https://orcid.org/0000-0003-1235-8950
Key Points
APC cleavage and A2 dissociation both contribute to in vivo FVIIIa inactivation
FIXa and FX modulate mechanisms of FVIIIa inactivation
Deficiency of (f)actor VIII causes hemophilia A and excess FVIII function increases venous thromboembolic risk. The phenotypic consequences of aberrant FVIII function underscore the importance of understanding mechanisms that downregulate (a)ctivated FVIII to inform disease pathology and therapeutic drug design. Spontaneous A2-domain dissociation and activated protein C (APC) proteolysis are established mechanisms of FVIIIa inactivation. However, we know very little about how FVIIIa binding interactions with FIXa and FX impact FVIIIa inactivation in vivo. Here we investigate this using recombinant FVIIIa variants to probe A2-domain dissociation (FVIIIa-D519V,E665V) and APC cleavage (FVIIIa-R336Q,R562Q) or both (FVIIIa-R336Q,R562Q/D519V,E665V) in biochemical assays and in HA mouse injury models. We found that FIXa binding to FVIIIa stabilized the A2 domain and increased the contribution of APC to FVIIIa inactivation. Additional studies using individual APC cleavage site variants (FVIIIa-R336Q and FVIIIa-R562Q) demonstrated that FIXa and FX can protect FVIIIa from APC cleavage at Arg562 and Arg336, respectively, in a manner that is incomplete in vivo. Data also demonstrate that APC inactivation of FVIIIa exceeds FVIII suggesting differential APC recognition of FVIIIa relative to FVIII. Hemostatic studies of FVIII variants with altered inactivation demonstrated that both A2-domain dissociation and APC cleavage contribute to in vivo FVIIIa regulation. Specifically, stabilizing the A2-domain, inhibiting APC cleavage, or both improved potency 2.4, 4.8, and >10-fold, respectively, over wild type FVIII in a mouse hemostatic assay. Data support that both mechanisms of FVIIIa inactivation and FIXa interactions could be leveraged to enhance FVIII function for therapeutic benefit.
