The pathophysiology of vascular injury in atherosclerosis is poorly understood. One of the earliest molecular events in the atherothrombotic lesion is the migration and proliferation of heterogeneous leukocyte subsets in the arterial intima. This mechanism is contributed by multiple molecular interactions at the surface of vascular cells, involving the recognition of specialized membrane receptors for growth factors/cytokines, chemoattractants, and blood clotting proteases. It has been recently suggested that in addition to its role in coagulation, the blood protease activated factor X (factor Xa) stimulates proliferation of smooth muscle cells in vitro. We have recently identified, cloned, and expressed a novel transmembrane molecule that functions as a receptor for factor Xa on various hematopoietic cells. Denominated Effector cell Protease Receptor-1 (EPR-1), we have shown that binding of factor Xa to this receptor induces lymphocyte proliferation in vitro. Furthermore, using monoclonal antibody strategy, we have also shown that EPR-1 plays a crucial regulatory role on clonotypic lymphocyte activation and proliferation both in vitro and in vivo. Therefore, a novel biological function for the interaction of factor Xa with hematopoietic cells is hypothesized. The present proposal is focused on the role of EPR-1, and of its ligand, in regulating activation and proliferation of hematopoietic cells. Synthetic peptidyl mimicry, sequence-specific antibodies, and targeted recombinant mutants will characterize the structure-function requirements of the interaction of factor Xa with EPR-1. The regulatory role of EPR-1 on lymphocyte proliferation will be dissected with respect to synthesis and release of activating cytokines (lL-2), generation of primary mitogens (thrombin, PDGF), and activation of accessory mechanisms of lymphocyte co-stimulation. Finally, the mitogenic signalling through EPR-1 will be elucidated in fluorescence analysis of intracellular second messengers, regulated mechanisms of protein phosphorylation, and receptor post-occupancy events. The overall project is designed to elucidate a novel pathway of leukocyte growth control that can directly contribute to vascular injury and to the early onset and development of the atherosclerotic lesion in vivo.
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