Pryzdial E L G, Meixner S C, Talbot K, Eltringham-Smith L J, Baylis J R, Lee F M H, Kastrup C J, Sheffield W P
Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.
J Thromb Haemost. 2016 Sep;14(9):1844-54. doi: 10.1111/jth.13402. Epub 2016 Aug 17.
Essentials Factor Xa (FXa) acquires cleavage-mediated tissue plasminogen activator (tPA) cofactor activity. Recombinant (r) tPA is the predominant thrombolytic drug, but it may cause systemic side effects. Chemically modified, non-enzymatic FXa was produced (Xai-K), which rapidly lysed thrombi in mice. Unlike rtPA, Xai-K had no systemic fibrinolysis activation markers, indicating improved safety.
Background Enzymatic thrombolysis carries the risk of hemorrhage and re-occlusion must be evaded by co-administration with an anticoagulant. Toward further improving these shortcomings, we report a novel dual-functioning molecule, Xai-K, which is both a non-enzymatic thrombolytic agent and an anticoagulant. Xai-K is based on clotting factor Xa, whose sequential plasmin-mediated fragments, FXaβ and Xa33/13, accelerate the principal thrombolytic agent, tissue plasminogen activator (tPA), but only when localized to anionic phospholipid. Methods The effect of Xai-K on fibrinolysis was measured in vitro by turbidity, thromboelastography and chromogenic assays, and measured in a murine model of occlusive carotid thrombosis by Doppler ultrasound. The anticoagulant properties of Xai-K were evaluated by normal plasma clotting assays, and in murine liver laceration and tail amputation hemostatic models. Results Xa33/13, which participates in fibrinolysis of purified fibrin, was rapidly inhibited in plasma. Cleavage was blocked at FXaβ by modifying residues at the active site. The resultant Xai-K (1 nm) enhanced plasma clot dissolution by ~7-fold in vitro and was dependent on tPA. Xai-K alone (2.0 μg g(-1) body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 μg g(-1) ), could be reduced by > 30-fold to restore blood flow with adjunctive Xai-K (0.5 μg g(-1) ). TNK-induced systemic markers of fibrinolysis were not detected with Xai-K (2.0 μg g(-1) ). Xai-K had anticoagulant activity that was somewhat attenuated compared with a previously reported analogue. Conclusion These results suggest that Xai-K may ameliorate the safety profile of therapeutic thrombolysis, either as a primary or tPA/TNK-adjunctive agent.
凝血因子Xa（FXa）可获得切割介导的组织纤溶酶原激活剂（tPA）辅因子活性。重组（r）tPA是主要的溶栓药物，但可能会引起全身副作用。制备了化学修饰的非酶促FXa（Xai-K），它能迅速溶解小鼠体内的血栓。与rtPA不同，Xai-K没有全身纤溶激活标志物，表明其安全性有所提高。
背景 酶促溶栓存在出血风险，必须通过与抗凝剂联合使用来避免再闭塞。为了进一步改善这些缺点，我们报告了一种新型双功能分子Xai-K，它既是一种非酶促溶栓剂又是一种抗凝剂。Xai-K基于凝血因子Xa，其连续的纤溶酶介导片段FXaβ和Xa33/13可加速主要溶栓剂组织纤溶酶原激活剂（tPA）的作用，但仅在定位于阴离子磷脂时才起作用。方法 通过比浊法、血栓弹性描记法和显色测定法在体外测量Xai-K对纤溶的作用，并通过多普勒超声在小鼠闭塞性颈动脉血栓形成模型中进行测量。通过正常血浆凝血测定以及在小鼠肝裂伤和断尾止血模型中评估Xai-K的抗凝特性。结果 参与纯化纤维蛋白纤溶的Xa33/13在血浆中被迅速抑制。通过修饰活性位点的残基，在FXaβ处的切割被阻断。所得的Xai-K（1 nM）在体外使血浆凝块溶解增强约7倍，且依赖于tPA。单独使用Xai-K（2.0 μg g(-1)体重）在小鼠中实现了治疗性再通。tPA变体替奈普酶（TNK；17 μg g(-1)）的最小初始剂量可减少>30倍，与辅助性Xai-K（0.5 μg g(-1)）一起恢复血流。使用Xai-K（2.0 μg g(-1)）未检测到TNK诱导的全身纤溶标志物。Xai-K具有抗凝活性，与先前报道的类似物相比有所减弱。结论 这些结果表明，Xai-K作为主要药物或tPA/TNK辅助药物，可能会改善治疗性溶栓的安全性。
