The first epidermal growth factor-like domains of factor Xa and factor IXa are important for the activation of the factor VII--tissue factor complex.

During tissue factor (TF)-induced coagulation, the factor (F)VIIa-TF complex activates factor (F)X and factor (F)IX. Through positive feedback, the generated FXa and FIXa activate FVII-TF. The first epidermal growth factor-like (EGF1) domains of FX and FIX serve as important TF-recognition motifs when FVIIa-TF activates FX or FIX. Here, we investigated the role of EGF1 domains of FXa and FIXa during the activation of FVII-TF and inhibition by tissue factor pathway inhibitor (TFPI). FXaPCEGF1 (EGF1 domain of FXa replaced with that of protein C), and FXaQ49P (EGF1 domain mutant with impaired calcium-binding), and the corresponding FIXa mutants were generated, and their abilities to activate FVII-TF were compared with the wild-type (WT) enzymes. In the absence of TF, the rates of FVII activation were similar between WT enzymes and mutant FXa and FIXa proteases. In the presence of either soluble TF (sTF) or relipidated TF, each mutant of FXa or FIXa activated FVII-TF at a slower rate than the corresponding WT enzyme. Kinetics of inhibition of the amidolytic activity of WT and the mutant FXa proteases by either two-domain or full-length TFPI were similar. However, compared with the complex of TFPI-FXaWT, the abilities of the complexes of TFPI-FXa mutants to inhibit FVIIa-TF were impaired. We conclude that the EGF1 domains of FXa and FIXa are important for the activation of FVII-TF and for the formation of FVIIa-TF-FXa-TFPI complex.