Substitution of the gla domain in factor X with that of protein C impairs its interaction with factor VIIa/tissue factor: Lack of comparable effect by similar substitution in factor IX

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of γ-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX_PCGla and FIX_PCGla (Gla domain replaced with that of protein C), FX_PCEGF1 and FIX_PCEGF1 (EGF1 domain replaced with that of protein C), as well as FX_PCGla/EGF1 and FIX_PCGla/EGF1 (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX_PCGla was minimally affected, and the reciprocal activation of FVII/TF by FIXa_PCGla was normal; however, both reactions were impaired for the FIX_PCEGF1 and FIX _PCGla/EGF1 mutants. Predictably, FXIa activation of FIX _PCEGF1 was normal, whereas it was impaired for the FIX _PCGla and FIX_PCGla/EGF1 mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.