Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer’s anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer’s activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

凝血辅助因子深刻地调节止血并且是抗凝剂的有吸引力的目标。然而，靶向此类蛋白质一直具有挑战性，因为它们缺乏活性位点。为了解决这个问题，我们分离出一种称为 T18.3 的 RNA 适配体，它以纳摩尔亲和力与因子 V (FV) 和 FVa 结合，并在血浆和全血中显示出临床相关的抗凝活性。该适配体还显示出与低分子量肝素的协同作用，并在从 2019 年冠状病毒病 (COVID-19) 患者收集的血浆中提供有效的抗凝作用。此外，使用硫酸鱼精蛋白可以快速有效地逆转适体的抗凝活性，这可能允许在给药后微调适体的活性。我们进一步表明，适体通过消除 FV/FVa 与磷脂膜的相互作用来实现其抗凝血活性。我们在生成靶向 FV/Va 的抗凝血适体方面的成功证明了使用辅因子结合适体作为治疗性蛋白质抑制剂的可行性，并通过中断蛋白质-膜相互作用揭示了适体的非常规工作机制。