Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets

Background Platelets ( PLT s) in stored PLT concentrates ( PLC s) release PLT extracellular vesicles ( PL ‐ EV s) induced by senescence and activation, resembling the PLT storage lesion. No comprehensive classification or molecular characterization of senescence‐induced PL ‐ EV s exists to understand PL ‐ EV heterogeneity. Study Design and Methods PL ‐ EV s from 5‐day‐stored PLC s from healthy individuals were isolated and subfractionated by differential centrifugation, filtration, and density gradient ultracentrifugation into five PLT microvesicle ( PL ‐ MV ) subfractions ( F raction [ F ]1‐ F 5) and PLT exosomes ( PL ‐ EX s). PL ‐ EV size, concentration, and composition were analyzed by nanoparticle tracking analysis, flow cytometry, and lipid and protein mass spectrometry. Protein data were verified by W estern blot. Results PL ‐ EV s showed overlapping mean particle sizes of 180 to 260 nm, but differed significantly in composition. Less dense, intermediate, and dense PL ‐ MV s enriched specific lipidomic and proteomic markers related to the plasma membrane, intracellular membranes, PLT granules, mitochondria, and PLT activation. α‐ S ynuclein (81% of total) accumulated in F 1 and F 2, amyloid‐β ( A β) precursor protein in F 3 and F 4 (84%), and apolipoprotein ( A po) E (88%) and A po J (92%) in F 3 to F 5. PL ‐ EX s enriched lipid species and proteins, with high abundance of lipid raft, PLT adhesion, and immune response–related markers. Conclusion Differential lipid and protein compositions of PL ‐ EV s suggest their unique cellular origins and functions, partly overlapping with PLT granule secretion. Dense PL ‐ MV s might represent autophagic vesicles released during PLT activation and apoptosis and PL ‐ EX s resemble lipid rafts, with a potential role in PLT aggregation and immunity. Segregation of α‐synuclein and A β precursor protein, A po E , and A po J into less dense and dense PL ‐ MV s, respectively, show their differential carrier role of neurologic disease–related cargo.