Platelet extracellular vesicles mediate transfusion-related acute lung injury by imbalancing the sphingolipid rheostat

Abstract Transfusion-related acute lung injury (TRALI) is a hazardous transfusion complication with an associated mortality of 5% to 15%. We previously showed that stored (5 days) but not fresh platelets (1 day) cause TRALI via ceramide-mediated endothelial barrier dysfunction. As biological ceramides are hydrophobic, extracellular vesicles (EVs) may be required to shuttle these sphingolipids from platelets to endothelial cells. Adding to complexity, EV formation in turn requires ceramide. We hypothesized that ceramide-dependent EV formation from stored platelets and EV-dependent sphingolipid shuttling induces TRALI. EVs formed during storage of murine platelets were enumerated, characterized for sphingolipids, and applied in a murine TRALI model in vivo and for endothelial barrier assessment in vitro. Five-day EVs were more abundant, had higher long-chain ceramide (C16:0, C18:0, C20:0), and lower sphingosine-1-phosphate (S1P) content than 1-day EVs. Transfusion of 5-day, but not 1-day, EVs induced characteristic signs of lung injury in vivo and endothelial barrier disruption in vitro. Inhibition or supplementation of ceramide-forming sphingomyelinase reduced or enhanced the formation of EVs, respectively, but did not alter the injuriousness per individual EV. Barrier failure was attenuated when EVs were abundant in or supplemented with S1P. Stored human platelet 4-day EVs were more numerous compared with 2-day EVs, contained more long-chain ceramide and less S1P, and caused more endothelial cell barrier leak. Hence, platelet-derived EVs become more numerous and more injurious (more long-chain ceramide, less S1P) during storage. Blockade of sphingomyelinase, EV elimination, or supplementation of S1P during platelet storage may present promising strategies for TRALI prevention.