Stored human platelets replicate increased EV formation, altered EV sphingolipid rheostat, and EV-mediated endothelial barrier failure. Group data show comparisons between EVs from human platelets stored for either 2 (D2-EVs) or 4 (D4-EVs) days in terms of EV number (#EVs) (A), EV size (B), EV composition in terms of concentrations of LCC (C), VLCC (D), and S1P (E), and the calculated AUC for TEER relative to initial monolayer resistance represented as endothelial permeability (F) from TEER data determined after treatment of HPMEC monolayers with volumetric doses of EVs. Data are depicted as mean plus or minus SD) for group data (outer bars) as well as individual data points with lines connecting values from the same sample at both time points, n = 5-7 each, *P < .05 vs D2-EVs (paired, 2-tailed Student t test).