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

Factor VIII (FVIII) functions as a cofactor of FIXa 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 the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9 +/- 0.7/6.3 +/- 0.3 nM). However, the Km value of FVIIIK1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was similar to 1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was similar to 2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was similar to 2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

Automated summary

This study aimed to identify mutated FVIII proteins that increase FVIIIa cofactor activity in the 1811-1818 region. The FVIII-K1813A mutant showed a lower affinity for FIXa binding but a significantly higher speed in FX activation compared to the wild-type. In coagulation assays, the addition of FVIII-K1813A displayed a clotting potential similar to the wild-type.