Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis

Background: Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in in flammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboin flammation. Objectives: Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboin flammatory analysis. Methods: Density -based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size -exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom -up proteomics. Results: G-UC ef ficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quanti fication, and apoB-100 detection con firmed the signi ficant reduction in lipoproteins from isolated EVs. Proteomic analysis identi fied similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboin flammatory responses of platelets and monocytes stimulated in vitro with EV isolates. Conclusion: Combination of G-UC + SEC signi ficantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.