Role of protease‐activated receptor‐4 in the phosphorylation of extracellular regulated kinase in vascular smooth muscle cells

Recent studies have shown that thrombin stimulates the activation of extracellular regulated kinase (ERK1/2) and Rho/Rho kinase, which are associated with cellular proliferation and migration in vascular diseases such as hypertrophy and atherosclerosis. Thrombin activates these kinases through protease‐activated receptors (PARs), which are a family of G‐protein coupled receptors activated by the enzymatic cleavage of the extracellular NH2 terminus. We immunoblotted with antibody specific for PAR‐4 to show that this receptor was expressed in rat aortic vascular smooth muscle cells (VSMCs). PAR‐4 activating peptide (AYPGKF) was used to stimulate the cells in a time‐ and concentration‐dependent manner. Using Western blot analysis, we observed that 25μM of the PAR‐4 activating peptide maximally phosphorylated ERK1/2 at a time point of 15 minutes. To confirm that our results were PAR‐4 specific, we pretreated VSMCs with the PAR‐4 inhibitor (tcy‐NH2) for one hour and then stimulated the cells with AYPGKF. ERK1/2 phosphorylation was significantly decreased in the presence of the PAR‐4 inhibitor. We conclude that PAR‐4 activates the ERK pathway, and therefore, may be a key mediator in VSMC migration and proliferation. NIH‐NIGMS‐SCORE