EVs form throughout platelet storage and elicit TRALI in LPS-primed mice. Group data show EV number (#EVs; A), EV size (B), and EV composition in terms of concentrations of LCC and VLCC chain length ceramide and S1P (C) for EVs of platelet pools stored for 1 (D1-EVs), 3 (D3-EVs), or 5 (D5-EVs) days. Wet-to-dry lung weight ratio (D), protein concentration in BAL (E), MPO activity in lung tissue (F), and histological features of lung injury (G) on a quantitative scale from 0 (no injury) to 1 (maximal) were determined in recipient BALB/c male mice 6 hours after transfusion of EVs. Mice were primed for 2 hours with LPS (2 mg/kg intraperitoneally) prior to transfusion of 10 mL/kg body weight of either normal saline (LPS group), EVs of platelet pools stored for 1 (D1-EVs) or 5 (D5-EVs) days, or day 5 platelet- and EV-depleted platelet pools (5 days non-EV). Representative images of hematoxylin-and-eosin–stained histological micrographs show progressive severity of lung injury with transfusion of EVs isolated from platelet pools with increasing storage time in LPS-primed mice. Scale bars, 50 μm (H). Group data for panels A-C are depicted as medians and 25% quartiles; n = 6-15 each, *P < .05 vs D1-EVs and #P < .05 vs D3-EVs (1-way analysis of variance and post hoc all pairwise Tukey test). Group data for panels D-G are given as mean ± SD; n = 5-8 each. *P < .05 vs LPS only, #P < .05 vs LPS plus D1-EVs and †P < .05 vs D5 non-EV (1-way analysis of variance and post hoc all pairwise Tukey test).