IDENTIFICATION OF A BINDING-SITE FOR BLOOD-COAGULATION FACTOR IXA ON THE LIGHT-CHAIN OF HUMAN FACTOR-VIII

The interaction between human factor IXa and factor VIII or its consti tuent units was investigated. Equilibrium binding studies were perform ed employing factor VIII light chain that was immobilized on a monoclo nal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (K-d = 14.8 +/- 3.2 nM) and approximately 1:1 stoic hiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution ef fectively inhibited binding of factor IXa to the immobilized light cha in (K-i = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and t he factor VIII heterodimer were equally effective in factor IXa bindin g, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be ineff icient (K-i greater than or equal to 1.2 mu M) in competing with facto r IXa, indicating that the high affinity for factor VII light chain wa s unique for factor IXa. The factor IXa-factor VIII light chain intera ction was inhibited by von Willebrand factor, but this effect was abol ished by cleavage of the factor VIII light chain by thrombin. An antib ody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of fa ctor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln(1778)-Asp(1840). We propo se that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand facto r complex.