부산대학교 도서관

Platelet integrin (alpha)IIb(beta)3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of (alpha)IIb(beta)3 signaling remain poorly understood. To better understand this process, we established conditions to study (alpha)IIb(beta)3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of (alpha)IIb(beta)3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virusmediated expression of isolated (beta)3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of (alpha)IIb(beta)3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2-deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of (alpha)IIb(beta)3 is controlled by NF-E2-regulated signaling pathways that mature late in megakaryocyte development and converge at the (beta)3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional (alpha)IIb(beta)3 signaling.