<b>The process is the product: Decoding the Healing Potential of Platelet-derived Extracellular Vesicles by a Multiomic Approach</b>.

Platelet-derived extracellular vesicles (pEV) have been shown to be the most potent fraction of platelet concentrates, being responsible for their therapeutic potential in regenerative medicine. This study compares pEV sourced from three different platelet concentrates: platelet lysate (PL), fresh platelets (fP), and aged platelets (aP). The aim was to determine how platelet activation and storage conditions of these platelet concentrates could determine characteristics, functionality and molecular cargo of pEV. Thus, PL, fP, and aP were prepared using various activation methods, including freeze-thaw cycles, CaCl₂platelet activation and platelet aging, respectively, followed by size exclusion chromatography (SEC) for pEV isolation. pEV were characterized using Transmission Electron Microscopy (TEM), Western Blotting (WB), and Nanoparticle Tracking Analysis (NTA). Functionality was assessed by wound healing assays, metabolic activity and cytotoxicity determination. Protein and miRNA profiling was conducted with LC-MS/MS and the GeneChip miRNA 4.0 Array respectively, followed by a bioinformatician analysis. Characterization indicated that PL-EV had the highest yield and purity, and the presence of typical pEV membrane and luminal markers (CD63, CD9 and HSC70). <i>In vitro</i> wound healing assays demonstrated enhanced fibroblast migration with PL-EV, essential for wound healing phases such as hemostasis, proliferation, and remodeling. Multi-omics analysis revealed three overexpressed miRNAs in PL-EV, notably miR-210-3p and the miR-320 family, which are implicated in wound healing processes. Differential protein analysis showed enrichment in immune response and wound healing functions in PL-EV. These findings highlight the importance of platelet concentrate preparation methods on pEV molecular cargo and functionality. The study identifies hsa-miR-320a, hsa-miR-320b, and hsa-miR-210-3p as key molecules in the wound healing efficacy of PL-EV, advocating for their clinical application in regenerative medicine.