Proteomic Validation of MEG-01-Derived Extracellular Vesicles as Representative Models for Megakaryocyte- and Platelet-Derived Extracellular Vesicles Sánchez-Manas, José Manuel Perales Romero, Sonia Martínez Navajas, Gonzalo Ceron-Hernández, Jorge López Vázquez, Cristina María Peralbo-Molina, Ángela Delgado, Juan Ramón Martínez-Galán, Joaquina Ramos Mejía, Verónica Chicano-Gálvez, Eduardo Hernández-Valladares, María Ortuño, Francisco Manuel Torres Perales, Carolina Real Luna, Pedro José Meg-01 cell line extracellular vesicles (EVs) Platelets Proteomics Microvesicles (MVs) Exosomes (EXOs) This study was funded by the FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (B-CTS-676-UGR) awarded to C.T., PID2023-152099OB-I00 from the Ministry of Economy and Competitiveness awarded to P.J.R. and C.T. and the Fundación Roberto Arnall support through a research grant leaded by J.R.D. and J.M.-G. We also received valuable support from the ROLUCAN Association (Rota Lucha contra el Cáncer), which made donations to help fund our laboratory’s research initiatives. J.M.S.-M. was supported by Grants for the Predoctoral Recruitment of Research Trainees-Predoctoral student (PREDOC_01765). G.M.-N. was supported by a PFIS fellow from the Spanish Health Institute Carlos III (FI17/00178). J.C.-H. was supported by a PhD program from the Ministry of Universities (FPU18/03410). J.M.S.-M., G.M.-N. and J.C.-H. were all PhD students in the Doctoral Program in Biomedicine at the University of Granada. 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 indicated that MEG-01-derived vesicles preserve key hemostatic, cytoskeletal, and immune pathways commonly associated with platelet EV biology. Ingenuity Pathway Analysis showed that PLPs exhibit proliferative transcriptional programs (elevated MYC/RB1/TEAD1, reduced GATA1), while plasma exosomes display minimal differential pathway activation compared to MEG-01 exosomes. Overall, these findings suggest that MEG-01-derived EVs approximate certain aspects of megakaryocyte-lineage exosomes and activated platelet-like states, although they do not fully replicate native platelet biology. Notably, plasma exosomes show strong proteomic convergence with MEG-01 exosomes, whereas platelet exosomes retain distinct activation-related features. 2025-12-09T07:43:38Z 2025-12-09T07:43:38Z 2025-12-05 journal article Sanchez-Manas, J.M.; Perales, S.; Martinez-Navajas, G.; Ceron-Hernandez, J.; Lopez, C.M.; Peralbo-Molina, A.; Delgado, J.R.; Martinez-Galan, J.; Ramos-Mejia, V.; Chicano-Galvez, E.; et al. Proteomic Validation of MEG-01-Derived Extracellular Vesicles as Representative Models for Megakaryocyte- and Platelet-Derived Extracellular Vesicles. Biomolecules 2025, 15, 1698. https://doi.org/10.3390/biom15121698 https://hdl.handle.net/10481/108639 10.3390/biom15121698 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional MDPI