Thrombin potently activates human umbilical vein endothelial cells (HUVEC) to synthesize prostacyclin (PGIs), a potent antithrombotic physiologic modulator that is a vasodilator and inhibitor of platelet function. However, the molecular mechanisms of thrombin action is still unclear. Most recently, Dr.Jaffe's data suggests that thrombin may activate cPLAs via a phosphorylation-dependent, calcium-independent pathway that my involve some of the following proteins which are involved in the activation of MAPK, ras, GAP, raf-1, MEK(MAP kinase), MEKK (MEK kinase), and MAP kinases. The goal of this proposal is to delineate the role of this pathway in HUVEC PGIs synthesis by studying phosphorylation of intracellular signaling proteins induced by thrombin and other agonists in HUVEC using both antiphosphotyrosine antibodies in Western blot and 22P04-labeling and correlating the induction of phosphorylation of specific proteins with functional activation of HUVEC as assessed by EIA of 6-keto-PGF1, the inactive breakdown product of PGI1. Proteins whose phosphorylation is induced by thrombin and other agonists that induce PGIs synthesis will be specifically identified using 22PO4- labeling, SDS-PAGE, and autoradiography and immunologic methods (immunoisolation and Western blotting with both protein specific and anti-phosphotyrosine antibodies). Isolated immunologic complexes will also be assayed for kinase activities. Attempts will be made to inhibit thrombin-induced activation and PGIs synthesis by HUVEC with anti-sense oligonuclotides to kinases delineated in experiments above as being involved in thrombin-induced stimulation of PGIs synthesis.
