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p38 mitogen-activated protein kinase regulates mitochondrial function and microvesicle release in riboflavin- and
Zhongming Chen1,2, Peter Schubert1,2,3, Sonia Bakkour4
1Centre for Innovation, Canadian Blood Services, Vancouver, British Columbia, Canada.
Background:
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.
Insights
Pathogen inactivation using riboflavin and UV light triggers p38 MAPK-dependent mitochondrial signaling and microvesicle release in platelet concentrates. This process impacts platelet functionality and quality during storage.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Understanding pathogen inactivation (PI) mechanisms in platelets (PLTs) is vital for assessing its impact on PLT functionality and quality.
- Biochemical analysis of PI-induced changes in PLTs is crucial for improving transfusion safety and efficacy.
Purpose of the Study:
- To investigate the biochemical mechanisms underlying platelet activation and microvesicle release following riboflavin and ultraviolet light (RF/UV) treatment.
- To determine the role of p38 mitogen-activated protein kinase (MAPK) in RF/UV-induced platelet responses.
Main Methods:
- Platelet concentrates (PCs) were treated with RF/UV, solvent control, or p38 MAPK inhibitor (SB203580) before RF/UV treatment.
- Flow cytometry assessed mitochondrial potential, reactive oxygen species (ROS) generation, and microvesicle (MV) release.
- Quantitative PCR (qPCR) measured extracellular mitochondrial DNA (mtDNA), and immunoblot analyzed mitochondrial protein translocation.
Main Results:
- RF/UV treatment increased mitochondrial translocation of Bax and Bid, cytochrome c release, loss of mitochondrial potential, and ROS generation.
- These effects were significantly inhibited by SB203580, indicating p38 MAPK dependency.
- RF/UV treatment elevated the release of MVs, mitochondria (MT), and MVs containing MT (MVs/MT), with partial inhibition by SB203580.
Conclusions:
- RF/UV treatment induces p38 MAPK-dependent mitochondrial signaling pathways in apheresis PCs.
- The study demonstrates that RF/UV-mediated PI triggers microvesicle release, contributing to changes in PLT functionality and quality.
