The Gla Domain of Factor IXa Binds to Factor VIIIa in the Tenase Complex

Abstract

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the γ-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1–47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1–47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1–479BPA, FIX1–4725BPA, and FIX1–4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1–4725BPA and to a much lesser degree with FIX1–4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46. During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the γ-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1–47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1–47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1–479BPA, FIX1–4725BPA, and FIX1–4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1–4725BPA and to a much lesser degree with FIX1–4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46. Factor IX is composed of a single polypeptide comprising an N-terminal γ-carboxyglutamic acid (Gla 1The abbreviations used are: Gla, γ-carboxyglutamic acid domain; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; BPA, benzoylphenylalanine.)-rich domain, an aromatic amino acid stack domain, two epidermal growth factor domains, and a C-terminal serine protease domain. The Gla domain contains 12 γ-carboxyglutamic acid residues a post-translationally modified glutamic acid residue. γ-Carboxylation occurs in the liver where the vitamin K-dependent carboxylase converts glutamic acid to γ-carboxyglutamic acid in an enzymatic reaction that requires vitamin K (1Furie B. Bouchard B.A. Furie B.C. Blood. 1999; 93: 1798-1808Crossref PubMed Google Scholar). In the presence of Ca2+, the Gla domain folds to become a membrane-binding structure (2Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 3Furie B. Furie B.C. N. Engl. J. Med. 1992; 326: 800-806Crossref PubMed Scopus (459) Google Scholar). In the absence of metal ions the Gla domain is largely unfolded and does not bind to membranes. Mg2+ ions, in the absence of Ca2+, support folding of residues 12–47 of factor IX but not folding of the membrane-interactive omega loop included in residues 1–11 (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), whereas Ca2+ ions support folding of the membrane-binding omega loop and are necessary for function of factor IXa (5Freedman S.J. Furie B.C. Furie B. Baleja J.D. Biochemistry. 1995; 34: 12126-12137Crossref PubMed Scopus (95) Google Scholar). The metal ion-specific conformational changes have provided the basis for developing antibodies that recognize the Ca2+-dependent 11-amino acid membrane binding region (6Liebman H.A. Furie B.C. Furie B. J. Biol. Chem. 1987; 262: 7605-7612Abstract Full Text PDF PubMed Google Scholar). Factor VIIIa is a cofactor for the factor IXa-mediated cleavage of factor X to factor Xa. Factor VIII is synthesized as a single polypeptide chain composed of three A domains (A1, A2, and A3), one B domain, and two C domains (C1 and C2) in the sequence, A1-A2-B-A3-C1-C2 (7Lenting P.J. van Mourik J.A. Mertens K. Blood. 1998; 92: 3983-3996Crossref PubMed Google Scholar). The A domains are homologous to ceruloplasmin, and the C domains are homologous to discoidin I, a phospholipid binding lectin (8Simpson D.L. Rosen S.D. Barondes S.H. Biochemistry. 1974; 13: 3487-3493Crossref PubMed Scopus (104) Google Scholar), and to a murine milk fat globule membrane protein (9Stubbs J.D. Lekutis C. Singer K.L. Bui A. Yuzuki D. Srinivasan U. Parry G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8417-8421Crossref PubMed Scopus (265) Google Scholar). The B domain has no known homology to any other protein. Factor VIII is variably cleaved, prior to its secretion, between the B and A3 domains to yield a heterodimeric protein composed of a variably sized heavy chain (A1-A2-B) and a light chain (A3-C1-C2). Thrombin activates factor VIII to factor VIIIa via several cleavages within both the heavy and light chains to yield a heterotrimeric protein (A1, A2, A3-C1-C2) that is non-covalently associated. The C2 domain of factor VIII/VIIIa contains the major membrane binding motif (10Arai M. Scandella D. Hoyer L.W. J. Clin. Invest. 1989; 83: 1978-1984Crossref PubMed Scopus (155) Google Scholar, 11Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Blood. 1990; 75: 1999-2004Crossref PubMed Google Scholar). Properties of the membrane-binding interaction include high affinity (12Gilbert G.E. Furie B.C. Furie B. J. Biol. Chem. 1990; 265: 815-822Abstract Full Text PDF PubMed Google Scholar), high specificity for membrane binding sites that include phosphatidylserine (13Gilbert G.E. Drinkwater D. Barter S. Clouse S.B. J. Biol. Chem. 1992; 267: 15861-15868Abstract Full Text PDF PubMed Google Scholar), and stereoselective interaction with phosphatidyl-l-serine (14Gilbert G.E. Drinkwater D. Biochemistry. 1993; 32: 9577-9585Crossref PubMed Scopus (88) Google Scholar). Factor IX is proteolytically activated to factor IXa, a serine protease. Factor VIII is activated to its cofactor form, factor VIIIa, via a proteolytic mechanism. Factor IXa and factor VIIIa assemble on phosphatidyl-l-serine-containing phospholipid membranes in the presence of Ca2+ to form an enzymatic complex known as the tenase complex (2Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 3Furie B. Furie B.C. N. Engl. J. Med. 1992; 326: 800-806Crossref PubMed Scopus (459) Google Scholar). The tenase complex converts factor X to factor Xa, the enzyme that converts prothrombin to thrombin leading to the conversion of fibrinogen to fibrin and the formation of a fibrin clot. Although much is known about the structures of both factor IXa and factor VIIIa from x-ray crystallography and NMR spectroscopy (5Freedman S.J. Furie B.C. Furie B. Baleja J.D. Biochemistry. 1995; 34: 12126-12137Crossref PubMed Scopus (95) Google Scholar, 15Brandstetter H. Bauer M. Huber R. Lollar P. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9796-9800Crossref PubMed Scopus (263) Google Scholar, 16Freedman S.J. Furie B.C. Furie B. Baleja J.D. J. Biol. Chem. 1995; 270: 7980-7987Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Nature. 1999; 402: 439-442Crossref PubMed Scopus (287) Google Scholar), details about the interaction of factor IXa with factor VIIIa have been limited to the analysis of naturally occurring mutations in hemophilic patients or site-specific mutagenesis studies (18Bajaj S.P. Thromb. Haemost. 1999; 82: 218-225Crossref PubMed Scopus (22) Google Scholar, 19Celie P.H. Van Stempvoort G. Fribourg C. Schurgers L.J. Lenting P.J. Mertens K. J. Biol. Chem. 2002; 277: 20214-20220Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20Lefkowitz J.B. Nuss R. Haver T. Jacobson L. Thompson A.R. Manco-Johnson M. Thromb Haemost. 2001; 86: 862-870Crossref PubMed Scopus (12) Google Scholar, 21Persson K.E. Villoutreix B.O. Thamlitz A.M. Knobe K.E. Stenflo J. J. Biol. Chem. 2002; 277: 35616-35624Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Analyses of factor IXa-factor VIIIa binding have revealed domain interactions between the serine protease domain of factor IXa and the A2 domain of factor VIIIa as well as the first epidermal growth factor domain of factor IXa and the A3 domain of factor VIIIa (22Lenting P.J. Van de Loo J.-W.H.P. Donath M.-J.S.H. van Moorik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Furthermore, a naturally occurring mutation of the Gla domain of factor IX leads to loss of factor VIIIa-mediated tenase activity (23Larson P.J. Stanfield-Oakley S.A. VanDusen W.J. Kasper C.K. Smith K.J. Monroe D.M. High K.A. J. Biol. Chem. 1996; 271: 3869-3876Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In this report, we use a synthetic analog of the factor IX Gla domain, FIX1–47 (Fig. 1), which we modify by replacing a specific hydrophobic amino acid with the photoactivatable amino acid, benzoylphenylalanine. With these Gla domain peptides, we provide direct evidence using a cross-linking strategy for the binding of the factor IX Gla domain to the light chain of factor VIIIa. Reagents—Factor IXa and factor X were purchased from Hematologic Technologies Inc. Recombinant human factor VIII formulated in sucrose (Kogenate FS) was received as a gift from Bayer Canada and activated to factor VIIIa as described previously (24Curtis J.E. Helgerson S.L. Parker E.T. Lollar P. J. Biol. Chem. 1994; 269: 6246-6251Abstract Full Text PDF PubMed Google Scholar). Bovine brain phosphatidylserine (PS), egg yolk phosphatidylcholine (PC), and dansylated phosphatidylethanolamine (PE) were obtained from Avanti Polar Lipids. Chromogenic substrate S-2765 was purchased from Diapharma Group Inc. ESH8 antibodies were purchased from American Diagnostica. Antibodies to the Gla domain of factor IXa were prepared as previously described (6Liebman H.A. Furie B.C. Furie B. J. Biol. Chem. 1987; 262: 7605-7612Abstract Full Text PDF PubMed Google Scholar). An antibody to γ-carboxyglutamic acid was a gift from Dr. Johan Stenflo (25Brown M.A. Stenberg L.M. Persson U. Stenflo J. J. Biol. Chem. 2000; 275: 19795-19802Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The FIX1–47BPA analogues were synthesized as previously described (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Enzymatic Assays—The activation of factor X by factor IXa was measured with a two-step amidolytic substrate assay. For inhibition experiments with the FIX1–47BPA peptides or with anti-FIX CaCl2-specific antibodies, factor IXa (20 nm) was incubated with 500 nm factor X, 4 nm factor VIIIa, and the specified concentrations of the FIX1–47 peptides or anti-FIX CaCl2-specific antibodies in 150 mm NaCl, 20 mm HEPES, pH 7.4, 5 mm CaCl2, and 0.1% bovine serum albumin for 60 min at 25 °C. The reaction was then stopped with 150 mm NaCl, 20 mm HEPES, pH 7.4, 5 mm EDTA. The amount of factor Xa generated was determined immediately using the chromogenic substrate S-2765 (0.3125 mg/ml) using a Molecular Devices enzyme-linked immunosorbent assay plate reader. Experiments conducted without factor VIIIa were incubated for 120 min. Measurements were carried out in duplicate, and IC50 values for FIX1–47BPA peptide inhibition of factor IXa activity in the presence of factor VIIIa were determined by non-linear least-square fitting of the values of factor Xa generation versus the logarithm of FIX1–47 peptide concentration using SigmaPlot 8.0 for Windows (SPSS). Preparation of Phospholipid Vesicles—Small unilamellar phospholipid vesicles (PC:PS, 75:25) were prepared by the method of Barenholz (26Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson R.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (753) Google Scholar). Phospholipids in chloroform were dried at 45 °C under N2, washed three times with methylene in 150 mm NaCl, mm pH 7.4, and in a the The of phospholipid vesicles was first at for min and then at for min in a using a The the unilamellar phospholipid Phospholipid concentrations were determined by analysis H. Chem. Scopus Google Scholar). fluorescence experiments and fluorescence quenching experiments were as previously described M. S.J. Furie B.C. Furie B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). were as described previously M.D. Jacobs M. Furie B. Furie B.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). cross-linking of FIX1–47 peptides to factor VIIIa was through of for specific hydrophobic residues in of the three peptides FIX1–4725BPA, or at a concentration of was with factor VIIIa nm) in mm pH 7.4, mm NaCl, and 5 mm The were with a for 20 min at a of protein-protein binding was by the of and and at °C for 5 min. The were on a and then to a The membrane was with a antibody to γ-carboxyglutamic acid, by the of a The were with an to the The cross-linking was carried out for peptide in the presence and absence factor VIIIa, and In cross-linking experiments were carried out in the presence of concentrations of factor experiments were carried out with a antibody to the C2 domain of factor VIIIa. and the were with to and by as described for the cross-linking The tenase complex, on phospholipid membranes, is composed of the factor IXa, and the factor VIIIa. To test the that the factor IXa Gla domain bind to factor VIIIa, inhibition of factor activation of factor X was in the presence or absence of factor VIIIa using an antibody to the of factor IXa The by this antibody is residues 1–11 within the Gla domain of factor antibodies inhibit factor IX binding to phospholipid membranes (6Liebman H.A. Furie B.C. Furie B. J. Biol. Chem. 1987; 262: 7605-7612Abstract Full Text PDF PubMed Google Scholar), phospholipid membranes were from this assay In the absence of phospholipid membranes, factor IXa the conversion of factor X to factor Xa at a antibodies inhibited the generation of factor Xa by factor IXa, factor VIIIa was (Fig. In the absence of factor VIIIa, there was no inhibition of the conversion of factor X to factor Xa by factor IXa (Fig. These results are with inhibition of the interaction of the Gla domain of factor IXa with factor VIIIa. A peptide comprising the Gla and aromatic amino acid stack domains of factor IXa, was used to study factor IXa Gla VIIIa FIX1–47 has been previously demonstrated to a for the of the factor IXa Gla domain M. S.J. Furie B.C. Furie B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). To demonstrate direct binding between the Gla domain of factor IXa and factor VIIIa, analogs of FIX1–47 benzoylphenylalanine were with structures within a at nm G. Biochemistry. 1994; PubMed Scopus Google Scholar). has been previously used to the residues that a phospholipid binding site in factor IXa (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). analogs were the of the in the Gla domain, and amino with (Fig. and Val-46 were replaced, respectively, in two peptides and and have been previously (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). A peptide was prepared with by three peptides bind to FIX1–47 M. S.J. Furie B.C. Furie B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), suggesting that the does not tertiary structure (Fig. fluorescence quenching at and for FIX1–479BPA, FIX1–4725BPA, and respectively, to with FIX1–47 M. S.J. Furie B.C. Furie B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), that the calcium-induced fluorescence FIX1–479BPA, FIX1–4725BPA, and bind to phospholipid membranes with binding and the binding is with as demonstrated previously (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The K for the binding of FIX1–4725BPA to phospholipid membranes was (Fig. to the binding of and FIX1–47 for phospholipid membranes and within of the (4Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The of the FIX1–47 peptides to inhibit the conversion of factor X to factor Xa by factor IXa in the presence and absence of factor VIIIa was determined in the absence of phospholipid membranes. A in inhibition of factor activation of factor X by FIX1–4725BPA in the presence (Fig. but not in the absence of factor VIIIa (Fig. These results suggest that FIX1–4725BPA bind to a specific site in factor VIIIa, factor VIIIa from binding to factor IXa and as a cofactor in the activation of factor X to factor Xa. The IC50 of this inhibition was The IC50 of the other peptides were as in Although activity was observed in the presence of concentrations of the FIX1–47 as in the in on the basis for this is of the factor VIIIa the assay was not in the absence of factor VIIIa (Fig. This the of this peptide was to tenase complex formation by the binding of factor VIIIa to factor by the peptides of the factor IXa-factor VIIIa complex in the absence of phospholipid in a To demonstrate direct binding between the factor IXa Gla domain and factor VIIIa and to the region on FIX1–47 that is or in the contact site that binds to factor VIIIa, cross-linking experiments were to which FIX1–47 bind to factor VIIIa. The photoactivatable peptides, FIX1–479BPA, FIX1–4725BPA, or were incubated with factor VIIIa in the presence of the protein were by analysis using an acid in FIX1–4725BPA was to factor VIIIa, whereas reaction FIX1–47 no is a in the cross-linking suggesting cross-linking is to a Furthermore, cross-linking was observed the reaction included FIX1–47 or peptides, ions, and factor VIIIa and the reaction was at The complex of FIX1–47 to factor VIIIa on in a that of the light chain of factor VIIIa, suggesting a complex between FIX1–47 and the light chain of factor VIIIa. To this we the with antibody to the C2 domain of factor VIIIa. The was on an and with an This in the that the region on factor VIIIa to which FIX1–4725BPA binds is the light chain of the complex with a factor VIII light chain Immunoprecipitation was with a antibody to the C2 domain of factor VIIIa. FIX1–4725BPA cross-linking to factor VIIIa in the presence of phospholipid membranes, the were incubated with ESH8 or a antibody to protein The to the was then to and with an antibody to γ-carboxyglutamic To the specificity of the cross-linking of FIX1–4725BPA to the light chain of factor VIIIa, factor IXa was at concentrations to inhibit in the of of factor IXa to inhibition of the cross-linking of FIX1–4725BPA to factor VIIIa. In the we demonstrate that the Gla domain of factor IXa with the light chain of factor VIIIa. A of this complex, on phospholipid membranes, is in contact sites on factor IXa have previously been in this fluorescence demonstrated a contact site between the A2 domain of factor VIIIa and the serine protease domain of factor This interaction was by an affinity of nm L.M. P.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. T. L.M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, P.J. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). contact site of affinity nm) has been between the A3 domain of factor VIIIa and the first epidermal growth factor domain of factor IXa P.J. Donath M.-J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In the we that the Gla domain of factor IXa and light chain of factor VIIIa, both bind to factor is homologous with factor IXa, the contact sites through which factor activates factor provide to the contact sites through which factor VIIIa activates factor using both fluorescence spectroscopy and demonstrate three sites between the factor and its A site of interaction between the Gla domain of factor and factor is one site of interaction to that in the This site is to of affinity in to the other sites of The that we for the factor IXa Gla domain complex are with that M. Persson M. U. Thromb. 2001; 86: Scholar). of the residues in factor that contact factor that these sites become from binding factor R. M. M. M. Persson U. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of evidence suggest that the contact between the light chain of factor VIIIa and the factor IXa Gla domain is a naturally occurring mutation in the Gla domain of factor IXa leading to B function in the presence of factor VIIIa. This with a of 12 by has a (23Larson P.J. Stanfield-Oakley S.A. VanDusen W.J. Kasper C.K. Smith K.J. Monroe D.M. High K.A. J. Biol. Chem. 1996; 271: 3869-3876Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). factor IXa-factor VIIIa interactions inhibited by a peptide comprising the Gla domain of factor IXa, suggest an interaction between the C-terminal of the Gla domain of factor IXa and the light chain of factor VIIIa. In the Gla domain is at the from the omega loop and phospholipid binding site and not from 46. The chains of and Val-46 from the of the Gla domain at an from one is that the chains of both residues with the light chain of factor VIIIa, from the cross-linking Val-46 (5Freedman S.J. Furie B.C. Furie B. Baleja J.D. Biochemistry. 1995; 34: 12126-12137Crossref PubMed Scopus (95) Google Scholar). The of to to factor VIIIa that the of the omega Phe-9, does not contact factor in the presence of membranes is in the phospholipid B. and B. C. In this we a region of the factor IXa Gla domain and Val-46 as a contact site with factor VIIIa. These contact sites are to the C-terminal of the Gla domain, which is to two homologous serine factor and protein C. The structure of the factor factor complex a direct interaction between the C-terminal region of the Gla domain of factor and factor A. C. A. Y. D. Nature. 1996; PubMed Scopus Google Scholar). In the of the protein complex, of the Gla domain of protein C with that of prothrombin protein C activity of protein its This protein to residues of the Gla domain of protein C M.D. L. T. S. A.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In studies demonstrate a specific interaction between the Gla domain of factor IXa and the light chain of factor VIIIa. that both the Gla domain of factor IXa and the C2 domain of factor VIIIa are membrane-binding we these to membranes and to the of this contact site activation of the factor IXa complex on phospholipid membranes to Johan and for gift of the acid antibody and Dr. for of the