Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Defining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist-induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1-/- and Par4-/- endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1-/- endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.