Activated Protein C has a Regulatory Role in Factor VIII Function

• Activated protein C contributes to the in vivo regulation of factor VIIIa.

• Factor VIIIa resistance to APC may be exploited to develop novel hemophilia A therapeutics.

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII and biochemical studies suggest APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein to an APC resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated a dose-dependent sensitivity to APC with or without protein S, while FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately 5-fold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, the tail clip assay was performed in homozygous HA/FV-Leiden mice infused with FVIII-QQ or FVIII-WT in the presence or absence of mAb1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FV-Leiden mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, supporting the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.