Endothelial cells, vWf cleavage, and thrombotic microangiopathies

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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).