This application seeks to examine the phenomenon of fibrinogen-bound GPIIb/IIIa internalization and cycling. The Applicant has observed that this receptor-ligand complex is rapidly internalized in activated platelets correlating with a failure of the cells to aggregate. These observations have led to the hypothesis that internalization and cycling represents a fundamental, yet unappreciated regulatory mechanism that modulates platelet function. The Applicant further hypothesizes that internalization of fibrinogen occurs via receptor-mediated endocytosis which serves to sequester fibrinogen in platelet alpha granules.
Three Specific Aims are proposed to investigate this phenomenon. 1) The kinetics of internalization in subcellular cycling in activated platelets will be examined. 2) The role of clathrin, clathrin-associated adapters and/or the platelet cytoskeleton in the internalization and cycling process will be examined. Site-directed mutagenesis within the cytoplasmic tails of GPIIb/IIIa will be performed to identify internalization signals. Detailed knowledge of GPIIb/IIIa receptor cycling would impart new perspective on classic ligand binding assays as well as provide information on the mechanism mediating adhesive protein uptake and targeting the secretory granules.
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