Portal de Periódicos da CAPES

C-terminal Residues 621–635 of Protein S Are Essential for Binding to Factor Va

1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês

10.1074/jbc.274.51.36187

1083-351X

Mary J. Heeb, Yumi Kojima, Jan Rosing, Guido Tans, John H. Griffin,

Heparin-Induced Thrombocytopenia and Thrombosis

Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621–635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K i ∼2 μm). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621–635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action. Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621–635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K i ∼2 μm). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621–635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action. Factor Va Factor Xa activated partial thromboplastin time protein S peptide Protein S is a vitamin K-dependent protein that can act as a cofactor for the anticoagulant functions of activated protein C (1DiScipio R.G. Davie E.W. Biochemistry. 1979; 18: 899-904Crossref PubMed Scopus (169) Google Scholar, 2Walker F.J. J. Biol. Chem. 1980; 255: 5521-5524Abstract Full Text PDF PubMed Google Scholar, 3Lundwall A. Dackowski W. Cohen E. Shaffer M. Mahr A. Dahlbäck B. Stenflo J.A. Wydro R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Crossref PubMed Scopus (149) Google Scholar). Heterozygous deficiency of protein S or protein C is associated with increased risks for both venous thrombosis (4Schwarz H.P. Fischer M. Hopmeier P. Batard M.A. Griffin J.H. Blood. 1984; 64: 1297-1300Crossref PubMed Google Scholar, 5Comp P.C. Nixon R.R. Cooper M.R. Esmon C.T. J. Clin. Invest. 1984; 74: 2082-2088Crossref PubMed Scopus (478) Google Scholar, 6Broekmans A.W. Bertina R.M. Reinalda-Poot J. Engesser L. Muller H.P. Leeuw J.A. Michiels J.J. Brommer E.J.P. Briët E. Thromb. Haemost. 1985; 53: 273-277Crossref PubMed Scopus (90) Google Scholar, 7Griffin J.H. Evatt B. Zimmerman T.S. Kleiss A.J. Wideman C. J. Clin. Invest. 1981; 68: 1370-1373Crossref PubMed Scopus (976) Google Scholar, 8Bertina R.M. Broekmans A.W. van der Linden I.K. Mertens K. Thromb. Haemost. 1982; 48: 1-5Crossref PubMed Scopus (177) Google Scholar) and ischemic stroke (9Wiesel M.-L. Charmantier J.-L. Freyssinet J.-M. Grunebaum L. Schuhler S. Cazenave J.-P. Thromb. Res. 1990; 58: 461-468Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 10Thommen D. Buhrfeind E. Felix R. Sulzer I. Furlan M. Lämmle B. Schweiz. Med. Wochenschr. 1989; 119: 493-499PubMed Google Scholar, 11Folsom A.R. Rosamond W.D. Shahar E. Cooper L.S. Nieto F.J. Rasmussen M.L. Wu K.K. Circulation. 1998; 98 (Abstr.): I-207Google Scholar). Homozygous deficiency of protein S, like that of protein C (12Branson H.E. Katz J. Marble R. Griffin J.H. Lancet. 1983; 2: 1165-1168Abstract PubMed Scopus (264) Google Scholar, 13Seligsohn U. Berger A. Abend M. Rubin L. Attias D. Zivelin A. Rapaport S.I. N. Engl. J. Med. 1984; 31: 559-562Crossref Scopus (348) Google Scholar), can lead to potentially fatal purpura fulminans in infancy unless aggressively treated (14Mahasandana C. Suvatte V. Marlar R.A. Manco-Johnson M.J. Jacobson L.J. Hathaway W.E. Lancet. 1990; 335: 61-62Abstract PubMed Scopus (0) Google Scholar). The physiological importance of protein S is underscored by the finding that infusions of protein S concentrates without activated protein C showed antithrombotic activity in a rabbit thrombosis model (15Schwarz H.P. Linnau Y. Pfeiler S. Molinari E. Thromb. Haemost. 1989; 62 (Abstr.): 25Google Scholar). Modes of antithrombotic action of protein S are, however, incompletely understood. Protein S can be anticoagulant even in the absence of activated protein C by inhibiting prothrombinase activity of the FVa-FXa complex in fluid phase and on phospholipid vesicles, endothelial cells, or platelets (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar). This inhibition presumably involves binding of protein S to Factor Va (FVa)1 and Factor Xa (FXa) as well as binding to the lipid membrane (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar, 20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar). Protein S binds FVa and FXa with apparent K d values of 33 and 18 nm, respectively (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar). In addition, protein S inhibits thrombin generation when subendothelial matrix is exposed to plasma under flow conditions (19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar). The relative anticoagulant potency of protein S has been correlated with its affinity for phospholipid vesicles, and protein S competes with prothrombinase components particularly at limiting phospholipid concentrations, e.g.0.2 μm (20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar). An assay has been developed for the activated protein C-independent activity of protein S in individual plasma samples (21van Wijnen M. van't Veer C. Meijers J.C.M. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). FV in combination with protein S enhances activated protein C anticoagulant activity (22Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar), and it was suggested that this complex phenomenon may involve binding of the sex hormone binding globulin region of protein S residues 270–635 to FV (23Nyberg P. Dahlbäck B. Garcia de Frutos P. FEBS Lett. 1998; 14: 28-32Crossref Scopus (41) Google Scholar). Studies presented here identify an essential binding site for FVa at the C terminus of protein S that contributes to the ability of protein S to inhibit prothrombinase activity.DISCUSSIONA novel mechanism of anticoagulant action of protein S involves its ability to function independently of activated protein C, via its binding to FVa, FXa, and/or phospholipids, thereby causing inhibition of prothrombinase activity (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar, 20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar, 21van Wijnen M. van't Veer C. Meijers J.C.M. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). This report explores the interaction of protein S with FVa and identifies an essential binding site for FVa on protein S involving the region of residues 621–635. Peptide PSP14 representing this region was a potent inhibitor of FVa binding to protein S and of FVa-dependent prothrombin activation and was anticoagulant in clotting assays. The inhibitory effect of peptide PSP14 was only observed in the presence of FVa, but the inhibition was not dependent on the presence or concentration of phospholipids. However, increasing FXa concentrations from 0.2 to 8 nm negated the inhibitory activity of PSP14, suggesting that PSP14 may compete with FXa for a binding site on FVa. This hypothesis was supported by a plot of 1/v versus[PSP14] at varying [FXa], which revealed the pattern for a competitive inhibitor with an apparent K i of ∼2 μm. Competition between FXa and protein S for binding to FVa has not been directly demonstrated but is consistent with the report that a FVa 15-mer peptide binds both protein S and FXa (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar) and with reports that protein S negates the ability of FXa to protect FVa from proteolytic inactivation by activated protein C (33Solymoss S. Tucker M.M. Tracy P.B. J. Biol. Chem. 1988; 263: 14884-14890Abstract Full Text PDF PubMed Google Scholar, 34Jane S.M. Hau L. Salem H.H. Blood Coag. Fibrinol. 1991; 2: 723-729Crossref PubMed Scopus (9) Google Scholar).The inhibitory potency of peptide PSP14 was enhanced by an internal disulfide bond introduced by the substitution of Cys for Ser635, because reduced and alkylated PSP14 and a peptide analog with Ser in position 635 were less potent than the cyclized peptide. Single amino acid substitutions in PSP14 analogs showed that Lys 630, 631, and 633 were each critical for prothrombinase inhibition. Although a short peptide with the linear sequence of residues 628–635 containing these Lys residues was not a potent inhibitor, cyclization of the peptide by addition of two Cys residues at the end of the peptide rendered it potently anticoagulant (Table II). Because the degrees of freedom that determine the relative populations of various conformations for a given peptide are greatly reduced in a cyclic peptide, it appears that some of the conformations of cyclized PSP14 and PSP629 may resemble those of native protein S.The anticoagulant and binding inhibitory effects of PSP14 were not due simply to its net positive charge per se, because a trilysine peptide, peptides with the same scrambled l-amino acid sequence (PSP14SCR) or with the same sequence ofd-amino acids and unrelated pentadecapeptides with four basic residues had little or no ability to inhibit binding of protein S to FVa or to inhibit prothrombinase or clotting assays.Other evidence for specificity of the inhibitory effects of peptide PSP14 was presented in several forms. Antibodies to PSP14 blocked protein S binding to FVa and neutralized the prothrombinase inhibitory activity of protein S. Neither PSP14 nor anti-PSP14 antibodies could block binding of biotin-FXa to protein S. Moreover, the ability of PSP14 to inhibit prothrombinase was entirely dependent on the presence of FVa. The fact that PSP14 was anticoagulant in protein C-depleted plasma is consistent with the concepts that protein S is anticoagulant in the absence of protein C and that the region of residues 621–635 is involved in protein S-FVa interactions.Residues 621–635 are not necessary for complexation of protein S with C4b-binding protein because peptide PSP14 did not inhibit binding of protein S to C4b-binding protein. Sites for interaction of protein S with C4b-binding protein have been reported in the steroid hormone binding globulin domain (35Chang G.T.G. Maas B.H.A. Ploos van Amstel H.K. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemost. 1994; 71: 461-467Crossref PubMed Scopus (29) Google Scholar) of protein S at residues 413–433 (29Fernández J.A. Heeb M.J. Griffin J.H. J. Biol. Chem. 1993; 268: 16788-16794Abstract Full Text PDF PubMed Google Scholar, 30Fernández J.A. Griffin J.H. Chang G.T.G. Stam J. Reitsma P.H. Bertina R.M. Bouma B.N. Blood Cells, Molecules, and Diseases. 1998; 24: 101-112Crossref PubMed Scopus (20) Google Scholar) and 447–460 (36He X. Shen L. Malmbourg A.-C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 37Linse S. Härdig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Peptides overlapping each of these regions in protein S did not inhibit prothrombinase activity (Table I), indicating that this experimental approach fails to implicate these sequences as essential for sites of interaction of protein S with FVa or FXa. The region in the FVa molecule with which residues 621–635 of protein S should interact remains a matter of speculation. A stretch of 14 amino acid residues in FVa (residues 493–506, GLLLICKSRSLDRR) potently inhibits prothrombinase activity and has been implicated in the binding of FVa to protein S, FXa, DEGR-FXa, and DIP-activated protein C but not to activated protein C or prothrombin (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar). It seems unlikely that this sequence in FVa interacts with residues 620–635 in protein S because of the likely electrostatic repulsion of the net positive charges of these sequences (+3 and +4, respectively). However, each peptide also contains a region of uncharged residues. It is likely that protein S and FVa have more than one site of molecular interaction. In the case of activated protein C interaction with FVa, three sites of molecular interaction have been suggested in the light and heavy chains of activated protein C, and two sites have been suggested in the light and heavy chains of FVa (24Mesters R.M. Houghten R.A. Griffin J.H. J. Biol. Chem. 1991; 266: 24514-24519Abstract Full Text PDF PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar, 39Mesters R.M. Heeb M.J. Griffin J.H. Protein Sci. 1993; 2: 1482-1489Crossref PubMed Scopus (26) Google Scholar, 40Mesters R.M. Heeb M.J. Griffin J.H. Biochemistry. 1993; 32: 12656-12663Crossref PubMed Scopus (32) Google Scholar, 41Walker F.J. Scandella D. Fay P.J. J. Biol. Chem. 1990; 265: 1484-1489Abstract Full Text PDF PubMed Google Scholar). For interactions between FXa and FVa, sites in both heavy and light chains of FVa are suggested (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar,42Tracy P.B. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2380-2384Crossref PubMed Scopus (66) Google Scholar, 43Annamalai A.E. Rao A.K. Chiu H.C. Wang D. Dutta-Roy A.K. Walsh P.N. Colman R.W. Blood. 1987; 70: 139-146Crossref PubMed Google Scholar, 44Kalafatis M. Xue J. Lawler C.M. Mann K.G. Biochemistry. 1994; 33: 6538-6545Crossref PubMed Scopus (38) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with specific implication of FVa residues 493–506 and 311–325 (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and FXa residues 263–274 (46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).It is of interest to elucidate direct anticoagulant modes of action of protein S, such as its interaction with FVa or FXa in the down-regulation of thrombin formation. The present identification of a binding site for FVa involving protein S residues 620–635 that does not bind FXa, protein C, or C4b-binding protein and is not dependent on the presence of phosholipids extends our understanding of the multiple anticoagulant mechanisms of protein S. Protein S is a vitamin K-dependent protein that can act as a cofactor for the anticoagulant functions of activated protein C (1DiScipio R.G. Davie E.W. Biochemistry. 1979; 18: 899-904Crossref PubMed Scopus (169) Google Scholar, 2Walker F.J. J. Biol. Chem. 1980; 255: 5521-5524Abstract Full Text PDF PubMed Google Scholar, 3Lundwall A. Dackowski W. Cohen E. Shaffer M. Mahr A. Dahlbäck B. Stenflo J.A. Wydro R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Crossref PubMed Scopus (149) Google Scholar). Heterozygous deficiency of protein S or protein C is associated with increased risks for both venous thrombosis (4Schwarz H.P. Fischer M. Hopmeier P. Batard M.A. Griffin J.H. Blood. 1984; 64: 1297-1300Crossref PubMed Google Scholar, 5Comp P.C. Nixon R.R. Cooper M.R. Esmon C.T. J. Clin. Invest. 1984; 74: 2082-2088Crossref PubMed Scopus (478) Google Scholar, 6Broekmans A.W. Bertina R.M. Reinalda-Poot J. Engesser L. Muller H.P. Leeuw J.A. Michiels J.J. Brommer E.J.P. Briët E. Thromb. Haemost. 1985; 53: 273-277Crossref PubMed Scopus (90) Google Scholar, 7Griffin J.H. Evatt B. Zimmerman T.S. Kleiss A.J. Wideman C. J. Clin. Invest. 1981; 68: 1370-1373Crossref PubMed Scopus (976) Google Scholar, 8Bertina R.M. Broekmans A.W. van der Linden I.K. Mertens K. Thromb. Haemost. 1982; 48: 1-5Crossref PubMed Scopus (177) Google Scholar) and ischemic stroke (9Wiesel M.-L. Charmantier J.-L. Freyssinet J.-M. Grunebaum L. Schuhler S. Cazenave J.-P. Thromb. Res. 1990; 58: 461-468Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 10Thommen D. Buhrfeind E. Felix R. Sulzer I. Furlan M. Lämmle B. Schweiz. Med. Wochenschr. 1989; 119: 493-499PubMed Google Scholar, 11Folsom A.R. Rosamond W.D. Shahar E. Cooper L.S. Nieto F.J. Rasmussen M.L. Wu K.K. Circulation. 1998; 98 (Abstr.): I-207Google Scholar). Homozygous deficiency of protein S, like that of protein C (12Branson H.E. Katz J. Marble R. Griffin J.H. Lancet. 1983; 2: 1165-1168Abstract PubMed Scopus (264) Google Scholar, 13Seligsohn U. Berger A. Abend M. Rubin L. Attias D. Zivelin A. Rapaport S.I. N. Engl. J. Med. 1984; 31: 559-562Crossref Scopus (348) Google Scholar), can lead to potentially fatal purpura fulminans in infancy unless aggressively treated (14Mahasandana C. Suvatte V. Marlar R.A. Manco-Johnson M.J. Jacobson L.J. Hathaway W.E. Lancet. 1990; 335: 61-62Abstract PubMed Scopus (0) Google Scholar). The physiological importance of protein S is underscored by the finding that infusions of protein S concentrates without activated protein C showed antithrombotic activity in a rabbit thrombosis model (15Schwarz H.P. Linnau Y. Pfeiler S. Molinari E. Thromb. Haemost. 1989; 62 (Abstr.): 25Google Scholar). Modes of antithrombotic action of protein S are, however, incompletely understood. Protein S can be anticoagulant even in the absence of activated protein C by inhibiting prothrombinase activity of the FVa-FXa complex in fluid phase and on phospholipid vesicles, endothelial cells, or platelets (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar). This inhibition presumably involves binding of protein S to Factor Va (FVa)1 and Factor Xa (FXa) as well as binding to the lipid membrane (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar, 20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar). Protein S binds FVa and FXa with apparent K d values of 33 and 18 nm, respectively (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar). In addition, protein S inhibits thrombin generation when subendothelial matrix is exposed to plasma under flow conditions (19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar). The relative anticoagulant potency of protein S has been correlated with its affinity for phospholipid vesicles, and protein S competes with prothrombinase components particularly at limiting phospholipid concentrations, e.g.0.2 μm (20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar). An assay has been developed for the activated protein C-independent activity of protein S in individual plasma samples (21van Wijnen M. van't Veer C. Meijers J.C.M. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). FV in combination with protein S enhances activated protein C anticoagulant activity (22Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar), and it was suggested that this complex phenomenon may involve binding of the sex hormone binding globulin region of protein S residues 270–635 to FV (23Nyberg P. Dahlbäck B. Garcia de Frutos P. FEBS Lett. 1998; 14: 28-32Crossref Scopus (41) Google Scholar). Studies presented here identify an essential binding site for FVa at the C terminus of protein S that contributes to the ability of protein S to inhibit prothrombinase activity. DISCUSSIONA novel mechanism of anticoagulant action of protein S involves its ability to function independently of activated protein C, via its binding to FVa, FXa, and/or phospholipids, thereby causing inhibition of prothrombinase activity (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar, 20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar, 21van Wijnen M. van't Veer C. Meijers J.C.M. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). This report explores the interaction of protein S with FVa and identifies an essential binding site for FVa on protein S involving the region of residues 621–635. Peptide PSP14 representing this region was a potent inhibitor of FVa binding to protein S and of FVa-dependent prothrombin activation and was anticoagulant in clotting assays. The inhibitory effect of peptide PSP14 was only observed in the presence of FVa, but the inhibition was not dependent on the presence or concentration of phospholipids. However, increasing FXa concentrations from 0.2 to 8 nm negated the inhibitory activity of PSP14, suggesting that PSP14 may compete with FXa for a binding site on FVa. This hypothesis was supported by a plot of 1/v versus[PSP14] at varying [FXa], which revealed the pattern for a competitive inhibitor with an apparent K i of ∼2 μm. Competition between FXa and protein S for binding to FVa has not been directly demonstrated but is consistent with the report that a FVa 15-mer peptide binds both protein S and FXa (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar) and with reports that protein S negates the ability of FXa to protect FVa from proteolytic inactivation by activated protein C (33Solymoss S. Tucker M.M. Tracy P.B. J. Biol. Chem. 1988; 263: 14884-14890Abstract Full Text PDF PubMed Google Scholar, 34Jane S.M. Hau L. Salem H.H. Blood Coag. Fibrinol. 1991; 2: 723-729Crossref PubMed Scopus (9) Google Scholar).The inhibitory potency of peptide PSP14 was enhanced by an internal disulfide bond introduced by the substitution of Cys for Ser635, because reduced and alkylated PSP14 and a peptide analog with Ser in position 635 were less potent than the cyclized peptide. Single amino acid substitutions in PSP14 analogs showed that Lys 630, 631, and 633 were each critical for prothrombinase inhibition. Although a short peptide with the linear sequence of residues 628–635 containing these Lys residues was not a potent inhibitor, cyclization of the peptide by addition of two Cys residues at the end of the peptide rendered it potently anticoagulant (Table II). Because the degrees of freedom that determine the relative populations of various conformations for a given peptide are greatly reduced in a cyclic peptide, it appears that some of the conformations of cyclized PSP14 and PSP629 may resemble those of native protein S.The anticoagulant and binding inhibitory effects of PSP14 were not due simply to its net positive charge per se, because a trilysine peptide, peptides with the same scrambled l-amino acid sequence (PSP14SCR) or with the same sequence ofd-amino acids and unrelated pentadecapeptides with four basic residues had little or no ability to inhibit binding of protein S to FVa or to inhibit prothrombinase or clotting assays.Other evidence for specificity of the inhibitory effects of peptide PSP14 was presented in several forms. Antibodies to PSP14 blocked protein S binding to FVa and neutralized the prothrombinase inhibitory activity of protein S. Neither PSP14 nor anti-PSP14 antibodies could block binding of biotin-FXa to protein S. Moreover, the ability of PSP14 to inhibit prothrombinase was entirely dependent on the presence of FVa. The fact that PSP14 was anticoagulant in protein C-depleted plasma is consistent with the concepts that protein S is anticoagulant in the absence of protein C and that the region of residues 621–635 is involved in protein S-FVa interactions.Residues 621–635 are not necessary for complexation of protein S with C4b-binding protein because peptide PSP14 did not inhibit binding of protein S to C4b-binding protein. Sites for interaction of protein S with C4b-binding protein have been reported in the steroid hormone binding globulin domain (35Chang G.T.G. Maas B.H.A. Ploos van Amstel H.K. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemost. 1994; 71: 461-467Crossref PubMed Scopus (29) Google Scholar) of protein S at residues 413–433 (29Fernández J.A. Heeb M.J. Griffin J.H. J. Biol. Chem. 1993; 268: 16788-16794Abstract Full Text PDF PubMed Google Scholar, 30Fernández J.A. Griffin J.H. Chang G.T.G. Stam J. Reitsma P.H. Bertina R.M. Bouma B.N. Blood Cells, Molecules, and Diseases. 1998; 24: 101-112Crossref PubMed Scopus (20) Google Scholar) and 447–460 (36He X. Shen L. Malmbourg A.-C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 37Linse S. Härdig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Peptides overlapping each of these regions in protein S did not inhibit prothrombinase activity (Table I), indicating that this experimental approach fails to implicate these sequences as essential for sites of interaction of protein S with FVa or FXa. The region in the FVa molecule with which residues 621–635 of protein S should interact remains a matter of speculation. A stretch of 14 amino acid residues in FVa (residues 493–506, GLLLICKSRSLDRR) potently inhibits prothrombinase activity and has been implicated in the binding of FVa to protein S, FXa, DEGR-FXa, and DIP-activated protein C but not to activated protein C or prothrombin (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar). It seems unlikely that this sequence in FVa interacts with residues 620–635 in protein S because of the likely electrostatic repulsion of the net positive charges of these sequences (+3 and +4, respectively). However, each peptide also contains a region of uncharged residues. It is likely that protein S and FVa have more than one site of molecular interaction. In the case of activated protein C interaction with FVa, three sites of molecular interaction have been suggested in the light and heavy chains of activated protein C, and two sites have been suggested in the light and heavy chains of FVa (24Mesters R.M. Houghten R.A. Griffin J.H. J. Biol. Chem. 1991; 266: 24514-24519Abstract Full Text PDF PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar, 39Mesters R.M. Heeb M.J. Griffin J.H. Protein Sci. 1993; 2: 1482-1489Crossref PubMed Scopus (26) Google Scholar, 40Mesters R.M. Heeb M.J. Griffin J.H. Biochemistry. 1993; 32: 12656-12663Crossref PubMed Scopus (32) Google Scholar, 41Walker F.J. Scandella D. Fay P.J. J. Biol. Chem. 1990; 265: 1484-1489Abstract Full Text PDF PubMed Google Scholar). For interactions between FXa and FVa, sites in both heavy and light chains of FVa are suggested (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar,42Tracy P.B. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2380-2384Crossref PubMed Scopus (66) Google Scholar, 43Annamalai A.E. Rao A.K. Chiu H.C. Wang D. Dutta-Roy A.K. Walsh P.N. Colman R.W. Blood. 1987; 70: 139-146Crossref PubMed Google Scholar, 44Kalafatis M. Xue J. Lawler C.M. Mann K.G. Biochemistry. 1994; 33: 6538-6545Crossref PubMed Scopus (38) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with specific implication of FVa residues 493–506 and 311–325 (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and FXa residues 263–274 (46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).It is of interest to elucidate direct anticoagulant modes of action of protein S, such as its interaction with FVa or FXa in the down-regulation of thrombin formation. The present identification of a binding site for FVa involving protein S residues 620–635 that does not bind FXa, protein C, or C4b-binding protein and is not dependent on the presence of phosholipids extends our understanding of the multiple anticoagulant mechanisms of protein S. A novel mechanism of anticoagulant action of protein S involves its ability to function independently of activated protein C, via its binding to FVa, FXa, and/or phospholipids, thereby causing inhibition of prothrombinase activity (16Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 17Heeb M.J. Rosing J. Bakker H.M. Fernández J.A. Tans G. Griffin J.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2728-2732Crossref PubMed Scopus (156) Google Scholar, 18Hackeng T.M. van't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 19van't Veer C. Hackeng T.M. Biesbroeck D. Sixma J.J. Bouma B.N. Blood. 1995; 85: 1815-1821Crossref PubMed Google Scholar, 20van Wijnen M. Stam J.G. van't Veer C. Meijers J.C.M. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1996; 76: 397-403Crossref PubMed Scopus (63) Google Scholar, 21van Wijnen M. van't Veer C. Meijers J.C.M. Bertina R.M. Bouma B.N. Thromb. Haemostas. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). This report explores the interaction of protein S with FVa and identifies an essential binding site for FVa on protein S involving the region of residues 621–635. Peptide PSP14 representing this region was a potent inhibitor of FVa binding to protein S and of FVa-dependent prothrombin activation and was anticoagulant in clotting assays. The inhibitory effect of peptide PSP14 was only observed in the presence of FVa, but the inhibition was not dependent on the presence or concentration of phospholipids. However, increasing FXa concentrations from 0.2 to 8 nm negated the inhibitory activity of PSP14, suggesting that PSP14 may compete with FXa for a binding site on FVa. This hypothesis was supported by a plot of 1/v versus[PSP14] at varying [FXa], which revealed the pattern for a competitive inhibitor with an apparent K i of ∼2 μm. Competition between FXa and protein S for binding to FVa has not been directly demonstrated but is consistent with the report that a FVa 15-mer peptide binds both protein S and FXa (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar) and with reports that protein S negates the ability of FXa to protect FVa from proteolytic inactivation by activated protein C (33Solymoss S. Tucker M.M. Tracy P.B. J. Biol. Chem. 1988; 263: 14884-14890Abstract Full Text PDF PubMed Google Scholar, 34Jane S.M. Hau L. Salem H.H. Blood Coag. Fibrinol. 1991; 2: 723-729Crossref PubMed Scopus (9) Google Scholar). The inhibitory potency of peptide PSP14 was enhanced by an internal disulfide bond introduced by the substitution of Cys for Ser635, because reduced and alkylated PSP14 and a peptide analog with Ser in position 635 were less potent than the cyclized peptide. Single amino acid substitutions in PSP14 analogs showed that Lys 630, 631, and 633 were each critical for prothrombinase inhibition. Although a short peptide with the linear sequence of residues 628–635 containing these Lys residues was not a potent inhibitor, cyclization of the peptide by addition of two Cys residues at the end of the peptide rendered it potently anticoagulant (Table II). Because the degrees of freedom that determine the relative populations of various conformations for a given peptide are greatly reduced in a cyclic peptide, it appears that some of the conformations of cyclized PSP14 and PSP629 may resemble those of native protein S. The anticoagulant and binding inhibitory effects of PSP14 were not due simply to its net positive charge per se, because a trilysine peptide, peptides with the same scrambled l-amino acid sequence (PSP14SCR) or with the same sequence ofd-amino acids and unrelated pentadecapeptides with four basic residues had little or no ability to inhibit binding of protein S to FVa or to inhibit prothrombinase or clotting assays. Other evidence for specificity of the inhibitory effects of peptide PSP14 was presented in several forms. Antibodies to PSP14 blocked protein S binding to FVa and neutralized the prothrombinase inhibitory activity of protein S. Neither PSP14 nor anti-PSP14 antibodies could block binding of biotin-FXa to protein S. Moreover, the ability of PSP14 to inhibit prothrombinase was entirely dependent on the presence of FVa. The fact that PSP14 was anticoagulant in protein C-depleted plasma is consistent with the concepts that protein S is anticoagulant in the absence of protein C and that the region of residues 621–635 is involved in protein S-FVa interactions. Residues 621–635 are not necessary for complexation of protein S with C4b-binding protein because peptide PSP14 did not inhibit binding of protein S to C4b-binding protein. Sites for interaction of protein S with C4b-binding protein have been reported in the steroid hormone binding globulin domain (35Chang G.T.G. Maas B.H.A. Ploos van Amstel H.K. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemost. 1994; 71: 461-467Crossref PubMed Scopus (29) Google Scholar) of protein S at residues 413–433 (29Fernández J.A. Heeb M.J. Griffin J.H. J. Biol. Chem. 1993; 268: 16788-16794Abstract Full Text PDF PubMed Google Scholar, 30Fernández J.A. Griffin J.H. Chang G.T.G. Stam J. Reitsma P.H. Bertina R.M. Bouma B.N. Blood Cells, Molecules, and Diseases. 1998; 24: 101-112Crossref PubMed Scopus (20) Google Scholar) and 447–460 (36He X. Shen L. Malmbourg A.-C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 37Linse S. Härdig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Peptides overlapping each of these regions in protein S did not inhibit prothrombinase activity (Table I), indicating that this experimental approach fails to implicate these sequences as essential for sites of interaction of protein S with FVa or FXa. The region in the FVa molecule with which residues 621–635 of protein S should interact remains a matter of speculation. A stretch of 14 amino acid residues in FVa (residues 493–506, GLLLICKSRSLDRR) potently inhibits prothrombinase activity and has been implicated in the binding of FVa to protein S, FXa, DEGR-FXa, and DIP-activated protein C but not to activated protein C or prothrombin (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 32Gale A.J. Heeb M.J. Griffin J.H. Thromb. Haemost. 1997; (Abstr.)PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar). It seems unlikely that this sequence in FVa interacts with residues 620–635 in protein S because of the likely electrostatic repulsion of the net positive charges of these sequences (+3 and +4, respectively). However, each peptide also contains a region of uncharged residues. It is likely that protein S and FVa have more than one site of molecular interaction. In the case of activated protein C interaction with FVa, three sites of molecular interaction have been suggested in the light and heavy chains of activated protein C, and two sites have been suggested in the light and heavy chains of FVa (24Mesters R.M. Houghten R.A. Griffin J.H. J. Biol. Chem. 1991; 266: 24514-24519Abstract Full Text PDF PubMed Google Scholar, 38Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Thromb. Haemostas. 1995; 73 (Abstr.): 1121Google Scholar, 39Mesters R.M. Heeb M.J. Griffin J.H. Protein Sci. 1993; 2: 1482-1489Crossref PubMed Scopus (26) Google Scholar, 40Mesters R.M. Heeb M.J. Griffin J.H. Biochemistry. 1993; 32: 12656-12663Crossref PubMed Scopus (32) Google Scholar, 41Walker F.J. Scandella D. Fay P.J. J. Biol. Chem. 1990; 265: 1484-1489Abstract Full Text PDF PubMed Google Scholar). For interactions between FXa and FVa, sites in both heavy and light chains of FVa are suggested (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar,42Tracy P.B. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2380-2384Crossref PubMed Scopus (66) Google Scholar, 43Annamalai A.E. Rao A.K. Chiu H.C. Wang D. Dutta-Roy A.K. Walsh P.N. Colman R.W. Blood. 1987; 70: 139-146Crossref PubMed Google Scholar, 44Kalafatis M. Xue J. Lawler C.M. Mann K.G. Biochemistry. 1994; 33: 6538-6545Crossref PubMed Scopus (38) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with specific implication of FVa residues 493–506 and 311–325 (31Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (48) Google Scholar, 45Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and FXa residues 263–274 (46Chattopadhyay A. James H.L. Fair D.S. J. Biol. Chem. 1992; 267: 12323-12329Abstract Full Text PDF PubMed Google Scholar, 47Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It is of interest to elucidate direct anticoagulant modes of action of protein S, such as its interaction with FVa or FXa in the down-regulation of thrombin formation. The present identification of a binding site for FVa involving protein S residues 620–635 that does not bind FXa, protein C, or C4b-binding protein and is not dependent on the presence of phosholipids extends our understanding of the multiple anticoagulant mechanisms of protein S. We are grateful for the excellent technical assistance of Yolanda Montejano, Benjamin Montoya, Ann Nicholson, and Cecille Dalanon Browne. We thank Dr. Harry Bakker for assistance in preparation of FV, Dr. José Fernández for performing binding tests with C4b-binding protein, Drs. Rolf Mesters and András Gruber for performing clotting tests with activated protein C, Dr. Subramanian Yegneswaran for purification of peptides, Drs. Tilman Hackeng and Stephen Kent for mass spectral analyses of peptides, and Dr. Bonno Bouma for helpful discussions. We thank Dr. Richard Houghten and James Winkle for the synthesis of peptides, and Dr. Zaverio Ruggeri, Jim Roberts, and Ben Gutierrez for production of antibodies.