Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD ⁺ and ATP Depletion

Abstract

Objectives: Carbon monoxide (CO) produced by haem oxygenases or released by CO releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO–induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry–based metabolomics were used. CORM-A1–induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1–induced delay of Krebs cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD + ) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD + –dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1–induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD + regeneration, pointing out to the key role of NAD + depletion in the inhibition of platelet bioenergetics by CORM-A1. Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration—attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis—ascribed to cytosolic NAD+ depletion.

Distinct Pharmacological Properties of Gaseous CO and CO-Releasing Molecule in Human Platelets

“…We demonstrated previously that CORM-A1 inhibited platelet aggregation through inhibition of platelet energy metabolism [ 11 ]. To compare directly the effects of CO G and CORM-A1 on platelet energy metabolism we measured oxygen consumption rate (OCR; reflecting mitochondrial respiration) and extracellular acidification rate (ECAR; reflecting glycolysis).…”

“…It is generally accepted that CO acts through the same mechanism in the same experimental system, irrespectively whether delivered as gaseous CO (CO G ) or released from CO–releasing molecules (CO-RMs). However, previous reports from independent groups claimed that antiplatelet effect of CO was [ 8 , 14 ] or was not [ 9 , 10 , 11 ] mediated by soluble guanylate cyclase (sGC). Remarkably, such discrepancy has not been explained until now.…”

“…Generally, in the field of CO research, it is accepted that either form of CO delivery induces biological effects by the same mechanism, which is determined by local concentration of CO. Carbon monoxide—gaseous or released by CO-RMs—induces antiplatelet effects in in vitro models [ 6 , 8 , 9 , 10 , 11 ]. However, previous reports provided by independent groups claimed that the antiplatelet effect of CO was mediated by soluble guanylate cyclase (sGC) when platelets were treated with gaseous CO [ 8 ], or independent of sGC when platelets were treated with CO-RMs [ 9 , 10 , 11 ].…”

“…Carbon monoxide—gaseous or released by CO-RMs—induces antiplatelet effects in in vitro models [ 6 , 8 , 9 , 10 , 11 ]. However, previous reports provided by independent groups claimed that the antiplatelet effect of CO was mediated by soluble guanylate cyclase (sGC) when platelets were treated with gaseous CO [ 8 ], or independent of sGC when platelets were treated with CO-RMs [ 9 , 10 , 11 ]. CO gas and CO-RMs are applied interchangeably; however, the mechanisms of antiplatelet effects induced by these two systems have not been compared directly as yet.…”

“…We demonstrated recently that CORM-A1 slowly releasing CO (t 1/2 = 21 min [ 12 ]) induced antiplatelet effect by the inhibition of oxidative phosphorylation and depletion of NAD + , resulting in the inhibition of glycolysis; subsequently simultaneous inhibition of these processes led to ATP depletion [ 11 ]. In turn, in another type of tissue, namely murine aortic rings, a vasodilatation induced by CO was independently of the source of CO mediated by sGC activation, as reviewed by Foresti et al [ 13 ].…”