regulate coagulation in vivo. For example, com positions can be used in a general way for the treatment of thrombosis. Altering only a few amino acid residues of the polypeptide as described above generally does not significantly affect the antigenicity of the mutant polypeptides. Vitamin K-dependent polypeptides that include modified G LA domes can be formulated into pharmaceutical compositions by mixing with non-toxic, pharmaceutically acceptable carriers or excipients. Such compounds and compositions can be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions in aqueous solutions of physiological buffers; for oral administration, particularly in the form of tablets or capsules, or for intranasal administration, particularly in the form of powders, nasal drops or aerosols. The positions for other administration routes can be prepared as desired using standard methods. Formulations for parenteral administration may contain, as excipients, sterile water, saline or saline, polyalkylene glycols., such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, biodegradable, biocompatible lactide polymer, lactide / glycolide copolymer or biodegradable polyoxyethylene-polyoxypropylene copolymers are examples of excipients for controlling the release of a compound of the invention in vivo. Other suitable parenteral delivery systems include ethylene-vinyl acetate acetate copolymer particles, osmotic pumps, im pallable infusion systems, and liposomes. Formulations for administration by inhalation may contain excipients such as lactose, if desired. The formulations for inhalation may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops. If desired, the compounds can be formulated as gels to be applied intranasally. Formulations for parenteral administration may also include glycocholate for oral administration. In an alternative embodiment, the invention also features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of a vitamin K-dependent polypeptide effective to increase clot formation in a mammal. The vitamin K dependent polypeptide includes a modified GLA domain with at least one amino acid substitution that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K dependent polypeptide. These pharmaceutical compositions can be useful for the treatment of coagulation disorders, such as, hemophilia A, hemophilia B and liver disease. In this embodiment, useful vitamin K-dependent polypeptides of the pharmaceutical compositions may include, without limitation, factor VII or the active form of factor VII, factor Vlla. The modified GLA domain of factor VII or factor Vlla can include substitutions at amino acid 11 and amino acid 33, for example, a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30) ). The pharmaceutical composition may further comprise soluble tissue factor. The factor VII is especially critical for blood coagulation due to its location at the initiation of the coagulation cascade, and its ability to activate two proteins, factors IX and X. Direct activation of factor X by factor Vlla is important for a possible treatment of the main forms of hemophilia, types A and B, because the steps involving factors IX and VIII are completely avoided. It has been found that the administration of factor VII to patients is effective for the treatment of some forms of hemophilia. Improving the membrane affinity of factor VII or Vlla by modifying the GLA domain provides the potential to make the polypeptide more responsive to many coagulation conditions, to decrease the Vll / Vlla dosages needed, to extend the intervals in which the Vll / Vlla factor must be administered and to provide additional qualitative changes that result in more effective treatment. In a global way, the improvement of the membrane contact site of the factor VII can increase both its activation rate and improve the activity of the factor Vl on the factor X or IX. These steps can have a multiplicative effect on global blood coagulation velocities in vivo, resulting in a very potent Vl factor for superior treatment of various blood coagulation disorders. Other vitamin K-dependent polypeptides useful for increasing clot formation include factor IX and factor IXa.

In another aspect, methods are described for reducing the formation of clots in a mammal. The method includes administering an effective amount of vitamin K-dependent polypeptide to inhibit the formation of clots in the mammal. The vitamin K-dependent polypeptide includes a modified GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. Protein C or modified APC, or modified active site Vl la, IXa, Xa and APC factors, can be used for this method. In another aspect, the invention also features methods for increasing clot formation in a mammal that includes administering an effective amount of vitamin K-dependent polypeptide to increase the formation of clots in the mammal. The vitamin K-dependent polypeptide includes a modi? Ed GLA domain that enhances the membrane binding affinity of the polypeptide relative to a corresponding natural vitamin K dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The modified factor VII or Vlla and modified factor IX or IXa can be used in this method. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 - Factor VII with enhanced activity and membrane affinity: It has been found that the membrane binding affinity of the factor VIL of human blood coagulation can be increased by site-directed mutagenesis. The properties of a P11Q.K33E mutant (referred to herein as Factor VIIQ11E33 or mutant Factor VII (SEQ ID NO: 30)) have been characterized. The membrane affinity was increased over the wild type protein by approximately 20 times. The autoactivation by the mutant was increased by at least 100 times over that of the natural type factor VII. The activated form of VIIQ11E33 (referred to as VllaQ11E33) showed approximately 10 times greater activity towards factor X. The coagulation activity of VllaQ11E33 with normal soluble tissue factor in normal plasma was approximately 10 times that of natural type Vlla. The coagulation activity of the zymogen, VIIQ11E33, with normal tissue factor (supplied as a 1: 100 dilution of thromboplastin-HS), was 20 times greater than the natural type factor VII. The degree to which the activity was intensified was dependent on the conditions, with VIIQ11E33 being especially active under conditions of low coagulation stimuli. In general, protein concentrations were determined by the Bradford assay using bovine serum albumin as the standard. Bradford, M.M., 1976, Analyt. Biochem. 248-254. Molar concentrations were obtained from the molecular weights of 50,000 for the factor VII and 55.00 for the factor X. Unless indicated, all measurements of activity were conducted in the standard buffer (Tris 0.05 M, pH 7.5, 100 mM NaCI). Production of mutant factor VII: mutant factor VII was generated from natural type factor VII cDNA (GenBank accession number M13232, NID g182799). Petersen et al., 1990, Biochemistry 29: 3451-3457. Mutation P11Q (change of amino acid 11 from a proline residue to a glutamine residue) and K33E mutation (change of amino acid 33 from a lysine residue to a glutamic acid residue) were introduced into the natural type factor VII cDNA by a polymerase chain reaction strategy, essentially as described by Vallette et al., 1989, Nucleic Acids Res. 17: 723-733. During this process, a restriction enzyme site Xmalll diagnosis of mutation was removed. Four PCR primers were designed to initiate the synthesis of two mutant fragments of M13232, one from Mlu \ a Sg / ll, positions 221 to 301, and the other from BglW to Ssrll, positions 302 to 787. These primers were used under PCR cyclisation conditions (GENEAMP, Perkin Elmer) to initiate fragment synthesis using 1 ng of the natural type factor VII cDNA as a template. The resulting fragments were gel purified and digested with Mlu \ and BglW or BglW and SstW. The two purified fragments were then ligated into the cDNA of factor VIII in the Zem219b expression vector from which the corresponding wild-type sequence had been removed as an M / vl-Ssfll fragment. Petersen et al., 1990 supra. Mutated fragments were sequenced in their entirety to confirm the P11Q and K33E substitutions, as well as to eliminate the possibility of other sequence changes induced by PCR. Transfection, selection and purification: Baby hamster kidney (BHK) cells were grown in modified Dubeccos Eagles supplemented with 10% fetal calf serum and penicillin-streptomycin. Subconfluent cells were transfected with the factor VII expression plasmid using lipofectAMINE (Gibco BRL) according to the manufacturer's recommendations. Two days post-transfection, the cells were trypsinized and diluted to selective medium containing 1 μM of methotrexate (MTX). Stably transfected BHK cells were subsequently cultured in serum-free Dubeccos modified Eagles medium, supplemented with penicillin-streptomycin, 5 μg / ml vitamin K, and 1 μM MTX, and conditioned medium was collected. The conditioned medium was applied twice to an immunoaffinity column composed of a calcium-dependent monoclonal antibody (CaFVII22) coupled to Affi-Gel 10. Nakagaki et al., 1991, Biochemistry, 30: 10819-10824. The final purified Factor VIIQ11E33 ran as a single band in the SDS polyacrylamide gel electrophoresis, without any evidence of factor Vlla in the preparation. Pure mutant VII (P11Q, K33E) showed 1400-2800 units of factor Vll / mg. Activation of factor VII: Factor VllaQ11E33 was activated by bovine factor Xa cut-off of VIIQ11E33 (weight ratio 1: 100, incubation for 1 h at 37 ° C). Alternatively, factor VllaQ11E33 was obtained by autoactivation (37 ° C, 20 min) in a mixture containing 7 μM of VIIQ11E33, 0.7 μM of sTF and phospholipid (phosphatidylserine / phosphatidylcholine (PS / PC), 25/75, 0.1 g / g of protein). The wild type factor Vlla was a homogeneous recombinant protein (NOVO Nordisk). Two preparations consisted of a commercial lyophilized product and non-lyophilized product. The last protein was further purified in mono-Q FPLC and showed a specific activity of 80,000 units / mg, calibrated with a George King NPP standard. Enhanced membrane interaction by factor VIIQ11E33: Phospholipid preparation, assay and measurement of protein-membrane binding was conducted by the method described by Nelsestuen and Lim, 1977, Biochemistry, 30: 10819-10824. Large unilamellar vesicles (LUVs) and small unilamellar vesicles (SUVs) were prepared by previously described methods. Hope, M.J. et al., Biochem. Biophvs. Minutes 812: 55-65; Huang, C, 1969, Biochemistry, 8: 344-352. Highly pure phosphatidylserine (bovine brain) and egg phosphatidylcholine (Sigma Chemical Co.) were mixed in chloroform. The solvent was removed by a stream of nitrogen gas. The dried phospholipids were suspended in buffer. The SUVs were formed by sonication and gel filtration, while the LUVs were formed by freeze-thaw and extrusion. Phospholipid concentrations were determined by organic phosphate assay, assuming a weight ratio of phosphorus: phospholipid compound of 25. SUVs were prepared either PS / PC (25/75) or PS / PC (10/90) . Protein was added to phospholipid in the proportions by weight shown in Fig. 1. The protein-membrane binding was assessed by light scattering at 90 ° by the method of Nelsestuen and Lim, 1977, supra. Briefly, the light scattering intensity of the phospholipid vesicles alone () and after the addition of protein (12) were measured and corrected by buffer support and unbound protein. The molecular weight ratio of the protein-vesicle complex (M2) to that of the vesicles alone (M 1) can be estimated from the relationship in equation 1, where dn / dc is the refractive index of the species respective.

I2 / I1 = (M2 / M1) 2 (dn / dCz / dn / dc 2 (eq.

If phospholipid and protein concentrations are known, the concentration of bound [P * PLm ax] and free protein [P] can be estimated. These values, together with the maximum protein binding capacity [P * PLmax] of the vesicles (assumed to be 1.0 g / g for all proteins) can be used to obtain the equilibrium constant for the protein-meme interaction by the relationship in equation 2, where all concentrations are expressed as molar protein or protein binding sites.

KD = [P] [P * PLma? -P * PL] / [P * PL] (eq.2) The binding to 5 mM calcium was assessed and expressed as the ratio, M2 / M1. Figure 1 shows the binding of wild-type Vlla (open circles) and factor VIIQ11E33 (filled circles) to memes of either PS / PC = 25/75, 25 μg / ml (Figure 1A) or PS / PC = 10/90 , 25 μg / ml (Figure 1B). VIIQ11E33 had much higher affinity than the wild-type protein. The binding to PS / PC (25/75) was at the quantitative level, so that [protein re] was essentially zero. Consequently, the Kd values could not be estimated from these data. The meme binding of bovine factor X (filled triangles) is shown in Figure 1 as a reference. Bovine factor X is one of the highest affinity proteins in this family, giving a Kd for PS / PC (20/80) at 2 mM calcium of 40 nM. McDonald et al., 1997, Biochemistry, 36: 5120-5127. The Kd for bovine factor X, obtained from the result at a protein / phospholipid ratio of 0.55 (Figure 1) was 0.025 μM. The binding of natural type and mutant factor VII to PS / PC memes (10/90) was also determined (Figure 1B). The VIIQ11E33 joined less than the quantitative level, which allowed us to estimate a binding constant from the relationship in equation 3.

Kd = [protein? L re] [binding site? L re] / [proteinun? Da] (eq.3) The [binding sites] were estimated from equation 4, assuming a maximum M2 / M1 of 1.0 (ie, [tta bond sites?] = [PhospholipidCOnc in Weight / proteinMw]). This is a common value observed for several proteins in this family. See McDonald et al., 1997, supra. [bres binding sites] = [binding sites totai] - [proteinun? da] (eq.4) Using these assumptions and data at a protein to phospholipid ratio of 0.37, the Kd values were 0. μM for bovine factor X, 5.5 μM for natural type factor VII and 0.23 μM for VIIQ11E33. Thus, it was clear that factor VIIQ11E33 was greatly improved in meme binding affinity over the natural type factor Vil and had one of the highest meme binding affinities among vitamin K dependent proteins. Enhanced factor activation VIIQ11E33: The first step in coagulation involves the activation of the factor VII. The autoactivation of Vil was conducted in a solution containing 100 nM of sTF (highly purified recombinant product of Dr. Walter Kisiel, Fiore et al., 1994, J. Biol. Chem., 269: 143-149), 36 nM of VIIQ11E33 and PS / PC (25/75, 22 μg / ml). The activity of VllaQ11E33 was estimated at several time intervals by adding 0.15 mm of substrate S-2288 (Kabi) and evaluating the release rate of p-nitrophenylphosphate product by absorbance change at 405 nm. The initial activity of the preparation of VIIQ11E33 was less than 4% that of VllaQ11E33 fully active. It was found that VIIQ11E33 was a much better substrate for activation than the natural type factor VII. Figure 2 shows the autoactivation of factor VIIQ11E33. The data were analyzed by the relationship in equation 5 (equation 7 by Fiore et al., 1994, supra).

Ln [VII], = ln [Vlla] o + kcat * and * t (eq.5) ln [Vlla] t is the concentration of factor Vlla at time t, kcat is the catalytic rate constant for factor Vlla acting on Vil and y is the fractional saturation of Vlla sites. For the Vlla type natural factor, this ratio and 1 μM of sTF gave a kcat of 0.0045 / s and a kcat / Km ratio of 7 * 103 M'V. See, Fiore et al., 194, supra. For enzyme VIIQ11E33, autoactivation was rapid (Figure 2) and it was only possible to estimate a lower limit for kcat. This was obtained from Vlla by doubling the time of approximately 25 seconds (kcat = (ln2) / t1 2). The resulting value (kcatmin = 0.03 / s), together with the substrate concentration of this reaction (3.6 * 10"8 M) and the assumption that y = 1.0, gave a value for kcat / [S] = 8 + 105 M * V1 This should be well below the real kcat / Km for VllaQ11E33, but was approximately 100 times greater than the kcat / Km value for the Vlla natural type / sTF factor estimated by Fiore et al., 1994, supra Thus, the combination of the enzyme VllaQ11E33 and the substrate of factor VIIQ11E33 was superior to the wild-type proteins in the activation step of coagulation, which suggested that VIIQ11E33 was superior to the natural type enzyme when the coagulation conditions were Minimal intensified activity of VllaQ11E33: Once generated, factor Vlla activates either factor X or factor IX.The activation of bovine factor X (0.1 μM) by factor Vlla was performed in 50 mM of Tris HCl buffer, pH 7.5 , containing 100 mM NaCl, 5 mM calcium, various amounts of phosphol Ipido (PS / PC, 25/75) and 1 mg / ml of bovine serum albumin at 22.5 ° C. The factor Vlla (0.06 nM of VllaQ11E33 or 0.6 nM of Vlla natural type) was added at time zero and the activity of Xa was determined in 1, 3 and 5 minutes. Aliquots of the reaction mixture (0.2 ml) were combined with buffer (0.2 ml) containing 10 mM EDTA and 0.4 mM S-2222 (Kabi), a chromogenic substrate for factor Xa. The change in absorbance at 405 nm was determined in a Beckman DU8 spectrophotometer. The amount of factor Xa generated from the extinction coefficient (1 + 104 M "1cm" 1) of the reaction product of p-nitrophenylphosphate and a velocity of 33 / s for substrate hydrolysis by purified bovine Xa under the conditions was calculated. of this essay. Figure 3 compares the capacity of the Vlla type natural factor (empty circles) and VllaQ11E33 (filled circles) to activate the X factor in a purified system. Again, VllaQ11E33 was much higher than the Vlla type natural factor in this reaction. The difference was greatest at low phospholipid concentrations and decreased to 2 times at 200 μg phospholipid per ml. This was expected from the fact that high membrane concentrations cause a larger portion of natural membrane to bind to the membrane. Again, the increased function of VllaQ11E33 was the highest under conditions of low phospholipid exposure. Top coagulation of VllaQ11E33: Blood coagulation assays were conducted at 37 ° C using the hand-cocked method to detect clot formation. The human plasma (0.1 ml) was allowed to equilibrate at 37 ° C for 1 minute. Several reagents were added in a volume of 0.1 ml of standard buffer. Soluble tissue factor (50 nM) and phospholipid (PS / PC, 10/90, 75 μg / ml) were added to the plasma, together with the factor Vlla concentration shown in Figure 4 (0.1 - 32 nM). Finally, 0.1 ml of 25 mM CaCl2 was added to start the reaction. The time to form a clot was measured. In most cases, the average and standard deviations of replicate samples were reported. Figure 4 shows the coagulation times of wild-type Vlla versus VllaQ11E33 in normal human plasma. The coagulation was supported by sTF and phospholipid vesicles added. The endogenous factor V, natural type, is approximately 10 nM in concentration and had virtually no impact on the coagulation times. The support coagulation was 120 seconds, with or without sTF. The VllaQ11E33 factor showed approximately 8 times greater activity than the wild type Vlla under these test conditions. Similar results were obtained with plasma deficient in factor VIII, suggesting that the main route for blood coagulation in this system involved the direct activation of factor X by factor Vlla. In a global form, the VllaQI 1E33 factor was superior to the wild type Vlla in procoagulant activity supported by membrane vesicles and soluble tissue factor. The wild-type zymogen had virtually no activity under these conditions, as indicated by similar support coagulation times of 2 minutes, whether or not sTF was added. Procoaqulant activity with normal tissue factor: The activity of Vlla and / or VIIQ11E33 was measured with sTF in normal human plasma. The endogenous factor Vil seemed to have no impact on the coagulation time in this assay; the coagulation time of support was 2 minutes for plasma with or without soluble tissue factor. The soluble tissue factor (50 nM final concentration) and Vlla were added to the plasma before the calcium solution. The coagulation time was evaluated for samples containing several levels of Vlla or VllaQ11E33. Two preparations of human, normal and deficient plasma of factor VIII were tested. The coagulation supported by normal tissue factor was evaluated with thromboplastin-HS of standard rabbit brain (HS = high sensitivity) containing calcium (Sigma Chemical Co.). This mixture contains both phospholipids and membrane-bound tissue factor. The thromboplastin-HS of rabbit brain was diluted 1: 100 in buffer and was used in the Vil test (added in the form of normal human plasma, which contains 10 nM of factor VII) and VIIQ11E33 (added as the pure protein ). The thromboplastin (0.2 ml) was measured and added to the plasma (0.1 ml) to start the reaction and the time required to form a blood clot. Full-strength thromboplastin assays were also conducted, as described by the manufacturer.

At optimal levels of human thromboplastin, natural type VIL showed a normal level of activity, approximately 1500 units per mg. This is approximately 25 times less than the activity of factor Vlla natural type (80,000 units per mg). The VIIQ11E33 gave approximately 1500-3000 units per mg under the standard conditions of the test, only 2 times more than the natural type Vil. The difference between natural type VII and VIIQ11E33 was much greater when the coagulation conditions were sub-optimal. Figure 5 shows coagulation times and zymogen concentrations in assays containing 0.01 times the normal level of thromboplastin. Under these conditions, VIIQ11E33 was approximately 20 times more than the natural type factor VII. Thus, the greater efficacy of the mutant VIIQ11E33 was especially evident when coagulation conditions were limited, which is relevant in many situations in vivo. Anticoagulant activities of DEGR-VllaQ11 E33: Standard coagulation assays were performed with normal human serum and human thromboplastin that was diluted 1:10 with buffer. The active site of factor VllaQ11E33 was modified by DEGR as described by Sorenson, B.B. et al., 1997, supra. Figure 6 shows the coagulation time of DEGR-VllaQ11 E33 (0-4 nm) incubated with thromboplastin, in calcium buffer, for 15 seconds before the addition of the plasma. The time to form a clot was determined with the hand-to-hand method. The coagulation time was approximately 45 seconds with approximately 1 nm of DEGR-VllaQ11 E33.

Example 2 - Circulation time of factor VIIQ11E33 in the rat: Two anaesthetized Sprague Dawley rats (3325-350 g) (sodium nembutol) were injected with 36 μg of factor VIIQ11E33 at time zero. The injection was through the jugular vein, in which a cannula had been placed. At the times shown in Figure 7, blood was withdrawn from the carotid artery, in which a cannula was inserted by surgery. The amount of factor VIIQ11E33 in the circulation was estimated from the time of coagulation of plasma deficient in human factor VII, to which 1 μl of a 1:10 dilution of the rat plasma was added. A 1: 100 dilution of thromboplastin-HS from rat brain (Sigma Chemical Co.) was used. Coagulation was assessed by the hand-tubing method as described in Example 1. The amount of factor VII activity in the plasma before injection of VIIQ11E33 was determined and subtracted as a blank. The concentration of factor VIIQ11E33 in the circulation is given as log nM. A substitute experiment was conducted in which a third animal received the operation and cannulation but without factor VIIQ11E33. The amount of factor VII activity in that animal did not change with the time of the experiment (100 minutes). At the end of the experiment, the animals were euthanized by excess sodium nembutol. The rats appeared normal throughout the experiment without any evidence of coagulation. Therefore, factor VIIQ11E33 did not induce indiscriminate coagulation, even in the post-operative rat. The circulation lifetime of VIIQ11E33 was normal (Figure 7), about 40% of the protein being cleared in approximately 60 minutes and even a slower disappearance of the remaining protein. This was similar for the rate of bovine prothrombin clearance of the rat. Nelsestuen and Suttie, 1971, Biochem. Biophys. Res. Commun., 45: 198-203. This is superior to a wild type recombinant Vlla factor, which gave a circulation time for functional tests of 20-45 minutes. Thomsen, M.K., et al., 1993, Thromb. Haemost., 70: 458-464. This indicated that factor VIIQ11E33 was not recognized as an abnormal protein and that it was not rapidly destroyed by coagulation activity. It appeared as a normal protein and should have a standard life time of circulation in the animal.

Example 3 - Intensification of the membrane site and protein C activity: Bovine and human protein C shows a high degree of homology in the amino acids of their GLA domains (44 amino terminal residues), despite approximately 10 times higher affinity of membrane of the human protein. Bovine protein C contains a proline at position 11 versus a histidine at position 11 of human protein C. The impact of replacing proline-11 on bovine protein C with histidine, and the inverse change in human protein C was examined. In both cases, the protein containing proline-11 showed lower membrane affinity, approximately 10 times for bovine protein C and 5 times for human protein C. Activated human protein C (hAPC) containing proline at position 11 showed 2.4 to 3.5 times less activity than wild-type hAPC, depending on the assay used. Bovine APC containing histidine-11 exhibited up to 15 times more activity than wild-type bAPC.

This demonstrates the ability to improve both membrane contact and activity by mutation. Mutagenesis of protein C: A full-length human protein C cDNA clone was provided by Dr. Johan Stenflo (Dept. of Clinical Chemistry, U niversity Hospital, Moalmo, Sweden). The bovine protein C cDNA clone was provided by Dr. Donald Foster (ZymoGenetics, I nc., USA). The GenBank accession number for the n-nucleotide sequence of bovine protein C is KO2435, N I D g 1 63486 and is K02059, N I D g 1 90322 for the nucleotide sequence of human protein C. Site-directed mutagenesis was performed by a PCR method. For. The mutagenesis of human C protein of histidine-1 to proline, the following oligonucleotides were synthesized: A, 5'-AAA TTA ATA CGA CTC ACT ATA GGG AGA CCC AAG CTT-3 '(SEQ ID NO: 7) (corresponding to nucleotides 860-895 in the vector pRc / CMV) to create a Hind lll site between pRc / CMV and protein C. B, 5 '-GCA CTC CCG CTC CAG GCT GCT GGG ACG GAG CTC CTC CAG GAA-3' (SEQ ID NO: 8) (corresponding to amino acid residues 4-1 7 in human protein C, the eighth residue in this sequence was subjected to aq ule amutation for human protein C to that of bovine protein c, as indicated by underlined). For the mutagenesis of bovine protein C from proline-1 to histidine, the following oligonucleotides were synthesized: A, (as described above); C, 5 '-ACG CTC CAC GTT GCC GTG CCG CAG CTC CTC TAG GAA-3' (SEQ ID NO: 9) (corresponding to amino acid residues 4-15 in bovine protein C, the sixth residue was subjected to mutation from aqela for bovine protein C to that of protein C h umana, as marked by underlining); D, 5'-TTC CTA GAG GAG CTG CGG CAC GGC AAC GTG GAG CGT-3 '(SEQ ID NO: 1 0) (corresponding to amino acid residues 4-1 5 in bovine protein C, the seventh amino acid was subjected to a mutation from that for bovine protein C to that of human protein C, nucleotides with mutation are underlined); E, 5'-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCC TCT AGA-3 '(S EQ ID NO: 1 1) (corresponding to nucleotides 984-101 9 in the vector pRc / CMV), creating an Xba site between pRC / CMV and protein C). Both human and bovine protein C cDNAs were cloned into the Hind lll and Xba I sites of the pRc / CMV expression vector. The human protein C cDNA containing the 5 'end to amino acid 1 7 was amplified by PCR with intact human protein C cDNA and primers A and B. The volume for the PCR reaction was 1 00 μl and contained 0.25 μg. of template DNA, 200 μM each of the four deoxyribonucleotide triphosphates, 0.5 mM of each injector and 2.5 U of Pwo-DNA polymerase (Boehringer Mannheim) in Tris-HCl buffer (1.0 mM Tris, 25 μM KCl, 5 mM (N H4) 2SO4, and 2 mM MgSO4, pH 8.85). The samples were subjected to 30 cycles of PCR consisting of a denaturation period 2 minutes at 94 ° C, a tempering period of 2 minutes at 55 ° C, and a lengthening period of 2 m inutes at 72 ° C. After amplification, the DNA was subjected to electrophoresis through a 0.8% agarose gel in 40 mM Tris-acetate buffer containing 1 mM EDTA. The PCR products were purified with JET Plasmid Miniprep-Kit (Saveen Biotech AB, Sweden). The human protein C cDNA containing respective mutations was cut by Hind lll and Bsr Bl, and then cloned into the pRc / CMV vector that was cut by Hind III / Xba I and which contained a human protein C fragment of Bsr Bl at 3 'end to produce a full-length cDNA, of human protein C, with the mutation. The bovine protein C cDNA, containing the 5 'end through amino acid 11, was amplified by PCR with intact human protein C cDNA and primers A and C. The bovine protein C cDNA from amino acid 11 to the 5' end was amplified with intact human protein c-DNA cDNA and primers D and E. These two cDNA fragments were used as templates to amplify the full-length bovine protein C cDNA containing amino acid with mutation with primers A and E. The reaction conditions of PCR were identical to those used for hPAPC. The bovine protein C cDNA containing the respective mutations was cut by Hind lll and Bsu 361, and the Hind fragment IHfBsu36l was cloned into the pRc / CMV vector containing intact bovine protein C fragments from Bsu 361 to the 3 'end to produce cDNA. of full-length bovine protein C containing the mutation. All mutations were confirmed by DNA sequencing before transfection. Cell Culture and Expression: The human kidney cell line transfected with adenovirus 293 was grown in DMEM medium supplemented with 10%? Fetal calf serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 U / ml of streptomycin and 10 μg / ml of vitamin Ki. The transfection was performed using the lipofectin method. Felgner, P.L. et al., 1987, Proc. Nati Acad. Sci. USA, 84: 7413-7417. Two μg of DNA were diluted to 0.1 ml with DMEM containing 2 mM L-glutamine medium. Ten μl of lipofectin (1 mg / ml) was added to 100 μl of DMEM containing 2 mM of L-glutamine medium. DNA and lipofectin were mixed and left at room temperature for 10-15 min. cell monolayers (25-50% confluence in 5 cm petri dishes) were washed twice in DMEM with 2 mM L-glutamine medium. The DNA / lipid mixture was diluted to 1.8 ml in DMEM containing 2 mM L-gltuamine medium, added to the cells and incubated for 16 hours. Cells were fed with 2 ml of complete medium containing 10% calf serum, allowed to recover for another 48-72 hours and then trypsinized and seeded in 10 cm dishes with selection medium (DMEM containing 10% of serum and 400 μg / ml of Geneticin) at 1: 5. Yan, S.C.B. et al., 1990, Bio / Technoloqy 655-661. Colonies resistant to Geneticin were obtained after 3-5 weeks of selection. Twenty-four colonies were chosen from each DNA transfection, grown to confluence and media were sorted by expression of protein C with a dot-blot assay using monoclonal antibody HPC4 (for human protein C) and monoclonal antibody BPC5 (for protein C bovine). Producer clones of high amounts of protein were isolated and grown to confluence in the presence of 10 μg / ml of vitamin K ^ The purification of bovine recombinant protein C and its mutant were based on the method previously described with some modifications.

Rezair, A.R., and Esmon, C.T., 1992, J. Biol. Chem., 267: 26104-26109. Serum-free medium conditioned from stably transfected cells was centrifuged at 5000 rpm at 4 ° C for 10 minutes. The supernatant was filtered through 0.45 μm cellulose nitrate membranes (Micro Filtration Systems, Japan). EDTA (5 mM, final concentration) and PPACK (0.2 μM, final concentration) were added to the conditioned medium from 293 cells, then passed through a Pharmacia FFQ anion exchange column at room temperature using Millipore With Sep LC100 (Millipore, USA). The protein was levigated with a gradient of CaCl2 (starting solution, 20 mM Tris-HCI / 150 mM NaCl, pH 7.4, limiting solution, 20 mM Tris-HCl / 150 mM NaCl / 30 mM CaCl2, pH 7.4.). After removal of CaCl2 by dialysis and Chelex 100 treatment, the protein was reabsorbed to a second FFQ column and then levigated with a gradient of NaCl (starting solution 20 mM Tris-HCl / 150 mM NaCl, pH 7.4; limiting 20 mM Tris-HCI / 500 mM NaCl, pH 7.4). At this point in the purification, recombinant bovine protein C mutant and wild-type were homogeneous as determined by SDS-PAGE. The first column used for purification of mutant and wild-type recombinant human protein C was the same as that described for bovine protein C. The chromatographic method described by Rezair and Esmon was employed with some modifications described for the protein purification method S. Rezair, A.R., and Esmon, C.T., 1992, supra; He, Z. et al., 1995, Eur. J. Biochem., 227: 433-440. Fractions containing protein C from anion exchange chromatography were identified by dot-blot. Positive fractions were deposited and applied to an affinity column containing the CaA-dependent HPC-4 antibody. The column was equilibrated with 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, containing 5 mM Benzamidine-HCI and 2 mM CaCl2. After application, the column was washed with the same buffer containing NaCl 1 M. Protein C was then levigated with 20 mM Tris-HCl, 150 mM NaCl and 5 mM EDTA, pH 7.4, containing 5 M Benzamidine-HCl. After purification, the purity of all preparations of recombinant human and bovine protein C was estimated by SDS-PAGE, followed by staining with silver. Proteins were dialyzed against buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.4) for 12 hours and stored at 70 ° C. Protein concentrations were measured by absorbance at 280 nm. Association of normal C protein molecules and membrane mutant: LUVs and SUVs were prepared by the methods described in Example 1. The light scattering at 90 ° was used for the incident light to quantify the protein-membrane binding as described for factor VII (25 μg / ml and PS / PC, (25/75) at 5 mM calcium (0.05 M buffer Tris-0.1M NaCl, pH 7.5) Bovine protein C containing histidine at position 11 interacted with membranes approximately 10 times more affinity than the wild-type protein When fitted to equation 2, the data gave KD values of 930 ± 80 nM for protein C-H11 and 9200 ± 950 nM for protein C of wild type ( Figure 8A) The difference in affinity corresponded to approximately 1.4 kcal / ml at 25 ° C. In fact, the membrane affinity of bovine protein C-H 1 1 was almost identical to that of natural human protein C (660 nM, Figure 8B) This suggested that Proline 1 1 formed a larger base for dif Efficiencies between the membrane binding site of human and bovine proteins. Inverse substitution, replacement of His-1 1 of human protein C with proline, decreased membrane affinity (Fig. 8B). When fitted to equation 2, these data gave KD values of 660 + 90 nM for wild type human protein C and 3350 ± 1 1 0 n M for human protein C-P 1 1. The impact of the introduction of proline was only slightly less than that of proline in the bovine proteins. I m of proline-1 1 in activated protein C activity: Activated protein C was generated by cutting thrombin, using identical conditions for both wild type and mutant proteins. Approximately 150 μg of the various protein C preparations (1 mg / μl) were mixed with bovine thrombin (3 μg) and incubated at 37 ° C for 5 hours. The reaction product was diluted to 0.025 M Tris-0.05M NaCl buffer and applied to one ml of S P-Sephadex C-50 column. The column was washed with one ml of the same buffer and the side-to-side flow was deposited as activated protein C. Approximately 65-80% of the protein applied to the column was recovered. The activity of APC was determined by proteolysis of S2366 (0. 1 mM) at 25 ° C. The preparations were compared with standard preparations obtained on a larger scale. The standard human APC was provided by Dr. Walter Kisiel. For bovine proteins, the standard was a large scale preparation of activated APC with thrombin. Bovine APC activity was consistent for all preparations of normal and mutant proteins (± 5%). Two preparations of bovine APC were used for comparisons. The human APC generated from trombina was 55 to 60% as active as the standard. The concentrations reported in this study were based on the activity towards S2366, in relation to that of the standard. A standard APTT test used bovine or human plasma and standard APTT reagent (Sigma Chemical Co.) according to the manufacturer's instructions. Alternatively, phospholipid was provided in the form of vesicles formed from highly purified phospholipids. In this assay, bovine plasma (0. 1 ml) was incubated with either kaolin (0. 1 ml of 5 mg / ml in 0.05 M Tris buffer, 0.1 M NaCl, pH 7.5) or ellagic acid (0. 1 mM in buffer) for 5 m inutes at 35 ° C. Coagulation was initiated by adding 0. 1 ml of phospholipid-containing buffer and the amounts of APC shown, followed by 0.1 ml of 25 mM calcium chloride. All reagents were in standard buffer containing 0.05 M Tris buffer, 0.1 M NaCl, pH 7.5. An average of a 14-fold higher concentration of natural bAPC was necessary to double the impact of mutant H 1 1. The coagulation time in bAPC-H 1 1 1 0 nM was greater than 1 20 minutes. Standard APTT reagent (Sigma Chemical C.) gave a coagulation time of approximately 61 seconds at 35 ° C with this plasma. The time required to form a clot was recorded by manual technique. The amount of phospholipid was designed to be the limiting component in the assay and to give the coagulation times shown. The phospholipids used were SUVs (45 μg / 0.4 ml in the final assay, PS / PC, 10/90) or LUVs (120 μg / 0.4 ml in the final assay, PS / PC, 25/75). The anticoagulant activity of activated protein C was tested in several tests. Figure 9 shows the impact in the APTT assay, conducted with limiting phospholipid. Under the conditions of this test, the coagulation times decreased in an inverse, almost linear relationship with the phospholipid concentration. It was necessary approximately 14 times as much natural bovine APC to equal the effect of bovine APC-H 1 1. Parts of the study in Figure 9 were repeated for membranes of PS / PC (25/75, LUV). Again, the activity was limited by phospholipid and its concentration was adjusted to give a control coagulation time of 360 seconds (1 20 μg of 25% PS in the 0.4 ml assay). Approximately 1 5 times more natural type enzyme was necessary to equalize the impact of mutant H 1 1. Finally, standard APTT reagent was used (Sigma Chemical Co., standard coagulation time 50 + 2 seconds). Approximately 1 0.0 ± 0.7 nM of natural type enzyme was necessary to double the clotting time to 102 ± 5 seconds. The same impact was produced by 2.2 ± 0. 1 nM of APC-H 1 1 vobina. The phospholipid was not rate limiting in the standard assay, so that a lower impact on membrane affinity can be expected. The results for human proteins are shown in Figure 8B. Approximately 2.5 times as much human APC containing proline-1 1 was required to prolong coagulation to the natural-type APC gage. A lower impact of the introduction of proline-11 may reflect the smaller differences in membrane affinity of human proteins (Figure 9B). Inactivation of factor Va: Inactivation of factor Va was assessed by the method of Nicolaes et al., 1996, Thrombosis and Haemostasis, 76: 404-410. Briefly, for bovine proteins, the bovine plasma was diluted 1000 times by 0.05 M Tris, 0.1 M NaCl, 1 mg / ml of bovine serum albumin and 5 mM of calcium at pH 7.5. Phospholipid vesicles (5 μg / 0.24 ml assay) were added and 5 μl of 190 nM thrombin were added to activated factor V. After incubation for 10 minutes at 37 ° C, APC was added and the incubation was continued for 6 minutes. Bovine prothrombin (at 10 μM final concentration) and factor Xa (0.3 nM final concentration) were added and the reaction was incubated for one minute at 37 ° C. A 20 μl sample of this activation reaction was added to 0.38 ml of buffer (0.05 M Tris, 0.1 M NaCl, 5 mM EDTA, pH 7.5) containing substrate S2288 (60 μM). The amount of thrombin was determined by the change in absorbance at 405 nM (e = 1.0 * 104 M "V \ kca, for thrombin = 100 / s.) For human proteins, 100-fold plasma deficient in human S protein was diluted (Biopool Canada, Inc.), factor Va was activated by human thrombin and the Va factor produced was titrated with the reagents used for the bovine proteins, bovine APC-H11 was 9.2 times more active than the wild type (Figure 10A) in inactivating factor. Va. As for membrane binding (anterior), the impact of proline-11 was lower with human proteins, averaging 2.4 times the difference between the curves drawn for natural and mutant type P-1 1 (Figure 1 0B ) Similar results were obtained with normal human plasma.

Example 4 - Identification of an archetype membrane affinity for the membrane contact site of vitamin K-dependent proteins: Comparison of several human and bovine protein C mutants and other vitamin K dependent polypeptides leads to an arq ueti proposed membrane contact site. The electrostatic archetype consists of an electropositive nucleus on a surface of the protein, created by bound calcium ions, surrounded by a halo of electronegative charge of protein amino acids. The closer a member of this family of proteins is to this electrostatic pattern, the greater its affinity for mem branes. Phospholipid vesicles, wild-type bovine protein C, membrane-protein interaction studies, activation and quantification of protein C and activity analysis were as described in Example 3. Recombinant mutant protein C was generated by the next procedures. Site-directed mutagenesis was performed using a PCR method. The following oligonucleotides were synthesized: A, as described in Example 3; F, 5'-GCA TTT AGG TGA CAC TAT AGA ATA GGC CCC TCT AGA -3 '(SEQ ID NO: 1 1) (Corresponding to nucleotides 984-101 9 in vector pRc / CMV), creating an Xba I site between pRc / CMV and protein C; G, 5 '-GAA GGC CAT TGT GTC TTC CGT GTC TTC GAA AAT CTC CCG AGC-3' (SEQ ID NO: 1 2) (corresponding to amino acid residues 40-27 in bovine protein C, amino acids 8 and 9 they were subjected to mutation from QN to ED as underlined); H, 5 '-CAG TGT GTC ATC CAC ATC TTC GAA AAT TTC CTT GGC-3' (SEQ ID NO: 1 3) (corresponding to amino acid residues 38-27 in the human protein, amino acids 6 and 7 in this sequence were subjected to mutation from QN to ED as indicated by underlining); I, 5'-GCC AAG GAA ATT TTC GAA GAT GTG GAT GAC ACA CTG-3 '(SEQ ID NO: 14) (corresponding to amino acid residues 27-38 in human protein C, amino acids 6 and 7 in this sequence they were submitted from QN to ED as indicated by underlining); J, 5 '-CAG TGT GTC ATC CAC ATT TTC GAA AAT TTC CTT GGC-3 (SEQ ID NO: 15) (corresponding to residues of amino acids 38-27 in human protein C, amino acid 7 in this sequence it was subjected to mutation from Q to E as indicated by underlining); K, 5'-GCC AAG GAA ATT TTC GAA AAT GTG GAT GAC ACA CTG-3 '(SEQ ID NO: 1 6) (corresponding to amino acid residues 27-38 in human protein C, the sixth amino acid in this The sequence was submitted from Q to E as indicated by underline); Both full length cDNAs, bovine and human protein C, were cloned into the Hind l l l site and Xba I of the pRc / CMV vector. To obtain bovine protein C33 mutant E33D34, the PCR amplification of the target DNA was performed as follows. Bovine protein C cDNA containing the 5 'end was amplified to the amino acid at position 40 with intact bovine protein C cDNA and the A and C inkers. The PCR reaction conditions were as described in Example 3. Our sample was subjected to 30 cycles of PCR consisting of a denaturation period of 2 m in at 94 ° C, a tempering period of 2 min at 55 ° C and a lengthening period of 2 m in at 72 ° C. After amplification, the DNA was subjected to electrophoresis through a 0.8% agarose gel in 40 mM Tris-acetate amrotiguador containing 1 mM EDTA. The PCR products were purified with the Geneclean II complex (Bio 1 01, I nc USA) and the PCR fragment of bovine protein C cDNA containing the respective mutations was cut by Hind I1 and Bbs I. The Hind III / Bbs I fragment and the human protein C fragment (Bbs I - 3 'end) were cloned into the H ind II and Xba I sites of the pRc / CMV vector to produce a full-length bovine protein C cDNA with the mutations. The Bovine Protein C mutant H 1 1 E33 D34 was created in the same manner, but bovine Protein C mutant H 1 1 was used as a template in the PCR reaction. The human protein C cDNA containing the 5 'end to amino acid 38 was amplified by PC R with intact human protein C cDNA and primers A and D. The human protein C cDNA from amino acid 27 to the 3' end was amplified with the intact human protein C cDNA and the B and E inners. These two cDNA fragments were used as templates to amplify full length bovine protein C cNA containing the amino acids with m utation (E33 D34) with the primers A and B. The human protein C mutant E33 was obtained by the following steps: cDNA of human protein C containing the 5 'end to amnacid 38 was amplified with intact human protein C cDNA and primers A and F. Human protein C cDNA from amino acid 27 to the 3 'end was amplified with full length bovine protein C cDNA containing amino acids with mutation (E33) with primers A and B. The mixture of PCR and program were described. cough before. The human protein C PCR products containing respective mutations were cut by Hi nd l l l and Sal I, and then the fragment (Hind l l l - Sal I) together with the intact human protein C fragment (Sal I - 3 'end) were cloned into the Hind ll l and Xba I sites of the pRc / CMV vector to produce the full-length human protein C cDNA with the respective mutations. All mutations were confirmed by DNA sequencing before transfection. The line of human kidney cells transfected with adenovirus was quantified and transfected as described in Example 3. Recombinant bovine and human protein C and mutants were purified as described in Example 3. The vitamin-dependent proteins. ina K were classified into four groups based on their affinities for a standard membrane (Table 4). Sequences of amino terminal residues of some relevant proteins including human protein C (hC), bovine protein C (bC), bovine prothrombin (bPT), bovine factor X (bX) and human factor Vi l (hVI I) are given by reference, where X is Gla (β-carboxyglutamic acid) or Glu. bPT: ANKGF XXVRK11GN XRXCLXX21PCSRXXAFXA31l-XSLSATDAF4lWAKY (SEQ ID NO: 17) bX ANS-F XXVKQuGNLXRXCLXXjjACSLXXARXVj, FXDAXQTDXF41WSKY (SEQ ID NO: 18) hC ANS-FLXXLRHjjSS XRXCIXXjjICDFXXAKX jjFQNVDDTLAF ^ WSKH (SEQ ID NO: l) bC ANS-F XX RPuGNVXRXCSXX ^ VCXFXXARXI ^ FQNTXDTMAF ^ WSFY (SEQ ID NO: 2) hVTI: ANA-FLXXLRPuGS XRXCKXXjiQCSFXXARXIjjFKDAXRTK F ^ ISY (SEQ ID NO: 3) TABLE 4 Loads and affinity 3 Greater affinity value equal to kdlsoC? Ac? On / 1 * 107 M "V; the denominator is a normal kdlsoc? Ac? On for other proteins.

In Table 4, mutants of vitamin K-dependent polypeptides are in bold. The total charge (residues 1-34) includes 7 calcium ions (+ 14) and the amino terminus (+ 1). Protein Z was assigned to class I at the base of its dissociation rate constant, which was 100 to 1000 times lower than that of other proeins. If protein Z exhibited a normal association rate constant (approximately 107 M "1s" 1), the KD would be approximately 10"10 M M. Wei, GJ et al., 1982, Biochemistry, 21: 1949-1959. The last affinity may be the maximum possible for vitamin K proteins. Class VI proteins differed from class III in the presence of proline-11, which may alter affinity by non-electrostatic means, although there was a relatively weak correlation between the membrane affinity and the net negative charge in residues 1-34, an excellent correlation was found when only residues 5, 11, 29, 33 and 34 were considered (Table 4) .The last amino acids are located on the surface of The protein A variety of proteins were modeled by amino acid substitution in the prothrombin structure and their electrostatic potentials were estimated by the DelPhi program A pattern sketch after the potential electrost The bovine protein Z protein is shown in Figure 11. The electronegative sites at 7, 8, 26, 30, 33, 34 and 11 produce an electronegative charge halo surrounding a cationic core produced by the calcium-coated pore (Figure 1). 1 ). The closer the protein structure is to this structure, the greater its affinity for the membrane. This correlation is evident from natural-type proteins, mutants and chemically modified proteins. The pattern for other structures can be extrapolated from the examination of load groups that are absent in other proteins. For example, bovine prothrombin Lys-1 1 and Arg-1 0 generate high electropositive regions in their vicinity; the lack of Gla-33 in protein C and factor Vi l creates less electronegativity in those regions of protein. In all cases, the highest affinity corresponded to a structure with an electropositive nucleus that was completely surrounded by the electronegative protein surface, as shown by the Z protein. The exceptions to this pattern are proteins with Pro-1 1 , which can diminish the affinity for a structural impact and ser-1 2 (human C protein), which is a single uncharged residue. To further test the hypothesis of an archetype for electrostatic distribution, site mutagenesis was used to replace Gln33Asn34 of bovine and human protein C with Glu33Asp34 (SEQ I D NO: 1 9). Glu33 should be further modified to Gla during protein processing. These changes altered the electrostatic potential of bovine protein C to that of bovine factor X. It was expected that the membrane affinity of the mutant protein was less than that of factor X due to the presence of proline-1. In fact, the bovine protein C moiety provided a membrane affinity similar to that of bovine prothrombin (Figure 12A) and slightly lower than that of bovine factor X (Table 4). It was more interesting that the inhibition of clot by APC was greater for the mutant than for the wild-type enzyme (Figure 12B, C). Inclusion of results for the Bovine protein C mutant P11H of Example 3 showed that a family of proteins could be produced, each with different membrane affinity and activity, by varying the amino acid substitutions at positions 11, 33 and 34. The human protein C mutants containing E33 and E33D34 resulted in a small increase in membrane binding affinity (Figure 13a). The activity of these mutants was slightly lower than the wild-type enzyme (Figure 13b). The results with bovine protein C mutants suggest that the failure of the E33D34 mutation in the human protein may arise from H11 and / or other amino acids unique in the protein. Figure 14A shows that the H11 mutant of the bovine protein C bound to the membrane with approximately 10 times higher affinity than the wild type protein, the mutant E33D34 bound with approximately 70 times the affinity, but that of the triple mutant, H11E33D34, it was only slightly better than mutant H 11. This relationship was reflected in the APC activity formed from these mutants (Figure 14B). This result suggested that the presence of H11 reduced the impact of E33D34 on membrane binding affinity. These results indicated that the introduction of E33D34 may not be optimal for all proteins. Accordingly, other mutations may be desirable to create human protein C that will use E33D34 and will have maximal membrane affinity increased. The result with bovine protein suggested that histidine 11 may be the primary cause of this phenomenon. Consequently, H11 can be altered to glutamine or another amino acid in human protein C, together with the E33D34 mutation. Another amino acid that can impact affinity is serine at position 12, an amino acid that is completely unique to human protein C. These additional changes should produce proteins with enhanced membrane affinity. The electrostatic archetype was also tested by comparison of human and bovine X factor. The presence of lysine-11 in human factor X suggests that it should have lower affinity than bovine factor X. This prediction was confirmed by the result shown in Figure 15. Previous studies had shown that the modification with trinitrobenzenesulfonic acid (TNBS) of bovine prothrombin fragment 1 and human had relatively little impact (0 to 5 times) on membrane affinity. Weber, D.J. et al., 1992, J. Biol. Chem., 267: 4564-4569; Welsch, D.J. et al., 1988, Biochemistry, 27: 4933-4938. The conditions used for the reaction resulted in derivatization of the amino terminus, a change that is bound to decreased membrane affinity. Welsch, D.J. and Nelsestuen, G.L., 1988 Biochemistry, 27: 4939-4945. Protein modification in the presence of calcium, which protects the amino terminus, resulted in protein modified with TNBS with much greater affinity for the membrane than natural fragment 1. The suggestion that the Z protein constitutes the archetype was based on its dissociation rate constant and that a normal association velocity would generate a KD = 1 0"1 0 M. If this value can be reached, it is uncertain. The slow association of protein Z is caused by inadequate protein misbinding, resulting in a low concentration of the membrane binding conformation.If conditions can be altered to improve protein folding, the Z protein association rates. In fact, the association rate constant for the Z protein was improved by pH alteration.The basis for this observation may be related to an unusual feature of the prothrombin estriento, which is the near placement of the extreme ino (+1 to pH 7.5) to calcium ions 2 and 3. The charge + 1 at the extreme end is responsible for the light electropositive region jus above Ca-1 in Figure 1 1. The charge repulsion between Ca and the amino terminal can destabilize protein folding and could be a serious problem for a protein that had low bending stability. Table 5 provides additional support for the model type. It shows the relationship between the distance of ion strontium ion strontium 1 and 8 groups (corresponding to calcium 1 and an extra divalent metal ion found in the prothrombin Sr X-ray crystal structure). The pattern suggests that the closer a ionic group is to these metal ions, the greater their impact on membrane affinity. The exception is Arg-16, which contributes to the charge of the electropositive nucleus. The higher affinity is correlated with the electronegative charge in all other places. This correlation also applies to G LA residuals.

TABLE 5 Distance to Sr-1, 8 and importance of ions a The distances are from this bovine prothrombin atom (prothrombin residue used in the measurement is given in parentheses) to strontium 1 and 8 of the structure of fragment 1 of Sr-prothrombin. Seshadri et al. 1 994, Biochemistry 33: 1 087-1092. b For all but 16-R, cations decrease affinity and anions increase affinity. c Thariath et al. 1 997 Biochem. J. 322: 309-31 5. d I mpact of Glu to Asp mutations, distances are averages for gamma-carboxyl-carbons. The KD (KM) data are from Ratcliffe et al. 1 993 J. Biol. Chem. 268: 24339-45. e The union was of less capacity or caused agigation, making comparisons less certain.

The results in Figure 1 5C show that the association rate for Z protein was substantially improved at pH 9, where an amino terminal should be uncharged. The velocity constant obtained from these data was approximately 12-fold higher at pH 9 than at pH 7.5 (Figure 1 5C).

Other modalities It will be understood that although the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the annexed subdivisions. Other aspects, advantages and modifications are within the scope of the following subdivisions.

LIST OF SEQUENCES < 110 > Reagents from the University of Minnesota < 120 > MODIFIED POLYPEPTIDES DEPENDENTS OF VITAMIN K < 130 > 09531 / 002WO1 < 150 > 08 / 955,636 < 151 > 1997-10-23 < 160 > 35 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 1 Wing Asn Ser Phe Leu Xaa Xaa Leu Arg His Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Wing Lys Xaa He Phe Gln 20 25 30 Asn Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 2 < 211 > 44 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 2 Wing Asn Ser Phe Leu Xaa Xaa Leu Arg ftr Gly Asn Val Xaa Arg Xaa 1 5 10- 15 Cys Ser Xaa Xaa Val Cys Xaa Phe Xaa Xaa Wing Arg Xaa He Phe Gln 20 25 30 Asn Thr Xaa Asp Thr Met Wing Phe Trp Ser Phe Tyr 35 40 < 210 > 3 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 3 Wing Asn Wing Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa He Phe Lys 20 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 4 < 211 > 44 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 4 Wing Asn Gly Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Arg Xaa Xaa Leu Cys Ser Phe Xaa Xaa Ala His Xaa He Phe Arg 20 25 30 Asn Xaa Xaa Arg Thr Arg Gln Phe Trp Val Ser Tyr 35 40 < 210 > 5 < 211 > 45 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 5 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cye Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 20 25 30 Xaa Asn Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 6 < 211 > 46 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 6 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 20 25 30 Xaa Asn Thr Xaa Lys Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 7 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 7 aaattaatac gactcactat agggagaccc aagctt 36 < 210 > 8 < 211 > 42 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 8 gcactcccgc tccaggctgc tgggacggag ctcctccagg aa 42 < 210 > 9 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 9 acgctccacg ttgccgtgcc gcagctcctc taggaa 36 < 210 > 10 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 10 ttcctagagg agctgcggca cggcaacgtg gagcgt 36 < 210 > 11 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 11 gcatttaggt gacactatag aatagggccc tctaga 36 < 210 > 12 < 211 > 42 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 12 gaaggccatt gtgtcttccg tgtcttcgaa aatctcccga ge 42 < 210 > 13 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 13 cagtgtgtca tccacatctt cgaaaatttc cttggc 36 < 210 > 14 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 14 gccaaggaaa ttttcgaaga tgtggatgac acactg 36 < 210 > 15 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 15 cagtgtgtca tccacatttt cgaaaatttc cttggc 36 < 210 > 16 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > mutagenic protein oligonucleotide C < 400 > 16 gccaaggaaa ttttcgaaaa tgtggatgac acactg 36 < 210 > 17 < 211 > 45 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 17 Wing Asn Lys Gly Phe Leu Xaa Xaa Val Arg Lys Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Leu Xaa Xaa Pro Cys Ser Arg Xaa Xaa Ala Phe Xaa Ala Leu 20 25 30 Xaa Ser Leu Ser Ala Thr Asp Ala Phe Trp Ala Lys Tyr 35 40 45 < 210 > 18 < 211 > 44 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 18 Wing Asn Ser Phe Leu Xaa Xaa Val Lys Gln Gly Asn Leu Xaa Arg Xaa 1 5 10 15 Cys Leu Xaa Xaa Wing Cys Ser Leu Xaa Xaa Wing Arg Xaa Val Phe Xaa 20 25 30 Asp Wing Xaa Gln Thr Asp Xaa Phe Trp Ser Lys Tyr 35 40 < 210 > 19 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 19 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg His Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 20 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 20 Wing Asn Ser Phe Leu Xaa Xaa Leu Arg Gln Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 21 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 21 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg Glu Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 22 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 22 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg Asp Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 23 < 211 > 44 < 212 > PRT < 213 > Bos taurus < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 23 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg His Gly Asn Val Xaa Arg Xaa 1 5 10 15 Cys Ser Xaa Xaa Val Cys Xaa Phe Xaa Xaa Wing Arg Xaa He Phe Gln 25 30 Asn Thr Xaa Asp Thr Met Wing Phe Trp Ser Phe Tyr 35 40 < 210 > 24 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 24 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg Gln Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 25 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 25 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg His Ser Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Wing Phe Xaa He Phe Glu 25 30 Asp Val Asp Aep Thr Leu Wing Phe Trp Ser Lys His 35 40 < 210 > 26 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 26 Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Glu Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa He Phe Lys 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 27 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 27 Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Asp Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa He Phe Lys 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 28 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 28 Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Phe Xaa He Phe Lys 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 29 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 29 Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa He Phe Asp 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 30 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 30 Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Gln Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa He Phe Glu 25 30 Asp Ala Xaa Arg Thr Lys Leu Phe Trp He Ser Tyr 35 40 < 210 > 31 < 211 > 45 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 31 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Asp Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 25 30 Xaa Asn Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 32 < 211 > 45 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 32 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Glu Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 25 30 Xaa Asn Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 33 < 211 > 45 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 33 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Phe Xaa Val Phe 25 30 Xaa Asn Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 34 < 211 > 45 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 34 Tyr Aen Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 1 5 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 25 30 Xaa Asp Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 < 210 > 35 < 211 > 44 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > MOD_RES < 222 > (0) ... (0) < 223 > Xaa = gamma carboxyglutamic acid or glutamic acid < 400 > 35 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg Glu Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys He Xaa Xaa He Cys Asp Phe Xaa Xaa Ala Lys Xaa He Phe Glu 25 30 Asp Val Asp Asp Thr Leu Wing Phe Trp Ser Lys His 35 40

Claims (1)

1. CLAIMS 1 A vitamin K dependent peptide comprising a modified GLA domain that enhances the membrane binding affinity of said polypeptide in relation to a corresponding natural vitamin K-dependent polypeptide, said modified GLA comprising at least one amino acid substitution in the residue 11, 12, 29 or 34 2 The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 11 or 34 The popeptide of claim 1, wherein said polypeptide further comprises an amino acid substitution at the amino acid The polypeptide of claim 1, wherein said polypeptide comprises an amino acid substitution at amino acid 11 and amino acid 33. The polypeptide of claim 1, wherein said polypeptide enhances the formation of clots. 1, wherein said polypeptide inhibits the formation of clots 7 The polypeptide of the claim 1, wherein said polypeptide comprises protein C or activated protein 8 The pohpeptide of claim 7, wherein said substitution comprises a glutamic acid residue at amino acid 33 and a residue of aspartic acid at amino acid 34 (SEQ ID NO. twenty) 9. The polypeptide of claim 8, wherein said amino acid substitution further comprises a glutamine at amino acid 11 (SEQ ID NO: 20). 10. The polypeptide of claim 8, wherein said amino acid substitution further comprises a glutamic acid at amino acid 11. (SEQ ID NO: 21). The polypeptide of claim 9 or 10, wherein said amino acid substitution further comprises a glycine at amino acid 12 (SEQ ID NO: 24 or SEQ ID NO: 35). 12. The polypeptide of claim 1, wherein said polypeptide comprises factor VII or factor Vlla. The polypeptide of claim 12, wherein said amino acid substitution comprises a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30). The polypeptide of claim 1, wherein said polypeptide comprises factor IX or factor IXa. 15. The polypeptide of claim 1, wherein said polypeptide comprises active site-modified factor Vlla. The polypeptide of claim 15, wherein said amino acid substitution comprises a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33 (SEQ ID NO: 30) 17. The polypeptide of claim 1, wherein said modified GLA domain comprises an amino acid sequence, which, in the calcium saturated state, forms a tertiary structure having a cationic core with an electronegative charge halo. A vitamin K-dependent polypeptide comprising a modified GLA domain that enhances the membrane binding affinity and the activity of said polypeptide relative to a corresponding natural vitamin K dependent peptide, said modified GLA domain comprising at least one substitution of amino acid, wherein said polypeptide is one that inhibits the formation of clots. The popeptide of claim 18, wherein said GLA domain is from amino acid 1 to about amino acid. The polypeptide of claim 18, wherein said amino acid substitution is at amino acid 11, 12, 29, 33 or 34 An isolated nucleic acid encoding a vitamin K-dependent polypeptide, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances binding affinity of membrane and the activity of said polypeptide in relation to a po dependent peptide of corresponding natural vitamin K 22 A mammalian host cell comprising a nucleic acid vector, said vector encoding a vitamin K-dependent polypeptide, said vitamin K dependent peptide comprising a modified GLA domain that enhances the membrane binding affinity and the activity of said polypeptide in relation to a corresponding natural vitamin K dependent peptide, said modified GLA domain comprising at least one amino acid substitution 23 A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of a vitamin K-dependent polypeptide effective to inhibit clot formation in a mammal, wherein said polypeptide Vitamin K dependent comprises a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding natural vitamin K-dependent polypeptide, said modified GLA domain comprising at least one amino acid substitution. . 24. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of a vitamin K-dependent polypeptide effective to increase clot formation, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances the affinity of Membrane binding of said polypeptide relates to a corresponding natural vitamin K dependent polypeptide, said modified GLA domain comprising at least one amino acid substitution at residue 11, 29, 29 or 34. 25. The pharmaceutical composition of claim 24, wherein said polypeptide further comprises an amino acid substitution at amino acid 33. 26. The pharmaceutical composition of claim 24, wherein said pharmaceutical composition further comprises soluble tissue factor. 27. A vitamin K-dependent polypeptide for use in treating a coagulation disorder, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide in relation to to a corresponding natural vitamin K-dependent polypeptide, said modified GLA domain comprising at least one amino acid substitution. 28. The use of a vitamin K-dependent polypeptide in the manufacture of a medicament for the treatment of a coagulation disorder, wherein said vitamin K-dependent polypeptide comprises a modified modified LA domino that enhances the binding affinity of membrane and activity of said polypeptide in relation to a corresponding natural vitamin K-dependent polypeptide, said modified GLA domain comprising at least one amino acid substitution. 29. A method for decreasing clot formation in a mammal comprising administering an amount of a vitamin K-dependent polypeptide effective to decrease the formation of clots in said mammal, wherein said vitamin K dependent polypeptide comprises a modified GLA domain that enhances the membrane binding affinity and activity of said polypeptide relative to a corresponding natural vitamin K-dependent polypeptide, said modified GLA domain including at least one amino acid substitution. 30. A method for increasing clot formation in a mammal comprising administering an amount of an effective vitamin K-dependent polypeptide to increase clot formation in said mammal, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances the membrane binding affinity of said polypeptide relative to a corresponding natural vitamin K-dependent polypeptide, said modified GLA domain comprising at least one amino acid substitution at residue 11, 12, 29 or 34.