ADAMTS13 structure and the molecular basis of VWF recognition and cleavage

ADAMTS13结构和VWF识别和裂解的分子基础

• 批准号: MR/M010260/1
• 负责人: Jonas Emsley
• 金额: $ 87.69万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FM010260%2F1
• 关键词: ADAMTS13; structure; molecular; basis; VWF

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.

血液凝固是对血管损伤的反应。这需要将血小板（专门的血细胞）募集到损伤部位，作为防止出血的第一个（许多）事件之一。这一过程高度依赖于一种在血液中循环的名为血管性血友病因子（VWF）的蛋白质。VWF完成这一任务的能力受到一种酶的调节，这种酶也存在于血液中，ADAMTS13，在非常单一的底物特定情况下，它会将VWF切割成更小的形式，从而更不能招募血小板。临床上，VWF缺乏是最常见的遗传性出血性疾病，而ADAMTS13缺乏的人患有危及生命的血栓性疾病，死亡率约为90%。血液中VWF和ADAMTS13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，也会影响心脏病发作和中风的可能性。ADAMTS13是一种高度特异性的蛋白水解酶，它只能在一个位点切割一种蛋白质（VWF），即使这样，也只能在非常特定的血液流动条件下进行。ADAMTS13由多个域组成。该酶的金属蛋白酶结构域包含切割VWF的活性位点，而另一个则可变地促进ADAMTS13与VWF的结合。尽管有这些知识，ADAMTS13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ADAMTS13的不同结构域片段的结构，包括自由形式和稳定复合物与特定抗体片段，可以帮助确定结构。此外，我们还将阐明ADAMTS13片段的结构，同时与VWF的相应片段结合。我们将通过ADAMTS13描述这些VWF片段的结合和裂解，并探讨钙结合对这一过程的影响。该项目的信息将为ADAMTS13在分子水平上如何发挥作用提供重要的见解，其独特的单一底物特异性切割VWF。该数据将为合理设计ADAMTS13提供机会，以提高其作为治疗剂的疗效，目前正在开发ADAMTS13作为治疗血栓性疾病的更特异性凝块抑制剂。