The HEECs used in this study were isolated, immortalized, and characterized as previously described 18 (link)–20 (link). Previous studies have demonstrated that these cells express the same factors and cytokines as the intact physiological endothelium 19 (link), 20 (link). Cell culture was performed as previously described 16 (link), 20 (link). For treatment experiments, HEECs were plated in 60mm tissue culture plates coated with 2% gelatin and grown until 70% confluent. Media was then replaced with serum-free OptiMEM (Gibco-Invitrogen; Grand Island, NY) with or without physiological concentrations of thrombin (Sigma-Aldrich, St. Louis, MO; 0.25 U/ml) and incubated for 1 hr. Without changing the media, cells were then treated with or without LPS (isolated from Escherichia coli 0111:B4; Sigma-Aldrich) at 1μg/ml, and the cells incubated for an additional 24 or 48 hrs. In subsequent treatment experiments, in place of thrombin, HEECs were treated with either a specific PAR1 agonist (TFLLR-NH2); a specific PAR2 agonist (SLIGKV-NH2); or a specific PAR4 agonist (AYPGKF-NH2) at 1mM (Sigma-Aldrich). After each time point, cell-free culture supernatants were collected and stored at −80°C. Cells were lysed for either RNA or protein isolation.