The sequence Glu(1811)-Lys(1818) of human blood coagulation factor VIII comprises a binding site for activated factor IX

In previous studies we have shown that the interaction between factors IXa and VIII involves the light chain of factor VIII and that this interaction is inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln(1778)-Asp(1840) (Lenting, P. J., Donath, M. J. S. H., van Mourik, J. A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu(1801) and Met(1823). Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VIII region Tyr(1786)-Ala(1834). Factor IXa binding was found to be particularly efficient to peptides corresponding to the factor VIII sequences Lys(1804)-Lys(1818) and Glu(1811)-Gln(1820). The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys(1804)-Lys(1818) and Glu(1811)-Gln(1820) interfere with binding of factor IXa to immobilized factor VIII light chain (K-i approximate to 0.2 mM and 0.3 mM respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (K-i approximate to 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent K-d approximate to 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys(1804)-Lys(1818) and Glu(1811)-Gln(1820) share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu(1811)-Lys(1818) comprises the minimal requirements for binding to activated factor IX.