Factor VIII mutated with Lys1813Ala within the factor IXa-binding region enhances intrinsic coagulation potential.

Factor VIII (FVIII) functions as a FIXa cofactor for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa-binding and that the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study was aimed to identify mutated FVIII protein(s) that increases FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells, followed by assessment using purified and global coagulation assays and hemophilia A (HA) mouse models. A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable to that of WT, while the kcat of this mutant was significantly greater than WT. Thrombin-catalyzed FVIII-K1813A activation was ~1.3-fold enhanced compared to WT, and the spontaneous decay of activated FVIII-K1813A was ~2.5-fold slower than that of WT. The heat-stability assay revealed that the decay rate of FVIII-K1813A was ~2.5-fold lower than that of WT. Thrombin generation assay and rotational thromboelastometry using HA patient blood samples demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited coagulation potential compatible with that of WT (1 nM). In the tail-clip assay of HA-mice, FVIII-K1813A showed a 2-4-fold higher hemostatic potential than WT. FVIII-K1813A, with higher FIXa binding affinity, enhances the global coagulation potential due to the stability of FVIII/FVIIIa molecules.