Regulation of von Willebrand Factor Reactivity

描述（由申请方提供）：血栓性血小板减少性紫癜（TTP）是一种灾难性和潜在致死性微血管血栓性疾病，与血浆金属蛋白酶ADAMTS-13的先天性或获得性缺陷相关。ADAMTS-13的唯一已知功能是切割血管性血友病因子（vonWillebrand factor，VWF），VWF是一种多聚体蛋白，当新分泌时，其自发地结合并激活血小板，导致全身血栓形成。新分泌的VWF是大的和巨大的粘附性，并已被命名为异常大或超大VWF（ULVWF）。ADAMTS-13将ULVWF转化为在血液中循环的更小和更少粘附的形式。虽然ADAMTS-13缺乏症是TTP临床表现所必需的，但它还不够，因为从酶先天性缺乏的患者通常直到生命的第二或第三个十年才表现出TTP的事实可以清楚地看出。我们已经表明，ULVWF存在于一个不同的构象比血浆VWF，构象，使其能够自发地结合血小板受体糖蛋白（GP）Ib 1。我们假设，这种替代构象是基于不同的模式的二硫键中的两种形式的VWF和高反应性的构象可以被转换为反应性较低的构象，不仅通过蛋白水解，但也通过二硫键异构化或还原。本申请中提出的实验将检验该假设，并将基于该假设评估TTP的潜在疗法。我们有三个具体目标：1）通过测定ULVWF A1-A2-A3区域的二硫键结构来确定VWF的高反应性形式的分子性质。我们将比较血浆VWF和ULVWF中A1-A2-A3区域内二硫键的排列，该区域包含GP Ib 1和胶原蛋白的结合位点以及ADAMTS-13切割位点。我们希望这将有助于阐明ULVWF高反应性的基础。2)检查VWF二硫键异构化/还原在VWF反应性中的作用。在这里，我们将研究不同的氧化还原条件和二硫键还原酶/异构酶将如何影响VWF和ULVWF的功能。3)进一步评价N-乙酰半胱氨酸治疗血栓性血小板减少性紫癜的疗效。我们有证据表明，N-乙酰半胱氨酸可以减少ULVWF多聚体的大小和调节血浆VWF和ULVWF的功能。我们将探索这种效应的分子基础，并研究这种药物是否可以预防或治疗ADAMTS-13缺乏症小鼠模型中诱导的TTP。公共卫生相关性：在本申请中，我们奋进找出为什么血浆蛋白，血管性血友病因子（VWF），是非常粘血小板时，它是第一次从细胞中释放，它是在其中。如果这种蛋白质不能正常加工，它会导致严重的凝血障碍，通常会导致受影响患者的死亡。我们提出的研究将帮助我们了解新的VWF与已经加工的VWF有何不同，我们将了解它是如何加工的。通过这种方式，我们将能够了解和治疗凝血障碍。

DESCRIPTION (provided by applicant): Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal microvascular thrombotic disorder associated with congenital or acquired deficiency of the plasma metalloprotease ADAMTS-13. The only known function of ADAMTS-13 is to cleave von Willebrand factor (VWF), a multimeric protein that when newly secreted spontaneously binds and activates platelets, accounting for the systemic thrombosis. The newly secreted VWF is large and tremendously adhesive, and has been named unusually large or ultra-large VWF (ULVWF). ADAMTS-13 converts ULVWF to smaller and less adhesive forms that circulate in the blood. Although ADAMTS-13 deficiency is necessary for the clinical manifestation of TTP, it is not sufficient, as is clear from the fact that patients congenitally deficient in the enzyme often do not manifest TTP until the second or third decade of life. We have shown that ULVWF exists in a different conformation than plasma VWF, a conformation that allows it to spontaneously bind the platelet receptor glycoprotein (GP) Ib1. We hypothesize that this alternative conformation is based on a different pattern of disulfide bonds in the two forms of VWF and that the hyperreactive conformation can be converted to the less reactive conformation not only by proteolysis but also by disulfide isomerization or reduction. The experiments proposed in this application will test that hypothesis and will evaluate a potential therapy for TTP based on that hypothesis. We have three Specific Aims: 1) To determine the molecular nature of the hyperreactive form of VWF by determining the disulfide-bonded structure of the ULVWF A1-A2-A3 region. We will compare the arrangement of disulfide bonds in plasma VWF and ULVWF within the A1-A2-A3 region, which contains binding sites for GP Ib1 and collagen, as well as the ADAMTS-13 cleavage site. We expect that this will shed light on the basis of ULVWF hyperreactivity. 2) To examine the role of VWF disulfide isomerization/reduction in VWF reactivity. Here, we will examine how different redox conditions and disulfide reductases/isomerases will affect the functions of VWF and ULVWF. 3) To evaluate further the effect of N-acetylcysteine as a potential treatment for thrombotic thrombocytopenic purpura. We have evidence that N-acetylcysteine can reduce the size of ULVWF multimers and modulate the functions of both plasma VWF and ULVWF. We will explore the molecular basis of this effect and examine whether this agent can prevent or treat induced TTP in a mouse model of ADAMTS-13 deficiency. PUBLIC HEALTH RELEVANCE: In this application, we endeavor to find out why a plasma protein, von Willebrand factor (VWF), is very sticky for platelets when it is first released from the cells in which it is made. If this protein is not normally processed, it causes a severe blood clotting disorder that often leads to death of the affected patient. The studies we propose will help us learn how the new VWF is different from that VWF that has been processes and we will learn how it is processed. In this way, we will be able to understand and treat disorders of blood clotting.