Effect of APC on the LPS-mediated release of HMGB1. (A) HUVECs were stimulated with indicated concentrations of LPS for 16 hours, and the release of HMGB1 was measured by an ELISA as described in “Methods.” (B) The LPS (100 ng/mL) mediated HMGB1 release by HUVECs was monitored after treating the cell monolayer with indicated concentrations of APC for 3 hours. (C) The same as panel B, except that cells were incubated with increasing concentrations of meizothrombin (MeizoTh; white bars) or PC-gla/MeizoTh (black bars). (D) The same as panels B or C, except that cells were preincubated with function-blocking Abs to PAR-1 or EPCR (25 μg/mL for 30 minutes) before treating cells with each protease (100nM APC, 2nM PC-gla/MeizoTh). All results are shown as means ± SDs of 5 different experiments. *P < .05 and **P < .01 compared with 0 (A) or LPS (B-D).