Factor VIII Aurora: A Naturally Occurring Gain of Function FVIII Variant with Enhanced FIXa Affinity

Abstract

Factor VIII Aurora (FVIII-R571S) is the first described naturally occurring enhanced-potency FVIII variant identified in a patient with recurrent thrombosis and early mortality. The patient's plasma exhibited increased procoagulant activity and reduced responsiveness to activated protein C (APC). To define the mechanism, we generated recombinant FVIII-R571S and performed in vitro and in vivo studies. Consistent with the clinical phenotype, FVIII-R571S demonstrated a 6-fold increase in one-stage assay activity, while chromogenic substrate assay activity was comparable to wild-type FVIII (FVIII-WT). This discrepancy was explained by biochemical studies showing that activated FVIII-R571S (FVIIIa-R571S) has 10-20-fold higher affinity for FIXa; notably, the chromogenic assay is insensitive to differences in FVIIIa-FIXa affinity. Additional analyses demonstrated that FVIII-R571S is inactivated by APC and protein S analogous to FVIII-WT, indicating that the variant is not intrinsically APC-resistant. However, the increased affinity of FVIIIa-R571S for FIXa confers FIXa-dependent reduced A2-domain dissociation and APC-mediated inactivation in purified and plasma-based studies. These enhanced biochemical properties translated in vivo to a more potent procoagulant phenotype in hemophilia A mice. In the tail clip assay, FVIII-R571S exhibited a 4-5-fold increase in potency compared to FVIII-WT. In a thrombosis model, FVIII-R571S promoted significantly increased platelet and fibrin accumulation relative to FVIII-WT at equivalent antigen levels. Collectively, these data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function. This same mechanism confers reduced A2 dissociation and functional APC resistance, providing a unifying explanation for the observed gain-of-function phenotype.