Biochemical analyses of mechanisms triggered in platelets (PLTs) upon pathogen inactivation (PI) are crucial to further understand the impact of PI on PLT functionality and, subsequently, quality.STUDY DESIGN AND METHODS:
PLT concentrates (PCs) were split into four small illumination bags: 1) untreated control, 2) treated with riboflavin and ultraviolet light (RF/UV), and spiked with 3) solvent control dimethyl sulfoxide and 4) p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 before RF/UV treatment. Flow cytometry was used to monitor PLT mitochondrial potential (ΔΨm); generation of intracellular reactive oxygen species (ROS); and release of microvesicles (MVs), mitochondria (MT), and MVs containing MT (MVs/MT). Quantitative polymerase chain reaction (qPCR) was used to quantify extracellular mitochondrial DNA (mtDNA). Translocation of selected mitochondrial proteins was analyzed in subcellular fractions by immunoblot.RESULTS:
RF/UV treatment triggered an increased mitochondrial translocation of both Bax and Bid (p < 0.05, Day 7) and cytochrome c release (p < 0.01, Day 7), loss of ΔΨm (p < 0.05, Day 5 and Day 7), and ROS generation (p < 0.01, Day 5 and Day 7) in PCs compared to the untreated control during storage. These PI-triggered changes were inhibited by SB203580 (p < 0.05). The release of MVs, MT, and MVs/MT was increased upon the RF/UV treatment during storage (p < 0.05) and, with the exception of MT, the release was decreased by the inhibitor (p < 0.05). qPCR analysis showed that RF/UV does not trigger mtDNA release during storage.CONCLUSION:
These findings further our understanding of mechanisms in PLTs initiated by the RF/UV treatment, demonstrating that this treatment induces p38 MAPK-dependent mitochondrial signaling and MV release in apheresis PCs.