aPC prevents inflammasome activation in cardiac resident cells and macrophages in vitro. (A-F) In mouse BMDMs (A-B), mouse neonatal cardiomyocytes (C-D), and mouse neonatal cardiac fibroblasts (E-F), inflammasome activation was induced by priming with LPS (500 ng/mL, 3 hours) followed by ATP (10 µM, 3 hours; control [cont]: PBS). Concomitant treatment with aPC (20 nM; added once 30 minutes before ATP stimulation) markedly reduced the LPS/ATP-mediated induction of Nlrp3 and cl-Casp1 and cl–IL-1β; representative immunoblots (A,C,E) and corresponding bar graphs summarizing results (B,D,F). Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (A,C,E). (B,D,F) The active form was quantified. (G-H) Nlrp3 expression and cl-Casp1 and cl–IL-1β are increased in mouse neonatal cardiomyocytes subjected to 6 hours of hypoxia (H; 1% oxygen) and serum and glucose deprivation (Hanks balanced salt solution medium), followed by 12 hours of reoxygenation (R; 21% oxygen) in complete medium. H/R induces inflammasome activation, which is prevented by aPC (20 nM; added once at the time of R); representative immunoblots of whole-cell lysates (G) and bar graph summarizing results (H), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (G) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β, respectively. (H) The active form was quantified. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments, each with at least 2 technical replicates (A-H); GAPDH as loading control (A,C,E,G). **P < .01; analysis of variance (B,D,F,H).