The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential

• The combined mutation of D519V/E665V and K1813A in FVIII increased FVIIIa stability and the association between FVIIIa and FIXa.

• The FVIII-D519V/E665V/K1813A mutant exhibited an ~8-fold enhanced coagulation potential compared with FVIII wild type.

The A2 domain dissociation in activated factor (F)VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. Aim is to assess the D519V/E665V/K1813A-FVIII mutation as a gain-of-function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a BHK cell system, and global coagulation potential of these mutants was compared with WT FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT. The WT-FVIII activity after thrombin activation increased by ~12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ~25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ~50-fold slower than WT in a one-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable to WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 µg/kg) was equal to WT (2 µg/kg). FVIII-D519V/E665V/K1813A mutant could provide an ~8-fold increase in hemostatic function of WT FVIII, due to increased FVIIIa stability and the association between FVIIIa and FIXa.