Optimizing platelet secretome production: Impact of prostaglandin I₂ during platelet isolation on platelet activation and secretome molecular composition

BACKGROUND: Platelet secretomes are emerging as promising therapeutic bioproducts for regenerative medicine and immunomodulation. However, methodological variability during platelet isolation and activation limits reproducibility and functional consistency. Prostaglandin I₂ (PGI₂) is commonly used to maintain platelet quiescence during processing, yet its impact on subsequent platelet responsiveness and the molecular profile of the obtained secretomes remains poorly characterized. METHODS: Washed platelets were isolated from buffy coats of healthy donors (n = 3) using standardized procedures, either in the absence (PLT-S) or presence of PGI₂ (PLT-S+PGI₂). Platelets were activated with TRAP-6 to generate secretomes. Platelet activation and degranulation were assessed by cytospin preparation and microscopy examination of platelet aggegates and CD62P (P-Selectin) membrane translocation by flow cytometry. Secretome composition was quantified using multiplex immunoassay targeting cytokines, chemokines, and growth factors. Paired t-test statistical analyses were performed. RESULTS: The addition of PGI₂ significantly reduced basal platelet activation and α-granule degranulation, as evidenced by decreased CD62P membrane expression, following TRAP-6 stimulation. Correspondingly, PLT-S+PGI₂ secretomes showed a consistent reduction in bioactive mediators compared to PLT-S, including a significant decrease in PDGF-BB, HGF, TPO, G-CSF, IFN-γ, IL-6, MCP-2 and MIP-1β. Secretome molecular content strongly correlated with the extent of platelet CD62P translocation. CONCLUSIONS: PGI₂ exposure during platelet isolation compromises subsequent platelet activation and significantly alters the molecular composition of platelet secretomes. Omission of PGI₂ enables more efficient degranulation and generation of biologically enriched secretomes, supporting improved standardization and functional quality of platelet-derived bioproducts for translational applications.