“CO”ping with a sticky situation

Carbon monoxide (CO) is a colorless, tasteless, odorless gas. Environmental CO is most commonly caused by incomplete carbon combustion from such sources as faulty furnaces, automobile engines, and fires. Hemoglobin has a 250 fold greater affinity for CO than for oxygen accounting for the approximately 50,000 cases of CO poisoning in the USA each year¹. However, in normal physiology, CO is a gasotransmitter, produced endogenously as a byproduct of heme catabolism via the activity of heme oxygenase (HO)². The biological benefits of low endogenous or low amounts of exogenous CO include cytoprotection, inflammation, and regulation of cell metabolism ³, as well as an intriguing antiplatelet effect⁴. Platelets, the key cellular modulator of thrombosis and hemostasis, lack a nucleus but contain fully functional mitochondria ⁵. Platelet mitochondria regulate the pro-thrombotic function of platelets through not only ATP generation, but also redox signaling and the initiation of apoptosis. Tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) and sodium boranocarbonate (CORM-A1), are the most well-known CO releasing molecules (CORM) which have been shown to simulate the bioactivities of gaseous CO ⁶. In the study published in this issue, the authors, Kaczara et al elegantly demonstrate, using Seahorse XFe technology and liquid chromatography tandem-mass spectrometry metabolomics, that ex vivo treatment of human platelets with CORM-A1 inhibits platelet aggregation through concomitant inhibition of mitochondrial respiration as well as glycolysis (Fig. 1). Consistent with other cell types⁷, the authors show that in platelets, the inhibition of mitochondrial respiration by CO is via the cytochrome c oxidase. The crystal structure of CO-bound cytochrome c oxidase was recently solved⁸. Kaczara el al, also identified the depletion of NAD+, leading to inhibition of glycolysis (at the level of NAD⁺-dependent GAPDH) by CORM-A1 released CO. Interestingly, the NAD+ depletion was found to be not a result of redox reaction; instead non-redox consumption of NAD+ has been proposed- however the mechanism remains unclear. The glycolysis pathway has previously been shown to be critical for platelet aggregation response and even compensates for synthetic inhibition of mitochondrial respiration after platelet stimulation⁹. This negative effect on glycolysis, together with inhibition of mitochondria respiration, was shown to ultimately reduce ATP production and thus platelet aggregation in response to thrombin and collagen. The authors demonstrate, using selective inhibitors, that combined inhibition of both glycolysis and mitochondrial respiration by CORM-A1 released CO is required for platelet aggregation inhibition.

Figure 1:

Carbon Monoxide Releasing Molecules (CORM-A1) in platelet bioenergetics and pathophysiology

Based on these results, the authors propose that the antiplatelet therapeutic strategies targeting metabolism, specifically through the CO pathway, could be beneficial. However there should be caution as excess CO can also lead to platelet activation by displacing NO from platelet surface hemoproteins¹⁰. Therefore, while the study convincingly describes the ex vivo effects of CORM-A1 application on isolated platelets and their bioenergetics; more in-depth animal model studies are needed to investigate whether similar effects are also observed in vivo. Circulating platelets in blood upon exposure to CORM-A1 may show a different phenotype as compared to ex vivo treatment model. An in vivo mouse study could answer some important questions including; 1) at low CO concentrations whether there is detrimental binding to hemoglobin and displacement of oxygen, 2) the concentration dependence of anti vs potential pro-aggregatory platelet effects, and 3) whether the application of CORM-A1 treatment modulates platelet lifespan in circulation or affects the platelet count through possible effects on megakaryocyte. Since platelet respiration machinery is involved in intraplatelet ROS generation, its inhibition by CORM-A1 may disrupt the platelet redox homeostasis and consequently platelet activation (Fig. 1). Moreover, as aging regulates platelet activation and redox homeostasis ¹¹, it would be interesting to further evaluate the antiplatelet effect of CORM-A1 with respect to different age groups.

Overall, the mechanistic understanding of the antiplatelet effects of CO through inhibition of glycolysis and mitochondrial respiration provides critical and much needed information that holds promise for CO as an antiplatelet therapeutic strategy. Further animal studies are however required due to the complex effects of CO on many cellular components including red blood cells.