Extracellular HMGB1 induces PS exposure and TF activation through caspase-11. (A-C, E, G-H) WT, caspase-11 KO, and GSDMD KO macrophages stimulated with ultrapure LPS (L, 1 μg/mL) or HMGB1 (H, 400 ng/mL) or luteinizing hormone (LH) for 10 hours. Representative confocal images of PS exposure labeled by Annexin V–fluorescein isothiocyanate are shown (A), PS exposure was quantified by using ImageJ software for the area percentage of cells that are positive (B and E), and TF activity was detected (C). (D) TF activity was measured at different time points after WT and caspase-11 KO macrophages were treated with ultrapure LPS plus HMGB1. (F) WT, caspase-11 KO, and GSDMD KO macrophages stimulated with ultrapure LPS (L, 1 μg/mL) or HMGB1 (H, 400 ng/mL), or LH for 2 hours. TF messenger RNA (mRNA) expression was detected by using quantitative polymerase chain reaction. Values are given as fold increase over unstimulated controls. (G) TF protein expression was detected by using western blot. (H) Quantitative analysis of TF protein expression. (I) TF activity was measured in HCV macrophages stimulated with HMGB1 plus ultrapure LPS with or without MFG-E8. PS exposure (J) and TF activity (K) were detected in LH-treated HCV macrophages transfected with control small interfering RNA (siRNA) or TMEM16F-specific siRNA. si1 and si2 represent 2 sequences of Tmem16f-specific siRNAs. (L) WT and GSDMD KO macrophages stimulated with ultrapure LPS (1 μg/mL) or crude LPS (1 μg/mL) for 10 hours. PS exposure was quantified (left) and TF activity was detected (right). HCV mice were used for the aforementioned TF activity tests. Data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. NS, not significant.