Assessing mitochondrial function and protein composition in platelet-derived extracellular vesicles.

Platelets, traditionally known for their role in hemostasis, also contribute to inflammation, cancer, and intercellular communication through the release of platelet-derived extracellular vesicles (or platelet-derived microparticles; PMPs). Among these vesicles, a subpopulation containing functional mitochondria, known as mitoMPs, can be transferred to recipient cells, thereby modulating their metabolism and biological responses. This mitochondrial transfer plays a key role in various pathological processes, where it may either restore metabolic functions or enhance cancer cell proliferation, survival, and metabolic plasticity. In this study, we developed a permeabilization protocol combined with high-resolution respirometry to assess mitochondrial respiration in both platelets and PMPs. First, we found that saponin was a more effective permeabilizing agent than digitonin to measure mitochondrial respiration in these models. Moreover, our analysis revealed distinct respiratory profiles between platelets and PMPs and demonstrated that freeze-thaw cycles severely compromise mitochondrial functions in PMPs. Additionally, we performed proteomic profiling of PMPs to characterize their protein cargo, which associate with specific molecular pathways, particularly those associated with mitochondrial metabolism. These results provide novel insights into the biological functions of PMPs and their potential involvement in disease processes. Together, these findings advance the understanding of PMP-mediated mitochondrial transfer and intercellular communication and establish a foundation for future biomedical and therapeutic investigations.