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 constituent units was investigated. Equilibrium binding studies were performed employing factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and the factor VIII heterodimer were equally effective in factor IXa binding, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with factor IXa, indicating that the high affinity for factor VIII light chain was unique for factor IXa. The factor IXa-factor VIII light chain interaction was inhibited by von Willebrand factor, but this effect was abolished by cleavage of the factor VIII light chain by thrombin. An antibody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of factor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln1778-Asp1840. We propose that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand factor complex.
Lenting
P J
PJ
Department of Blood Coagulation, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Donath
M J
MJ
van Mourik
J A
JA
Mertens
K
K
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015949:Factor IXa / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism*; D006801:Humans; D009994:Osmolar Concentration; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014841:von Willebrand Factor; D005169:Factor VIII; D015949:Factor IXa; D002118:Calcium