Abstract
Factor VIII (FVIII) circulates as an inactive procofactor and is converted to its active form (FVIIIa) by proteolytic cleavage at Arg372, Arg740, and Arg1689. Although these cleavage events are well characterized, their individual contributions to FVIII activation and hemostatic function remain incompletely defined. To address this, we engineered a FVIII variant (FVIII-2RKR) in which the B-domain and acidic region 3 region were replaced with a PACE/furin processing site, generating a molecule that requires only cleavage at Arg372 for conversion to FVIIIa. FVIII-2RKR exhibited markedly increased apparent activity in plasma-based assays, shortened thrombin generation lag time, and enhanced factor Xa (FXa) generation compared with B-domain-deleted FVIII, whereas both proteins were functionally equivalent following thrombin activation. FVIII-2RKR was cleaved more rapidly at Arg372 by thrombin and FXa, demonstrating that processing at Arg740 and Arg1689 regulates the rate of Arg372 cleavage rather than directly contributing to cofactor activity. In hemophilia A mice, FVIII-2RKR consistently enhanced early hemostatic function across ferric chloride, laser injury, and tail bleeding models, achieving improved efficacy at lower doses. Despite reduced half-life due to impaired von Willebrand factor (vWF) binding, FVIII-2RKR remained effective in vWF-deficient mice, indicating that vWF is not required for local FVIII function at sites of vascular injury. Together, these findings define distinct roles for FVIII activation cleavage sites and demonstrate that processing at Arg740 and Arg1689 regulates activation through Arg372, linking the rate of FVIII activation to hemostatic efficacy in vivo.