Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

中文翻译：

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Microlyse：一种溶栓剂，以 VWF 为靶点，清除微血管血栓。


血栓性微血管病的特征是播散性微血管血栓形成的发作。在血栓性血小板减少性紫癜 (TTP) 中，这是由于 ADAMTS13 缺陷导致血栓形成的超大冯维勒布兰德因子 (VWF) 多聚体增加所致。我们之前报道过，系统性纤溶酶原激活在 TTP 小鼠模型中具有治疗作用。与其天然激活剂（即组织纤溶酶原激活剂和尿激酶纤溶酶原激活剂[uPA]）相反，纤溶酶原可以直接与VWF结合。为了获得最佳功效和安全性，我们的目标是集中并加速微血管闭塞部位的纤溶酶原激活。我们在这里描述了 Microlyse 的开发和表征，这是一种融合蛋白，由针对 VWF 的 CT/CK 结构域和 uPA 的蛋白酶结构域的高亲和力 VHH 组成，用于微血栓上的局部纤溶酶原激活。在激活的内皮细胞上和在凝集研究中，Microlyse 会触发纤溶酶对血小板-VWF 复合物的靶向破坏。在等摩尔浓度下，Microlyse 降解微血栓的速度比用二价人源化 VHH（caplacizumab*）阻断血小板-VWF 相互作用的速度快七倍。最后，在 ADAMTS13-/- TTP 小鼠模型中，Microlyse 比 caplacizumab* 更有效地减轻血小板减少症和组织损伤（表现为血浆乳酸脱氢酶活性增加，以及 PAI-1 和纤维蛋白原水平），且不影响尾部止血。夹子出血模型。这些发现表明，Microlyse 对 VWF 进行靶向溶栓是治疗 TTP 的有效策略，并且可能对其他形式的 VWF 驱动的血栓性疾病有价值。