Regulation of von Willebrand Factor Reactivity

描述(由申请人提供)：血栓性血小板减少性紫癜(TTP)是一种灾难性的、可能致命的微血管血栓形成疾病，与先天性或获得性血浆金属蛋白酶ADAMTS-13缺陷有关。ADAMTS-13唯一已知的功能是切割von Willebrand因子(VWF)，这是一种新分泌的多聚体蛋白，当新分泌时自发结合并激活血小板，导致全身血栓形成。新分泌的VWF很大，具有极强的粘附性，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、更少粘连的形式。虽然ADAMTS-13缺乏对于TTP的临床表现是必要的，但这是不够的，从以下事实可以清楚地看出，先天缺乏酶的患者通常直到第二或第三个十年才表现出TTP。我们已经证明ULVWF以不同于血浆VWF的构象存在，这种构象允许它自发地与血小板受体糖蛋白(GP)Ib1结合。我们假设，这种替代构象是基于两种形式的VWF中不同的二硫键模式，并且高活性的构象不仅可以通过蛋白质分解，而且可以通过二硫异构化或还原来转化为活性较低的构象。本申请中提出的实验将检验这一假设，并将基于该假设评估TTP的潜在治疗方法。我们有三个具体的目标：1)通过确定ULVWF A1-A2-A3区域的二硫键结构来确定VWF高活性形式的分子性质。我们将比较A1-A2-A3区域内血浆VWF和ULVWF中二硫键的排列，该区域包含GP Ib1和胶原的结合部位，以及ADAMTS-13裂解部位。我们期望这将有助于揭示ULVWF高反应性的基础。2)研究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-乙酰半胱氨酸可以缩小ULVWF多聚体的大小，并调节血浆VWF和ULVWF的功能。我们将探索这种作用的分子基础，并检查该药物是否可以预防或治疗ADAMTS-13缺陷小鼠模型中诱导的TTP。公共卫生相关性：在这项应用中，我们努力找出为什么血浆蛋白von Willebrand因子(VWF)在第一次从制造它的细胞中释放出来时对血小板非常粘性。如果这种蛋白质没有被正常加工，它会导致严重的血液凝结障碍，通常会导致患者死亡。我们提出的研究将帮助我们了解新的VWF与已被处理的VWF有何不同，我们将了解它是如何处理的。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.