Role of Factor VIII C2 Domain in Factor VIII Binding to Factor Xa
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.31000
ISSN1083-351X
AutoresKeiji Nogami, Midori Shima, Kazuya Hosokawa, Toyoaki Suzuki, Takehiko Koide, Evgueni L. Saenko, Dorothea Scandella, Masaru Shibata, Seiki Kamisue, Ichiro Tanaka, Akira Yoshioka,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoFactor 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 produces 54-kDa (A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg1689 removes 40 amino-terminal acidic peptides from the A3 domain (16Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar) and produces a 72-kDa fragment. Cleavage by FXa at Arg1721 produces a 67-kDa LCh fragment (17Donath M.J.S.H. Lenting P.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1995; 270: 3648-3655Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Proteolysis at Arg372 and Arg1689 is essential for generating FVIIIa cofactor activity (18Shima M. Ware J. Yoshioka A. Fukui H. Fulcher C.A. Blood. 1989; 74: 1612-1617Crossref PubMed Google Scholar, 19Kamisue S. Shima M. Nishimura T. Tanaka I. Nakai H. Morichika S. Takata N. Kuramoto A. Yoshioka A. Br. J. Haematol. 1994; 86: 106-111Crossref PubMed Scopus (20) Google Scholar, 20Tuddenham E.G.D. Schwaab R. Seehafer J. Millar D.S. Gitschier J. Higuchi M. Bidichandani S. Connor J.M. Hoyer L.W. Yoshioka A. Peake I.R. Olek K. Kazazian H.H. Lavergne J.M. Giannelli F. Antonarakis S.E. Cooper D.N. Nucleic Acids Res. 1994; 22: 3511-3518Crossref PubMed Scopus (80) Google Scholar). FXa-dependent FVIII activation is different from thrombin-dependent FVIII activation in several ways (21Neuenschwander P. Jesty J. Arch. Biochem. Biophys. 1992; 296: 426-434Crossref PubMed Scopus (25) Google Scholar, 22Parker E.T. Pohl J. Blackburn M.N. Lollar P. Biochemistry. 1997; 36: 9365-9373Crossref PubMed Scopus (25) Google Scholar). The procoagulant activity of FVIIIa produced by FXa is 4-fold lower and is more stable than that generated by thrombin (22Parker E.T. Pohl J. Blackburn M.N. Lollar P. Biochemistry. 1997; 36: 9365-9373Crossref PubMed Scopus (25) Google Scholar). Furthermore, the presence of vWF moderates the activation of FVIII by FXa but not by thrombin (23Koedam J.A. Hamer R.J. Beeser-Visser N.H. Bouma B.N. Sixma J.J. Eur. J. Biochem. 1990; 189: 229-234Crossref PubMed Scopus (75) Google Scholar). Recently, Lapan and Fay (24Lapan K.A. Fay P.J. J. Biol. Chem. 1997; 272: 2082-2088Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) localized a factor X (FX) binding site within the A1 domain. The role of FXa-dependent FVIII activation in vivo is still uncertain, however. In the present study, we demonstrated that an anti-C2 monoclonal antibody, containing an epitope within residues Val2248–Gly2285, inhibited FXa cleavage of FVIII in the absence of PL. Furthermore, the C2 domain competed with FVIII for FXa cleavage of the LCh and bound directly to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine, indicating that the C2 domain contains a FXa binding site. FVIII was affinity-purified using monoclonal antibody NMC-VIII/10, recognizing the FVIII A3 domain. Elution from the monoclonal antibody column was performed with 1 m KI and 40% ethylene glycol as described previously (25Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar). The specific activity of the purified FVIII was 2700 units/mg. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the purified FVIII was free of vWF antigen (26Shima M. Yoshioka A. Yoshikawa N. Tanaka I. Imai S. Tsubura Y. Fukui H. J. Nara Med. Assoc. 1986; 31: 672-679Google Scholar). LCh and HCh fragments of FVIII, together with A1, A2, and thrombin-cleaved 72-kDa LCh fragments, were prepared from plasma FVIII as described previously (27Lollar P. Fay P.J. Fass D.N. Methods Enzymol. 1993; 222: 128-143Crossref PubMed Scopus (94) Google Scholar, 28Regan L.M. Fay P.J. J. Biol. Chem. 1995; 270: 8546-8552Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 29Thompson A.R. Murphy M.E.P. Liu M.L. Saenko E.L. Healey J.F. Lollar P. Scandella D. Blood. 1997; 90: 1902-1908Crossref PubMed Google Scholar). Recombinant C2 domain preparations were produced and purified as previously reported (30Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13826-13833Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). vWF was purified from a commercially available FVIII/vWF concentrate (Confact F®; Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) using gel filtration on a column of Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden) as previously reported (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). Residual FVIII was removed using immunobeads coated with immobilized anti-FVIII monoclonal antibody. ELISA confirmed that FVIII antigen was not present in the vWF fraction (26Shima M. Yoshioka A. Yoshikawa N. Tanaka I. Imai S. Tsubura Y. Fukui H. J. Nara Med. Assoc. 1986; 31: 672-679Google Scholar). Purified human FXa (specific activity 125 units/mg) was obtained from American Diagnostica Inc. (Greenwich, CT). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine andl-α-lecithin egg phosphatidylcholine were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). PL vesicles were prepared as a sonicated phosphatidylserine/phosphatidylcholine mixture (20:80 molar ratio) in 20 mm Tris-HCl, 150 mm NaCl, pH 7.4, as described previously (31Gilbert G.E. Furie B.C. Furie B. J. Biol. Chem. 1990; 265: 815-822Abstract Full Text PDF PubMed Google Scholar). FVIII activity was assayed in a one-stage clotting assay. Anti-FVIII activity was quantified using the Bethesda assay (32Kasper C.K Aledort L.M. Counts R.B. Edson J.R. Frantatoni J. Green D. Hampton J.W. Hilgartner M.W. Lazerson J. Levine P.H. McMillan C.W. Pool J.G. Shapiro S.S. Sculman N.R. van Eyes J. Thromb. Diath. Haemorrh. 1975; 34: 869-872PubMed Google Scholar). One Bethesda unit (BU)/ml was defined as the concentration of antibody that inhibited 50% of the FVIII activity contained in 1 ml of normal plasma after a 2-h incubation at 37 °C. The specific anti-FVIII activity of each monoclonal antibody was expressed as BU/mg of IgG. Ranges of monoclonal antibodies against different epitopes of FVIII were utilized. Two monoclonal antibodies against the FVIII C2 domain, ESH8 (American Diagnostica Inc.) and NMC-VIII/5, recognize amino acid residues 2248–2285 and 2170–2327, respectively (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). NMC-VIII/5 inhibits FVIII binding to vWF and PL (11Shima M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 67: 240-246Google Scholar). In contrast, ESH8 does not prevent FVIII binding to vWF or PL (12Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar). Its FVIII inhibitory activity is attributed to the inhibition of release of vWF from FVIII following 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). Monoclonal antibody NMC-VIII/10, recognizing residues 1675–1684 of the A3 domain, inhibits vWF binding, but it has no effect on PL binding (25Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 33Shima M. Yoshioka A. Nakajima M. Nakai H. Fukui H. Br. J. Haematol. 1992; 81: 533-538Crossref PubMed Scopus (20) Google Scholar). Monoclonal antibody C5, recognizing the carboxyl-terminal acidic region of the A1 domain, was kindly provided by Dr. C. A. Fulcher (Scripps Clinic Research Institute, La Jolla, CA) (34Foster P.A. Fulcher C.A. Houghten R.A. de Graaf Mahoney S. Zimmerman T.S. J. Clin. Invest. 1988; 82: 123-128Crossref PubMed Scopus (78) Google Scholar). Monoclonal antibody JR8 (JR8 Scientific Inc., Woodland, CA) recognizes the A2 domain. C5 and JR8 have no effect on FVIII binding to either vWF or PL. 2K. Nogami, M. Shima, K. Hosokawa, T. Suzuki, T. Koide, E. L. Saenko, D. Scandella, M. Shibata, S. Kamisue, I. Tanaka, and A. Yoshioka, unpublished data. These properties of the monoclonal antibodies are summarized in TableI. The IgG of each monoclonal antibody was fractionated by protein A-Sepharose affinity chromatography (Amersham Pharmacia Biotech). F(ab)′2 fragments of IgG antibodies were prepared using immobilized pepsin (Pierce) and protein A-Sepharose.Table IProperties of anti-FVIII monoclonal antibodiesAntibodyanti-FVIII:CFVIII domains bound in immunoblottingInhibitory effects on FVIII bindinga+ and −, antibody inhibits or does not inhibit, respectively, FVIII binding to vWF or PL.vWF (IC50)PL (IC50)BU/mgμg/mlNMC-VIII/590C2+ (2.0)+ (5.0)ESH81,020C2−−NMC-VIII/1024A3+ (5.0)−C5450A1−−JR810,600A2−−NMC-VIII/10 and C5 bind to the acidic region of the A3 and A1 domains, respectively.a + and −, antibody inhibits or does not inhibit, respectively, FVIII binding to vWF or PL. Open table in a new tab NMC-VIII/10 and C5 bind to the acidic region of the A3 and A1 domains, respectively. Five μg of purified FVIII was radiolabeled by incubation with 0.5 mCi of Na125I (Amersham Pharmacia Biotech) using IODO-GEN® (Pierce) for 3 min as described previously (35Fraker P.J. Speck J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar). Remaining free Na125I was removed by chromatography on a PD-10 column (Amersham Pharmacia Biotech). The specific radioactivity of 125I-FVIII was 10 μCi/μg protein. The activity of125I-FVIII determined in a one-stage clotting assay was similar to that of unlabeled FVIII. Aliquots of radiolabeled FVIII were stored at −80 °C for up to 1 month. Anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine, was prepared as described for the preparation of anhydrothrombin (36Ashton R.W. Scheraga H.A. Biochemistry. 1995; 34: 6454-6463Crossref PubMed Scopus (10) Google Scholar). In outline, FXa was chemically modified with phenylmethylsulfonyl fluoride (PMSF; Wako Pure Chemical Industries Ltd., Osaka, Japan). To convert the phenylmethylsulfonyl residues of the modified FXa to dehydroalanine residues, the product was diluted with 0.05 m NaOH and incubated for 12 min at 0 °C, and the pH was adjusted to 7.5. After dialysis against 50 mm Tris-HCl, pH 7.5, containing 1m NaCl, anhydro-FXa was purified by benzamidine-Sepharose 4B column chromatography (Amersham Pharmacia Biotech). The purified anhydro-FXa demonstrated 95%). FVIII (100 nm) was diluted in veronal buffer (50 mm sodium acetate, 7 mm sodium barbital, 0.1m NaCl), containing 2% bovine serum albumin (BSA; Bovine Fraction V®, Katayama Chemical, Osaka, Japan) and was incubated together with FXa (2 nm), PL vesicles (10 μm), and CaCl2 (2.5 mm) at 37 °C. At timed intervals, samples (10 μl) were taken from the mixture, and FXa action was immediately quenched by 1000-fold dilution in 1 mm PMSF in veronal buffer at 4 °C. Each sample was tested for FVIIIa coagulant activity using a one-stage clotting assay. To assess the inhibitory effects of monoclonal antibodies on FVIII activation by FXa, each antibody was mixed with FVIII prior to FXa activation and incubated for 2 h at 37 °C. The anti-FVIII activity of each antibody was adjusted to 2 BU/ml. Control experiments indicated that the presence of FXa and PMSF in the diluted samples did not influence the FVIII activity during the coagulation assay. 125I-FVIII (10 nm) in 20 mm Tris-HCl, 150 mm NaCl, pH 7.4, was mixed together with FXa (20 nm) and CaCl2 (2.5 mm) in the presence or absence of PL vesicles (10 μm). The mixture was incubated at 37 °C for 30 min in the presence of PL and for 1 h in the absence of PL. At timed intervals, samples (20 μl) were taken, and FXa action was quenched by adding an equal volume of 0.4% SDS and immediately heating the samples to 100 °C for 5 min. Each sample was analyzed on 7.5% SDS-PAGE (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), followed by autoradiography of the dried gels. To assess the inhibitory effects of antibodies on FVIII cleavage by FXa, an equal volume of each antibody (5 μg) was mixed with 125I-FVIII for 2 h at 37 °C prior to incubation with FXa, as described above. To examine the cleavage of FXa in the presence of vWF,125I-FVIII (10 nm) was incubated with vWF (50 nm) for 1 h at 37 °C prior to the addition of FXa in the absence of PL. Microtiter wells (NUNC-Immuno Plate MaxiSorp, NUNC, Denmark) were coated overnight at 4 °C with 2 μg of each monoclonal antibody (NMC-VIII/5, ESH8, C5, or JR8) per well in 100 μl of coating buffer (0.1 m sodium bicarbonate, pH 9.6). After washing three times with washing buffer (phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20), the wells were then blocked for 2 h at 37 °C by the addition of coating buffer containing 4% BSA. After washing, FVIII (10 nm) in washing buffer containing 4% BSA was added to each well and incubated for 2 h at 37 °C. FXa (20 nm) and CaCl2 (2.5 mm) were then added at 37 °C. At timed intervals, the supernatants were removed, and FXa action was quenched by the addition of 1 mm PMSF. Bound FVIII was detected by incubation with peroxidase-conjugated NMC-VIII/10, which recognizes the amino-terminal acidic region of LCh, followed by the addition of o-phenylenediamine dihydrochloride substrate dissolved in 25 mm citric acid, 50 mm Na2HPO4, 0.03% hydrogen peroxide. After 2 m H2SO4 was added as a quenching solution, the absorbance was read at 492 nm (Labsystem Multiskan Multisoft, Helsinki, Finland). Control experiments indicated that the presence of PMSF did not influence this system. Furthermore, SDS-PAGE confirmed that NMC-VIII/10, which was used to detect bound FVIII, did not itself inhibit FXa cleavage. The rate of FVIII LCh cleavage was calculated as follows: (1 − (bound − nonspecific A492/bound at time zero − nonspecific A492)) × 100(%). The absorbance reading in the absence of FVIII was regarded as nonspecific. In competitive inhibition experiments using FVIII fragments (72-kDa LCh, A1, A2, or recombinant C2 domain), 2 μg of NMC-VIII/10 was coated onto microtiter wells as described above. In this assay, a 100 nm concentration of each FVIII fragment was added simultaneously with FXa prior to FXa action, and peroxidase-conjugated NMC-VIII/5 was used for detection of bound FVIII. Six μg of anhydro-FXa in 20 mm Tris-HCl, 150 mm NaCl (TBS), pH 7.4, were immobilized onto each well of a microtiter plate. After blocking with 4% BSA, serially diluted FVIII fragments in TBS, containing 2.5 mm CaCl2 and 4% BSA, were added and incubated for 2 h at 37 °C. Bound LCh or HCh fragment was detected by peroxidase- conjugated NMC-VIII/5 or JR8, respectively. In competitive inhibition experiments using FVIII fragments, serially diluted 125I-FVIII was added to the immobilized anhydro-FXa. Bound 125I-FVIII was measured in a γ-counter. In this assay, serially diluted FVIII fragments were mixed with 125I-FVIII (100 nm) prior to adding to the anhydro-FXa. The percentage inhibition was calculated as follows: (1 − (bound − nonspecific count)/(maximum − nonspecific)) × 100 (%). Radioactive counts in the absence of 125I-FVIII were regarded as nonspecific. The kinetics of FVIII and anhydro-FXa interaction were determined by surface plasmon resonance using a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden). Anhydro-FXa was covalently bound to an activated carboxymethyldextran-coated CM5 sensor chip surface by amine coupling according to the manufacturer's recommendations (39O'Shannessy D.J. Bringham-Burke M. Soneson K.K. Hensley P. Brooks I. Anal. Biochem. 1993; 212: 4457-4468Google Scholar, 40Karlsson R. Anal. Biochem. 1994; 221: 142-151Crossref PubMed Scopus (215) Google Scholar). Binding (association) of all ligands were monitored in TBS, pH 7.4, containing 2.5 mm CaCl2, 0.005% Tween 20 at a flow rate of 10 μl/min for 4 min. Dissociation was monitored over a 5–10 min range after return to buffer flow. After each analysis, regeneration of the chip surface was achieved by 0.1 mglycine, pH 2.0, for 1 min. The values of association rate constants (k a) and dissociation rate constants (k d) were determined by nonlinear regression analysis as described previously (39O'Shannessy D.J. Bringham-Burke M. Soneson K.K. Hensley P. Brooks I. Anal. Biochem. 1993; 212: 4457-4468Google Scholar, 40Karlsson R. Anal. Biochem. 1994; 221: 142-151Crossref PubMed Scopus (215) Google Scholar) using the evaluation software provided by Biacore AB. The values of equilibrium dissociation constants (K d) were calculated ask d/k a. Each overlapping peptide was serially diluted and mixed with 100 nm recombinant C2 domain or 72-kDa LCh fragment prior to addition to the immobilized anhydro-FXa. Bound FVIII was detected using peroxidase-conjugated NMC-VIII/5. Control experiments indicated that none of the synthetic peptides affected binding of FVIII fragments to NMC-VIII/5. The percentage inhibition was calculated as follows: (1 − (bound − nonspecific A492)/(maximum − nonspecific A492)) × 100(%). Absorbance at 492 nm in the absence of FVIII was regarded as nonspecific. Five min after incubation of FVIII with FXa there was an initial 4.5-fold increase in FVIII coagulant activity (Fig.1 A). Peak activity was followed by inactivation, and the base-line level was reached within 45 min of incubation. In order to examine the influence of anti-FVIII monoclonal antibodies, FXa-dependent FVIII activation in the presence of each antibody was performed in the presence of PL. Monoclonal antibody ESH8 recognizing the FVIII C2 domain strongly prevented the activation at a concentration of 2 BU/ml (Fig.1 A), and the inhibitory effect was dose-dependent (Fig. 1 B). In contrast, the alternative anti-C2 monoclonal antibody, NMC-VIII/5, tended to enhance FVIII activation by FXa when compared with the pattern in the absence of antibody. None of the other monoclonal antibodies, anti-A1 antibody (C5), anti-A2 antibody (JR8), and anti-A3 antibody (NMC-VIII/10), inhibited FXa-dependent activation of FVIII at concentrations of 2 BU/ml (Fig. 1 A) or 5 BU/ml (not shown). We postulated that the inhibitory effect of the anti-C2 monoclonal antibody ESH8 on FVIII activation by FXa might be caused by either a change in the cleaved FVIIIa molecule that alters its coagulant activity or by a direct inhibition of the proteolytic cleavage of FVIII by FXa. To distinguish between these possibilities, we incubated125I-labeled FVIII together with FXa for 1 h at 37 °C in the absence or presence of FVIII antibodies and then examined the cleavage pattern by SDS-PAGE. FVIII cleavage by FXa in the absence of antibodies resulted in the conversion of the 90–210 kDa fragments of the HCh into 54- and 44-kDa fragments and proteolysis of the 80-kDa fragment of the LCh into 72- and 67-kDa fragments (Fig.2 A, lane 2). In this instance, however, the 54-kDa fragment was observed only very weakly, and 47- and 40-kDa faint fragments as well as strong bands at dye front were also visible in the lower part oflane 2, suggesting that the 54-kDa fragment had been extensively proteolyzed by FXa. ESH8 completely blocked the cleavage of the 80-kDa LCh fragment and reduced the formation of the 54- and 44-kDa fragments (Fig. 2 A, lane 3). These results suggested that ESH8 completely prevented proteolytic cleavage at Arg1689 and Arg1721 in the LCh and partially inhibited Arg372 in the HCh. In contrast, NMC-VIII/5 did not block the cleavage of the 80-kDa LCh fragment; rather, it tended to promote proteolysis, since the 72-kDa LCh fragment was more strongly evident than in other reactions (Fig.2 A, lane 4). Moreover, the 54-kDa fragment was markedly stronger and the 90-kDa fragment was not observed in the presence of NMC-VIII/5. These findings suggested that the antibody enhanced FXa-induced proteolysis of the HCh as well as the LCh, although selective inhibition of cleavage at Arg336 by NMC-VIII/5 could not be excluded. NMC-VIII/10, C5, and JR8 did not interfere with FXa cleavage of FVIII (Fig. 2 A,lanes 5–7, respectively). The C2 domain contains a PL binding site, and several anti-C2 antibodies are known to inhibit FVIII binding to PL (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). Furthermore, since FVIII cleavage by FXa occurs at a faster rate in the presence of PL than in its absence, inhibition of FVIII binding to PL results in indirect inhibition of FVIII cleavage by FXa. Therefore, we further examined the effects of monoclonal antibodies in the absence of PL. ESH8 again completely blocked the cleavage of the 80-kDa LCh fragment and delayed the formation of the 54- and 44-kDa fragments from the 90-kDa HCh fragment (Fig. 2 B, lane 3). This finding indicated that the inhibitory effect of ESH8 was not due to the presence of PL. Also, the cleavages by FXa in the presence of NMC-VIII/5 in the non-PL system were similar to those obtained in the presence of PL and tended to be more marked in the presence of the antibody than in its absence (Fig. 2 B, lane 4). Moreover, NMC-VIII/10, C5, and JR8 did not inhibit FVIII cleavage in the absence of PL (Fig. 2 B, lanes 5–7, respectively). All of these findings indicated that the inhibitory effect of
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