Proteolytic Regulation of Factor VIII Before and After Activation

Abstract Maintaining factor VIII (FVIII) in the normal range is critical for normal hemostasis. Deficiency of FVIII results in the bleeding disorder hemophilia A (HA) and excess FVIII confers prothrombotic risk. While the impact of serine protease cleavage on FVIII function has been extensively studied, the contribution of proteolysis on plasma FVIII levels is largely unexplored. Further, we demonstrated that the significance of proteolytic inactivation of activated FVIII (FVIIIa) has been underestimated. The overall objective of this proposal is to comprehensively investigate the proteolytic mechanisms that govern FVIII regulation before and after activation. Factor VIII circulates in plasma with its carrier protein von Willebrand factor (vWF). Prior to activation, FVIII levels in plasma are impacted by endothelial stimulation (where FVIII is synthesized and stored) and binding to vWF, which regulates FVIII clearance. We generated preliminary data in multiple model systems that support proteolysis of FVIII contributes, in part, to plasma FVIII concentration. A major goal of this application is to understand how proteolysis of FVIII or FVIII/vWF modulates plasma FVIII concentration, which will be investigated in Aim 1. Following activation, downregulation of FVIIIa function occurs through spontaneous dissociation of the A2-domain and cleavage by activated protein C (APC). Kinetic analysis suggests that A2 dissociation surpasses the rate of APC-mediated proteolysis leading to the general conclusion that APC minimally, if at all, contributes to FVIIIa inactivation. However, we demonstrated that a FVIII variant resistant to APC cleavage exhibits a 4-5 fold enhanced hemostatic effect and poses a greater prothrombotic risk in vivo compared to wild-type FVIII (FVIII-WT). Thus, our findings underscore the physiological relevance of APC inactivation of FVIIIa, challenging established mechanistic concepts entrenched in the field. This may be partly attributed to experimental constraints of studying FVIIIa inactivation in vitro. For example, purified system analysis of the kinetics of APC cleavage of FVIIIa have been predominantly studied on FVIII that underestimates the rate of APC cleavage of FVIIIa or the challenge of accurately modeling macromolecular interactions that impact FVIIIa inactivation. Another major objective is to comprehensively elucidate the mechanism underlying the proteolytic inactivation of FVIIIa that will be investigated in Aim 2. Our research will provide new mechanistic insight into FVIII biology, physiologic FVIIIa regulation and inform FVIII associated prothrombotic risk. These findings will directly inform the safety and efficacy of our and others’ work that aims to overcome current limitations of HA gene therapy limitations by using rationally engineered gain-of-function FVIII variants that bypass mechanisms of FVIIIa regulation.