Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant

Abstract

Summary Background Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia. Objectives To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A. Methods Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice. Results and conclusions FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). ‘superFVa,’ a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVLeiden(Arg506Gln), or FVaLeiden(A2-SS-A3). Furthermore, superFVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, superFVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose superFVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, superFVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that superFVa enhances not only clot formation but also clot stabilization. Thus, superFVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

An Engineered Factor Va Prevents Bleeding Induced by Anticoagulant wt Activated Protein C

“…The mechanism by which super FVa prevents APC-induced bleeding is consistent with FVa activity augmentation within the prothrombinase complex [1]. It is well described that the presence of FVa in the prothrombinase complex potently enhances the rate of thrombin generation to approximately 10,000-fold [2], [29].…”

“…Both super FVa and wt-FVa dose-dependently normalized the aPTT in normal human plasma in the presence of 10 nM APC ( Figure 1a ) . There was an approximately two orders of magnitude difference in efficacy between super FVa and wt-FVa, which cannot be explained by the approximately 1.5 to 3-fold higher specific activity of super FVa in the prothrombinase assay [1]. This result suggests that the APC cleavage site mutations in super FVa were likely responsible for the increased efficacy of super FVa to normalize aPTT clotting times.…”

“…The biochemical characterization of super FVa and its efficacy of bleed control in a hemophilic mouse model after tail clip was previously published [1]. Because B-domain deleted FV has some inherent cofactor activity, only activated FVa's were compared.…”

“…We hypothesize that super FVa, an engineered FVa-variant that efficiently normalizes hemostasis in hemophilia A [1], fits the criteria for a prohemostatic biologic and may provide beneficial effects for bleeding associated with treatment of activated protein C (APC). As an anticoagulant enzyme, APC proteolytically inactivates activated factor V (FVa) and FVIIIa.…”

Safety, Stability and Pharmacokinetic Properties of superFactor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding

“…The molecule was engineered to be resistant against irreversible proteolytic inactivation by activated protein C (APC) involving mutation of all three APC cleavage sites at Arg506, Arg306 and Arg679. In addition, an engineered interdomain disulfide bond (involving His609Cys-Glu1691Cys) connecting the A2 and A3 domains (A2-SS-A3) was introduced (1), which confers increased specific activity, presumably by preventing domain dissociation as the final step of FVa inactivation (1,2). Super FVa is a potent molecule to achieve “FVa activity augmentation”, which is emerging as a new concept to enhance hemostasis (2).…”