The coronavirus SARS-CoV-19 that causes coronavirus disease 2019 (COVID-19) has spread to almost 100 countries, infected over 10 million patients and resulted in 505K deaths worldwide as of 30th June 2020 (1). The major clinical feature is acute Respiratory Distress Syndrome (ARDS) with a key complication being heart and multi-functional failure abnormal blood oxygen saturation is at least 95% in most lung diseases, such as pneumonia. Whilst decreasing oxygen saturation accompanies other change, such as stiff or oedematous lungs, increasing levels of carbon dioxide are usually seen in COVID-19 patients with pneumonia (2). Thus, many COVID-19-infected patients with pulmonary involvement have hypoxia and dyspnea as important hallmarks of disease. In COVID-19 patients, despite the respiratory system insufficiently oxygenating the blood, these patients are often alert and feeling relatively well and can easily talk (3).
Red blood cells (RBCs) are highly adapted cells for blood gas transport. At the high oxygen tensions (PO2) prevailing in the pulmonary system, the blood is normally completely saturated with oxygen and hemoglobin (Hb) will formed an R structure. When the blood enters the microcirculation, the PO2 is attenuated promoting oxygen dissociation from hemoglobin and a shift to the T form (4).
Clinical examination of severe cases of COVID-19 patients shows a decreased ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2 ratio) with concomitant hypoxia and tachypnea in most cases (5). Nitric oxide (NO) plays a key role in controlling the vascular system by regulating vascular tone and blood flow following activation of soluble guanylate cyclase (sGC) within the vascular smooth muscle. NO also controls mitochondrial oxygen consumption by inhibiting cytochrome c oxidase (6). RBCs have long been considered as powerful scavengers of endothelial cell-derived NO, participating in systemic NO metabolism mainly by limiting NO bioavailability (7). RBCs passing through the microcirculation sense tissue oxygen conditions via their degree of deoxygenation and couple this information to the release of vasodilatory compounds including ATP and NO to enhance blood flow to hypoxic tissues (8). NO is a free radical and has a critical pathophysiological role in infectious diseases.
RBC intracellular NO is derived from three sources: a) entry from the cell exterior by binding to the highly conserved β-globin chain cysteine 93 residue to form bioactive S-nitrosohemoglobin (SNO–Hb) (9), b) formation from nitrite entering RBC due to the reductive potential of deoxyhemoglobin (10) and c) intracellular production of NO by RBC derived from an active and functional eNOS-like enzyme (RBC NOS). This is localized in the RBC membrane and cytoplasm and has similar properties to eNOS in terms of phosphorylation sites controlling enzymatic activity and its activity dependence on intracellular calcium and L-arginine concentrations (11).
Transfer of NO from SNO–Hb to the membrane-bound anion exchanger (AE1) is required for transfer of NO out of the RBC and is dependent on both the SNO–Hb state (T or R) and the SNO–Hb concentration. Therefore, the ability of SNO–Hb to transfer NO to AE1 or other proteins (e.g., glutathione) are limiting factors in respiratory efficiency. The kinetics and allosteric regulation of Hb nitrosylation by oxygen and pH are consistent with the physiologic mechanisms that modulate tissue blood flow, namely acidosis, hypoxemia and tissue hypoxia lead to NO generation by the RBC via SNO–protein transfer of NO activity (12). In addition, insults such as cellular stress activates RBC NOS, leading to NO release and vasodilation of vessel segments under hypoxic conditions. Together, this supports a prominent role of RBC-derived NO in the regulation of local blood flow (13).
Therefore, the erythrocrine function of RBCs i.e. the release of bioactive molecules including NO, NO metabolites, and ATP are likely to be important in tissue protection and regulation of cardiovascular homeostasis by RBCs. Despite this clear role of NO in vasodilation, there is little evidence regarding the role of NO in COVID-19 particularly in ‘happy hypoxic’ patients. To examine the hypothesis that NO is important in regulating vasodilation during hypoxia in these subjects we studied intracellular levels of NO in COVID-19 patients.