OC 49.5 - FVIII mutated with D519V/E665V/K1813A enhances coagulation potentials by high affinity for FIXa and stabilizing A2 domain

Background: Factor (F)VIII A2 dissociation predominantly contributes to activated FVIII (FVIIIa) inactivation. Previous studies demonstrated that FVIII mutants (D519VE665V and K1813A) showing the slower A2 dissociation increased the global coagulation potential. We hypothesized, therefore, that mutated FVIII by combining these mutations might enhance coagulant activity.

Aims: To create the gain-of-function FVIII mutant.

Methods: FVIII mutants with K1813A, D519V/E665V and D519V/E665V/K1813A were expressed in BHK cell system and were evaluated using purified and global coagulation assays. The global coagulation potential of these mutants was compared with that of WT FVIII using rotational thromboelastometry and tail-clip assays in hemophilia A mice in vivo.

Results: Kinetic analyses indicated that the Km for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the Vmax for D519V/E665V/K1813A was significantly greater than WT. The Vmax/Km of D519V/E665V/K1813A for FIXa was 3.7-fold higher relative to WT. As for thrombin-catalyzed activation, the FVIII activity (FVIII:C) of WT (10 nM) increased by 12-fold within 5 min after addition of thrombin (0.4 nM), and subsequently decreased to the initial levels within 30 min. The FVIII:C of D519V/E665V/K1813A increased with time after thrombin activation and showed 25-fold elevation at 120 min. The FVIII A2 dissociation of D519V/E665V/K1813A after thrombin activation was 50-fold slower than that of WT. Thrombin generation assay demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited similar coagulation potential to WT (1 nM) in plasma with hemophilia A. 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).

Conclusion(s): D519V/E665V/K1813A showed an ~8-fold increase in hemostatic function of WT-FVIII due to enhanced FVIIIa stability and high binding affinity for FIXa.