EVs from stored murine platelets elicit endothelial barrier dysfunction. (A) Group data show TEER (measured in ohms) as ratio relative to initial monolayer resistance of primary HPMECs cultured for 4 hours with PBS, EVs from platelets stored for 1 (D1-EVs) or 5 (D5-EVs) days. EVs were dosed volumetrically (diamonds) using a similar strategy as in the in vivo experiments. (B) For each individual TEER measurement, the area under the curve (AUC) relative to the baseline y = 1.0 was calculated and is given as group data for endothelial permeability, with increased AUC indicating loss of resistance (increased permeability), whereas 0 or negative values indicate maintenance of or increased endothelial barrier function. (C) Group data show TEER in HPMEC monolayers cultured with PBS, or equal numbers (circles; 5 × 10⁵ EVs each instead of volumetric dosing as seen in panels A-B) of D1-EVs, D3-EVs, or D5-EVs. Day 5 platelet- and EV-depleted platelet pools (D5 non-EV) were administered in identical volumes (5 µL) as D5-EVs Corresponding calculated AUCs are given in panel D. Group data are depicted as mean ± SD; n = 5-8 each. *P < .05 vs PBS only, #P < .05 vs D1-EVs and †P < .05 vs D5 non-EV (1-way analysis of variance and post hoc all pairwise Tukey test).