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

Objective
An 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 Results
SuperFVa 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.
Conclusions
Our 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.

研究目标
接受活化蛋白C（activated protein C, APC）治疗的患者可出现出血风险升高的情况，该现象或成为新型APC疗法临床应用的限制因素。由于APC的治疗效应通常依赖其对细胞的细胞保护活性，而非其抗凝活性，因此若能开发一种特异性拮抗APC的抗凝作用、却不影响其细胞保护作用的制剂，便可为控制出血风险顾虑提供有效手段。我们提出假说：superFVa——一种可恢复血友病患者止血功能的工程化活化凝血因子Va（activated factor Va, 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）引入点突变，并在A2与A3结构域之间构建二硫键（Cys609-Cys1691），成功工程化构建了SuperFVa。该改造可增强其生物学活性，并使其对APC产生高抗性。在野生型（wild-type, wt）FVa未显现活性的浓度条件下，SuperFVa可恢复APC延长的凝血时间，并修复人及小鼠血浆中被APC抑制的凝血酶生成。向BALB/c小鼠静脉注射APC后，于体外全血样本中加入SuperFVa（而非野生型FVa），可显著恢复全血凝血功能。在BALB/c小鼠接受APC静脉给药前预先静脉给予SuperFVa，可显著降低小鼠尾部夹伤或肝脏撕裂后的失血量。此外，SuperFVa可消除APC治疗小鼠因肝脏撕裂后过度出血引发的死亡风险（该模型下小鼠死亡率约50%）。

研究结论
本研究结果证实了SuperFVa可有效预防APC诱导的出血，且在出血风险较高的多种临床场景中，作为促止血制剂可带来潜在治疗获益。