Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Japan.
Blood Adv. 2023 Apr 25;7(8):1436-1445. doi: 10.1182/bloodadvances.2022008187.
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.
VIII 因子（FVIII）作为 FIXa 的辅因子在内在 tenase 复合物中激活 FX。已经观察到 FVIII A3 结构域中的 1811-1818 区域有助于 FIXa 结合，并且 K1813A/K1818A 突变体增加了与 FIXa 的结合亲和力。本研究旨在鉴定增加 1811-1818 区域中 FVIIIa 辅因子活性的突变 FVIII 蛋白。在仓鼠肾细胞中表达具有 K1813A、K1818A 和 K1813A/K1818A 的 FVIII 突变体，并通过使用纯化和全局凝血测定法在具有血友病 A（HA）的小鼠模型中进行评估。基于表面等离子体共振的测定法显示，FVIII-K1813A 与 FIXa 相互作用的 Kd 值低于野生型（WT）（3.9±0.7/6.3±0.3 nM）。然而，FVIII-K1813A 对 tenase 活性上的 FIXa 的 Km 值与 WT 相当，而该突变体的 kcat 显著大于 WT。凝血酶催化的 FVIII-K1813A 激活比 WT 增强约 1.3 倍，并且激活的 FVIII-K1813A 的自发衰变比 WT 慢约 2.5 倍。热稳定性测定法显示，FVIII-K1813A 的衰减率比 WT 慢约 2.5 倍。使用来自 HA 患者的血液样本进行的凝血酶生成测定和旋转血栓弹性测定表明，添加 0.5 nM 的 FVIII-K1813A 表现出与 WT（1 nM）兼容的凝血潜能。在 HA 小鼠的尾巴夹测定中，FVIII-K1813A 表现出比 WT 高两到四倍的止血潜能。FVIII-K1813A 具有更高的 FIXa 结合亲和力，由于 FVIII/FVIIIa 分子的稳定性，增强了整体凝血潜能。
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