Page 204 - Haematologica May 2022

Letters to the Editor
compensatory augmentation in glycolysis. In agreement, treatment with either antimycin/oligomycin or CCCP was associated with enhanced lactate release from thrombin-stimulated platelets (Figure 3C), with antimycin being the most effective among them. These findings are strongly suggestive of a compensatory surge in glycolytic rate in platelets upon mitochondrial inhibi- tion which sustains aIIbβ3 platelet integrin activation,
aggregation and phosphatidylserine exposure. Aggregation induced by threshold concentrations of thrombin and collagen was unaffected by oligomycin pre-treatment (Online Supplementary Figure S1F, J, K), which further bolsters the hypothesis that glycolytic ATP is necessary and sufficient to fuel platelet aggregation.
The above observations led us to consider that the molecular events underpinning the effects of CCCP on
AB
CDE
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Figure 3. Effects of pre-treatment with mitochondrial inhibitors (antimycin, oligomycin) or an uncoupler (CCCP) on platelet ATP, reactive oxygen species, cal- cium and lactate release rates. Washed human platelets were pre-treated with vehicle (control), antimycin (2 μg/mL), oligomycin (10 μg/mL) or CCCP (100 μM) for 15 min at room temperature, followed by addition of thrombin (0.5 U/mL) for 5 min (mitochondrial calcium), 15 min (cellular ATP), or 30 min (lactate release rates, mitochondrial reactive oxygen species [ROS] and intracellular ROS). (A-C) Bar diagrams indicating cellular ATP levels, mitochondrial calcium and lactate release rates, respectively. (F, G) Histogram overlay plots representing MitoSOX (Invitrogen #M36008) and dichlorofluorescein (DCF) (Sigma #D6883) fluores- cence intensities, respectively. (D, E) Corresponding bar diagrams indicating mitochondrial and intracellular ROS levels, respectively. Each dot represents an independent observation. Data are presented as mean ± standard error of mean *P<0.05 with respect to vehicle-treated thrombin-stimulated platelets. #P<0.05 with respect to vehicle-treated unstimulated platelets. Statistical significance of difference of means was tested by repeated measures analysis of variance and the Dunnett multiple comparison test. RP: resting platelets; Thr, thrombin.
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