Thrombin initiates a molecular dialogue between platelets, the endothelium and other coagulation proteins that is fundamental to the ultimate control of the hemostatic response. In reactive cells, alpha- thrombin initiates an array of activation-dependent responses including phosphoinositide hydrolysis with resultant elevations in cytosolic calcium ([Ca+2}i), inhibition of adenylate cyclase and mitogenesis. We have isolated, cloned and stably expressed a thrombin receptor (TR) from a human umbilical vein endothelial cell (HUVEC) library, and demonstrated that it is identical to its recently identified platelet-derived homologue. Initial genomic characterization using the radiolabeled CDNA as probe has confirmed that the TR is present as a single-copy gene in the human genome, whose entire coding region is contained within two exons. Isolation of partial clones from a human genomic library has successfully identified the splice junction site for the large about 3.2 kb exon, and will be utilized to further characterize the TR gene and its promoter. Activation-dependent responses to alpha-thrombin and TR42-55 (the new N-terminus generated after thrombin cleavage) have been studied in transfected CHO cells, HUVEC's and platelets using an antibody (anti- TR1-160) generated by expressing the N-terminal 160 amino acids of the TR as a chimeric protein in E. coli. Consistent abrogation of [Ca+2]i transients to TR42-55 has been demonstrated in all three cells using fura2-loading and microspectrofluorimetry, suggesting that a recognition sequence (Arg70-Ser99) critical for ligand-mediated activation is located within the N-terminal extension. Deletional and in vitro mutagenesis studies will be completed to further characterize the critical regulatory mechanism involved in mediating the early steps of thrombin-associated signal transduction. Elucidation of the regulatory mechanisms of thrombin-mediated cell activation will have broad implications for furthering our understanding of critical events governing the thrombotic response.
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