Ultra-Large VWF and Thrombotic Microangiopathy

描述（由申请人提供）：血栓性血小板减少性紫癜（TTP）是一种由血浆中内皮源性超大型VWF （ULVWF）多聚体积累引起的严重血栓性疾病。这些ULVWF多聚体在与其受体相互作用时具有高度反应性。ULVWF的积累是由于VWF切割蛋白酶的缺陷，该蛋白酶最近被表征为ADAMTS 13金属蛋白酶。这种蛋白酶将ULVWF多聚体切割成更小的血浆形式，但切割发生的方式和位置仍然完全未知。我们最近发现，ULVWF多聚体分泌后，粘附在内皮表面，形成极长的串状结构。在流体剪切应力作用下，ADAMTS13金属蛋白酶快速剪切了这些VWF串。基于这些结果，我们假设ADAMTS13金属蛋白酶在流体剪切应力下切割内皮表面的EC ULVWF多聚体。为了进一步了解这一蛋白质水解过程，我们建议在本申请中回答两个具体问题。首先，VWF字符串是如何形成的，又是如何固定在内皮细胞上的？我们发现p -选择素抗体阻止了VWF链的形成，并假设VWF链的形成是通过p -选择素通过前肽和膜锚定ULVWF自结合的结果。其次，ADAMTS13金属蛋白酶切割这些VWF链需要什么条件？我们发现VWF的切割被RGD肽和血栓反应蛋白阻断，这表明金属蛋白酶也可能被锚定在内皮表面。我们假设金属蛋白酶通过与内皮整合素（avb3）和VWF串形成复合物来切割VWF。我们为此专门设计了实验来检验这些可能性。这些研究对于从多个方面了解ULVWF多聚体的分裂和TTP的病理生理具有重要意义。首先，它们将帮助我们阐明ec衍生的ULVWF多聚体被ADAMTS13金属蛋白酶切割的机制；因此，提供关于ADAMTS13家族其他成员如何发挥作用的信息。虽然这个金属蛋白酶家族的14个成员已经被表征，但只有两个成员的功能是已知的，包括ADAMTS13。其次，我们的研究可能会揭示其他类型的微血管病变是如何发展的，特别是由药物和感染引起的溶血性尿毒综合征和TTP。最后，它们使我们能够开发一种临床试验来测试vwf切割蛋白酶的活性，从而提供诊断TTP的能力。这种检测方法将快速且更符合生理学。

DESCRIPTION (provided by applicant):Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic disease caused by accumulation of the endothelium-derived ultra-large VWF (ULVWF) multimers in plasma. These ULVWF multimers are hyper-reactive in interacting with their receptors. The accumulation of ULVWF is due to defects in the VWF cleaving protease that has been recently characterized as the ADAMTS 13 metalloprotease. This protease cleaves ULVWF multimers to smaller plasma form, but how and where the cleavage occurs remains completely unknown. We have recently found that the ULVWF multimers, upon secretion, adhered to the endothelial surface, forming extremely long string-like structures. These VWF strings were rapidly cleaved by the ADAMTS13 metalloprotease under fluid shear stress. Based on these results, we hypothesize that the ADAMTS13 metalloprotease cleaves the EC ULVWF multimers on the endothelial surface under fluid shear stress. To further our understanding of this proteolytic process, we propose in this application to answer two specific questions. First, how do VWF strings form and how are they anchored to endothelial ceils? We found that P-selectin antibody prevented the formation of VWF strings and hypothesize that VWF strings form as a result of ULVWF self-association through propeptides and membrane anchorage through P-selectin. Second, what are required to cleave these VWF strings by the ADAMTS13 metalloprotease? We found that the cleavage of VWF was blocked by a RGD peptide and thrombospondin, suggesting that the metalloprotease may also be anchored to the endothelial surface. We hypothesize that the metalloprotease cleaves VWF by forming a complex with an endothelial integrin (avb3) and VWF strings. We have designed experiments in this specific aim to examine these possibilities. The proposed studies are very important for understanding the cleavage of ULVWF multimers and the pathophysiology of TTP in several ways. First, they will help us to elucidate mechanisms through which the EC-derived ULVWF multimers are cleaved by the ADAMTS13 metalloprotease and; therefore, provide information as to how other members of the ADAMTS13 family may function. Although 14 members of this family of metallprotease have been characterized, functions of only two, including ADAMTS13, are known. Second, our studies may shed light on how other types of microangiopathies develop, especially HUS and TTP caused by drugs and infections. Finally, they allow us to develop a clinical assay to test the activity of the VWF-cleaving protease thus giving the ability to diagnose TTP. This assay will be fast and more physiologically relevant.