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Platelet adhesion and aggregation are involved in hemostatic maintenance of vascular tone, regulating blood flow turbidity, and inducing inflammatory immune responses to injury. Platelets have been found to secrete redox gasotransmitters, nitric oxide (NO) and hydrogen sulfide (H2S), to regulate their thrombolytic functions. High concentrations of NO can competitively inhibit oxygen binding to cytochrome c oxidase, suppressing oxidative phosphorylation. Nitric oxide can react with superoxide radicals (O2•–) to form peroxynitrite (ONOO•–), a potent oxidant that can damage mitochondrial electron transport proteins. Conversely, H2S can antagonize the deleterious effects of NO through inhibiting O2•– formation and scavenging ONOO•–. Previous work from our lab has found that platelet activation is primarily dependent on glycolysis, but oxidative phosphorylation plays a secondary role for normal functions. Therefore, we propose the presence of a gasotransmitter-mediated mechanism protecting against spontaneous platelet activation during the induction of ROS/RNS. In this study, DetaNONOate (an NO donor) in conjunction with diallyl disulfide (DADS), a H2S donor, was used to evaluate the potential roles of redox transmitters in platelet metabolism using the Seahorse Extracellular Flux Analyser. Our findings corroborated previous understandings of the mitochondrially toxic effects of NO, as elevated DetaNONOate inhibited oxygen consumption rate (OCR) in a dose-dependent manner, suggesting that electron transport machinery was being suppressed or damaged; this dose-dependence was also found with DADS pre-treatment. Damage was reversed when platelets were pre-treated with DADS prior to DetaNONoate exposure; DADS resulted in increased glycolytic flux, leading to recovery of mitochondrial function. These data demonstrate the platelets’ abilities to compensate for acute damage to oxidative phosphorylation machinery using H2S.

Acetaminophen (APAP) is a common over-the-counter pain reliever with high hepatotoxic potential. Although APAP hepatotoxicity has been recognized for years, the mechanisms remain elusive. APAP is metabolized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome p450 and conjugated with GSH for detoxification, causing oxidative stress that impairs mitochondrial function. In a previous study, we found that APAP decreases rat hepatocyte respiration, and this effect is exacerbated in alcohol-treated rats. Recently, it has been reported that following APAP overdose, platelets and monocytes accumulate in the liver contributing to hepatotoxicity. In the present study, we report the inhibition of mitochondrial function of platelets and monocytes, as well as the oxidative burst capacity of monocytes and neutrophils treated with APAP. Platelets, monocytes, and neutrophils were isolated from blood of healthy human volunteers. The cells were seeded into XF96 microplates and treated with APAP in a dose-response manner for 30 min prior to mitochondrial stress test (MST). In platelets and monocytes, APAP decreases mitochondrial function in a dose-dependent manner revealed by decreased basal oxygen consumption rates (OCR), as well as decreased maximal OCR, reserve capacity, and ATP-linked OCR. In addition to MST, we tested the platelets activation with 0.2 U/ml of thombin, and monocytes and neutrophiles oxidative burst measured following activation with phorbol 12-myristate 13-acetate (PMA). APAP (5 mM) completly abolished thrombin-activation of platelets. Similarly, APAP inhibited the oxidative burst in neutrophils in a dose-dependent manner. Taken together, these data show that within an hour, APAP causes significant bioenergetic changes of platelets, monocytes and decreases the oxidative burst magnitude of neutrophils and monocytes. This work was supported by National Institute of Health grants R21AA023273 (to VDU and AS).