Endothelial cells, vWf cleavage, and thrombotic microangiopathies

内皮细胞、vWf 裂解和血栓性微血管病

• 批准号: 6584924
• 负责人: JOEL L MOAKE
• 金额: $ 21.2万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-15 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584924
• 关键词: bone marrow transplantation; drug screening /evaluation; enzyme activity; hemodynamics; hemolytic anemia; human subject; metalloendopeptidases; microcirculation; patient oriented research; peripheral blood vessel disorder; platelet aggregation; platelet aggregation inhibitors; protein protein interaction; protein purification; protein structure function; proteolysis; renal failure; shear stress; thrombin receptor; thrombocytopenic purpura; thrombosis; vascular endothelium; von Willebrand factor

At elevated levels of fluid shear stress in vitro, platelet aggregation occurs directly without a requirement for preceding platelet-surface adhesion. This high shear stress-induced platelet aggregation is mediated by the binding of large of large and usually large (UL) von Willebrand factor (vWF) multimers to the platelet surface glycoprotein (GP) complexes, GPIbalpha-IX-V and GPIIb-IIIalpha (aIIb/b3) in the presence of adenosine diphosphate (ADP). In vivo, high shear stress-induced microvascular aggregation mediated by large vWF/ULvWf multimers is the probably cause of systemic platelet aggregation in thrombotic thrombocytopenic purpura (TTP), the most extensive and threatening of all human platelet clumping disorders. The failure to cleave proteolytically large/ULvWf multimers, via a vWF metalloproteinase is the underlying critical defect in most types of TTP. Current techniques for measuring vWF metalloproteinase interaction with large/ULvWf multimers, which is unknown, is the subject of Specific Aim A. In Aim A, we will determine the effects of shear stress on the cleavage of large vWF/unusually large ((UL) vWF multimers by vWF metalloproteinase and, specifically, whether or not surface membranes (endothelial cells, platelets) are required for the enzyme-substrate reaction to proceed. The hemolytic-uremic syndrome (HUS) and bone marrow transplantation (BMT)/chemotherapy-related thrombotic microangiopathy share some clinical characteristics with TTP. In contrast to most types of TTP, the vWF metalloproteinase activity (measured by currently available fluid phase assays) is normal in diarrhea-associated HUS and BMT/chemotherapy-related thrombotic microangiopathy. Nevertheless, plasma vWF multimeric abnormalities in some patients with these disorders suggest that platelet aggregation in renal and other areas of the high shear arterial circulation may be vWF-mediated. We will determine whether or not this it so in Specific Aim B. Although the majority of patients with the various types of TTP are treated effectively by plasma infusion/exchange, may continue to die or suffer crippling cardiovascular complications because they are refractory to plasma manipulation. Furthermore, no therapy is to die or suffer crippling cardiovascular complications because they are refractory to plasma manipulation. Furthermore, no therapy is consistently effective in HUS or BMT/chemotherapy-related thrombotic microangiopathy. Development of additional therapeutic options is needed urgently, and steps in the direction are the goals of Specific Aim C. Specifically, we will evaluate agents ex vivo that inhibit events in shear-induced, vWF- mediated platelet aggregation, and we will devise a simple purification procedure for vWF metalloproteinase. In several portions of this project, there is important experimental collaboration with Drs. Lopez (SCOR PI) and Project 1), Dong (Core B), Kroll (Project 2), Bray (Project 3) and Thiagarajan (Project 5).

在体外流体剪切应力水平升高时，血小板直接发生聚集，无需事先进行血小板表面粘附。这种高剪切应力诱导的血小板聚集是通过在二磷酸腺苷 (ADP) 存在下，大量的、通常是大的 (UL) 冯维勒布兰德因子 (vWF) 多聚体与血小板表面糖蛋白 (GP) 复合物、GPIbalpha-IX-V 和 GPIIb-IIIalpha (aIIb/b3) 的结合来介导的。在体内，由大 vWF/ULvWf 多聚体介导的高剪切应力诱导的微血管聚集可能是血栓性血小板减少性紫癜 (TTP) 中全身性血小板聚集的原因，TTP 是所有人类血小板聚集疾病中最广泛和最具威胁性的疾病。无法通过 vWF 金属蛋白酶裂解大蛋白/ULvWf 多聚体是大多数类型 TTP 的潜在关键缺陷。当前测量 vWF 金属蛋白酶与大/ULvWf 多聚体相互作用的技术是未知的，是特定目标 A 的主题。在目标 A 中，我们将确定剪切应力对 vWF 金属蛋白酶裂解大 vWF/异常大 ((UL) vWF 多聚体) 的影响，特别是，是否需要表面膜（内皮细胞、血小板） 酶-底物反应继续进行。溶血尿毒症综合征（HUS）和骨髓移植（BMT）/化疗相关的血栓性微血管病与TTP有一些共同的临床特征。与大多数类型的 TTP 相比，腹泻相关 HUS 中的 vWF 金属蛋白酶活性（通过当前可用的液相测定法测量）正常 BMT/化疗相关的血栓性微血管病。然而，一些患有这些疾病的患者血浆 vWF 多聚体异常表明，肾脏和高剪切动脉循环其他区域的血小板聚集可能是 vWF 介导的。我们将在具体目标 B 中确定是否如此。尽管大多数患有各种类型 TTP 的患者都可以通过血浆得到有效治疗 输注/交换后，可能会继续死亡或遭受严重的心血管并发症，因为它们对血浆操作是难治的。此外，没有任何疗法会导致死亡或遭受严重的心血管并发症，因为这些并发症对血浆操作是难治的。此外，对于 HUS 或 BMT/化疗相关的血栓性微血管病，没有任何治疗方法始终有效。开发额外的治疗选择 迫切需要这一方向，具体目标 C 的目标是朝着这个方向迈出的步伐。具体来说，我们将评估体外抑制剪切诱导的 vWF 介导的血小板聚集事件的药物，并且我们将为 vWF 金属蛋白酶设计一种简单的纯化程序。在该项目的几个部分中，与博士进行了重要的实验合作。 Lopez (SCOR PI) 和项目 1)、Dong（核心 B）、 Kroll（项目 2）、Bray（项目 3）和 Thiagarajan（项目 5）。