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

ObjectiveAn increased risk of bleeding is observed in patients receiving activated protein C (APC), which may be a limiting factor for the application of novel APC therapies. Since APC's therapeutic effects often require its cytoprotective activities on cells but not APC's anticoagulant activities, an agent that specifically antagonizes APC's anticoagulant effects but not its cytoprotective effects could provide an effective means to control concerns for risk of bleeding. We hypothesized that superFVa, an engineered activated FVa-variant that restores hemostasis in hemophilia could reduce APC-induced bleeding.Approach and ResultsSuperFVa was engineered with mutations of the APC cleavage sites (Arg506/306/679Gln) and a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains, which augment its biological activity and cause high resistance to APC. SuperFVa normalized APC-prolonged clotting times and restored APC-suppressed thrombin generation in human and murine plasma at concentrations where wild-type (wt) FVa did not show effects. Following intravenous injection of APC into BALB/c mice, addition to whole blood ex vivo of superFVa but not wt-FVa significantly normalized whole blood clotting. Blood loss following tail clip or liver laceration was significantly reduced when superFVa was administered intravenously to BALB/c mice prior to intravenous APC-treatment. Furthermore, superFVa abolished mortality (∼50%) associated with excessive bleeding following liver laceration in mice treated with APC.ConclusionsOur results provide proof of concept that superFVa is effective in preventing APC-induced bleeding and may provide therapeutic benefits as a prohemostatic agent in various situations where bleeding is a serious risk.

研究背景：接受活化蛋白C（activated protein C）治疗的患者会出现出血风险升高的情况，这可能成为新型APC疗法应用的限制因素。由于APC的治疗效果通常依赖其对细胞的细胞保护活性，而非抗凝活性，因此一种能够特异性拮抗APC的抗凝作用、却不影响其细胞保护作用的制剂，将为控制出血风险相关的担忧提供有效手段。本研究假设：superFVa——一种可在血友病中恢复止血功能的工程化活化FVa变体——能够降低APC诱导的出血风险。研究方法与结果：本研究通过对APC切割位点（Arg506/306/679Gln）进行突变，并在A2与A3结构域之间引入二硫键（Cys609-Cys1691），成功构建了superFVa，该变体可增强其生物学活性并获得对APC的高抗性。在野生型（wt）FVa无法发挥作用的浓度下，superFVa能够校正APC延长的凝血时间，并恢复APC抑制的人源和鼠源血浆中的凝血酶生成。在向BALB/c小鼠静脉注射APC后，体外全血中加入superFVa（而非野生型FVa）可显著校正全血凝血功能。在APC治疗前向BALB/c小鼠静脉给予superFVa，可显著降低小鼠尾剪或肝撕裂后的失血量。此外，在接受APC治疗的肝撕裂小鼠中，superFVa完全消除了因过度出血导致的约50%死亡率。结论：本研究结果证实了superFVa可有效预防APC诱导的出血，且可作为促止血制剂在各类存在严重出血风险的场景中发挥治疗益处。