Regulation of von Willebrand Factor Reactivity

冯维勒布兰德因子反应性的调节

• 批准号: 8077284
• 负责人: Jose Aron Lopez
• 金额: $ 46.59万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8077284
• 关键词: ADAMTS; Accounting; Acetylcysteine; Adhesives; Affect; Binding; Binding Sites; Blood; Blood Clot; Blood Coagulation Disorders; Blood Platelets; Blood coagulation; C-terminal; Cells; Cessation of life; Cleaved cell; Clinical; Collagen; Data; Disease; Disulfides; Enzymes; Glycoprotein Ib; Glycoproteins; Health; Isomerase; Learning; Life; Light; Metalloproteases; Molecular; Molecular Conformation; Mus; Names; Nature; Oxidation-Reduction; Oxidoreductase; Patients; Pattern; Plasma; Plasma Proteins; Preparation; Process; Proteins; Proteolysis; Regulation; Role; Site; Structure; Testing; Thrombosis; Thrombotic Thrombocytopenic Purpura; base; disulfide bond; mouse model; prevent; receptor; research study; von Willebrand Factor

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.

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，潜在的致命性微血管血栓形成障碍，与先天性或血浆金属蛋白酶的缺乏相关的是金属蛋白酶ADAMTSS-13。 ADAMTS-13的唯一已知功能是裂解von Willebrand因子（VWF），这是一种多聚体蛋白，当新分泌的自发分泌时，它会自发结合并激活血小板，从而考虑了全身性血栓形成。新近分泌的VWF很大且非常粘合，并被称为异常大或超大的VWF（ULVWF）。 Adamts-13将ULVWF转换为循环中较小且较少的粘合剂形式。尽管ADAMTS-13缺乏对于TTP的临床表现是必要的，但这还不是足够的，从以下事实中可以清楚地看出，直到生命的第二十年或第三十年，先天患者在酶中的患者通常才表现出TTP。我们已经表明，ULVWF与血浆VWF不同的构象存在，该构象使其能够自发结合血小板受体糖蛋白（GP）IB1。我们假设这种替代构象基于两种形式的VWF中的二硫键键的不同模式，并且过度反应构象可以不仅通过蛋白水解，而且通过二硫化物异构化或减少来转化为反应性较低的构象。本应用程序中提出的实验将检验该假设，并将根据该假设评估TTP的潜在疗法。我们有三个特定的目的：1）通过确定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和胶原蛋白的结合位点，以及ADAMTS-13裂解位点。我们预计这将根据ULVWF的过度反应性来阐明。 2）检查VWF二硫化物异构化/降低VWF反应性的作用。在这里，我们将研究不同的氧化还原条件和二硫化物还原酶/异构酶将如何影响VWF和ULVWF的功能。 3）进一步评估N-乙酰半胱氨酸作为血小板性血小板减少性紫癜的潜在治疗方法。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，并调节等离子体VWF和ULVWF的功能。我们将探索这种效果的分子基础，并检查该药物是否可以预防或治疗ADAMTS-13缺乏的小鼠模型中的诱导TTP。公共卫生相关性：在此应用中，我们努力找出为什么血浆von Willebrand因子（VWF）为何在血小板中首次从制造的细胞中首次释放时非常粘。如果该蛋白质通常未进行处理，则会引起严重的血液凝血疾病，通常导致受影响的患者死亡。我们提出的研究将帮助我们了解新的VWF与流程的VWF有何不同，我们将学习如何处理。这样，我们将能够理解和治疗血液凝结的疾病。