The N-terminal EGF domain of coagulation factor IX: Probing its functions in the activation of factor IX and factor X with a monoclonal antibody.

SUMMARY Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor (EGF)-like domains and a serine protease domain. In this study the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by FXIa, but activation by FVIIa/tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent kcat for FX both in the presence and absence of FVIIIa. Based on these results, we produced a preliminary model of the structure of the FIXa-FVIIIa-AW complex on the surface of phospholipid. The model suggests that in the Xase complex EGF1 of FIXa is not involved in direct binding to FVIIIa. Studies of the interaction of antibody AW with a mutated FIX molecule (Arg94Asp) also suggest that the Glu78-Arg94 salt-bridge is not important for maintaining the structure of FIX.