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 FVIII-K1813A 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 ∼1.3-fold more enhanced than that of the 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 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.

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© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.