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The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

1997; Elsevier BV; Volume: 272; Issue: 29 Linguagem: Inglês

10.1074/jbc.272.29.18007

ISSN

1083-351X

Autores

Evgueni L. Saenko, Dorothea Scandella,

Tópico(s)

Blood Coagulation and Thrombosis Mechanisms

Resumo

A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649–1689, also controls fVIII·vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII·vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14- and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nm) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170–2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf. A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649–1689, also controls fVIII·vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII·vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14- and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nm) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170–2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf. The plasma glycoprotein factor VIII (fVIII) 1The abbreviations used are: fVIII, factor VIII; LCh, light chain of fVIII; HCh, heavy chain of fVIII; A3-C1-C2, fVIII residues 1690–2332; 14-kDa fragment, fVIII residues 1672–1794; 63-kDa fragment, fVIII residues 1795–2332; vWf, von Willebrand factor; mAb, monoclonal antibody; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: fVIII, factor VIII; LCh, light chain of fVIII; HCh, heavy chain of fVIII; A3-C1-C2, fVIII residues 1690–2332; 14-kDa fragment, fVIII residues 1672–1794; 63-kDa fragment, fVIII residues 1795–2332; vWf, von Willebrand factor; mAb, monoclonal antibody; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid. functions as a cofactor for factor IXa in the factor X activation enzyme complex of the intrinsic pathway of blood coagulation (1van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar). fVIII internal protein sequence homology has led to the designation of six domains arranged in the order A1-A2-B-A3-C1-C2 (Ref. 2Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (652) Google Scholar; Fig. 1). The heavy chain (HCh) of fVIII consists of the A1, A2, and B domains, whereas the light chain (LCh) consists of the A3, C1, and C2 domains. Three regions rich in acidic amino acids are located at the carboxyl termini of the A1 and A2 domains and the amino terminus of the A3 domain.Maintenance of a normal fVIII level in the circulation is dependent on its complex formation with von Willebrand factor (vWf) since patients with severe von Willebrand's disease, who have a complete deletion of the vWf gene or mutations which reduce binding between fVIII and vWf, have a secondary deficiency of fVIII. When fVIII is bound to vWf, a stable association of its HCh and LCh is maintained (3Wise R.J. Dorner A.J. Krane M. Pittman D.D. Kaufman R.J. J. Biol. Chem. 1991; 266: 21948-21955Abstract Full Text PDF PubMed Google Scholar), and fVIII is prevented from binding to phospholipid vesicles (4Fay P.J. Coumans J.-V. Walker F.J. J. Biol. Chem. 1991; 266: 2172-2177Abstract Full Text PDF PubMed Google Scholar), platelets (5Nesheim M. Pittman D.D. Giles A.R. Fass D.N. Wang J.H. Slonosky D. Kaufman R.J. J. Biol. Chem. 1991; 266: 17815-17820Abstract Full Text PDF PubMed Google Scholar), or factor IXa, functions required for its procoagulant activity. In addition, vWf protects fVIII from activation by activated factor X (6Koppelman S.J. Koedam J.A. van Wijnen M. Stern D.M. Nawroth P.P. Sixma J.J. Bouma B.N. J. Lab. Clin. Med. 1994; 123: 585-593PubMed Google Scholar) and from protein C-catalyzed inactivation (4Fay P.J. Coumans J.-V. Walker F.J. J. Biol. Chem. 1991; 266: 2172-2177Abstract Full Text PDF PubMed Google Scholar). fVIII binds to vWf through the LCh (7Hamer R.J. Koedam J.A. Beeser-Visser N.H. Bertina R.M. van Mourik J.A. Sixma J.J. Eur. J. Biochem. 1987; 166: 37-43Crossref PubMed Scopus (64) Google Scholar, 8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). The reported binding stoichiometry is 1 fVIII molecule/vWf monomer (9Lollar P. Parker C.G. J. Biol. Chem. 1987; 262: 17572-17576Abstract Full Text PDF PubMed Google Scholar, 10Vlot A.J. Koppelman S.J. Meijers J.C.M. Damas C. van den Berg H.M. Bouma B.N. Sixma J.J. Willems G.M. Blood. 1996; 87: 1809-1816Crossref PubMed Google Scholar).Thrombin, the principal physiological activator of fVIII, cleaves the protein at Arg372 and Arg740 in the HCh and at Arg1689 in the LCh (11Pieneman W.C. Reitsma P.H. Briët E. Thromb. Haemostasis. 1993; 69: 473-475Crossref PubMed Scopus (9) Google Scholar). Activated fVIII (fVIIIa) is a heterodimer of 50-, 43-, and 73-kDa subunits, all of which are required for procoagulant activity (12Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). Cleavage of the LCh at Arg1689, which releases the acidic region (fVIII residues 1649–1689), is responsible for dissociation of fVIIIa from vWf (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar,13Hill-Eubanks D.C. Parker C.G. Lollar P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6508-6512Crossref PubMed Scopus (70) Google Scholar). The importance of the LCh acidic region for fVIII binding to vWf was suggested by the observations that several anti-acidic region monoclonal antibodies (mAbs) with epitopes within residues 1670–1689 (14Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Thromb. Haemostasis. 1990; 63: 403-406Crossref PubMed Scopus (16) Google Scholar, 15Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 16Precup J.W. Kline B.C. Fass D.N. Blood. 1991; 77: 1929-1936Crossref PubMed Google Scholar, 17Leyte A. Verbeet M.P. Brodniewicz-Proba T. van Mourik J.A. Mertens K. Biochem. J. 1989; 257: 679-683Crossref PubMed Scopus (104) Google Scholar) inhibit fVIII binding to vWf, as does complete deletion of the acidic region (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). In contrast, deletion of the fVIII B domain and part of the acidic region (1649–1669) did not abolish vWf binding (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar), suggesting that the acidic region residues 1669–1689 are critical for fVIII·vWf binding. Mutants of fVIII with partial or complete deletions of the acidic region have normal procoagulant activity (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar), which suggests that the loss of this region has no effect other than the elimination of vWf binding. The presence of post-translationally sulfated Tyr1680 was shown to be essential for vWf binding. However, synthetic peptide 1673–1689 failed to inhibit fVIII binding to vWf, regardless of whether Tyr1680 was sulfated (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). Thus, the exact function of the acidic region is not known, since its direct binding to vWf has not been demonstrated.We demonstrated that a glutathione S-transferase-C2 fusion protein binds to immobilized vWf in a dose-dependent, saturable fashion (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar), which indicates that the C2 domain also contains a binding site for vWf. Synthetic peptide 2303–2332, corresponding to the previously identified fVIII phosphatidylserine binding site (20Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Blood. 1990; 75: 1999-2004Crossref PubMed Google Scholar), prevented this interaction (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar, 21Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13286-13833Abstract Full Text Full Text PDF Scopus (91) Google Scholar), demonstrating that the C2 region 2303–2332 is involved in fVIII interaction with both phosphatidylserine and vWf.In this study we describe methods for isolating several proteolytic LCh fragments. We have used these fragments for quantitative measurements of their binding to vWf to further elucidate the roles of the acidic region and the C2 domain.DISCUSSIONSeveral previous studies had identified mAbs that recognize epitopes in the acidic region of the fVIII LCh (residues 1649–1689) and that also prevent the formation of the fVIII·vWf complex (14Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Thromb. Haemostasis. 1990; 63: 403-406Crossref PubMed Scopus (16) Google Scholar, 15Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 16Precup J.W. Kline B.C. Fass D.N. Blood. 1991; 77: 1929-1936Crossref PubMed Google Scholar, 17Leyte A. Verbeet M.P. Brodniewicz-Proba T. van Mourik J.A. Mertens K. Biochem. J. 1989; 257: 679-683Crossref PubMed Scopus (104) Google Scholar). Deletion of the entire acidic region (1649–1689) eliminated vWf binding, whereas deletion up to 1668 did not (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). However, synthetic peptide 1673–1689 at a molar excess of 2,500 over fVIII was not able to inhibit its binding to vWf (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). Our approach to this apparent contradiction was to assume that the synthetic peptide did not have the appropriate conformation for vWf binding and that a larger proteolytic LCh fragment might be more suitable. We were able to cleave the fVIII LCh with S. aureus V8 protease to generate a 14-kDa fragment consisting of amino acids 1672 to approximately 1794, and we have shown in the present study that it does indeed bind to vWf.In our study we used a surface plasmon resonance phenomenon for direct real time measurement of association and dissociation of unlabeled proteins and subsequent determination of the corresponding rate constants (42Yeung D. Gill A. Maule C.H. Davies R.J. Trends Anal. Chem. 1995; 14: 49-56Google Scholar). Since one binding partner must be immobilized to a matrix, the kinetic parameters derived from optical biosensor kinetic measurements may potentially show deviations from those determined in a fluid phase assay. We were able to demonstrate, however, that theK d values determined for fVIII, LCh, 14-kDa, and C2 binding to vWf using the biosensor method (Table I) were similar to theK i values of 0.38, 4.1, 84, and 470 nmfor the respective fragments derived from the fluid phase assay in which binding between 125I-fVIII and vWf was competed by unlabeled ligands. This result indicates that vWf was not altered by immobilization and therefore the kinetic parameters for binding of fVIII and its derivatives to vWf derived in our experiments are valid. The high sensitivity of this technique and its capability to register fast dissociation kinetics has allowed us to determine up to 2,000-fold lower affinities of fVIII proteolytic fragments for vWf than that of fVIII.All LCh derivatives lacking the acidic region (A3-C1-C2, C2, and 63-kDa fragment) had greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm) (Table I). In addition, the similar affinities of A3-C1-C2, 63-kDa fragment, C2, and HCh/A3-C1-C2 for vWf indicated that the LCh contains no vWf binding site other than the one within C2. The 14-kDa fragment lacking the C2 domain also had a lower affinity (K d 72 nm) for vWf than LCh. In contrast, the proteolytically cleaved but undissociated complex containing both the acidic region and C2 (14 kDa/63 kDa) had an affinity identical to that of the intact LCh. Reassociation of the 14-kDa/63-kDa complex from the individual 14- and 63-kDa fragments and the demonstration that its binding to vWf is similar to that of the original 14-kDa/63-kDa complex indicated that the above fragments were not altered by purification under denaturing conditions. The previous hypotheses that the LCh acidic region contains a vWf binding site and that both this region and the C2 domain are essential for high affinity binding are therefore confirmed. Our data also demonstrate that the two binding sites must be simultaneously present, although not necessarily covalently linked, for maximal affinity vWf binding to occur. Our data also demonstrate that the residues 1649–1671 are not involved in vWf binding, which is consistent with previous findings (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). We demonstrated that neither the acidic region peptide 1672–1689 nor the COOH-terminal 11.5-kDa fragment derived by the thrombin cleavage of 14-kDa fragment were able to bind vWf in biosensor experiments or inhibit fVIII·vWf interaction in fluid phase assays. We hypothesize, therefore, that the acidic region vWf binding site extends COOH-terminal to the thrombin cleavage site at residue 1689 and that it is destroyed by thrombin cleavage. This hypothesis could explain why synthetic peptide 1673–1689 (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar) did not bind vWf. The decreased affinity of the 14-kDa fragment affinity for vWf is due to a lower association rate constant (k on) than that for LCh (Table I) since the dissociation rate constants determined for the 14-kDa fragment and LCh complexes with vWf were similar. For the C2 domain the k off value was 10-fold greater than that for LCh or 14-kDa fragment. These results suggest that the interaction of the LCh acidic region with vWf is the rate-determining step for the dissociation of LCh·vWf and fVIII·vWf complexes in the absence of thrombin activation. The loss of the acidic region leads to a 160-fold increased k off and a >1000-fold increased K d for HCh/A3-C1-C2 heterodimer binding to vWf compared with that of fVIII. This would predict a similar reduction of fVIII affinity upon thrombin activation, allowing efficient fVIIIa binding to the phospholipid surface required for its maximal activity in the factor Xase enzyme complex.We demonstrated that the LCh acidic region not only directly participates in vWf binding, but it is probably also required to maintain the normal conformation of the C2 binding site. We used anti-C2 mAb NMC-VIII/5, which prevents fVIII·vWf binding (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar,22Shima M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 69: 240-246Crossref PubMed Scopus (174) Google Scholar), as a probe to detect possible conformational changes in C2 upon removal of the acidic region. The reduced affinities for NMC-VIII/5 of LCh derivatives lacking the acidic region indicated that such a change occurs. Similar affinities of NMC-VIII/5 for the LCh and the 14-kDa/63-kDa complex demonstrated that noncovalent association of the amino-terminal 14-kDa fragment with the carboxyl-terminal 63-kDa fragment appeared to be sufficient to maintain the C2 conformation similar to that within intact LCh. In contrast, the conformation of the acidic region does not depend on the presence of C2, since the affinity of the anti-acidic region mAb NMC-VIII/10 for the 14-kDa fragment and the LCh was similar.Our results are consistent with the hypothesis that the light chain acidic region and C2 are in close proximity and together form one high affinity binding site for vWf. The computer modeling of the three-dimensional structure of the fVIII A domains, based on their structure in ceruloplasmin, predicts that the carboxyl and amino termini of A3 are in close proximity (43Pemberton S. Lindley P. Zaitsev V. Card G. Tuddenham E.G.D. Kemball-Cook G. Blood. 1997; 89: 2413-2421Crossref PubMed Google Scholar). In addition, the disulfide bond determined between Cys2021 and Cys2169 and that proposed between Cys2174 and Cys2326 (44McMullen B.A. Fujikawa K. Davie E.W. Hedner U. Ezban M. Protein Sci. 1995; 4: 740-746Crossref PubMed Scopus (67) Google Scholar) demonstrate that the amino and carboxyl termini of the C1 (residues 2019–2172) and C2 (residues 2173–2332) domains, respectively, are also spatially close. These findings suggest that the acidic region located at the amino terminus of the LCh and the carboxyl terminus of the C2 domain may also be close together in the three-dimensional structure of the LCh. Our findings that the LCh acidic region and C2 together form the high affinity vWf binding site is consistent with this model. The inhibition of high affinity fVIII binding to mature vWf residues 1–116 (45Lavergne J.-M. Piao Y.-C. Ferreira V. Kerbinou-Nabias D. Bahnak B.R. Meyer D. Biochem. Biophys. Res. Commun. 1993; 194: 1019-1024Crossref PubMed Scopus (16) Google Scholar) by both anti-LCh acidic region and anti-C2 mAbs (21Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13286-13833Abstract Full Text Full Text PDF Scopus (91) Google Scholar) would also fit this model.The K d for LCh binding to vWf (3.8 nm) is 9.5 times higher than that for fVIII (0.4 nm), demonstrating that the HCh is required for the maximal affinity of fVIII for vWf. The higher affinity of fVIII·vWf interaction than that of LCh·vWf is mainly due to the lower dissociation rate of the former complex (Table I). The participation of the HCh in fVIII·vWf binding is indirect because the HCh itself did not bind to vWf in our experiments or as previously demonstrated by ultracentrifugation (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). Recently Sudhakar and Fay observed that dissociation of the HCh·LCh complex led to conformational changes in each chain (46Sudhakar K. Fay P.J. J. Biol. Chem. 1996; 271: 23015-23021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It is therefore possible that the LCh has to be associated with HCh to have the optimal conformation for its binding to vWf. Once the LCh acidic region is removed by thrombin cleavage, the HCh cannot enhance the binding of A3-C1-C2 to vWf. The plasma glycoprotein factor VIII (fVIII) 1The abbreviations used are: fVIII, factor VIII; LCh, light chain of fVIII; HCh, heavy chain of fVIII; A3-C1-C2, fVIII residues 1690–2332; 14-kDa fragment, fVIII residues 1672–1794; 63-kDa fragment, fVIII residues 1795–2332; vWf, von Willebrand factor; mAb, monoclonal antibody; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid. 1The abbreviations used are: fVIII, factor VIII; LCh, light chain of fVIII; HCh, heavy chain of fVIII; A3-C1-C2, fVIII residues 1690–2332; 14-kDa fragment, fVIII residues 1672–1794; 63-kDa fragment, fVIII residues 1795–2332; vWf, von Willebrand factor; mAb, monoclonal antibody; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid. functions as a cofactor for factor IXa in the factor X activation enzyme complex of the intrinsic pathway of blood coagulation (1van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar). fVIII internal protein sequence homology has led to the designation of six domains arranged in the order A1-A2-B-A3-C1-C2 (Ref. 2Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (652) Google Scholar; Fig. 1). The heavy chain (HCh) of fVIII consists of the A1, A2, and B domains, whereas the light chain (LCh) consists of the A3, C1, and C2 domains. Three regions rich in acidic amino acids are located at the carboxyl termini of the A1 and A2 domains and the amino terminus of the A3 domain. Maintenance of a normal fVIII level in the circulation is dependent on its complex formation with von Willebrand factor (vWf) since patients with severe von Willebrand's disease, who have a complete deletion of the vWf gene or mutations which reduce binding between fVIII and vWf, have a secondary deficiency of fVIII. When fVIII is bound to vWf, a stable association of its HCh and LCh is maintained (3Wise R.J. Dorner A.J. Krane M. Pittman D.D. Kaufman R.J. J. Biol. Chem. 1991; 266: 21948-21955Abstract Full Text PDF PubMed Google Scholar), and fVIII is prevented from binding to phospholipid vesicles (4Fay P.J. Coumans J.-V. Walker F.J. J. Biol. Chem. 1991; 266: 2172-2177Abstract Full Text PDF PubMed Google Scholar), platelets (5Nesheim M. Pittman D.D. Giles A.R. Fass D.N. Wang J.H. Slonosky D. Kaufman R.J. J. Biol. Chem. 1991; 266: 17815-17820Abstract Full Text PDF PubMed Google Scholar), or factor IXa, functions required for its procoagulant activity. In addition, vWf protects fVIII from activation by activated factor X (6Koppelman S.J. Koedam J.A. van Wijnen M. Stern D.M. Nawroth P.P. Sixma J.J. Bouma B.N. J. Lab. Clin. Med. 1994; 123: 585-593PubMed Google Scholar) and from protein C-catalyzed inactivation (4Fay P.J. Coumans J.-V. Walker F.J. J. Biol. Chem. 1991; 266: 2172-2177Abstract Full Text PDF PubMed Google Scholar). fVIII binds to vWf through the LCh (7Hamer R.J. Koedam J.A. Beeser-Visser N.H. Bertina R.M. van Mourik J.A. Sixma J.J. Eur. J. Biochem. 1987; 166: 37-43Crossref PubMed Scopus (64) Google Scholar, 8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). The reported binding stoichiometry is 1 fVIII molecule/vWf monomer (9Lollar P. Parker C.G. J. Biol. Chem. 1987; 262: 17572-17576Abstract Full Text PDF PubMed Google Scholar, 10Vlot A.J. Koppelman S.J. Meijers J.C.M. Damas C. van den Berg H.M. Bouma B.N. Sixma J.J. Willems G.M. Blood. 1996; 87: 1809-1816Crossref PubMed Google Scholar). Thrombin, the principal physiological activator of fVIII, cleaves the protein at Arg372 and Arg740 in the HCh and at Arg1689 in the LCh (11Pieneman W.C. Reitsma P.H. Briët E. Thromb. Haemostasis. 1993; 69: 473-475Crossref PubMed Scopus (9) Google Scholar). Activated fVIII (fVIIIa) is a heterodimer of 50-, 43-, and 73-kDa subunits, all of which are required for procoagulant activity (12Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). Cleavage of the LCh at Arg1689, which releases the acidic region (fVIII residues 1649–1689), is responsible for dissociation of fVIIIa from vWf (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar,13Hill-Eubanks D.C. Parker C.G. Lollar P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6508-6512Crossref PubMed Scopus (70) Google Scholar). The importance of the LCh acidic region for fVIII binding to vWf was suggested by the observations that several anti-acidic region monoclonal antibodies (mAbs) with epitopes within residues 1670–1689 (14Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Thromb. Haemostasis. 1990; 63: 403-406Crossref PubMed Scopus (16) Google Scholar, 15Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 16Precup J.W. Kline B.C. Fass D.N. Blood. 1991; 77: 1929-1936Crossref PubMed Google Scholar, 17Leyte A. Verbeet M.P. Brodniewicz-Proba T. van Mourik J.A. Mertens K. Biochem. J. 1989; 257: 679-683Crossref PubMed Scopus (104) Google Scholar) inhibit fVIII binding to vWf, as does complete deletion of the acidic region (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). In contrast, deletion of the fVIII B domain and part of the acidic region (1649–1669) did not abolish vWf binding (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar), suggesting that the acidic region residues 1669–1689 are critical for fVIII·vWf binding. Mutants of fVIII with partial or complete deletions of the acidic region have normal procoagulant activity (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar), which suggests that the loss of this region has no effect other than the elimination of vWf binding. The presence of post-translationally sulfated Tyr1680 was shown to be essential for vWf binding. However, synthetic peptide 1673–1689 failed to inhibit fVIII binding to vWf, regardless of whether Tyr1680 was sulfated (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). Thus, the exact function of the acidic region is not known, since its direct binding to vWf has not been demonstrated. We demonstrated that a glutathione S-transferase-C2 fusion protein binds to immobilized vWf in a dose-dependent, saturable fashion (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar), which indicates that the C2 domain also contains a binding site for vWf. Synthetic peptide 2303–2332, corresponding to the previously identified fVIII phosphatidylserine binding site (20Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Blood. 1990; 75: 1999-2004Crossref PubMed Google Scholar), prevented this interaction (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar, 21Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13286-13833Abstract Full Text Full Text PDF Scopus (91) Google Scholar), demonstrating that the C2 region 2303–2332 is involved in fVIII interaction with both phosphatidylserine and vWf. In this study we describe methods for isolating several proteolytic LCh fragments. We have used these fragments for quantitative measurements of their binding to vWf to further elucidate the roles of the acidic region and the C2 domain. DISCUSSIONSeveral previous studies had identified mAbs that recognize epitopes in the acidic region of the fVIII LCh (residues 1649–1689) and that also prevent the formation of the fVIII·vWf complex (14Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Thromb. Haemostasis. 1990; 63: 403-406Crossref PubMed Scopus (16) Google Scholar, 15Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 16Precup J.W. Kline B.C. Fass D.N. Blood. 1991; 77: 1929-1936Crossref PubMed Google Scholar, 17Leyte A. Verbeet M.P. Brodniewicz-Proba T. van Mourik J.A. Mertens K. Biochem. J. 1989; 257: 679-683Crossref PubMed Scopus (104) Google Scholar). Deletion of the entire acidic region (1649–1689) eliminated vWf binding, whereas deletion up to 1668 did not (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). However, synthetic peptide 1673–1689 at a molar excess of 2,500 over fVIII was not able to inhibit its binding to vWf (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). Our approach to this apparent contradiction was to assume that the synthetic peptide did not have the appropriate conformation for vWf binding and that a larger proteolytic LCh fragment might be more suitable. We were able to cleave the fVIII LCh with S. aureus V8 protease to generate a 14-kDa fragment consisting of amino acids 1672 to approximately 1794, and we have shown in the present study that it does indeed bind to vWf.In our study we used a surface plasmon resonance phenomenon for direct real time measurement of association and dissociation of unlabeled proteins and subsequent determination of the corresponding rate constants (42Yeung D. Gill A. Maule C.H. Davies R.J. Trends Anal. Chem. 1995; 14: 49-56Google Scholar). Since one binding partner must be immobilized to a matrix, the kinetic parameters derived from optical biosensor kinetic measurements may potentially show deviations from those determined in a fluid phase assay. We were able to demonstrate, however, that theK d values determined for fVIII, LCh, 14-kDa, and C2 binding to vWf using the biosensor method (Table I) were similar to theK i values of 0.38, 4.1, 84, and 470 nmfor the respective fragments derived from the fluid phase assay in which binding between 125I-fVIII and vWf was competed by unlabeled ligands. This result indicates that vWf was not altered by immobilization and therefore the kinetic parameters for binding of fVIII and its derivatives to vWf derived in our experiments are valid. The high sensitivity of this technique and its capability to register fast dissociation kinetics has allowed us to determine up to 2,000-fold lower affinities of fVIII proteolytic fragments for vWf than that of fVIII.All LCh derivatives lacking the acidic region (A3-C1-C2, C2, and 63-kDa fragment) had greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm) (Table I). In addition, the similar affinities of A3-C1-C2, 63-kDa fragment, C2, and HCh/A3-C1-C2 for vWf indicated that the LCh contains no vWf binding site other than the one within C2. The 14-kDa fragment lacking the C2 domain also had a lower affinity (K d 72 nm) for vWf than LCh. In contrast, the proteolytically cleaved but undissociated complex containing both the acidic region and C2 (14 kDa/63 kDa) had an affinity identical to that of the intact LCh. Reassociation of the 14-kDa/63-kDa complex from the individual 14- and 63-kDa fragments and the demonstration that its binding to vWf is similar to that of the original 14-kDa/63-kDa complex indicated that the above fragments were not altered by purification under denaturing conditions. The previous hypotheses that the LCh acidic region contains a vWf binding site and that both this region and the C2 domain are essential for high affinity binding are therefore confirmed. Our data also demonstrate that the two binding sites must be simultaneously present, although not necessarily covalently linked, for maximal affinity vWf binding to occur. Our data also demonstrate that the residues 1649–1671 are not involved in vWf binding, which is consistent with previous findings (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). We demonstrated that neither the acidic region peptide 1672–1689 nor the COOH-terminal 11.5-kDa fragment derived by the thrombin cleavage of 14-kDa fragment were able to bind vWf in biosensor experiments or inhibit fVIII·vWf interaction in fluid phase assays. We hypothesize, therefore, that the acidic region vWf binding site extends COOH-terminal to the thrombin cleavage site at residue 1689 and that it is destroyed by thrombin cleavage. This hypothesis could explain why synthetic peptide 1673–1689 (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar) did not bind vWf. The decreased affinity of the 14-kDa fragment affinity for vWf is due to a lower association rate constant (k on) than that for LCh (Table I) since the dissociation rate constants determined for the 14-kDa fragment and LCh complexes with vWf were similar. For the C2 domain the k off value was 10-fold greater than that for LCh or 14-kDa fragment. These results suggest that the interaction of the LCh acidic region with vWf is the rate-determining step for the dissociation of LCh·vWf and fVIII·vWf complexes in the absence of thrombin activation. The loss of the acidic region leads to a 160-fold increased k off and a >1000-fold increased K d for HCh/A3-C1-C2 heterodimer binding to vWf compared with that of fVIII. This would predict a similar reduction of fVIII affinity upon thrombin activation, allowing efficient fVIIIa binding to the phospholipid surface required for its maximal activity in the factor Xase enzyme complex.We demonstrated that the LCh acidic region not only directly participates in vWf binding, but it is probably also required to maintain the normal conformation of the C2 binding site. We used anti-C2 mAb NMC-VIII/5, which prevents fVIII·vWf binding (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar,22Shima M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 69: 240-246Crossref PubMed Scopus (174) Google Scholar), as a probe to detect possible conformational changes in C2 upon removal of the acidic region. The reduced affinities for NMC-VIII/5 of LCh derivatives lacking the acidic region indicated that such a change occurs. Similar affinities of NMC-VIII/5 for the LCh and the 14-kDa/63-kDa complex demonstrated that noncovalent association of the amino-terminal 14-kDa fragment with the carboxyl-terminal 63-kDa fragment appeared to be sufficient to maintain the C2 conformation similar to that within intact LCh. In contrast, the conformation of the acidic region does not depend on the presence of C2, since the affinity of the anti-acidic region mAb NMC-VIII/10 for the 14-kDa fragment and the LCh was similar.Our results are consistent with the hypothesis that the light chain acidic region and C2 are in close proximity and together form one high affinity binding site for vWf. The computer modeling of the three-dimensional structure of the fVIII A domains, based on their structure in ceruloplasmin, predicts that the carboxyl and amino termini of A3 are in close proximity (43Pemberton S. Lindley P. Zaitsev V. Card G. Tuddenham E.G.D. Kemball-Cook G. Blood. 1997; 89: 2413-2421Crossref PubMed Google Scholar). In addition, the disulfide bond determined between Cys2021 and Cys2169 and that proposed between Cys2174 and Cys2326 (44McMullen B.A. Fujikawa K. Davie E.W. Hedner U. Ezban M. Protein Sci. 1995; 4: 740-746Crossref PubMed Scopus (67) Google Scholar) demonstrate that the amino and carboxyl termini of the C1 (residues 2019–2172) and C2 (residues 2173–2332) domains, respectively, are also spatially close. These findings suggest that the acidic region located at the amino terminus of the LCh and the carboxyl terminus of the C2 domain may also be close together in the three-dimensional structure of the LCh. Our findings that the LCh acidic region and C2 together form the high affinity vWf binding site is consistent with this model. The inhibition of high affinity fVIII binding to mature vWf residues 1–116 (45Lavergne J.-M. Piao Y.-C. Ferreira V. Kerbinou-Nabias D. Bahnak B.R. Meyer D. Biochem. Biophys. Res. Commun. 1993; 194: 1019-1024Crossref PubMed Scopus (16) Google Scholar) by both anti-LCh acidic region and anti-C2 mAbs (21Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13286-13833Abstract Full Text Full Text PDF Scopus (91) Google Scholar) would also fit this model.The K d for LCh binding to vWf (3.8 nm) is 9.5 times higher than that for fVIII (0.4 nm), demonstrating that the HCh is required for the maximal affinity of fVIII for vWf. The higher affinity of fVIII·vWf interaction than that of LCh·vWf is mainly due to the lower dissociation rate of the former complex (Table I). The participation of the HCh in fVIII·vWf binding is indirect because the HCh itself did not bind to vWf in our experiments or as previously demonstrated by ultracentrifugation (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). Recently Sudhakar and Fay observed that dissociation of the HCh·LCh complex led to conformational changes in each chain (46Sudhakar K. Fay P.J. J. Biol. Chem. 1996; 271: 23015-23021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It is therefore possible that the LCh has to be associated with HCh to have the optimal conformation for its binding to vWf. Once the LCh acidic region is removed by thrombin cleavage, the HCh cannot enhance the binding of A3-C1-C2 to vWf. Several previous studies had identified mAbs that recognize epitopes in the acidic region of the fVIII LCh (residues 1649–1689) and that also prevent the formation of the fVIII·vWf complex (14Foster P.A. Fulcher C.A. Houghten R.A. Zimmerman T.S. Thromb. Haemostasis. 1990; 63: 403-406Crossref PubMed Scopus (16) Google Scholar, 15Shima M. Yoshioka A. Nakai H. Tanaka I. Sawamoto Y. Kamisue S. Terada S. Fukui H. Int. J. Hematol. 1991; 54: 515-522PubMed Google Scholar, 16Precup J.W. Kline B.C. Fass D.N. Blood. 1991; 77: 1929-1936Crossref PubMed Google Scholar, 17Leyte A. Verbeet M.P. Brodniewicz-Proba T. van Mourik J.A. Mertens K. Biochem. J. 1989; 257: 679-683Crossref PubMed Scopus (104) Google Scholar). Deletion of the entire acidic region (1649–1689) eliminated vWf binding, whereas deletion up to 1668 did not (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). However, synthetic peptide 1673–1689 at a molar excess of 2,500 over fVIII was not able to inhibit its binding to vWf (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). Our approach to this apparent contradiction was to assume that the synthetic peptide did not have the appropriate conformation for vWf binding and that a larger proteolytic LCh fragment might be more suitable. We were able to cleave the fVIII LCh with S. aureus V8 protease to generate a 14-kDa fragment consisting of amino acids 1672 to approximately 1794, and we have shown in the present study that it does indeed bind to vWf. In our study we used a surface plasmon resonance phenomenon for direct real time measurement of association and dissociation of unlabeled proteins and subsequent determination of the corresponding rate constants (42Yeung D. Gill A. Maule C.H. Davies R.J. Trends Anal. Chem. 1995; 14: 49-56Google Scholar). Since one binding partner must be immobilized to a matrix, the kinetic parameters derived from optical biosensor kinetic measurements may potentially show deviations from those determined in a fluid phase assay. We were able to demonstrate, however, that theK d values determined for fVIII, LCh, 14-kDa, and C2 binding to vWf using the biosensor method (Table I) were similar to theK i values of 0.38, 4.1, 84, and 470 nmfor the respective fragments derived from the fluid phase assay in which binding between 125I-fVIII and vWf was competed by unlabeled ligands. This result indicates that vWf was not altered by immobilization and therefore the kinetic parameters for binding of fVIII and its derivatives to vWf derived in our experiments are valid. The high sensitivity of this technique and its capability to register fast dissociation kinetics has allowed us to determine up to 2,000-fold lower affinities of fVIII proteolytic fragments for vWf than that of fVIII. All LCh derivatives lacking the acidic region (A3-C1-C2, C2, and 63-kDa fragment) had greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm) (Table I). In addition, the similar affinities of A3-C1-C2, 63-kDa fragment, C2, and HCh/A3-C1-C2 for vWf indicated that the LCh contains no vWf binding site other than the one within C2. The 14-kDa fragment lacking the C2 domain also had a lower affinity (K d 72 nm) for vWf than LCh. In contrast, the proteolytically cleaved but undissociated complex containing both the acidic region and C2 (14 kDa/63 kDa) had an affinity identical to that of the intact LCh. Reassociation of the 14-kDa/63-kDa complex from the individual 14- and 63-kDa fragments and the demonstration that its binding to vWf is similar to that of the original 14-kDa/63-kDa complex indicated that the above fragments were not altered by purification under denaturing conditions. The previous hypotheses that the LCh acidic region contains a vWf binding site and that both this region and the C2 domain are essential for high affinity binding are therefore confirmed. Our data also demonstrate that the two binding sites must be simultaneously present, although not necessarily covalently linked, for maximal affinity vWf binding to occur. Our data also demonstrate that the residues 1649–1671 are not involved in vWf binding, which is consistent with previous findings (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar). We demonstrated that neither the acidic region peptide 1672–1689 nor the COOH-terminal 11.5-kDa fragment derived by the thrombin cleavage of 14-kDa fragment were able to bind vWf in biosensor experiments or inhibit fVIII·vWf interaction in fluid phase assays. We hypothesize, therefore, that the acidic region vWf binding site extends COOH-terminal to the thrombin cleavage site at residue 1689 and that it is destroyed by thrombin cleavage. This hypothesis could explain why synthetic peptide 1673–1689 (18Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar) did not bind vWf. The decreased affinity of the 14-kDa fragment affinity for vWf is due to a lower association rate constant (k on) than that for LCh (Table I) since the dissociation rate constants determined for the 14-kDa fragment and LCh complexes with vWf were similar. For the C2 domain the k off value was 10-fold greater than that for LCh or 14-kDa fragment. These results suggest that the interaction of the LCh acidic region with vWf is the rate-determining step for the dissociation of LCh·vWf and fVIII·vWf complexes in the absence of thrombin activation. The loss of the acidic region leads to a 160-fold increased k off and a >1000-fold increased K d for HCh/A3-C1-C2 heterodimer binding to vWf compared with that of fVIII. This would predict a similar reduction of fVIII affinity upon thrombin activation, allowing efficient fVIIIa binding to the phospholipid surface required for its maximal activity in the factor Xase enzyme complex. We demonstrated that the LCh acidic region not only directly participates in vWf binding, but it is probably also required to maintain the normal conformation of the C2 binding site. We used anti-C2 mAb NMC-VIII/5, which prevents fVIII·vWf binding (19Saenko E.L. Shima M. Rajalakshmi K.J. Scandella D. J. Biol. Chem. 1994; 269: 11601-11605Abstract Full Text PDF PubMed Google Scholar,22Shima M. Scandella D. Yoshioka A. Nakai H. Tanaka I. Kamisue S. Terada S. Fukui H. Thromb. Haemostasis. 1993; 69: 240-246Crossref PubMed Scopus (174) Google Scholar), as a probe to detect possible conformational changes in C2 upon removal of the acidic region. The reduced affinities for NMC-VIII/5 of LCh derivatives lacking the acidic region indicated that such a change occurs. Similar affinities of NMC-VIII/5 for the LCh and the 14-kDa/63-kDa complex demonstrated that noncovalent association of the amino-terminal 14-kDa fragment with the carboxyl-terminal 63-kDa fragment appeared to be sufficient to maintain the C2 conformation similar to that within intact LCh. In contrast, the conformation of the acidic region does not depend on the presence of C2, since the affinity of the anti-acidic region mAb NMC-VIII/10 for the 14-kDa fragment and the LCh was similar. Our results are consistent with the hypothesis that the light chain acidic region and C2 are in close proximity and together form one high affinity binding site for vWf. The computer modeling of the three-dimensional structure of the fVIII A domains, based on their structure in ceruloplasmin, predicts that the carboxyl and amino termini of A3 are in close proximity (43Pemberton S. Lindley P. Zaitsev V. Card G. Tuddenham E.G.D. Kemball-Cook G. Blood. 1997; 89: 2413-2421Crossref PubMed Google Scholar). In addition, the disulfide bond determined between Cys2021 and Cys2169 and that proposed between Cys2174 and Cys2326 (44McMullen B.A. Fujikawa K. Davie E.W. Hedner U. Ezban M. Protein Sci. 1995; 4: 740-746Crossref PubMed Scopus (67) Google Scholar) demonstrate that the amino and carboxyl termini of the C1 (residues 2019–2172) and C2 (residues 2173–2332) domains, respectively, are also spatially close. These findings suggest that the acidic region located at the amino terminus of the LCh and the carboxyl terminus of the C2 domain may also be close together in the three-dimensional structure of the LCh. Our findings that the LCh acidic region and C2 together form the high affinity vWf binding site is consistent with this model. The inhibition of high affinity fVIII binding to mature vWf residues 1–116 (45Lavergne J.-M. Piao Y.-C. Ferreira V. Kerbinou-Nabias D. Bahnak B.R. Meyer D. Biochem. Biophys. Res. Commun. 1993; 194: 1019-1024Crossref PubMed Scopus (16) Google Scholar) by both anti-LCh acidic region and anti-C2 mAbs (21Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13286-13833Abstract Full Text Full Text PDF Scopus (91) Google Scholar) would also fit this model. The K d for LCh binding to vWf (3.8 nm) is 9.5 times higher than that for fVIII (0.4 nm), demonstrating that the HCh is required for the maximal affinity of fVIII for vWf. The higher affinity of fVIII·vWf interaction than that of LCh·vWf is mainly due to the lower dissociation rate of the former complex (Table I). The participation of the HCh in fVIII·vWf binding is indirect because the HCh itself did not bind to vWf in our experiments or as previously demonstrated by ultracentrifugation (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar). Recently Sudhakar and Fay observed that dissociation of the HCh·LCh complex led to conformational changes in each chain (46Sudhakar K. Fay P.J. J. Biol. Chem. 1996; 271: 23015-23021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It is therefore possible that the LCh has to be associated with HCh to have the optimal conformation for its binding to vWf. Once the LCh acidic region is removed by thrombin cleavage, the HCh cannot enhance the binding of A3-C1-C2 to vWf. We are grateful to Dr. Kenneth Ingham for critical review of the manuscript and helpful discussions.

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