Schematic concept how EVs and imbalances of the sphingolipid rheostat conjointly cause endothelial barrier failure. (A) As platelets are stored over time, they form increasing amounts of EVs which become larger, enriched in long chain ceramides, and S1P deplete. This abundance in EVs with an imbalanced sphingolipid rheostat leads to endothelial barrier failure in vitro and in vivo. (B) Inhibition of ASM by pharmacological blockade (10 μg/mL ARC39) or genetic absence (Smpd1^−/−) of ASM leads to less numerous but similarly ceramide enriched and S1P deplete D5-EVs after 5 days of platelet storage. Volumetric transfusion of these EVs causes less TRALI in vivo due to lower abundance, although equal doses of these EVs still cause barrier failure in vitro due to an imbalanced sphingolipid rheostat. (C) The presence of elevated EV-ceramide alone is insufficient to cause barrier failure in vitro, as platelets stored for 1 day with SM release ceramide-enriched but also S1P-rich EVs, which fail to cause barrier failure when dosed in equal numbers in vitro. (D) Corroborating that the presence of S1P can prevent barrier failure by LCC-rich EVs, supplementation of S1P prevented barrier failure in response to D5-EVs.