Sligkv nh2

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.

Mastoparan was purchased from American Peptide (Sunnyvale, CA, USA). Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), SLIGKV-NH_2, α-lactalbumin from bovine milk, and calmodulin bovine were all purchased from Sigma-Aldrich (St. Louis, MO, USA). DSP-d₀ was bought from Thermo Scientific (Grand Island, NY, USA) and DSP-d₈ was purchased from ProteoChem (Loves Park, IL, USA). Deionized water used for sample preparation was obtained from a Nanopure Diamond Barnstead purification system (Barnstead International, Dubuque, IA, USA). HPLC-grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (FA) was purchased from Spectrum Chemical Mfg. Corp (Gardena, CA, USA).