Htf 1

Endothelial cells were washed and put into serum-free media for 1 h. To determine the role of TF-mediated signaling, EVs were pretreated with FVIIa and FX (10 and 150 nM, respectively, unless otherwise noted; Hematologic Technologies, Essex Junction, VT, USA). To inhibit TF, EVs were pretreated with 10 μg/ml HTF-1 (BD Bioscience, Franklin Lake, NJ, USA) or isotype control for 30 min. As positive controls, endothelial cells were treated with bovine thrombin (1 U/ml, Sigma-Aldrich, St. Louis, MO, USA), or FXa (80 nM, Hematologic Technologies). To inhibit FXa, FPRCK or EGRCK (40 μM, Hematologic Technologies) was added. To determine the role of endothelial PAR-1 and PAR-2 signaling, PAR-1 or PAR-2 agonist peptides (100 μM; TFLLR, Sigma-Aldrich, and SLIGKV, Abcam, Cambridge, UK, respectively) and scramble peptides (RLLFT, American Peptide Company, Sunnyvale, CA, USA, and VKGILS, Bachem, Torrance, CA, USA, respectively) were used with amastatin (10 μM, Santa Cruz Biotechnology, Dallas, TX, USA) to prevent peptide degradation (28 (link)). To inhibit PAR-1 and PAR-2, endothelial cells were pretreated with 0.1 μM E5555 (Axon Med Chem, Reston, VA, USA) and 50 μM FSLLRY (Bachem) for 30 min at 37°C. We did not use SCH79797, which induced cell death at the concentration required for PAR-1 inhibition (29 (link), 30 (link)). Untreated endothelial cells were used as negative controls.