Ultra-Large VWF and Thrombotic Microangiopathy

血栓性血小板减少性紫癜(TTP)是一种严重的血栓性疾病，由血凝素蓄积引起 血浆内皮源性超大VWF(ULVWF)多聚体。这些ULVWF多聚体在 与它们的受体相互作用。ULVWF的积累是由于VWF裂解酶存在缺陷，该酶具有 最近被鉴定为ADAMTS 13金属蛋白酶。这种蛋白酶将ULVWF多聚体切割成更小的 但卵裂如何发生以及发生在哪里仍是完全未知的。我们最近发现， ULVWF多聚体在分泌时附着于内皮细胞表面，形成极长的线状结构。 这些VWF链在流体剪切力下被ADAMTS13金属蛋白酶迅速切割。基于这些 结果：我们推测ADAMTS13金属蛋白酶切割内皮细胞上的ULVWF多聚体 在流体剪应力作用下的表面。为了加深我们对这一蛋白质分解过程的理解，我们建议在本申请中 来回答两个具体的问题。首先，VWF串是如何形成的，它们是如何锚定到内皮细胞上的？我们 发现P-选择素抗体阻止了VWF串的形成，并假设VWF串形成为 ULVWF通过前肽自结合和通过P-选择素的膜锚定的结果。第二，什么是 需要被ADAMTS13金属蛋白酶切割这些VWF弦吗？我们发现VWF的切割是 被RGD多肽和Thrabospondin阻断，这表明金属蛋白酶也可能锚定在 内皮表面。我们假设金属蛋白水解酶通过与内皮细胞形成复合体来裂解血管内皮因子。 整合素(Avb3)和VWF串。我们已经为这个特定的目标设计了实验来检验这些可能性。这个 所提出的研究对于了解ULVWF多聚体的切割和ULVWF多聚体的病理生理学非常重要。 TTP有几种方式。首先，它们将帮助我们阐明欧共体派生的ULVWF 多聚体被ADAMTS13金属蛋白酶切割，因此，提供了关于如何其他 ADAMTS13家族的成员可以发挥作用。尽管这个MetaUp酶家族的14个成员已经被 经过鉴定，目前只有两个已知的功能，包括ADAMTS13。其次，我们的研究可能会揭示出 其他类型的微血管病变也会发生，特别是由药物和感染引起的HUS和TTP。最后，他们允许 美国开发了一种临床测试方法来测试VWF裂解酶的活性。因此，赋予诊断的能力 TTP。这项测试将是快速的，更具生理学意义。

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 mulfimers 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 throrabospondin, 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 mulfimers 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 metaUoprotease 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.