Blood platelets are required for hemostasis and abnormal platelet activation leads to thrombosis. Cellular and molecular mechanisms of platelet assembly within, and their release from, megakaryocytes (MKs) remain largely unknown. Current understanding builds on cell biological studies, genetic analysis in mice, and appreciation of congenital human thrombocytopenias such as the Myh9- related disorders. At the conclusion of an elaborate maturation process, MKs elaborate long, branched cytoplasmic extensions called proplatelets. Blood platelets assemble within these structures before release into the circulation. As a result of an R01 award now in its 10th year, our group has scrutinized this remarkable process, which depends on activity of the transcription factor NF-E2. We seek to renew funding to continue with studies that will significantly advance appreciation of how MKs make platelets. First, we recently reported on the surprising result that the Myh9 gene product, non-muscle myosin heavy chain IIA, regulates proplatelet formation negatively and seems to receive signals through the small-GTPase Rho. Our preliminary studies suggest two specific hypotheses: (1) that myosin-IIA deficiency promotes precocious platelet assembly within immature MKs, and (2) that myosin-IIA inhibition of platelet release is normally lifted when mature MKs encounter the chemokine Sdf-1 (CXCL12), which down-regulates cellular Rho activity.
In Specific Aim 1 we propose critical tests of these hypotheses. We will use multi-photon intravital microscopy to visualize platelet release in living Myh9-null mice, and biochemical and functional analysis of signal transduction in cultured primary mouse MKs to evaluate the role of Sdf-1. Second, although NF-E2 is an essential effector of late MK maturation and platelet biogenesis, satisfactory understanding of its mechanisms and transcriptional targets has been elusive. We have combined chromatin immunoprecipitation with hybridization to tiled genome arrays (ChIP-chip) to identify the complete complement of genes that NF-E2 may regulate in MKs.
In Specific Aim 2 we will characterize in depth the NF-E2 "cistrome" and the breadth of cellular processes recruited for thrombopoiesis. Preliminary studies underscore the feasibility of the approach and its potential to generate powerful hypotheses about NF-E2's regulatory mechanisms, which we will test.
In Specific Aim 3 we will use computational methods to identify recurring patterns, especially groupings of transcription factor-binding motifs, within NF-E2-occupied cis-elements in MK-expressed genes. We will test the hypothesis that genes activated by NF-E2 have a cis-element structure distinct from that for repressed genes. We will also determine if NF-E2 occupies distinct loci in immature and terminally differentiated MKs, and if cis-element structures differ between these site classes. The sum of these studies should lead to new insights into physiologic and molecular control of thrombopoiesis.
Circulating blood platelets prevent bleeding but also mediate diverse disease processes, including clotting (a central factor in strokes and heart attacks) and inflammation. Acute clotting in the heart, brain and other sites is a major cause of suffering and death;conversely, cancer and leukemia treatments frequently reduce platelet numbers, causing therapy to be aborted or suspended. Although blood platelet numbers are therefore regulated precisely, we know little about the process by which bone marrow megakaryocytes produce and release platelets. Studies proposed in this application seek to extend more than a decade's worth of research focused on cellular and molecular mechanisms of platelet formation. Characterizing key molecular pathways of platelet formation should lead to better control of circulating platelet counts and to implementation of less toxic treatments for cancer.
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