One of the most important medical advances of the 20th century was the development of cardiopulmonary bypass (CPB) which paved the way to open-heart surgery [1]. While being an irreplaceable part of cardiothoracic surgery, it is well established that extracorporeal circulation systems are associated with the activation of inflammatory and oxidative stress responses [2,3]. Moreover, the exposure of blood to the synthetic surfaces of the heart–lung machine as well as to non-physiological shear stress due to mechanical pumping are responsible for the perioperative hemostatic abnormalities; both bleeding and thrombotic events were reported as major causes of morbidity and mortality in these patients [4,5]. In particular, the CPB circuit determines several structural and functional platelets abnormalities. The activation of platelets that remain in circulation by the bypass circuit will render them hypo-responsive to stimulation with various agonists, which may impair their hemostatic properties [6].

The last decade has witnessed an increased interest for another pathomechanism in the setting of open-heart surgery, namely mitochondrial dysfunction. During CPB, cardiac mitochondrial dysfunction results from systemic inflammation, surgical trauma, and ischemia/reperfusion injury, and in turn, impairs postoperative myocardial contractility and predisposes one to arrhythmias [7]. Not only cardiac but also cerebral mitochondrial dysfunction was reported to occur during CPB. A recent study performed in piglets subjected to 4 h of CPB showed that, even in the absence of local markers of ischemia, CPB was associated with decreased mitochondrial respiration and increased mitochondrial ROS formation in brain cortex homogenates [8].There is one pioneering study in the literature that assessed the mitochondrial respiration of intact platelets isolated from adult patients before and after CPB using the extracellular flux analyzer Seahorse XF24; these authors found neither changes in respiratory parameters nor an association between platelet respiration and aggregation/postoperative bleeding [9]. Of note, the latter observation has been also recapitulated in chronic conditions by the group of Cezar Watala; in a recent elegant study performed in the experimental model of type 1 diabetes, they reported that elevated platelet mitochondria respiration occurred later in the evolution of the disease and was not related to the earlier observed changes in platelet activation/reactivity [10].

However, the intraoperative impairment of platelets due to blood exposure to the CPB circuit occurs in cardiac surgery dependent on its duration, and whether their damage can be rescued has not been investigated so far. Nowadays, several pharmacological strategies for mitochondrial protection or recovery after injury are currently under investigation. Cell-permeable succinates are prodrugs able to pass over the cellular membrane, release succinate in the cytosol, and enhance mitochondrial ATP production. They have been developed to bypass mitochondrial complex I defects [11] and have been reported to support electron transport in various pathologies associated with complex I dysfunction/inhibition [12,13].

The present pilot study aimed to assess whether a first-generation cell-permeable succinate, NV118 (diacetoxymethyl succinate), is able to modulate platelets bioenergetics in patients undergoing cardiopulmonary bypass. Here, we report that NV118 elicited a global improvement in the respiratory function of intact platelets isolated both prior to and after cardiopulmonary bypass.

DMSO was used as the solvent for NV118, and its effect on ROUTINE respiration was firstly assessed. As depicted in Figure 1, no differences in platelet physiological OCR were found prior to (8.98 ± 0.36 vs. 8.96 ± 0.36 pmol × s⁻¹ × 2 × 10⁻⁸ cells—Figure 1A) or after (11.50 ± 0.58 vs. 11.51 ± 0.56 pmol × s⁻¹ × 2 × 10⁻⁸ cells—Figure 1B) cardiopulmonary bypass in the presence of DMSO in the oxygraph chambers.

Furthermore, the effect of NV118 (as compared to DMSO) on intact platelets, isolated both prior to and after CPB, was assessed by recording the above-mentioned respiratory parameters: ROUTINE respiration, LEAK respiration, ET capacity, and ETII.

The main finding of this pilot study is the improvement (via NV118) of mitochondrial bioenergetics in intact platelets isolated from patients prior to and after cardiopulmonary bypass. To our knowledge, this is the first study which investigated the effect of cell-permeable succinate on platelets exposed to extracorporeal circulation. Here, we report a significant increase in all respiratory parameters, in the presence of NV118, in isolated platelets, regardless of the moment of their harvesting.

In particular, routine respiration is dependent on the existent mitochondrial matrix succinate that platelets retain while ex vivo. Since the addition of a membrane-permeable succinate increased oxygen consumption both pre- and post-CPB, this indicates that platelet oxidative metabolism is limited by the concentration of succinate as an energetic substrate. The addition of a cell-permeable succinate would correct this in any condition. From a translational perspective, this observation may have significant clinical implications. For example, the administration of permeable succinates in extracorporeal circulation will correct the substrate deficit and improve platelet bioenergetics.

As previously reported by the Gary Fiskum group in their pioneering work that analyzed platelet respiration in the setting of CPB using the Seahorse extracellular flux analyzer [9], we did not find significant differences in platelet respiration prior to or after CPB using the HRR technique in this pilot study (data not shown). The mean duration of CBP was 1.5 h in this group. We cannot exclude the possibility that longer periods of extracorporeal circulation and/or the presence of metabolic comorbidities (reported to be associated with mitochondrial dysfunction) in patients undergoing open-heart surgery might elicit platelet mitochondrial respiratory dysfunction.

A wide range of pharmacological approaches have been studied to counteract the pathomechanisms responsible for the potential deleterious effects of CPB, namely to mitigate systemic inflammation [17], oxidative stress [18], and ischemia/reperfusion injury lesions [19] and provide metabolic support [20].

In recent years, the use of peripheral platelets as an easy obtainable source of viable mitochondria has been brought to light, which allows the analysis of mitochondrial respiratory dys/function [21] in either acute pathologies such as sepsis [22], cardiac arrest [23], acute leukemia [24], and preeclampsia [25] or chronic diseases such as diabetes [26], obesity [27], and asthma [28]. Additionally, the past few decades witnessed an increasing interest in the development of drugs capable of supporting mitochondrial function, and cell-permeable succinates have emerged as such compounds. Succinate is the substrate of succinate dehydrogenase, which is complex II (embedded in the inner mitochondrial membrane), being crucial for oxidative phosphorylation as it couples the tricarboxylic cycle with the electron transport system activity. Succinate causes the ETS to run sequentially from complex II (i.e., it bypasses CI) all the way down to complex IV, where oxygen is converted to water [29]. Thus, it is particularly useful in pathological conditions associated with dysfunctional CI either due to genetic mutations or damage due to increased oxidative stress or drug toxicity (more frequent in practice). However, succinate is not cell-permeable (being an anion in living organisms), so it has limited intracellular uptake when exogenously administered. To date, cell-permeable succinate prodrugs have emerged as alternative fuel sources in order to prevent energetic dysfunction in various pathological conditions. Accordingly, succinate prodrugs have been reported to improve mitochondrial respiration in cellular models of drug toxicity [12,13,30], peripheral blood mononuclear cells from acute poisoning with carbon monoxide [31], and fibroblasts cultured from a patient with Leigh syndrome (mitochondrial disease with CI defect) [32].

Whether cardiac and cerebral mitochondrial dysfunction reported to occur in some of the patients undergoing CPB can be alleviated by cell-permeable succinates awaits future investigation.

We acknowledge the small number of subjects investigated, and thus, the limited statistical power as an important limitation of this pilot study.

NV118, a cell-permeable succinate prodrug, improved platelet bioenergetics in the setting of cardiopulmonary bypass. Whether this effect can support platelet function during CPB is yet another area of enquiry for future investigation. This finding may also be a voice in the discussion on new pharmacological approaches of organ protection during cardiac surgery.