Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa(EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation wit h zymogen-like properties. Association with tissue factor (TF), locally exp osed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by r eplacing residues believed to function as determinants for the inherent zym ogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appe ar to retain the substrate specificity of the parent enzyme and can be furt her stimulated by TF. Insights into the mechanism behind the increased acti vity of the mutants, presumably also pertinent to the TF-induced, allosteri c stimulation of FVIIa activity, were obtained by studying their calcium de pendence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficaci ous treatment of bleeding episodes especially in hemophiliacs with inhibito ry antibodies precluding conventional replacement therapy.