The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa

Abstract

The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181-310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181-310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IX(K214F), factor IX(K228R), factor IX(E240Q), factor IX(K247V), and factor IX(N260K)) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IX(D275K) nor factor IX(R248H) bound to FXC008. Factor IX(R252V) had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.