Involvement of Caspase-2 in EMP release. (A) Inhibition of thrombin-induced-EMP release by caspase inhibitors. Serum-starved HMEC-1 cells were preincubated for 1 hour with 2 μM Pan-caspase inhibitor (Z-VAD-FMK) or each of the specific caspase inhibitor. Caspase-1 inhibitor: Z-YVAD-FMK; caspase-2 inhibitor: Z-VDVAD-FMK; caspase-3 inhibitor: Z-DVED-FMK; caspase-6 inhibitor: Z-VEID-FMK; caspase-8 inhibitor: Z-IETD-FMK; caspase-9 inhibitor: Z-LEHD-FMK. HMEC-1 were kept untreated or stimulated with 1 IU/mL thrombin in presence of the same concentrations of inhibitors for 18 hours. EMP release was determined as described in “Materials and methods.” Bar graphs represent mean values ± SEM for 3 independent experiments. *P < .05, **P < .01, ***P < .001; NS indicates not significant. (B) Activation of caspase-2 by thrombin. Caspase-2 activity was determined in triplicate wells by a fluorescent assay. Results are expressed in relative fluorescent units. The curves represent the mean values ± SEM from 3 independent experiments. Upper panel: time-dependent increase in caspase-2 activity. HMEC-1 cells were kept unstimulated (•) or stimulated with thrombin (♦) for different times and lysed. **P < .01 versus unstimulated cells at the same time. Lower panel: HMEC-1 cells were preincubated with 2 μM Z-VDVAD-FMK or 1 μM Y27632 and stimulated for 3 hours with 1 IU/mL thrombin in presence of the inhibitors. ***P < .001 versus thrombin-stimulated HMEC cells. (C) Absence of apoptosis in HMEC-1 cells stimulated by thrombin. Nucleosomes enrichment determined by cell death ELISA. Serum-starved HMEC-1 cells were kept untreated (control) or were stimulated for 18 hours with thrombin (1 IU/mL) or 25 ng/mL TNF-α and 2.5 μg/mL cyloheximide (TNF/CHX). ***P < .001; NS indicates not significant.