Role of ceramide and S1P in endothelial barrier regulation by murine platelet EVs. (A) Group data show calculated AUC for TEER relative to initial monolayer resistance in HPMECs cultured for 4 hours with equal volumes of EVs attained from 5-day (Smpd1^−/−) platelets treated throughout storage (on day 0, 2, and 4) with 0.1 U/mL SM or with equivalent volume of normal saline. (B-C) Group data show TEER relative to initial monolayer resistance in HPMECs cultured for 4 hours with PBS, EVs from 1 day of storage with (D1-EVs [SM]) or without (D1-EVs [WT]) addition of 0.1 U/mL sphingomyelinase (SM), or EVs from 5 days of platelet storage with (D5-EVs [ARC39]) or without (D5-EVs [WT]) 10 μM/L of the ASM inhibitor ARC39, or EVs from 5 days of storage of Smpd1^−/− platelets (D5-EVs [Smpd1^−/−]) with equal numbers of EVs (circles; 5 × 10⁵ EVs each) (B), and corresponding calculated AUCs (C). (D-E) Group data show TEER in HPMECs cultured with PBS, with 50 ng/mL S1P, or EVs from 5 days stored platelets with (D5-EVs [S1P]) or without (D5-EVs) 50 ng/mL S1P with equal numbers of EVs (circles each; 5 × 10⁵ EVs) (D), and corresponding calculated AUCs (E). Group data are depicted as mean ± SD; n = 5-8 each. *P < .05 vs (D5-EVs [Smpd1^−/−]) (A); *P < .05 vs PBS, #P < .05 vs D1-EVs (WT) (C); and *P < .05 vs PBS, #P < .05 D5-EVs (S1P) (E) (1-way analysis of variance and post hoc all pairwise Tukey test).