LAUSR

Significance Coagulation factor VIIa (FVIIa) is an intrinsically poor serine protease that requires assistance from its cofactor tissue factor (TF) to trigger the extrinsic pathway of blood coagulation. TF stimulates FVIIa through allosteric maturation of its active site and by facilitating substrate recognition. The surface dependence of the latter property allowed us to design a potent membrane-triggered activity switch in FVIIa by engineering a disulfide cross-link between an allosterically silent FVIIa variant and soluble TF. These results show that optimization of substrate recognition remote from the active site represents a promising new route to simultaneously enhance and localize the procoagulant activity of FVIIa for therapeutic purposes. Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.