Engineered recombinant factor VII Q(217) variants with altered inhibitor specificities

Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VII Q217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrate d 100%, VIIQ217A 15%, and VIIQ217G <1% clotting activities relative to wild -type VII. Binding of VIIQ217A and VIIQ217G to TF was comparable to that of wild-type VII to TF. All the variants were readily activated by factor Xa. Autoactivation in the presence of TF was efficient with VIIQ217E, slow wit h VIIQ217A, but undetected with VIIQ217G. Relative to wild-type VII added a t the same concentration, VIIQ217E had no effect on the PT of normal plasma , whereas VIIQ217A slightly and VIIQ217G dramatically prolonged the clottin g time in a dose-dependent manner. Activation of macromolecular substrates paralleled this functional inhibition. The k(cat)/K-M values for factor X a ctivation in the presence of TF were 2.4 for VIIaQ217E as compared to 1.9 ( M-1 s(-1) x 10(7)) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VI TaQ217G. 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 demonstr ated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less effic iently by TFPI either in the presence or in the absence of factor Xa. In co ntrast, VIIaQ217A association with antithrombin III in the presence of hepa rin was the fastest among the variants with a second-order rate constant of 2.31 (x 10(3) M-1 min(-1)), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q(217) is important in regulating substrate and, more importantly, inhibitor recog nition by VIIa.