Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100%, VIIQ217A 15%, and VIIQ217G -1 s-1 x 107) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x 103 M-1 min-1), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q217 is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.
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