Proteomic validation of MEG-01-Derived Extracellular Vesicles as Biologically Representative Models for Megakaryocyte- and Platelet-Derived Extracellular Vesicles.

Platelets and their extracellular vesicles (EVs) have emerged as promising liquid biopsy biosources for cancer detection and monitoring. The megakaryoblastic MEG-01 cell line offers a controlled system for generating platelet-like particles (PLPs) and EVs through valproic acid induced differentiation. Here, we performed comprehensive characterization and proteomic validation of MEG-01-derived populations, native human platelets and their EVs using nanoparticle tracking analysis, transmission electron microscopy, imaging flow cytometry and quantitative proteomics. MEG-01 megakaryocytic differentiation is characterized by polylobulated nuclei, proplatelet formation and elevated CD41/CD42a expression. PLPs predominantly exhibit an activated-like phenotype (CD62P+, degranulated morphology), while microvesicles (100-500 nm) and exosomes (50-250 nm) displayed size distributions and phenotypic markers consistent with native platelet-derived EVs. Proteomics identified substantial core proteomes shared across fractions and fraction-specific patterns consistent with selective cargo partitioning during EV biogenesis. Functional enrichment revealed that MEG-01-derived vesicles retained hemostatic, cytoskeletal and immune pathways characteristic of physiological platelet EVs. Ingenuity Pathway Analysis demonstrated that PLPs maintained proliferative transcriptional programs (elevated MYC/RB1/TEAD1, reduced GATA1), while plasma exosomes showed minimal differential pathway activation relative to MEG-01 exosomes. These findings validate MEG-01-derived EVs as representative of megakaryocyte-lineage exosomes and activated platelet-like states; plasma exosomes converge proteomically with EXOs MEG-01, whereas platelet exosomes retain distinct activation-associated features.