This application is intended to address several questions related to the structure and function of von Willebrand factor (vWF) and its platelet receptors in patients with chronic renal disease. Since the occurrence of abnormal bleeding is a major complication observed in the course of uremia, the proposed studies are relevant to exploring mechanisms of hemostasis that may contribute to the hemorrhagic tendency in the patients described. First, the structure of the circulating vWF protein, as well as the platelet-derived vWF, will be explored with respect to global multimeric composition, structure of individual multimers, and subunit composition. A variety of electrophoretic techniques, in agarose or polyacrylamide gels, under non-reducing and reducing conditions, will be employed. By using a number of specific monoclonal antibodies and immunoradiological detection methods, any derangement of vWF structure occurring in chronic renal disease will become apparent. Secondly, the interaction between vWF and glycoprotein (GP)Ib will be analyzed both in the presence and in the absence of ristocetin. Studies will be performed in uremic platelet rich plasma, in order to evaluate the possible effect of substances present in the circulation on the reactions being explored. For this purpose, vWF in plasma will be tagged with radiolabeled monoclonal antibody, and the binding to platelets studied in the presence of ristocetin. Alternatively, desialylated normal vWF, or a 52/48 kDa proteolytic fragment of native vWF containing the GPIb binding domain, will be used to perform binding studies in the absence of ristocetin. Any abnormality in the vWF-GPIb interaction should become apparent. Thirdly, the structural integrity of GPIb and the functional integrity of GPIIb/IIIa will be evaluated. A monoclonal antibody that reacts with reduced and denatured GPIb will be used to study the structure of the GPIb Alpha-chain in uremic platelets. Proteolytic fragments of GPIb will also be sought in the circulation. The number of GPIb and GPIIb/IIIa molecules on the membrane of uremic platelets will be determined, both under resting conditions or following stimulation. Finally, the binding of fibrinogen and vWF to GPIIb/IIIa will be analyzed. The proposed studies will provide information relevant to the understanding of the pathophysiology of uremic bleeding and to the definition of better therapeutic strategies to prevent the occurrence of hemorrhagic complications in the course of chronic renal disease.
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