Partitioning of platelet-derived mitochondrial DNA between microparticle (MP)-associated and MP/platelet-depleted fractions during apheresis platelet storage: Predominance of a conserved MP-associated fraction

BACKGROUND: Mitochondrial damage due to platelet lesions can cause mtDNA release. However, platelet microparticles (MPs) can also transfer mtDNA in a conserved, protected form that may not directly participate in inflammatory signaling. This study investigated storage-dependent partitioning of platelet-derived mtDNA between an MP-enriched fraction and an MP/platelet-depleted supernatant and its relationship with oxidative stress and mitochondrial lesions in apheresis platelets. METHODS: Six apheresis platelet concentrates were analyzed by flow cytometry for P-selectin expression, phosphatidylserine (PS) exposure, reactive oxygen species (ROS), MP formation, and mitochondrial membrane potential (MMP; JC-1 Red/Green-GeoMFI ratio) during storage. Platelet-derived mtDNA was quantified by real-time polymerase chain reaction (PCR) in an MP-enriched pellet and the corresponding MP/platelet-depleted supernatant. RESULTS: Unlike MMP, platelet activation markers increased during storage. Supernatant mtDNA increased during storage but remained approximately 13-fold lower than mtDNA in the corresponding MP-enriched fraction. Supernatant mtDNA levels were associated with platelet activation, oxidative stress, and MMP loss. As experimental controls, CCCP, a mitochondrial uncoupler, induced greater MMP loss than ABT-737, a BCL-2/BCL-xL inhibitor, whereas ABT-737 produced higher supernatant mtDNA levels. These responses support assay sensitivity to mitochondrial perturbation without establishing a specific cell-death or release mechanism. CONCLUSION: Microparticles represent a major particulate-associated source of platelet-derived mtDNA during storage, whereas the supernatant signal represents an operationally MP/platelet-depleted fraction with substantially lower mtDNA levels. MP-associated mtDNA may represent a biologically conserved form, whereas its accessibility and inflammatory activity may be limited. The pathophysiological significance and precise release mechanism of mtDNA in the supernatant remain to be determined.