Macromolecular assembly and generation of serine proteases on cellular surfaces is critically involved in regulation of hemostatic, inflammatory, or fibrinolytic pathways. The concept that a number of these serine proteases may effect cellular activation and proliferative responses has engendered an emerging paradigm focusing on the molecular mechanisms regulating cellular/protease interaction. Previous data suggest that some of these cellular responses are mediated by a novel class of G protein-coupled proteolytically activated receptors (PARs), exemplified by the thrombin receptor (TR, PAR-1), and most recently, PAR-2 and PAR-3. Work in the applicant's laboratory has confirmed that all three PARs isolated to date co-localize in the human genome, an observation that was used for the independent isolation of the PAR-3 gene (a second thrombin receptor). Accordingly, the proposal represents an expanded version of the initial grant application, whose major themes include the identification, characterization, and elucidation of regulatory features of PAR activation and signaling. Endothelial cells (known to express PARs 1-3) and megakaryocytes/platelets (known to express PAR-1 and PAR-3) provide optimal model systems for these studies. This proposal will focus on four themes.
Specific Aim 1 focuses on the molecular characterization of the PAR-3 gene, further outlining approaches for the identification of other putative PARs that may be clustered with this region of the genome.
In Specific Aim 2, the applicant will dissect thrombin-mediated activation and proliferation pathways using endothelial cells and platelets as in vitro model systems.
Specific Aim 3 focuses on molecular interactions of PARs on endothelial cells, exploring the hypothesis that cell-specific differences in PAR activation and processing may be regulated by cell-surface or intracellular receptor interactions in cells co-expressing these receptors.
Specific Aim 4 focuses on the possibility that PAR-1 and PAR-3 may be developmentally regulated on megakaryocytes (suggesting discrete receptor functions), and will initiate studies to elucidate the fundamental roles of these two receptors in endothelial cell and megakaryocytic proliferation and survival.
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