The long-term goal of this research is to understand at the molecular level the biogenesis of platelet dense granules. Deficiencies in these granules are associated with congenital and acquired disorders that manifest with prolonged bleeding. Understanding how platelet dense granules are formed, and which elements are involved in this process, may thus lead to the development of appropriate treatments for these disorders. In addition, the molecular machinery that generates platelet dense granules is a potential target for the development of drugs that could help reduce the risk of myocardial infarction and stroke. Although the mechanism of platelet dense granule biogenesis remains largely unknown, recent genetic studies have revealed that adaptor protein 3 (AP-3), a protein complex conserved among all eukaryotes, plays a critical role in this process. This finding has opened new avenues for the identification of additional elements involved. This application proposes to identify human proteins that interact physically with AP-3 and test the hypothesis that they are components of the molecular machinery that generates platelet dense granules. AP-3-interacting proteins will be isolated and characterized by biochemical methods, and their putative role in AP-3-dependent events will be assessed using a recently developed assay that allows detection of AP-3 function defects in various human cell lines. Subsequently, the direct involvement of these AP-3-interacting proteins in platelet dense granule biogenesis will be tested by mutational analyses in mouse models of platelet dense granule deficiency or, alternatively, by targeted disruption of the encoding gene in mice.
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