AbstractThrombin exerts pleiotropic effects on endothelial cells, including the release of microparticles (EMPs) that disseminate and exchange information with vascular cells. Nevertheless, the mechanisms leading to their generation are not elucidated. We performed microarray analysis to identify genes involved in EMP release by the endothelial cell line HMEC-1 in response to thrombin. We identified a group of genes linked to the cytoskeleton reorganization family. Among these, the Rho-kinase ROCK-II presented a high transcription rate. ROCK-I, another Rho-kinase isoform, was not modulated by thrombin. Pharmacologic inhibition of Rho-kinases or specific depletion of ROCK-II by short interfering (si) RNA inhibited thrombin-induced EMP release. In contrast, ROCK-I mRNA silencing did not modify EMP generation by thrombin. Exposure of HMEC-1 to thrombin in presence of the caspase-2 selective inhibitor Z-VDVAD-FMK prevented ROCK-II cleavage and inhibited the thrombin-induced EMP release. These events were observed in absence of cell death. Our data clearly identified ROCK-II as a target of thrombin in EMP generation. They indicated that the 2 Rho-kinases did not share identical functions. The involvement of caspase-2 in ROCK-II activation independently of cell death points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in EMP generation in response to cell activation.