ADAMTS13 structure and the molecular basis of VWF recognition and cleavage

血液凝固是对血管损伤的反应。这需要血小板（专门的血细胞）的特定募集到损伤部位，作为预防出血的第一个（许多）事件之一。这个过程高度依赖于一种在血液中循环的蛋白质，称为血管性血友病因子（VWF）。VWF执行这一任务的能力是由一种酶调节的，这种酶也存在于血液中，ADAMTS 13，并且在非常单一的底物特异性情况下，将VWF切割成较小的形式，这些形式不太能够招募血小板。临床上，VWF缺乏是最常见的遗传性出血性疾病，而ADAMTS 13缺乏的人患有危及生命的血栓性疾病，死亡率约为90%。VWF和ADAMTS 13的血液水平/功能的更细微差异也是个体出血和血栓形成风险的重要决定因素，并且也影响心脏病发作和中风的可能性。ADAMTS 13是一种高度特异性的蛋白水解酶，仅切割一种蛋白质（VWF），并且仅在单一位点切割，即使如此，也仅在非常特定的血流条件下切割。ADAMTS 13由多个域组成。该酶的金属蛋白酶结构域含有切割VWF的活性位点，而其他的酶则有助于ADAMTS 13与VWF的结合。尽管有这些知识，但ADAMTS 13如何识别和切割VWF仍然不清楚。为了在分子水平上理解这一点，我们将确定ADAMTS 13的不同结构域片段的结构，无论是游离形式还是与特异性抗体片段的稳定复合物，都可以帮助确定结构。此外，我们还将阐明ADAMTS 13片段的结构，同时结合到相应的VWF片段。我们将研究ADAMTS 13对这些VWF片段的结合和切割，并探讨钙离子结合对这一过程的影响，这一研究结果将为ADAMTS 13在分子水平上如何发挥其独特的单一底物特异性切割VWF的功能提供重要的见解，为合理地改造ADAMTS 13以提高其作为治疗药物的疗效提供机会。目前正在开发其作为治疗血栓性疾病的更特异性的凝块剂。

Blood clotting occurs in response to blood vessel damage. This requires the specific recruitment of platelets (specialised blood cells) to the site of injury as one of the first (of many) events that prevents bleeding. This process is highly dependent upon a protein known as von Willebrand factor (VWF) that circulates in blood. The ability of VWF to perform this task is regulated by an enzyme that is also present in the blood, ADAMTS13, and that, under very single substrate specific circumstances, cleaves VWF into smaller forms that are less capable of recruiting platelets. Clinically, deficiency in VWF is the most common inherited bleeding disorder, whereas people with ADAMTS13 deficiency suffer from a life-threatening thrombotic disorder with a ~90% mortality rate. More subtle differences in the blood levels/function of VWF and ADAMTS13 are also important determinants of an individual's risk of bleeding and thrombosis, and also influence the likelihood of both heart attack and stroke. ADAMTS13 is a very highly specific proteolytic enzyme that cleaves only one protein (VWF) and does so at just a single site, and even then, only under very specific conditions of blood flow. ADAMTS13 is made up of multiple domains. The metalloprotease domain of this enzyme contains the active site that cleaves VWF, whereas the other variably contribute to the binding of ADAMTS13 to VWF. Despite this knowledge, how ADAMTS13 recognises and cleaves VWF so specifically remains unclear. To understand this at a molecular level, we will ascertain the structure of different domains fragments of ADAMTS13, both in free forms and in stabilising complexes with specific antibody fragments that can aid in determining structures. In addition, we will also elucidate the structure of ADAMTS13 fragments whilst bound to the corresponding fragments of VWF. We will characterise the binding and cleavage of these VWF fragments by ADAMTS13 and also explore the influence of calcium binding to this process.The information from this project will provide important insights into how ADAMTS13 functions at a molecular level its unique single substrate specific cleavage of VWF.This data will provide the opportunity to rationally engineer ADAMTS13 to improve its efficacy as a therapeutic agent, for which it is currently under development as a more specific clotbuster for the treatment of thrombotic disease.