Endothelial cells, vWf cleavage, and thrombotic microangiopathies

在体外，当流体剪切力水平升高时，直接发生血小板聚集，而不需要预先进行血小板-表面黏附。这种高切应力诱导的血小板聚集是通过在二磷酸腺苷(ADP)存在下，大的和通常大的(UL)von Willebrand因子(VWF)多聚体与血小板表面糖蛋白(GP)复合体GPIbalpha-IX-V和GPIIb-IIIpha(AIIb/b3)结合而介导的。在体内，大vWF/ULvWf多聚体介导的高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)系统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。在大多数类型的TTP中，未能通过vWF金属蛋白酶切割蛋白水解性大/ULvWf多聚体是潜在的关键缺陷。目前测量vWF金属蛋白酶与大/超大vWf多聚体相互作用的技术尚不清楚，这是特定目标A的主题。在目标A中，我们将确定剪切力对vWF金属蛋白酶切割大vWF/异常大(UL)vWF多聚体的影响，特别是是否需要表膜(内皮细胞、血小板)来进行酶-底物反应。溶血性尿毒症综合征(HUS)和骨髓移植(BMT)/化疗相关的血栓性微血管病变与TTP有一定的临床特征。与大多数类型的TTP相比，腹泻性HUS和BMT/化疗相关的血栓性微血管病变患者的vWF金属蛋白酶活性(通过现有的液体相检测)是正常的。然而，在一些患有这些疾病的患者中，血浆vWF多聚体的异常表明，肾脏和其他高切动脉循环区域的血小板聚集可能是由vWF介导的。我们将在特定的目标B中确定是否如此。尽管大多数不同类型的TTP患者通过血浆输注/交换得到有效治疗，但由于他们对血浆操作无效，可能会继续死亡或遭受严重的心血管并发症。此外，任何治疗方法都不会导致死亡或遭受严重的心血管并发症，因为它们对血浆操作无效。此外，对于HUS或BMT/化疗相关的血栓性微血管病变，没有任何治疗方法是一贯有效的。迫切需要开发更多的治疗方案，朝着这个方向的步骤是特定目标C的目标。具体地说，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，并设计一种简单的vWF金属蛋白酶的纯化程序。在该项目的几个部分中，与洛佩兹博士(SCOR Pi)和项目1、董博士(核心B)、Kroll博士(项目2)、Bray博士(项目3)和Thiagarajan博士(项目5)进行了重要的实验合作。

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).