EVs cause platelet-dependent inflammasome activation in trophoblast cells. (A) Representative images (double immunofluorescence staining) showing colocalization (yellow) of cleaved Casp-1 (red) and Gcm1 (green, syncytiotrophoblast marker) indicating inflammasome activation in trophoblasts after EV injections. Size bar represents 80 µm. (B-C) Platelets enhance EV-mediated (mouse endothelial cell–derived EVs) inflammasome activation in murine trophoblast cells. Representative immunoblots showing enhanced EV-mediated inflammasome activation in trophoblast cells in the presence of PRP (EV+PRP) compared with EVs only (EV) or EVs with PPP (EV+PPP). Control cells were exposed to the supernatant obtained after the last PBS wash during EV isolation (B, representative immunoblots; C, bar graph summarizing results). (D) Immunoblots showing no effect on inflammasome activation in p45-NF-E2^+/− and Gαq^+/− placenta after EV treatment in p45-NF-E2^−/− or Gαq^−/− pregnant female mice, respectively (representative immunoblots). Arrowheads indicate inactive (proform, white arrowheads) and the active (cleaved form, black arrowheads) form of Casp-1 or IL-1β, respectively (B,D). Only the active form was quantified (C). Data shown represent mean ± SEM from 5 independent experiments. *P < .05 (relative to control, C); #P < .05 (relative to EV); §P < .05 (relative to EV+PRP). (C) ANOVA.