Role of Factor VIII C2 Domain in Factor VIII Binding to Factor Xa

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa. Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa. factor VIII factor Xa phospholipid von Willebrand factor heavy chain of FVIII light chain of FVIII activated FVIII factor X enzyme-linked immunosorbent assay phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis Tris-buffered saline bovine serum albumin Bethesda unit(s) determined by inhibitor assay Factor VIII (FVIII)1 is a glycoprotein cofactor that accelerates the generation of factor Xa (FXa) by factor IXa in the presence of Ca2+ and negatively charged phospholipid (PL) expressed on a membrane surface (1van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar). Quantitative and qualitative deficiencies of FVIII result in the congenital bleeding disorder, hemophilia A. FVIII is noncovalently bound to von Willebrand factor (vWF) in plasma. vWF regulates the synthesis, the cofactor activity, and the transport of FVIII to the site of vascular injury (2Hoyer L.W. Blood. 1981; 58: 1-13Crossref PubMed Google Scholar, 3Weiss H.J. Sussman I.I. Hoyer L.W. J. Clin. Invest. 1977; 60: 390-404Crossref PubMed Scopus (330) Google Scholar, 4Kaufman R.J. Wasley L.C. Davies M.V. Wise R.J. Israel D.I. Dorner A.J. Mol. Cell. Biol. 1989; 9: 1233-1242Crossref PubMed Scopus (145) Google Scholar). Mature FVIII is synthesized as a single chain polypeptide consisting of 2332 amino acid residues (5Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.G.D. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (522) Google Scholar, 6Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster W.B. Coe M.L. Knutson G.J. Fass D.N. Hewick R.M. Nature. 1984; 312: 342-347Crossref PubMed Scopus (663) Google Scholar). Based on internal homologies of the amino acid sequence, FVIII has three types of domains arranged in the order of A1-A2-B-A3-C1-C2 (7Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar). FVIII circulates in the plasma as a heterodimer of a heavy chain (HCh) consisting of the A1, A2, and heterogeneous fragments of partially proteolyzed B domains, together with a light chain (LCh) consisting of A3, C1, and C2 domains (6Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster W.B. Coe M.L. Knutson G.J. Fass D.N. Hewick R.M. Nature. 1984; 312: 342-347Crossref PubMed Scopus (663) Google Scholar, 7Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar). Several findings have indicated that the structure and function of the C2 domain is important for the expression and regulation of FVIII. The C2 domain contains a PL binding site (8Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Blood. 1990; 75: 1999-2004Crossref PubMed Google Scholar, 9Scandella D. Gilbert G.E. Shima M. Nakai H. Eagleson C. Felch M. Prescott R. Rajalakshmi K.J. Hoyer L.W. Saenko E. Blood. 1995; 86: 1811-1819Crossref PubMed Google Scholar) and a vWF binding site (10Shima M. Nakai H. Scandella D. Tanaka I. Sawamoto Y. Kamisue S. Morichika S. Murakami T. Yoshioka A. Br. J. Haematol. 1995; 91: 714-721Crossref PubMed Scopus (62) Google Scholar, 11Shima M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar, 12Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar) together with a common epitope for FVIII inhibitor alloantibodies, which develop in patients with severe hemophilia A (13Prescott R. Nakai H. Saenko E.L. Scharrer I. Nilsson I.M. Humphries J.E. Hurst D. Bray G. Scandella D. Blood. 1997; 89: 3663-3671Crossref PubMed Google Scholar). Furthermore, residues Val2248–Gly2285within the C2 domain contain the epitope for a monoclonal antibody ESH8, which reduces the rate of FVIII/vWF dissociation after thrombin activation of FVIII (14Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). FVIII is transformed into an active form (FVIIIa) by limited proteolysis by two serine proteases, thrombin and FXa (15Lollar P. Knutson G.J. Fass D.N. Biochemistry. 1985; 24: 8056-8064Crossref PubMed Scopus (77) Google Scholar, 16Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar). Cleavage at Arg372 and Arg740 of the 90-kDa HCh fragment containing the A1 and A2 domains and Cleavage of the LCh fragment at peptides the domain D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar) and a 72-kDa Cleavage by FXa at a LCh fragment J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). at Arg372 and is for cofactor M. J. Yoshioka A. Fukui H. Fulcher C.A. Blood. 1989; PubMed Google Scholar, S. Shima M. T. Tanaka I. Nakai H. Morichika S. A. Yoshioka A. Br. J. Haematol. 1994; 86: PubMed Scopus Google Scholar, E.G.D. R. J. Gitschier J. M. S. J.M. Hoyer L.W. Yoshioka A. J.M. F. D.N. 1994; PubMed Scopus Google Scholar). FVIII activation is FVIII activation in P. J. PubMed Scopus Google Scholar, J. P. Biochemistry. 1997; PubMed Scopus Google Scholar). The of by FXa is and is more than that by thrombin J. P. Biochemistry. 1997; PubMed Scopus Google Scholar). Furthermore, the presence of vWF the activation of FVIII by FXa not by thrombin R.J. J.J. J. 1990; PubMed Scopus Google Scholar). and J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) a factor X binding site the A1 The role of FVIII activation in is the demonstrated that an anti-C2 monoclonal containing an epitope residues inhibited FXa cleavage of FVIII in the absence of Furthermore, the C2 domain with FVIII for FXa cleavage of the LCh and bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site that the C2 domain contains a FXa binding FVIII using monoclonal antibody the FVIII the monoclonal antibody with and as M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. J. Google Scholar). The of the FVIII immunosorbent assay demonstrated that the FVIII of vWF M. Yoshioka A. Tanaka I. S. Y. Fukui H. J. 1986; Scholar). LCh and HCh fragments of together with A1, A2, and 72-kDa LCh were plasma FVIII as P. Fass D.N. 1993; PubMed Scopus Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M.L. Saenko E.L. P. Scandella D. Blood. 1997; PubMed Google Scholar). C2 domain were and as E.L. Scandella D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). vWF a FVIII/vWF using gel on a of as (10Shima M. Nakai H. Scandella D. Tanaka I. Sawamoto Y. Kamisue S. Morichika S. Murakami T. Yoshioka A. Br. J. Haematol. 1995; 91: 714-721Crossref PubMed Scopus (62) Google Scholar). FVIII using with immobilized monoclonal that FVIII not in the vWF M. Yoshioka A. Tanaka I. S. Y. Fukui H. J. 1986; Scholar). FXa were PL were as a in as G.E. B. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). FVIII in a clotting assay. using the Bethesda assay J. D. J. P.H. J. Thromb. Google Scholar). Bethesda as the of antibody that inhibited of the FVIII in of plasma after a at The of monoclonal antibody expressed as of of monoclonal epitopes of FVIII were monoclonal the FVIII C2 ESH8 and amino acid residues 2248–2285 and M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar, 12Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar). FVIII binding to vWF and PL M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar). ESH8 does not FVIII binding to vWF or PL E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar). FVIII is to the of of vWF FVIII thrombin activation (14Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). antibody residues of the vWF has on PL binding M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. J. Google Scholar, M. Yoshioka A. M. Nakai H. Fukui H. Br. J. Haematol. PubMed Scopus Google Scholar). antibody the of the A1 by C. A. Fulcher P.A. Fulcher C.A. Houghten R.A. S. Zimmerman T.S. J. Clin. Invest. PubMed Scopus Google Scholar). antibody recognizes the A2 and have on FVIII binding to vWF or M. T. T. E. D. M. S. I. and A. of the monoclonal in The of monoclonal antibody by affinity fragments of were using immobilized and of monoclonal domains bound in on FVIII and antibody or does not FVIII binding to vWF or and bind to the of the and A1 domains, and antibody or does not FVIII binding to vWF or in a and bind to the of the and A1 domains, respectively. of FVIII by with of using for as PubMed Scopus Google Scholar). by on a The of The determined in a clotting assay to that of FVIII. of FVIII were at for to a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site as for the of Biochemistry. 1995; PubMed Scopus Google Scholar). FXa with phenylmethylsulfonyl fluoride the phenylmethylsulfonyl residues of the FXa to dehydroalanine the with and for at and the to containing anhydro-FXa by The anhydro-FXa demonstrated activity, and to by SDS-polyacrylamide gel electrophoresis to that of the FXa not consisting of overlapping amino and corresponding to the epitope of monoclonal antibody ESH8 (residues were synthesized by the of peptide as R.A. S. A. 1985; PubMed Scopus Google Scholar). were and by FVIII in containing bovine serum albumin and together with FXa PL and at were the and FXa by in in at for using a clotting assay. the of monoclonal on FVIII activation by antibody with FVIII to FXa activation and for at The of antibody to indicated that the presence of FXa and in the did not the FVIII the coagulation assay. in together with FXa and in the presence or absence of PL The at for in the presence of PL and for in the absence of were and FXa by an of and the to for on Nature. PubMed Scopus Google by of the the of on FVIII cleavage by an of antibody with for at to with as the cleavage of FXa in the presence of with vWF for at to the of FXa in the absence of were at with of monoclonal antibody ESH8, or in of three with containing the were for at by the of containing FVIII in containing to and for at FXa and were at the were and FXa by the of FVIII by with which recognizes the of by the of in as a the at indicated that the presence of did not Furthermore, that which to bound did not inhibit FXa The rate of FVIII LCh cleavage as at The in the absence of FVIII as competitive using FVIII fragments A1, A2, or recombinant C2 of as a of FVIII fragment with FXa to FXa and for of bound FVIII. of anhydro-FXa in were immobilized of a with FVIII fragments in containing and were and for at LCh or HCh fragment by or respectively. competitive using FVIII to the immobilized anhydro-FXa. in a FVIII fragments were with to to the anhydro-FXa. The as in the absence of were as The of FVIII and anhydro-FXa were determined by surface using a bound to an activated surface by to the D.J. M. P. I. 1993; Scholar, R. 1994; PubMed Scopus Google Scholar). of were in containing at a rate of for a after to of the surface by for The values of association rate and dissociation rate d) were determined by analysis as D.J. M. P. I. 1993; Scholar, R. 1994; PubMed Scopus Google Scholar) using the by The values of dissociation (K d) were overlapping peptide and with recombinant C2 domain or 72-kDa LCh fragment to to the immobilized anhydro-FXa. FVIII using indicated that of the synthetic peptides binding of FVIII fragments to The as at in the absence of FVIII as after of FVIII with FXa an in FVIII by and the of order to the of monoclonal FVIII activation in the presence of antibody in the presence of antibody ESH8 the FVIII C2 domain the activation at a of and the the anti-C2 monoclonal to FVIII activation by FXa with the in the absence of of the monoclonal antibody antibody and antibody inhibited activation of FVIII at of or that the of the anti-C2 monoclonal antibody ESH8 on FVIII activation by FXa by a in the that or by a direct of the proteolytic cleavage of FVIII by FXa. between FVIII together with FXa for at in the absence or presence of FVIII and the cleavage by FVIII cleavage by FXa in the absence of in the of the fragments of the HCh into and fragments and proteolysis of the fragment of the LCh into and fragments the fragment and and fragments as as at were in the that the fragment proteolyzed by FXa. ESH8 the cleavage of the LCh fragment and the of the and fragments results that ESH8 proteolytic cleavage at and in the LCh and partially inhibited Arg372 in the did not the cleavage of the LCh to the 72-kDa LCh fragment more than in the fragment and the 90-kDa fragment not in the presence of findings that the antibody proteolysis of the HCh as as the of cleavage at by not and did not with FXa cleavage of FVIII The C2 domain contains a PL binding and anti-C2 to inhibit FVIII binding to PL D. Gilbert G.E. Shima M. Nakai H. Eagleson C. Felch M. Prescott R. Rajalakshmi K.J. Hoyer L.W. Saenko E. Blood. 1995; 86: 1811-1819Crossref PubMed Google Scholar). Furthermore, FVIII cleavage by FXa at a rate in the presence of PL than in of FVIII binding to PL results in of FVIII cleavage by FXa. the of monoclonal in the absence of ESH8 the cleavage of the LCh fragment and the of the and fragments the 90-kDa HCh fragment indicated that the of ESH8 not to the presence of the cleavages by FXa in the presence of in the were to in the presence of PL and to more in the presence of the antibody than in absence and did not inhibit FVIII cleavage in the absence of PL of findings indicated that the of ESH8 on activation of FVIII to a in of FVIII LCh proteolysis at and have that the monoclonal antibody the dissociation of FVIII the M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google ESH8 dissociation of FVIII and vWF (14Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). the results that ESH8 and on FXa activation and cleavage of the C2 the C2 domain is in FVIII binding to the of two C2 monoclonal on FVIII cleavage by FXa in the presence of vWF by vWF the cleavage of LCh and the cleavage of the HCh findings indicated that the vWF of the FVIII the proteolytic of FXa. ESH8 to FVIII in the presence of vWF to the of the of vWF and as in the absence of cleavage of the LCh and proteolysis of the HCh to the of the of vWF and the LCh and HCh were proteolyzed to fragments results were in with findings that FVIII vWF by M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar) and to proteolysis by FXa. order to the of on FVIII cleavage by an in the absence of for to the of does not bind to the and LCh and does not with FXa binding the LCh is at or The in of with bound FVIII a of cleavage of the cleavage of LCh in the presence of ESH8 for after the of FXa in the presence of and cleavages of the LCh at were at proteolysis of LCh by FXa in the presence of to more than in the presence of or the results of the anti-C2 antibody ESH8 inhibited proteolysis and the that the C2 domain is an domain for the association between FVIII and FXa. FVIII fragments A1, A2, or C2 were for to for the proteolysis of FVIII LCh by FXa in the The 72-kDa LCh fragment inhibited FXa cleavage of FVIII LCh by Similarly, the recombinant C2 domain inhibited cleavage by competitive using the A1 and A2 domains findings a direct role for the C2 domain in FVIII and FXa the between the C2 domain and an to the direct binding of FXa to FVIII the cleavage of FXa which proteolytic using or not to inhibit FXa for LCh or respectively. showed that FVIII bound to immobilized anhydro-FXa in a Similarly, the 80- and 72-kDa LCh fragments demonstrated a binding and to bind more than the FVIII the C2 domain bound to anhydro-FXa in a in the binding that of the LCh The HCh fragment and the A2 domain showed or binding to anhydro-FXa binding of FXa to FVIII in a competitive assay using FVIII fragments and The 80- and 72-kDa LCh fragments inhibited anhydro-FXa binding of by and the C2 domain inhibited binding by The did not inhibit binding of FVIII to anhydro-FXa findings that the C2 domain is with the between FVIII and FXa. The and d) for binding of FVIII to anhydro-FXa were by surface analysis and in for FVIII values for the 80- and 72-kDa LCh fragments and nm, were than that of the FVIII. The for the C2 domain 560 The HCh did not with for binding of FVIII fragments to immobilized not not in a the anti-C2 monoclonal ESH8, inhibited cleavage of FVIII by FXa and the recombinant C2 domain bound to anhydro-FXa, on the epitope structure of ESH8 to the FXa binding of the synthetic designated EP-2 (residues inhibited binding of the recombinant C2 domain to anhydro-FXa. The of the synthetic peptide EP-2 inhibited binding of the 72-kDa LCh fragment to anhydro-FXa Similarly, the synthetic peptide (residues inhibited binding of the C2 domain and the 72-kDa LCh to anhydro-FXa to a and and (residues partially inhibited binding to (residues (residues and (residues demonstrated or A peptide containing the as EP-2 in a did not with the binding of FVIII fragments to anhydro-FXa binding findings indicated that the FXa binding site is residues of the C2 domain of of FVIII fragment binding to anhydro-FXa by synthetic overlapping synthetic peptide and with a of the C2 domain or 72-kDa LCh fragment to with the immobilized anhydro-FXa. FVIII fragment by The findings that the of the C2 domain in the association between FVIII and FXa. the anti-C2 monoclonal ESH8, inhibited cleavage of FVIII by that the antibody inhibited FXa binding to the C2 domain or that antibody binding a that FXa the C2 domain as as the 72-kDa LCh fragment of FVIII inhibited FXa cleavage of the FVIII indicated that the C2 domain is for binding of FVIII to FXa than for cleavage of FVIII by FXa. the C2 domain bound to a catalytically inactive anhydro-FXa, more that the C2 domain contains the FXa binding and synthetic corresponding to the epitope of ESH8, inhibited the binding of the C2 domain and the 72-kDa LCh fragment of FVIII to anhydro-FXa. amino acid residues the C2 domain as for FXa order to more the between the of the monoclonal antibody and C2 epitope ESH8 with anti-C2 monoclonal which FVIII. were were ESH8, which has an epitope the inhibited activation and cleavage of FVIII by FXa did not inhibit FVIII binding to PL (14Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). which has an epitope residues and PL binding M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google the activation and cleavage of FVIII by FXa. results that the for the association between FVIII and FXa is more to the C2 domain than in the PL binding Furthermore, the of ESH8 on FVIII cleavage by FXa not to of FVIII binding to The cleavage of FVIII by FXa inhibited by the of vWF in of vWF on activation were by R.J. J.J. J. 1990; PubMed Scopus Google Scholar). the of the HCh fragment in the absence of with the of of and that the of the FVIII proteolytic cleavage by FXa. The of the PL binding to FVIII. the vWF and PL binding have in the C2 domain (8Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Blood. 1990; 75: 1999-2004Crossref PubMed Google Scholar, 12Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google results the of the C2 domain in the association between FVIII and FXa. The findings and that a vWF binding site is to the FXa binding the of of FXa to FVIII is with vWF The two anti-C2 monoclonal ESH8 and in FVIII and vWF ESH8 FVIII vWF (14Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google FVIII the and vWF binding M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar). the did not inhibit the cleavage by FXa to Furthermore, the of vWF on proteolysis of FVIII at and in the presence of not in the presence of FVIII at in the and is not inhibited in the presence of vWF R.J. J.J. J. 1990; PubMed Scopus Google Scholar). is that activation of FVIII by FXa is by the presence of vWF and that the binding for FXa is that of at a binding site for the of FVIII at the of the A1 domain J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). determined in binding using immobilized FVIII by the monoclonal ESH8, which inhibited FXa cleavage in results using FVIII immobilized on an monoclonal antibody and the of the FVIII LCh in on FXa of FVIII is activated by FXa. is not that FXa to FVIII at a site FXa binding in factor which is with FVIII and has a domain were the HCh and LCh M. J. Biochemistry. 1994; PubMed Scopus Google Scholar). Furthermore, a membrane to the LCh of the factor has as a membrane for FXa in D. T.S. J. 1990; Google Scholar). order to more that the C2 domain contains the FXa binding a catalytically derivative of FXa in binding anhydro-FXa, in which the active-site serine by and the phenylmethylsulfonyl serine to dehydroalanine by of phenylmethylsulfonyl using that binding of the derivative is Biochemistry. 1995; PubMed Scopus Google Scholar). the recombinant C2 domain or the LCh fragments and 72 kDa) bound to anhydro-FXa in a and for the C2 domain than for the 80- and 72-kDa LCh fragments and nm, Several for affinity of the C2 domain that at the 72-kDa LCh is for of the C2 The for the binding to vWF of the C2 domain is than that for the LCh E.L. Scandella D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). for the LCh fragment to that for the 72-kDa that of the domain for FXa. is that the HCh did not bind to anhydro-FXa. Furthermore, proteolytic cleavage at Arg372 in the HCh inhibited by the anti-C2 monoclonal antibody ESH8, than that at and in the results that the role of the C2 domain is more than that of the HCh in FXa direct evidence for the presence of the FXa binding site in the C2 domain by competitive using overlapping synthetic peptides on the epitope of of the peptides corresponding to residues inhibited binding of the C2 domain and partially inhibited binding of the 72-kDa LCh fragment to anhydro-FXa. a containing the amino with a did not inhibit the peptide corresponding to residues inhibited binding of the C2 domain to anhydro-FXa. that residues an important binding site for FXa in the C2 provide evidence for the presence of a binding site for FXa in the C2 domain of the of the presence of an binding site in the Our findings provide the first direct evidence that the C2 domain of FVIII contains a FXa binding FXa cleavage in the FXa bind the C2 domain at a site active Our results that of FXa proteolytic a FVIII to the role of FXa binding to FVIII. J. C. for