TRANSCRIPTION FACTOR NF-E2 IN ALPHA IIB BETA 3 SIGNALING

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Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering inward signals that regulate the cytoskeleton. Since current understanding of alphaIIbbeta3 signaling is limited, we will study alphaIIbbeta3 signaling in primary murine megakaryocytes, cells that naturally express alphaIIbbeta3 and, unlike platelets, are amenable to genetic manipulation. We have discovered that agonist-induced fibrinogen binding to alphaIIbbeta3 occurs only in the largest, most mature megakaryocytes from normal mice. In contrast, large megakaryocytes from mice genetically deficient in the transcription factor NF-E2 p45 subunit (NF-E2-/-) fail to bind soluble fibrinogen and they adhere poorly to immobilized fibrinogen, despite normal expression of alphaIIbbeta3. This suggests that one or more NF-E2-regulated genes are required for inside-out alphaIIbbeta3 signaling. The goal of this project is to identify and characterize NF-E2-regulated genes that control alphaIIbbeta3 function. First, the functional defect in NF-E2-/- megakaryocytes will be characterized in detail by examining cytoskeletal reorganization in fibrinogen-adherent cells, by extending the analysis to other integrins, and by determining whether recombinant expression of p45 in NF-E2-/- megakaryocytes reconstitutes normal alphaIIbbeta3 signaling. Recombinant p45 will be expressed using retroviral and Sindbis virus systems. Second, NF-E2-regulated genes, one or more of which may re regulate alphaIIbbeta3 function, will be identified by examining differential gene expression in wild-type and NF-E2-/- megakaryocytes using a series of complementary approaches. Third, candidate genes identified by differential expression will be evaluated for their role(s) in alphaIIbbeta3 signaling by using viral vectors to express them, or relevant mutants, in megakaryocytes and determining their effects on alphaIIbbeta3 function. The unique window on alphaIIbbeta3 signaling provided by NF-E2-/- megakaryocytes promises to fill major gaps in our understanding of bidirectional integrin signaling. Taken together with the complementary work on thrombin and alphaIIbbeta3 signaling proposed by our collaborators, this mechanistic information may facilitate the development of a new class of antithrombotic drugs that inhibit alphaIIbbeta3 function from within the platelet.