Dose and time-dependent release of endothelial microparticles (EMP) by thrombin. (A) Flow cytometric detection and quantification of EMP. Conditioned cell culture media (CCM) were recovered from serum-starved HMEC-1 untreated (control) or stimulated for 18 hours with thrombin (1 IU/mL) or TNF-α (100ng/mL). Size-selected events 1μm were plotted according to fluorescence for specific annexin V–FITC binding (FL1) on FL1/side scatter histograms (top row). Positive labeled events are included in gate D and considered as annexin V–positive EMP (bottom row). Representative count of EMP versus fluorescence intensity. These plots are representative of 4 independent experiments. (B) Effect of thrombin, PAR-1 agonist TRAP, inactive thrombin-PPACK and TNF-α on EMP release by HMEC-1. Serum-starved HMEC-1 cells were kept unstimulated (control) or stimulated for 18 hours with increasing doses of thrombin and EMP were assayed in CCM, as described in “Materials and methods.” The bar graph represented the mean value of EMP per 1 × 10⁶ HMEC-1 cells ± SEM from 10 independent experiments. (C) Time response curve of thrombin-induced EMP release. EMPs were recovered from CCM from serum-starved HMEC-1 cells kept untreated (▴) or stimulated for different times with 1 IU/mL thrombin (•). The curves represent the mean values of EMP per 1 × 10⁶ cells ± SEM from 6 independent experiments. *P < .01, ***P < .001, for thrombin-stimulated versus unstimulated (control) HMEC-1 cells. NS indicates not significant for thrombin-versus TRAP-stimulated HMEC-1 cells.