We have determined that human platelets and megakaryocytes have receptors for the Pc portion of IgG encoded by the FcgammaRIIA gene. Engagement of the platelet Fcgamma receptor by immune complexes and anti-platelet antibodies leads to accelerated platelet clearance and platelet activation, the release of biologically active products and thrombosis. Therapy to intervene in these pathologic processes will require understanding the regulation of receptor expression level and the mechanism of the transmission of the receptor's activation signals. The goal of this research project is to gain a greater understanding of the biologic role of the platelet Fcgamma receptor in platelet activation. The first specific aim examines the regulation of expression of the platelet Fcgamma receptor. Alterations in the level of platelet surface FcgammaRIIA expression leads to alterations in receptor mediated activation of platelets. We have identified cis-acting sequences and trans-acting factors important for FcgammaRIIA basal transcription and will identify those responsible for the modulation of FcgammaRIIA transcription by cytokines and glucocorticoids using reporter gene assays in megakaryotic cells. Our transgenic mouse model will enable determination of the effect of these regulatory molecule on platelet FcgammaRIIA expression in vivo. The second specific aim examines structure/function relationships in platelet/megakaryocyte Fcgamma receptor signaling. FcgammaRIIA has three cytoplasmic tyrosines in an ITAM-(immunoreceptor tyrosine-based activation motif)-like sequence. We have established that all three cytoplasmic tyrosines and the tyrosine kinase Syk are important in the early events following receptor crosslinking. Syk antisense oligodeoxynucleotides will be used to identify the effect of Syk ablation on protein tyrosine phosphorylation and Ca2+ signaling in megakaryocytic cells. These signal transduction events will be studied in megakaryocytic cells transfected with wild-type and mutant FcgammaRIIA cytoplasmic domain sequences. We will also use platelets from our FcgammaRIIA transgenic mice to study the FcgammaRIIA mediated secretory response. Finally, the third specific aim examines the role of FcgammaRIIA in platelet clearance and activation in vivo. Our hypothesis is that the expression of human FcgammaRIIA leads to more extensive thrombocytopenia triggered by anti-platelet antibodies and by immune complexes and to enhanced intravascular platelet activation. Following challenge with anti-platelet antibody and immune complex, platelet counts and a marker of intravascular platelet activation in our FcgammaRIIA transgenic mice will be compared to wild-type mice and mice expressing FcgammaRIIA only in platelets.
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