Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.
Geoffrey Jefferson Brain Research Centre, Manchester Academic Health Science Centre, Northern Care Alliance National Health Service (NHS) Group-University of Manchester, Manchester, United Kingdom.
Blood. 2022 Mar 10;139(10):1575-1587. doi: 10.1182/blood.2021012787.
Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.
我们对 ADAMTS13 结构及其完全活性所需构象变化的认识的进步，重新燃起了将其用作溶栓治疗的可能性。我们已经测试了一种新型的 Ala1144Val ADAMTS13 变体（组成型活性[ca] ADAMTS13），该变体表现出组成型活性，使用 ADAMTS13 活性的体外测定进行了表征，并在 2 种缺血性中风的啮齿动物模型中极大地增强了溶栓活性，即远端 FeCl3 大脑中动脉闭塞（MCAo）模型和伴有全身炎症和缺血/再灌注损伤的短暂性大脑中动脉闭塞（tMCAO）。这两种模型中的主要疗效衡量标准是恢复 MCA 区域的局部脑血流（rCBF），这是通过激光散斑对比成像来确定的。与野生型（wt）ADAMTS13 相比，caADAMTS13 变体表现出组成型活性构象和对荧光共振能量转移底物 von Willebrand 因子 73（FRETS-VWF73）的五倍增强活性。此外，caADAMTS13 在亚生理浓度下抑制 VWF 介导的血小板捕获，并增强 t-PA/纤溶酶对纤维蛋白（原）的溶解，而 wtADAMTS13 则没有观察到这两种作用。用 caADAMTS13 治疗的动物观察到 rCBF 的显著恢复和病变体积的减少。在 FeCl3 MCAo 后 1 小时给予 caADAMTS13 变体时，caADAMTS13 变体可显著减少 MCA 中的残留 VWF 和纤维蛋白沉积物、血小板聚集形成和中性粒细胞募集。在 tMCAo 模型中再灌注 4 小时后给予 caADAMTS13 变体时，caADAMTS13 变体诱导血小板聚集物的显著溶解，并减少由此产生的组织低灌注。由于其组成型活性构象导致的增强的体外和体内活性，caADAMTS13 变体代表了一种有希望的治疗急性缺血性中风以及其他血栓形成适应症的治疗选择。
