Please cite this paper as:

Loh, D. 2020. The potential of melatonin in the prevention and attenuation of oxidative hemolysis and myocardial injury from cd147 SARS-CoV-2 spike protein receptor binding. Melatonin Research. 3, 3 (Jun. 2020), 380-416. DOI:https://doi.org/https://doi.org/10.32794/mr11250069.

Review

The potential of melatonin in the prevention and attenuation of oxidative hemolysis and myocardial injury from cd147 SARS-CoV-2 spike protein receptor binding

Doris Loh^{1 *}

¹ Independent researcher

*Correspondence: doris@evolutamente.it   Tel: +15127632332

Running Title: Melatonin for hemolysis in COVID-19

Received: April 27, 2020; Accepted: June 8, 2020

ABSTRACT

     The rapid escalation of pandemic health threats associated with the novel, pathogenic SARS-CoV-2 coronavirus poses unprecedented challenges as proven effective vaccines and drugs have yet to be produced. Refractory hypoxemia and myocardial injury have been observed as two of the major causes of fatality in COVID-19 patients. SARS-CoV-2 spike (S) protein binding to broadly expressed CD147 receptors on erythrocytes causes oxidative hemolysis that may result in refractory hypoxemia and myocardial injury. Both of these life-threatening conditions are further exacerbated by imbalance in ACE2 from spike (S) protein receptor binding. Dysregulation in the CD147-cyclophilin A signaling pathway, together with altered calcium signaling from SARS-CoV-2 ion channel activities, may contribute to hypercoagulation, thrombosis, and cardiac remodeling resulting in heart failure. Melatonin is an ancient pleiotropic molecule with recognized antioxidant properties that is essential for the protection of erythrocytes from oxidative hemolysis. Found in erythrocytes, melatonin can reverse hemolytic anemia, normalize heme synthesis, restore lymphocytes and platelet counts, and reduce vessel permeability during an acute hemolytic crisis by maintaining intracellular calcium homeostasis and reduction of oxidative stress. In acute hypoxic conditions, melatonin is cardioprotective via blunting of cardiopulmonary response to hypoxia and suppressing hypoxia pathways. Melatonin normalizes endothelial-dependent nitric oxide production to prevent multiple organ damage from hypercoagulability, thrombosis, and hypertension associated with oxidative hemolysis and ACE2 deficiency, protecting cardiomyocytes from hypertrophy. This review discusses the full potential of melatonin as a safe and effective therapeutic intervention for the prevention and attenuation of hemoglobinopathies, refractory hypoxemia and myocardial injury during critical COVID-19 infections. 

Key words: melatonin, COVID-19, CD147, ACE2, hemolysis, hypoxia, myocardial injury, erythrocytes, platelets.

_____________________________________________________________________________

1. INTRODUCTION

     The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein exhibits high affinity to angiotensin-converting enzyme 2 (ACE2) (1, 2). ACE2 is a homologue of ACE that degrades angiotensin I into angiotensin 1-9 and converts angiotensin II into angiotensin 1-7. Host cell entry and membrane fusion begins with the successful binding of spike protein RBD to ACE2 receptors using ACE2 molecules captured by RBDs (3). Furin enzyme cleavage at S1/S2 site may precede the priming of spike protein by host serine protease TMPRSS2 (4). ACE2 is highly expressed in cardiomyocytes, cardiac fibroblasts and coronary endothelial cells. An imbalance in ACE2 can result in blood pressure dysregulation and acceleration of heart failure progression. The possible downregulation of ACE2 protein expression as a result of SARS-CoV infection (5) may contribute to acute myocardial injury and cardiovascular system damage observed in COVID-19 patients (6-9). The negative correlation between ACE2 expression and COVID-19 fatality observed in an analysis of ACE2 expression quantitative loci (eQTLs) from GTEx in 30 tissues from thousands of individuals (10) further emphasizes the important essential physiological functions of ACE2 that could protect COVID-19 patients during infections.

     The abundant expression of ACE2 receptors in differentiated airway epithelia (11) supports the initial observation of prevalent pneumonia that could evolve rapidly into acute respiratory distress syndrome (ARDS) in COVID-19 infections (11, 12,).  Recent accounts from northern Italy describing COVID-19 patients who fulfilled the Berlin criteria of ARDS revealed atypical presentation of the ARDS syndrome. The severity of hypoxemia in these patients was disproportionate to their relatively normal lung capacities.  The unexpected discrepancy between relatively high respiratory compliance of 50.2 ± 14.3 ml/cmH2O indicating acceptable lung gas volume and shunt fraction of 0.50 ± 0.11 has never been reported before in ARDS patients (13). In severe ARDS, the level of hypoxemia would be linearly associated with low respiratory system compliance (14).  

    While atypical ARDS features are being reported in COVID-19 patients in different countries (15), two cohort studies representing early data from hospitalized COVID-19 patients in Wuhan, China, identified the association between myocardial injury and high mortality rates (51.2%, 42/82) (16). Myocardial injury resulting in cardiac dysfunction and arrhythmia was found in 27.8% of COVID-19 patients in a case series study involving 187 patients (17). Both cohort studies found elevated troponin T (TnT) and troponin I (TnI) correlated with high mortality rates (59.6%, 31) compared to patients with normal troponin levels (16, 17). Mortality rate was unusually high (37.5%, 6/16) in COVID-19 patients without any prior history of cardiac disease (17).   

     The binding of ACE2 to SARS-CoV-2 spike protein RBD may explain the development of ARDS and myocardial injury in infected patients. The unexpected dissociation in severe hypoxemia and relatively normal lung capacity in critical COVID-19 patients remained largely unelucidated. The report by Liu et al. (18) of an in silico demonstration of the coordinated attacks by SARS-CoV-2 ORF1ab, ORF10, and ORF3a proteins on the 1-beta chain of heme in hemoglobin and the subsequent dissociation of iron ion, docking of viral protein ORF8 in porphyrin, presented the first working hypothesis that supports hemolysis in COVID-19 infection. The subsequent in vitro identification of CD147 as a novel receptor of SARS-CoV-2 spike protein (SP) by Wang et al. employing immuno-electron microscope to determine the localization and CD147-SP interaction demonstrated viral invasion of Vero E6 cells was facilitated by CD147/SP interactions (19). The development of refractory hypoxemia and myocardial injury can be the direct result of CD147-SP interactions as CD147 are highly expressed in erythrocytes (20) and cardiomyocytes (21). The invasion of host cells by SARS-CoV-2 through CD147 as a novel receptor of SP could result in myocardial injury and extensive pathologies associated with oxidative hemolysis. Even though Meplazumab (anti-CD147 antibody) has been found to inhibit SARS-CoV-2 invasion by blocking CD147 (22), its use should be carefully considered.

2. DIRECT EFFECTS OF CD147/SPIKE PROTEIN INTERACTIONS

     CD147, also known as Basigin or EMMPRIM, is a transmembrane glycoprotein with diverse pleiotropic functions, regulating both physiological processes in vision, spermatogenesis, and pathological processes in cancer involving apoptosis, cell proliferation and migration (23). The pleiotropic effects of CD147 is uniquely demonstrated in its ability to promote either lysis or fibrosis dependent on cell microenvironment and cell-cell interactions. Inflammation induces glycosylation of CD147, promoting adhesion, migration and enhancement of inflammatory signaling (NF‐κB) and synthesis of matrix metalloproteinases (MMPs) (24, 25). CD147 has been deeply implicated in thrombotic and inflammatory pathways in coronary artery disease (CAD) progression due to its wide expression on many cell types including hematopoietic, endothelial cells, leukocytes, platelets, to name a few (26). In erythrocytes, CD147 promotes P. falciparum malaria parasite invasion of red blood cells serving as direct receptor for the parasite ligand PfRh5, in a way not dissimilar to the binding to spike protein of SARS-CoV-2 (27).

2.1.  Mature erythrocyte plasma circulation and leukocytosis.

     CD147 is an adhesion molecule that is expressed in all stages during the differentiation and maturation of erythrocytes. CD147 is essential for the expression of monocarboxylate transporter 1 (MCT1) on erythrocytes, forming a complex with MCT1 (28, 29). MCT1 facilitates the transport of lactate and pyruvate (30). Dysregulation in CD147/MCT1 can lead to the accumulation of lactate in erythrocytes, resulting in the disturbance of the lactate/pyruvate ratio required for normal glycolytic flow in mature erythrocytes (31). Blocking CD147 adhesion on plasma surfaces of mature erythrocytes completely disrupts their circulation, resulting in anemia as erythrocytes become trapped in the spleen, resulting in splenomegaly (32). In the inflammatory microenvironment of splenomegaly, CD147 can induce liver injury and fibrosis (33), as inflammation induces glycosylation of CD147 that can promote adhesion and migration (24). During inflammation, the spleen recruits leukocytes directly from the blood (34). The development of leukocytosis (14.1 x 103/uL) in COVID-19 infection has been reported (35). Leukocytosis in either normal or leukemic cells is often correlated with leukocyte adhesiveness/aggregation in the peripheral blood (36). During inflammation, leukocytes upregulate CD147, exacerbating cell-cell aggregation to cause significant fibrosis and liver injury. Blocking of CD147 in vivo can reduce leukocyte aggregation and significantly attenuate liver injury and fibrosis (37). In severe COVID-19 patients, liver dysfunctions marked by abnormal elevation in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have also been observed (38). In view of the possible diverse effects of CD147 spike protein binding on red and white blood cells, further research and clarification of the exact role and function is urgently needed.

2.2. Lymphopenia.

     Lymphopenia (lymphocytopenia) in COVID-19 patients can accurately predict disease severity

and mortality (39). COVID-19 non-survivors have significantly lower lymphocyte counts than survivors, as functional exhaustion of lymphocytes is correlated to disease progression (40, 41). Critical COVID-19 patients were found with low lymphocyte counts (0.8 vs 1.0 ×10⁹; P < 0.001), higher neutrophil-to-lymphocyte ratio (5.5 vs 3.2; P < 0.001) that reflected high neutrophil counts (4.3 vs 3.2 ×10⁹; P < 0.001) (42). Lymphopenia in severe COVID-19 patients may be due to the active destruction of lymphocytes. Studies have demonstrated that SARS-CoV-2 could invade T-lymphocytes through spike protein receptor binding to CD147 (22, 43). The destruction of lymphocytes by CD147 SP invasion is extremely plausible since ACE2 is entirely absent in lymphocytes, while CD147 is highly expressed on activated T-lymphocytes (44). Lymphopenia is often accompanied by coagulopathy in COVID-19 patients (39). A high platelet to lymphocyte ratio has been correlated to COVID-19 disease severity, where patients with distinctly elevated platelet counts had longer average hospitalization (45).

2.3. Thrombocytopenia, coagulopathy and myocardial injury.

     Hypercoagulability leading to widespread microvascular thrombosis, characterized by the depletion of platelets and coagulation proteins, results in life-threatening bleeding in coagulopathy (46). A meta-analysis of four studies involving 1,427 COVID-19 patients found thrombocytopenia (OR, 5.1; 95% CI, 1.8-14.6) to be correlated with a five-fold increase in risk of disease severity. A subgroup analysis detected mortality to be associated with an even lower platelet count (WMD, -48×10⁹/L; 95% CI, -57 to -39×10⁹/L) (47). A separate large-scale study involving 1,476 COVID-19 patients revealed that thrombocytopenia is strongly correlated to in-hospital mortality rate. A 92.1% mortality rate was associated with the group with the lowest platelet count (0, 50 x 10⁹/L), and only 4.7% with the group with normal platelet count (150- x 10⁹/L).  Patients with platelet counts below 50 x 10⁹/L had the highest mortality relative risk of 13.68 (95% CI 9.89‐18.92) compared to 3.42 (95% confidence interval [CI] 2.36‐4.96) for those with platelet counts between 100-150 x 10⁹/L (48). Von Willebrand factor (VWF) is a platelet-adhesive protein produced by endothelial cells and megakaryocytes (49). The coagulation protein factor VIII must be complexed with VWF to avoid proteolysis and clearance (50). VWF is often found to be elevated, together with factor VIII in severe COVID-19 patients (51, 52). ADAMTS13 is a metalloprotease that cleaves von Willebrand factor to prevent thrombocytopenia. ADAMTS13 is downstream of CD147 (53, 54). CD147 is a novel platelet surface receptor that activates platelets (55). In humans, 20.45% +/- 1.63% of circulating platelets express CD147 (56). Binding of SP to CD147 can deactivate CD147, inhibiting ADAMTS13, while at the same time, CD147 SP receptor binding on platelets may induce platelet degranulation, leading to dysregulation in coagulation and possible destruction of platelets (57). CD147-Cyclophilin A (CypA) signaling can activate platelets, causing increased adhesion and thrombus formation in vitro and in vivo (57). 90% of COVID-19 inpatients with pneumonia with elevated D-dimer showed increased coagulation activity. A D-dimer value greater than 1 μg/mL (18·42, 2·64–128·55; p=0·0033) was associated with high mortality. 81% of non-survivors (44/54) had D-dimer values over 1 μg/mL, compared to 24% (28/118) in survivors (58). Hypercoagulation in COVID-19 patients (59) can be the result of hemolysis which has been shown to activate platelets in vitro and in vivo (60).

     Myocardial ischemia/reperfusion injury is exacerbated by upregulation of platelet surface receptors such as CD147 that results in cardiac damage via activation of immune cells or modification of cardiovascular endothelium (61, 62). CD147 has recently been demonstrated to promote cardiac fibroblast proliferation. Elevated serum levels of CD147 in patients with heart failure predicted poor prognosis as CD147 is essential in the pathogenesis of cardiac hypertrophy, fibrosis and failure in humans and mice (63). In COVID-19 patients, 20% of non-survivors (11/54) were found to have platelet counts of <100 × 10⁹ per L, compared to only 1% (2/137) in survivors (58), while elevated platelet counts also indicated poor prognosis for COVID-19 patients (64). In a retrospective single-center study, 11.8% (32 of 271) of enrolled COVID-19 patients, 71/9% of whom were elderly with low lymphocyte count on admission, developed delayed-phase thrombocytopenia 3 to 4 weeks after symptom onset. These patients exhibited mean nadir platelet count at 86.0 × 10⁹ /L (SD = 37.48) (65).

3. CD147/SP INTERACTIONS INDUCE ACUTE OXIDATIVE HEMOLYSIS

     Erythrocytes are now understood to affect immune function with no less than 46 pro- and anti-inflammatory cytokines detected in red blood cell lysates of healthy individuals (66). The cytokine interleukin 1 beta (IL-1β) and its receptor IL1R1 have been demonstrated to enhance megakaryocyte maturation and activate platelets under proinflammatory, prothrombotic conditions in humans and mice (67).  Extracellular hemoglobin (ECHb), when oxidized, is able to upregulate expression of NLRP3 inflammasome, activating IL-1β production in human umbilical vein endothelial cells (HUVECs) (68, 69).  In vivo, hemolysis and heme have been shown to activate NLRP3 inflammasome in macrophages (70, 71).  Elevated platelet counts in COVID-19 patients have been associated with increased cytokines that stimulate the release of platelets (72).  Elevated levels of fibrinogen and fibrin degradation product D-dimer, as well as VWF and factor VIII, may indicate a dysregulation in the balance between coagulation and fibrinolysis (73). In critically ill COVID-19 patients, shutdown of fibrinolysis is significantly correlated with thrombotic complications (74). Patients with higher levels of hemolysis have increased buildup of platelet clots as extracellular Hb can increase platelet adhesion and thrombosis by interacting with the A1 domain of VWF (75).  Injury to lung tissue and pulmonary endothelial cells can cause the activation, aggregation and retention of platelets in the lung (76).  The lung is a major site where mature megakaryocytes release platelets. Approximately 50% of total platelets produced in the human body, or about 10 million platelets per hour, are generated in the lung (77). Lung injury or damage to pulmonary capillary beds can block megakaryocyte ruptures (78), resulting in reduction of platelet release and platelet synthesis (79). During acute lung injury or ventilator-induced lung injury, CD147 expression in lung endothelial and epithelial cells may be significantly upregulated (80, 81).  

     The invasion of host cells including lymphocytes by SARS-CoV-2 spike protein receptor binding to CD147 receptors has been clearly identified (19, 22, 43). The broad expression of CD147 on erythrocytes and platelets imply that SARS-CoV-2 can readily bind and fuse with erythrocytes and platelets. It is likely that spike protein fusion peptides and internal fusion peptides participate in cell entry by coronaviruses (82), causing hemolysis. Hemolysis describes the pathological condition when disruption or damage of erythrocyte plasma membrane results in the release of hemoglobin or other intracellular components to surrounding plasma.  More than 30 grams of cell-free heme may be released from erythrocytes during hemolysis (83).

     Erythrocytes are dependent upon a robust antioxidant system to maintain hemoglobin (Hb) in a reduced state that binds and transports oxygen and carbon dioxide throughout the body (84). Iron ions in the center of heme groups within hemoglobin can only bind oxygen in the reduced ferrous (Fe²⁺) state (85). During hemolysis when the cell membranes of erythrocytes are disrupted or damaged, such as during an attack by SARS-CoV-2 spike protein receptor binding with CD147, Hb is rapidly released from ruptured erythrocytes, forming cell-free hemoglobin. Without adequate antioxidants in plasma, Hb becomes destabilized and participates in oxidative reactions resulting in formation of methemoglobin in the ferric (Fe3⁺) state which can release free heme at much higher rates (86, 87). Under oxidative conditions with un-neutralized H₂O₂, ferrous iron (Fe²⁺) in free hemoglobin may react with H₂O₂ as a “Fenton” reagent that catalyzes the generation of dangerous hydroxyl radicals, causing significant cellular and tissue damage (88). The in silico model of heme attack by SARS-CoV-2 implies an acceleration in the release of oxidized Hb (18), leading to acute oxidative hemolytic crisis in COVID-19 infection. In COVID-19 patients, median (IQR) serum ferritin levels for non-survivors (n=54) was extremely high at 1435.3 μg/L, compared to 503.2 μg/L in survivors (n=137) (58). High serum ferritin is often detected in patients with hemolysis (89) as iron is processed and recycled at exceedingly elevated rates during active hemolysis than under normal conditions (90).   COVID-19 patients with anemia were found to have a higher mortality rate — 26% in non-survivors versus 11% in survivors (58).  In a meta-analysis involving 1,210 COVID-19 patients, hemoglobin levels were found to be dramatically lower in patients with severe disease (224, 18.5%), and the decrease in hemoglobin levels was correlated to deterioration in clinical progression. (91)

3.1. Acute pulmonary embolisms in COVID-19.

     Hemolysis is associated with pathologies including acute and chronic vascular disease, thrombosis, endothelial dysfunction, renal impairment/failure, immune dysregulation, and inflammation. Extravascular translocation of Hb during hemolysis can cause oxidative reactions with physiological oxidants such as hydrogen peroxide and lipid peroxides (92). Extracellular heme (ECHb) has devastating effects on the vasculature as ECHb scavenges nitric oxide (NO) to promote pathogenesis of clinical events associated with reduced NO bioavailability (83) – hypercoagulability in pulmonary hypertension where downregulation of platelet activation results in platelet aggregation and vascular clot formation. Hemolysis has another route in the promotion of thrombosis via the binding of extracellular Hb with von Willebrand factor (VWF) to competitively block cleavage by ADAMTS13 in vitro (93). The receptor binding of CD147 by SP may inactivate the protein, further inhibiting downstream ADAMTS13 activation (54).

     Acute Pulmonary Embolisms (APE) is the third most common cause of death from cardiovascular disease after myocardial infarction and stroke (94). Pulmonary hypertension often develops after a pulmonary embolism event (95). Pulmonary hypertension and thrombotic complications are increasingly associated with hemolysis which can deplete nitric oxide by ECHb resulting in hypercoagulability (96, 83). Hypercoagulability in COVID-19 patients in a retrospective study from China has been associated with increased risk for venous thromboembolism (VTE) (97). VTE (deep vein thrombosis or pulmonary embolism) has been positively correlated with myocardial injury in that both VTE and thromboembolic arterial diseases involve the formation of clots within blood vessels (98). A study of 94 COVID-19 patients in Wuhan, China, identified significantly deranged coagulation functions, where D-dimer and fibrin/fibrinogen degradation products (FDP) were higher in patients with severe SARS-CoV-2 compared to healthy controls (99). COVID-19 medical emergencies are further complicated by diagnostic challenges presented in the overlap of symptoms in ARDS and pulmonary embolism (100). In general, patients with severe, acute pulmonary embolism (PE) have arterial hypoxemia (101).

3.2. Refractory hypoxemia and hypoxia.

     COVID-19 patients under mechanical ventilation for the treatment of ARDS symptoms may experience refractory hypoxemia where adequate levels of inspired oxygen cannot compensate for inadequate arterial oxygenation. Mortality rate is extremely high from refractory hypoxemia.  Physiologically, an increase in partial pressure of oxygen in arterial blood (PaO₂) of <5 mmHg if fraction of inspired oxygen (FiO₂) is increased by 0.1 is generally recognized as refractory hypoxemia (102). In COVID-19 patients, the unexpected severity of refractory hypoxemia that is dissociated from lungs exhibiting relatively normal lung functions can only be explained by acute oxidative hemolytic crisis from SARS-CoV-2 damage of erythrocytes (18), where tissue hypoxia eventually results from increased methemoglobin (MetHb) levels and the concomitant reduction of viable heme that can bind and transport oxygen and carbon dioxide.

     Hypoxemia occurs when arterial oxygen tension (PaO₂) is below normal. Hypoxemia can lead to systemic hypoxia where inadequate oxygen supply restricts functions in tissues and cells. Myocardial infarction results from coronary artery obstruction leading to heart tissue ischemia. Heart failure and mortality is significantly increased by tissue necrosis from hypoxia. Myocardial injury can result from hypoxemia, where a decrease in oxygen partial pressure (pO₂) is observed (103, 104). Under normal physiological conditions, hypoxia induces gene expressions that initiate erythropoiesis, activating the maturation and proliferation of hematopoietic progenitor cells (hematopoietic stem cells) present in peripheral blood and bone marrow (105). However, it is imperative that the precise role of SARS-CoV-2 SP binding to CD147 on hematopoietic progenitor cells (HPC) be clarified as CD147 has recently been demonstrated to be important for the proliferation of HPC (106). Bone marrow and umbilical mesenchymal stem cells all express CD147 (107). It has been proposed that the loss of airway epithelial cells during COVID-19 infections is caused by defective cellular repair/renewal from viral infection of regenerative stem cells as a result of CD147/SP interactions (108, 109). The binding and membrane fusion of SP to HPC can cause membrane permeabilization, depolarization and apoptosis (110), not only in HPC, but platelets, erythrocytes and other cells that express CD147 and ACE2. Varga et al. introduced histological evidence of direct viral infection by SARS-CoV-2 of endothelial cells that caused extensive inflammation and apoptosis in COVID-19 patients (111).

4. VIROPORIN ION CHANNEL ACTIVITIES — INDIRECT EFFECTS OF CD147/SP INTERACTIONS

     SARS-CoV-2 encodes viroporin transmembrane proteins that display ion channel activities, just like SARS-CoV-1 and other coronaviruses (112). Coronavirus viroporins are pore-forming proteins that modulate cellular ion channels in order to regulate and facilitate multiple stages of viral entry, assembly, release, and pathogenesis (113). Viroporins ORF3a and E protein in SARS-CoV-1 have been found to be indispensable for maximum replication and virulence (114, 115, 116). E proteins on the viral envelope form protein-lipid channels in cell membranes that allow passage of calcium ions, promoting virulence (117, 118). Increased intracellular calcium results in ionic imbalance that activates the NLRP3 inflammasome, initiating release of proinflammatory cytokines (119, 120). E protein ion channel activities in SARS-CoV-1 enhanced pulmonary damage, edema accumulation and death (121). E proteins are highly conserved in coronaviruses. A functional pangenome analysis revealed that the SARS-CoV-2 E protein differs from that of SARS-CoV-1 by only three substitutions and one deletion. These substitutions and insertions are all positioned in flexible cytoplasmic regions where they would be unlikely to affect the binding proteins and ion channel functions (122). An in silico model was also able to demonstrate the similarity in functions between SARS-CoV-1 and SARS-CoV-2 E proteins, whereby the a-helix and loops present in SARS-CoV-2 E protein modulate ion channel activities, increasing pathogenesis in humans (123). The inhibition of E protein in SARS-CoV-2 has now been proposed as a viable treatment alternative for COVID-19 (122). ORF3a in SARS-CoV-1 has been shown to increase NLRP3 inflammasomes (124). Similar to the E protein, ORF3a in SARS-CoV-2 have also been found to have highly conserved domains that are linked to virulence, infectivity, ion channel formation and viral release (125). ORF3a in SARS-CoV-1 is known for its K⁺ ion channel activities that cause apoptosis (126). Domain III in ORF3a of SARS-CoV-2 has been shown to consist of a K⁺ ion channel (125). K+ ion channel activities from SARS-CoV-2 infection could be the reason behind hypokalemia observed in COVID-19 patients (127). Potassium ion imbalance can also cause calcium influx (128). ORF3a in SARS-CoV-1 has been shown to bind to calcium in cytoplasmic domains whereby significant changes in their protein conformation were observed upon calcium binding (129). It is of interest to note that hemolysis can also result from cellular leakage of potassium (130).  

     A scholarly interpretation of a paper that presented the first native RNA sequence of SARS-CoV-2 (MT007544.1) transcriptome and epitranscriptome using Oxford Nanopore Technology (131), indicated an unexpectedly high number of reads in SP for CD147 (67 reads for CD147 compared to 1 read for ACE2). The possibility that SARS-CoV-2 SP predominantly binds to CD147 over ACE2 further provides explanation of the different severe pathologies observed along with acute hemolytic crisis from erythrocyte damage.

4.1. Calcium ion influx initiates apoptotic events.

     Calcium mobilization from viroporin ion channel activities after successful receptor binding creates a high gradient that allows leakages of Ca²⁺ into the cytoplasm (85). Calcium influx causes Ca²⁺ overload and subsequent collapse of ionic gradients leading to membrane permeability and apoptosis (132, 126). Calcium ion influx into endothelial cells which express CD147 (26) increased permeability and activated changes in gene expression in lung endothelial cells (133). Removal of extracellular calcium blocked the permeability increase in microvessels exposed to bradykinin (134).

4.2. Calcium overload in erythrocytes and platelets.

     Calcium overload in erythrocytes contributes to hemolytic anemia through enhanced clearance from circulation (135). In vitro models demonstrated a dose-dependent increase in erythrocyte hemolysis with an increase in calcium concentration in medium. Inhibition of calcium influx via the use of calcium channel blockers terminated hemolysis (136). CD147 is a novel platelet surface receptor that activates platelets (55). Platelet activation is also dependent upon calcium signaling. Yet Ca²⁺ overload can lead to disturbances in platelets. Thrombosis and related disorders have been associated with platelets with enhanced calcium influx and elevated free cytoplasmic calcium. Calcium-loaded erythrocytes adhere strongly to endothelium-derived von Willebrand factor, enhancing the formation of erythrocyte-rich thrombi (137). Patients with thromboses, hypertension, thrombocytopenia, hemolytic anemia, myelofibrosis all exhibited significant elevation of calcium levels. The use of calcium channel blocker nifedipine increased platelet count in patients with immune thrombocytopenia (138). Thrombocytopenia can also be reversed by melatonin (139) which has the ability to maintain calcium homeostasis by modulating ion channel currents, reducing calcium overload. In addition, melatonin inhibits apoptosis, protecting cells and mitochondria by increasing membrane potential, reversing the apoptotic effects of depolarization (140, 141). The potential effects of melatonin on CD147 SARS-CoV-2 Spike protein mediated damages are discussed in details below.

5. MELATONIN

     Melatonin (N-acetyl-5-methoxytryptamine) is a mitochondria-targeted molecule that exists in cells of all tested eucarya and bacteria (142). Mainly synthesized in mitochondria (143), it is estimated that 99% of melatonin in vertebrates is most likely not produced in the pineal gland, nor released into the circulation upon pineal production (144). The total amount of extra pineal melatonin synthesized is estimated to exceed those in the pineal gland and in the circulation by more than two orders of magnitude, equivalent to an increase of 100-fold (145). Melatonin is a unique free radical scavenger with characteristics different from other antioxidants (146) All of the secondary and tertiary metabolites of the indoleamine can quench a diverse universe of free radicals (147). One molecule of melatonin can effectively scavenge up to ten reactive oxygen species (ROS), compared to classic antioxidants that can only neutralize one or less molecules of ROS (148). Melatonin has been extensively shown to protect mitochondria by scavenging reactive oxygen species and inhibiting mitochondrial permeability transition pore (mPTP) opening (149). The ability of melatonin to deliver systemic antioxidant protection; preserve cardiac, mitochondrial and lymphocyte functions; restore erythropoiesis; reverse hemolysis, thrombocytopenia, hypercoagulation, hypertension, and hypoxia, renders this indoleamine to be a safe and invaluable therapeutic agent during COVID-19 infections.

5.1.  Melatonin protects cardiomyocyte from hypertrophy. 

     SARS-CoV-2 SP RBD binds to CD147 receptors that are widely expressed in cardiomyocytes (21) and endothelial cells (26). During COVID-19 infections, increased pro-inflammator cytokines can further enhance the expression of CD147 in cardiomyocytes (150). SARS-CoV-2 viroporin ion channel activities mobilize calcium ion influx into cytoplasm (117). Excessive external calcium influx increases internal calcium but depletes external serum calcium (151). Lower serum calcium is strongly correlated to sudden cardiac arrest (152). A retrospective cross-sectional study involving 241 COVID-19 patients in China found hypocalcemia (mean serum calcium levels of 2.12 (IQR, 2.04-2.20) mmol/L) in 74.7% of patients upon hospital admission. Lower calcium levels (≤2.0 mmol/L) were associated with clinical severity and prognosis, with increased organ injury, septic shock and mortality rate (153). In addition, serum levels of CD147 are often found to be highly elevated in patients with heart failure (154). It is currently unknown how SARS-CoV-2 SP binding to CD147 in cardiomyocytes affects CD147-CypA signaling which has been demonstrated to cause cardiac hypertrophy that develops into heart failure (155). 

     Melatonin has been demonstrated to exert therapeutic effects in the protection of cardiomyocytes from hypertrophy induced by angiotensin II (Ang II) and CD147-CypA signaling pathways. It is possible that SARS-CoV-2 SP binding to ACE2 and CD147 may disrupt Ang II and CD147-CypA activities by lowering ACE2 and upregulating CD147 to induce cardiomyocyte hypertrophy via elevated production of reactive oxygen species (ROS). Melatonin pre-treatment of H9c2 cells blocked ROS production promoted by CD147-CypA-dependent Ang II, in a concentration-dependent manner, protecting cardiomyocytes from hypertrophy (156).  Although CD147 is a requisite receptor for the CypA ligand, CD147, which is highly expressed in the left ventricle (LV), has been demonstrated to promote platelet activation and pulmonary hypertension (157, 158). In a transverse aortic constriction rodent model, CD147^+/- mice displayed less hypertrophy and less LV dilation than CD147^+/+ mice (63).  The ability of melatonin to exert myocardial protection is not limited to inhibition of ROS production.

     Excessive calcium influx into cardiomyocyte cytosol from possible SARS-CoV-2 ion channel activities can induce hypercontracture mediating cell death during early reperfusion after brief ischemia (159). Melatonin maintains calcium homeostasis in cardiomyocytes through modulation of IP3R and SERCA2a (Fig. 1), protecting cells against reperfusion injury in vitro (160). Melatonin is a mitochondria-targeted molecule that exists in every cell of every known species (142). Mitochondria provides ATP to endothelial cells while modulating intracellular calcium and the production of ROS. Survival of endothelial cells can be compromised by the opening of mitochondrial permeability transition pore (mPTP) and activation of mitochondrial apoptotic pathways (161). Increased influx of Ca²⁺ ions can suppress mitochondrial membrane potential (ΔΨm), opening mPTP and activating mitochondrial-dependent apoptotic pathways. Melatonin, as the ancient mitochondrial-targeted molecule, protects survival of cardiac microvascular endothelial cells (CMECs) by blocking Ca²⁺ overload in mitochondria via stimulation of MAPK/ERK, inactivating CREB and inhibiting IP3R/VDAC upregulation. Melatonin preserves both structural and functional integrity of mitochondria to prevent apoptosis leading to death of CMECs (162). 

5.2.  ACE2 cardioprotection enhanced by melatonin.

     ACE2 is highly expressed in cardiomyocytes, cardiac fibroblasts and coronary endothelial cells. SARS-CoV infections have been shown to downregulate ACE2 protein expression (5). It is currently unknown how SARS-CoV2 SP binding to ACE2 receptors may contribute to pathologies associated with ACE2 deficiency and imbalance (6-9). ACE2 is a negative regulator of the renin-angiotensin system (RAS) by converting Ang II into Ang 1-7. Loss of ACE2 increases heart failure vulnerability (8), while adequate ACE2 protected mice from ARDS associated lung injury induced by sepsis (163). The importance of nitric oxide deficiency in myocardial injury as a result of ACE2 imbalance was clearly demonstrated in an ACE2-deficient rodent model where impairment in vascular function was caused by decreased nitric oxide, and reduced eNOS expression at protein and mRNA levels (164). Nitric oxide is antifibrotic (165) and can lower blood pressure, protecting the cardiovascular system (166). Melatonin modulates blood pressure biological rhythms (167), having a profound effect on cardioprotection by reducing blood pressure and attenuating organ damage from hypertension. Melatonin regulates both central and peripheral blood pressure by increasing antioxidant defenses and modulating expression of different nitric oxide synthases (168, 169). Melatonin administered to rodents under nitric oxide depletion from L-NAME (nitric oxide synthase inhibitor) treatment, rescued animals from hypertension by lowering systolic blood pressure, insoluble and total collagen concentration and content in the left ventricle (170). Nitric oxide deficiency during neutrophil activation can exaggerate neutrophil adhesion and endothelial cell damage (171). Under inflammatory conditions, various cell types including macrophages, dendritic cells, neutrophils, and airway epithelial cells express inducible nitric oxide synthase (iNOS) (172, 173) iNOS activation can result in the synthesis of a considerable amount of reactive nitrogen oxide species (RNOS) that mediates inflammatory processes (174). Nitric oxide derived from iNOS has been shown to cause proinflammatory and profibrotic alterations induced by particulate matter in lungs of mice (175). Melatonin is a pleiotropic immune modulator with known pro- and anti-inflammatory regulatory effects. Under inflammatory conditions, melatonin has been observed to downregulate iNOS (176). In rodent models of ventilator-induced lung injury (VILI), upregulation of IL-10 by melatonin significantly suppressed iNOS expression induced by VILI. Treatment with luzindole (melatonin receptor antagonist) counteracted the protective effect of melatonin (177).

5.3. Melatonin rescues neutrophil net induced vascular permeability and hypercoagulation.

     One theory identified neutrophil extracellular traps (NETs) as a possible cause for hypercoagulation activities in severe COVID-19 patients. High blood levels of NETs, composed of extracellular DNA fibers that bind pathogens, are correlated with thrombin levels that predict adverse cardiac events that may lead to major organ damages in lungs, heart and kidneys (178). A retrospective analysis of 452 COVID-19 patients found a significant elevation of neutrophil counts (4.3 vs 3.2 ×10⁹; P < 0.001) in severe cases (42). NETs are able to modify endothelial barrier structures, causing vascular endothelial permeability that reduces anti-thrombotic and anti-inflammatory features (179, 180). An in vivo rodent experiment demonstrated that local injection of melatonin (14 or 140 pg) could effectively reduce endothelial cell vascular permeability induced by leukotriene B4-activated neutrophils. The reduction in vascular permeability was likely mediated through the inhibition of endothelial cell hyper-adhesiveness by melatonin (181). In humans, oral melatonin administration (3 mg) resulted in an inverse relationship with procoagulant measures in 46 healthy young men, where increased plasma melatonin predicted lower levels of FVIII:C (P = 0.037) and fibrinogen (P = 0.022) (182). Hypercoagulability and thrombotic complications are frequently observed in patients with hemolytic anemia (183).

5.4. Melatonin attenuates hypoxemia from nitric oxide dysregulation in oxidative hemolysis.

     Hypercoagulability and thrombosis are frequently associated with increased hemolysis as a result of nitric oxide depletion via several mechanisms. Compartmentalized hemoglobin within erythrocytes or haptoglobin have limited ability to scavenge nitric oxide (NO) (184). In vitro, NO reaction time with free hemoglobin (Hb) to form Hb-NO products is 1,000-fold faster than with erythrocytes (185). Vasoconstriction and coagulopathy from the dysregulation of NO during hemolysis is exacerbated by the release of arginase which converts L-arginine into ornithine, further diminishing NO production by reducing NO substrate L-arginine (186). Platelets are an important source of NO. Platelet dysregulation from CD147/SP interactions on platelet membranes can cause NO dysregulation, facilitating platelet activation, adhesion, and aggregation that eventually results in arterial thrombosis (187). Nitric oxide gas inhalation to increase NO availability is now under clinical trial as possible treatment for COVID-19 (188). The use of inhaled nitric oxide should be carefully monitored in the context of oxidative hemolysis due to the possible induction of diffusion hypoxemia, commonly known as the “Fink” or “Third Gas Effect” where PaO₂ can be significantly lowered when O₂ is below 21% saturation (189). A study discovered an alarming correlation between long-term nitrogen dioxide (NO₂) and COVID-19 fatality where 83% of fatalities (3,701/4443 cases) examined occurred in areas where maximum NO₂ concentration was above 100 μmol/m2, while only 1.5% (51/4443 cases) of all COVID-19 fatalities occurred in areas where maximum NO₂ concentration was below 50 μmol/m2 (190). Higher concentration of NO delivered during inhaled NO therapy may also shift the oxygen dissociation curve to the left as nitric oxide increases oxygen affinity of Hb, decreasing heme-heme interactions and increasing formation of MetHb while further reducing oxygen-carrying capacity of hemoglobin in blood (191-193). Yet nitric oxide can exert allosteric effects on Hb to either increase or decrease its affinity to oxygen through different biochemical reactions that yield different Hb products. Increased Met-Hb and SNO-Hb shifts the oxygen dissociation curve towards the left, while increased HbFe²⁺NO shifts the curve the opposite direction to the right (194). Hb-NO product formed as a result of NO dysregulation and subsequent reactivity with target molecules such as Hb can exacerbate endothelial dysfunctions and associated myocardial injury in COVID-19 patients (195). Optimal regulation of NO production and curtailing downstream effects from NO dysregulation is best achieved through the use of melatonin.

     Melatonin is an ancient pleiotropic molecule that can regulate NO dysregulation achieving different biological effects under different situations. Melatonin is well known for its circadian antihypertensive effects. Gene chip analysis of umbilical endothelial cells after 6 hours of melatonin treatment revealed 121 upregulated genes and 214 downregulated genes that resulted in a significant elevation of NO levels and endothelial nitric oxide synthase (eNOS) activity, together with a decrease in endothelin and Ang II (196). While in rodents with metabolic syndrome, melatonin treatment (10 mg/kg/day for 3 weeks) effectively decreased blood pressure by upregulating nitric oxide synthase (eNOS) protein expression and activities in the brain but not the heart (197). On the other hand, under hypobaric hypoxic conditions where rats were exposed to partial oxygen concentration similar to being at 9000 m elevation, pretreatment with melatonin (100mg/kg) inhibited hypoxia-induced increase of nNOS, eNOS, iNOS that would have resulted in neuropsychological dysfunctions including insomnia, dizziness and memory deficiencies (198).

     Increasing evidence showed cumulative incidence of venous and arterial thrombotic complications among COVID-19 patients admitted to ICU, with pulmonary embolism (PE) being the most common insult (199). Most patients with severe, acute pulmonary embolism (PE) have arterial hypoxemia (101). Hypoxemia can lead to systemic hypoxia where inadequate oxygen supply restricts functions in tissues and cells, leading to pulmonary hypertension and vascular remodeling. Daily administration of melatonin (10mg/kg) to hypoxic rats attenuated pulmonary hypertension and vascular remodeling by dramatically increasing eNOS phosphorylation in the lung that restored NO production (200). Hypoxia not only affects NO homeostasis (201), it can alter erythrocyte membrane protein band 3 to initiate hemolysis (202).

6. MELATONIN PROTECTS ERYTHROCYTES FROM OXIDATIVE HEMOLYSIS

     Erythrocytes are able to neutralize endogenous and exogenous reactive oxygen species (ROS) using a robust antioxidant system. Under normal conditions, methemoglobin (MetHb) – hemoglobin that is oxidized into the ferric (Fe³⁺) form, unable to bind and transport oxygen – is reduced back to hemoglobin by NADH–cytochrome b5–metHb reductase. Although Hb auto-oxidation rate is quite slow at around 0.5% to 3% daily, the process generates intracellular ROS H₂O₂, where insufficient intracellular reducants (glutathione, catalase) can cause oxidative damage to membranes via lipid peroxidation (203). The formation of ferryl hemoglobin and oxoferry hemoglobin when intracellular H₂O₂ oxidizes Hb can cause hemoglobin denaturation in erythrocytes (204). Oxidative denaturation of hemoglobin produces membrane damage via the release of hemin that results in the alteration of structural and functional properties of membranes, leading eventually to the destruction of erythrocytes (205). Addition of melatonin in vitro to erythrocytes exposed to oxidative stress that would have resulted in 100% hemolysis in 180 minutes, inhibited the formation of protein carbonyls and hemin precipitation for 20 mins and 10 mins respectively. During oxidative hemolysis, melatonin was observed to be rapidly absorbed into the cytosol and consumed by erythrocytes, delaying Hb denaturation and release of hemin (206).

     Melatonin can effectively reduce both ferryl hemoglobin (Fe⁴⁺= O²⁻) and oxoferryl hemoglobin, the Hb oxidation products that cause Hb denaturation and heme destruction, back into MetHb in the ferric (Fe³⁺) form (207, 208).  Heme in the ferric (Fe³⁺) form has been shown to dissociate from Hb at much higher rates than in the ferryl (Fe⁴⁺) form, as MetHb is less stable due to the occupancy of water coordinated to the iron ion (87). Yet ferryl hemoglobin is highly oxidative as it is associated with radical proteins that can cause lipid peroxidation and generate cytotoxic bioactive molecules (209). The timely reduction of MetHb into functional heme in the ferrous (Fe²⁺) form by NADH–cytochrome b5–metHb reductase is essential in the prevention of rapid heme loss (203).

6.1. Melatonin recycles NAD⁺ to maintain NADH-cytochrome b5 reductase.

     90% of heme degradation products in hemolysis are found on membranes of erythrocytes (204). The integrity of the antioxidant redox system such as NADH–cytochrome b5 reductase (NADH–cytochrome b5–metHb reductase) on membranes preserves Hb functionality and survival.  Patients with recessive hereditary methemoglobinemia have been associated with deficits in this enzyme (210-212).  The human CYB5R3 gene encodes two forms of NADH-cytochrome b5 reductase. Only the erythrocyte-restricted soluble form that is bound tightly to the inner face of the erythrocyte membrane has been shown to be active in methemoglobin reduction (213). The ubiquitous membrane-associated form involved in lipid metabolism is expressed in subcellular compartments such as the endoplasmic reticulum, the mitochondrial outer membrane, and the plasma membrane (214). NADH-cytochrome B5 reductases are the default mechanisms responsible for the reduction of MetHb back into functional heme in the ferrous (Fe²⁺) form in erythrocytes (203), using NADH as principal reductant.  All CYB5R enzymes use NADH as electron donors in one-electron transfer cellular reduction-oxidation reactions (214). The ability of melatonin to recycle NAD is perhaps one of the main reasons why this indoleamine has been found to be actively utilized by erythrocytes under oxidative stress conditions, protecting erythrocytes from hemolysis as a result of hemoglobin denaturation and the release of free heme (206).

     Melatonin is an ideal electron donor due to its electron-rich aromatic indole ring (215) Melatonin has been demonstrated in both cell-free and cultured PC12 cells to preserve NADH levels under oxidative stress where melatonin readily donates an electron to reduce the NAD radical (216).  By recycling NAD⁺, the oxidized form of NAD, melatonin maintains a constant, adequate, intracellular supply of NADH to support the reduction of MetHb by NADH-cytochrome b5 reductase. Since erythrocytes are highly sensitive to oxidative stress, the fact that the active metabolites generated as a result of melatonin oxidation such as N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and N¹-acetyl-5-methoxykynuramine (AMK) are also effective free radical scavengers, renders this electron-rich indolamine to be a unique antioxidant (217).

     The protective role of melatonin in blood may also be linked to its association with a class of two-electron quinone reductase called NRH:quinone oxidoreductase 2 (NQO2).  The NQO gene encodes NQO1 and NQO2 — the two major quinone reductases in mammalian systems (218). NQO1 is not expressed in blood under normal conditions (219), whereas NQO2 is expressed at higher levels in blood (220). The nomenclature of NQO1 and NQO2 may sometimes be confused with NADH-cytochrome b5 reductase due to the common use of the term ‘diaphorase’ for both classes of enzymes more than 50 years ago (221).  The catalytic activity of NQO2, a known human target of the antimalarial drug chloroquine (CQ), can be inhibited by binding within active sites to chloroquine, resveratrol, and melatonin (222, 223). Even though NQO2 has been regarded by some as the third binding site for melatonin (MT3) (224), its affinity to the enzyme is much lower when compared to potent flavonoid inhibitors like quercetin and resveratrol (225). Flavonoids are not endogenous inhibitors of NQO2.  Plants that produce flavonoids have not been identified with NQO genes, whereas vertebrates expressing NQO genes synthesize melatonin but not flavonoids (221).  It is possible that the inhibition of NQO2 by melatonin is meant to occur only under specific conditions as melatonin has been shown to bind to only the free oxidized (FAD) form of NQO2 (223), and NQO2 is known for its ability to generate reactive oxygen species such as superoxide (226, 227). The discovery in 2017 by Cassagnes et al. that anti-malarial indolone-type derivatives could serve as substrates of human NQO2 instead of acting as inhibitors such as chloroquine (228), revives the hypothesis that melatonin could be a naturally occurring co-substrate for NQO2 (229). This hypothesis could not be confirmed by another group employing nuclear magnetic resonance studies (230).  In view of the significance of melatonin as a potential treatment for COVID-19, further studies on the relationship between melatonin and NQO2 are necessary.

6.2. Melatonin protects Band 3 membrane protein in erythrocytes.

     Band 3 protein is the essential anion exchanger on erythrocyte membranes responsible for ion balance, maintaining the shape and integrity of erythrocytes, and optimizing oxygen delivery to cells and tissues (231, 232). The Band 3 glycoprotein or anion exchanger 1 (AE1) encoded by SLC4A1, plays a critical role in efficient respiration by facilitating the electroneutral exchange of chloride and bicarbonate across erythrocyte plasma membranes (231). Band 3, being the most abundant protein (about 25%) in the erythrocyte plasma membrane, is responsible for the largest ion-specific flux known for any single cell in the body (233). Carbon dioxide is converted into bicarbonate after it is diffused into erythrocytes. Band 3 protein transports the bicarbonate anion out of the cell in exchange for a chloride anion.  This exchange process is reversed when Band 3 transports the bicarbonate anion into the cell in exchange for a chloride anion. The bicarbonate anion is then converted into carbon dioxide which will diffuse out of the cell to be expelled by the lung (231). A hypoxic microenvironment can upregulate genetic expression of CD147 via HIF-1a mediated transcription factors (234). Elevated intracellular calcium as a result of CD147/SP interactions induces band 3 protein tyrosine phosphorylation promoted by dissociation of protein tyrosine phosphatase (PTP) from band 3 (235). Protracted oxidative stress-induced phosphorylation of band 3 initiates a series of events that eventually results in oxidative hemolysis (236, 237). In vitro, melatonin at 100 µM concentration protects band 3 protein from H₂O₂-induced oxidative damage to membrane conformation and arrangement, preserving erythrocyte morphology integrity supporting deformability and cell shape to allow for optimal gas diffusion (238-240). Melatonin has been observed to protect band 3 protein even in the absence of catalase, restoring antioxidant enzyme activities in erythrocytes challenged by severe oxidative stress (241). During oxidative hemolysis, heme is the major source of reactive oxygen species (ROS). Free heme can become a potent cytotoxic pro-oxidant due to the iron (Fe) atom contained within its protoporphyrin ring. Hemolytic products including heme and hemin have been shown to directly target the endothelium to promote endothelial cell pro-inflammatory activation in the lung (92, 242-244). Free iron-protoporphyrin in hemin liberated during hemolysis, can promote lipid peroxidation. It has been suggested that endothelial cell death and vascular dysfunction may be the result of mitochondrial dysfunctions caused by posttranslational modification of proteins generated by lipid and reactive oxygen species produced by hemin during hemolytic events (245). A ‘multiple-hit’ model described how hemolysis initiated by a pathological event can release toxic products that are potent endothelial stressors leading to the development of proinflammatory and prothrombotic environments resulting in endothelial cell damage or cell death (246).  SARS-CoV-2 is now believed to induce endotheliitis in multiple organs as viral elements have been identified in endothelial cells that exhibited high levels of inflammation and cell death (111). Children infected by COVID-19 have been found to display pediatric hyperinflammatory syndrome with symptoms similar to Kawasaki disease (247) Kawasaki disease is strongly associated with endothelial cell damage and dysfunction (248-251). Melatonin may attenuate oxidative damage to erythrocytes during hemolysis by protecting Band 3 protein, and chelating free iron to inhibit iron overload in erythrocytes, hepatic, and renal tissues. Even though chelation of iron by melatonin has been reported in literature (252, 253), the exact mechanisms involved remain unclear.

6.3. Melatonin preserves endothelial cells calcium homeostasis during hypoxia.

     Viroporin-induced ion channel activities as a result of CD147 receptor binding to spike protein can increase Ca²⁺ influx, initiating oxidative hemolysis (26, 126, 132, 136). Hypoxia as a result of hemolysis in turn exacerbates influx of Ca²⁺ in endothelial cells (254). Calcium overload resulting in oxidative stress-mediated impaired Ca²⁺ handling in the sarcoplasmic reticulum (SR) leads to deterioration of myocardial functions. Rodents exposed to 10% oxygen for 4 weeks but injected with melatonin showed elevated cardioprotection against myocardial injury under hypoxia by preserving sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) expression, thus improving SR Ca²⁺ handling and calcium homeostasis (255). Hypoxia can increase HIF-1ɑ-mediated migration speed of endothelial cells by 1.4-fold (256). Melatonin downregulates HIF-1ɑ signaling pathways (ERK/Rac1), suppressing HIF-1ɑ expression and stabilization to prevent endothelial cell migration under hypoxic conditions (257, 258). Suppression of HIF-1ɑ expression and stabilization can also reduce increased CD147 activation under hypoxic conditions (234), possibly reducing SARS-CoV-2 virulence and replication. Calcium mobilization can activate NLRP3 inflammasomes, leading to cytokine storms (120). Extracellular Hb from oxidative hemolysis has been demonstrated to activate NLRP3 inflammasomes, initiating platelet activation, aggregation and consumption, resulting in platelet derangement (68, 69, 72). ECHb also enhances VWF platelet adhesion, disturbing the balance between coagulation and fibrinolysis (75). NLRP3 inflammasome is a known target of melatonin (259). Melatonin inhibits NLRP3 inflammasomes through enhancement of Nrf2 expression (260).  Melatonin supplementation may reduce inflammatory effects of cytokine storms induced by SARS-CoV-2 infection by reversing aerobic glycolysis in immune cells (261). The ability of melatonin to increase erythropoiesis, protect platelets and lymphocytes, as well as its capacity to regenerate NADH supplying essential electrons to protect erythrocytes against oxidative hemolysis as a result of CD147/SP interactions, further supports its use during COVID-19 infections (206, 216).

7. MELATONIN ENHANCES ERYTHROPOIESIS, PROTECTS LYMPHOCYTES AND PLATELETS

     Melatonin has been shown in vitro to be readily absorbed by erythrocytes via GLUT1 transporters. The addition of glucose to the growth medium can markedly enhance uptake (262). Melatonin’s association with glucose is closely related to its ability to enhance the expression of glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme in the pentose phosphate pathway (PPP). The PPP consists of an oxidative phase that generates NADPH and a nonoxidative phase that interconverts phosphorylated sugars (263). NADPH produced by G6PD in the PPP is required for the regeneration of oxidized glutathione (GSSG), Without adequate reduced glutathione (GSH), hemoglobin cannot be maintained in its soluble form under oxidative stress (264). Hemolytic events can be easily triggered under conditions of G6PD deficiencies (265, 266). Oral and IV glutathione treatments given to COVID-19 pneumonia patients have been shown to substantially alleviate dyspnea and respiratory distress (267).

7.1. Melatonin upregulates G6PD activity during erythropoiesis.

     G6PD is essential during differentiation of hematopoietic stem cells, protecting these progenitors from oxidative stress (268, 269). During erythropoiesis, after erythroblasts switch to Hb composition during late maturation (270), definitive erythrocytes after hemoglobin switch can undergo apoptosis in mice deficient in G6PD enzymes. Apoptosis of definitive erythrocytes can only be prevented by the restoration of G6PD activity which is indispensable for definitive erythropoiesis (271). Melatonin at pharmacological doses has been shown in both in vitro (0.08 mM to 0.1mM) and in vivo (10 mg/kg) animal models to markedly enhance G6PD enzyme activity in human erythrocytes and in Sprague-Dawley type rats respectively (272). In animal models of lead-induced suppression of the heme synthesis pathways, daily intragastric pretreatment with melatonin (30mg/kg) prevented lead toxicity-induced inhibition of heme-synthesizing enzymes and iron deficiency, restoring enzymatic and non-enzymatic antioxidant levels, while normalizing zinc and copper levels in the liver (273). Anemic patients with chronic kidney disease exhibit erythropoietin (EPO) hyporesponsiveness where there is a significant decrease in Hb or failure to raise Hb despite adequate EPO dose (274). Erythropoietin hyporesponsive anemic patients receiving 5 mg of melatonin showed an impressive increase in Hb, serum iron, and transferrin saturation ratio (TSAT) compared to baseline. When compared to control groups and baseline, patients receiving melatonin displayed significantly reduced inflammatory biomarkers (275).

7.2. Melatonin restores normal lymphocyte functions.

     COVID-19 non-survivors have significantly lower lymphocyte counts than survivors, as functional exhaustion of lymphocytes is correlated to disease progression (40, 41). Glutathione is required for T-lymphocyte proliferation. Inhibition of glutathione can reduce T-lymphocyte DNA synthesis by more than 75% (276). T-lymphocytes produce glutathione using the pentose phosphate pathway where G6PD is the rate-limiting enzyme (277). Glutathione has also been shown to increase production of IL-2 in human lymphocytes. (278). Resting and phytohemagglutinin-stimulated human lymphocytes have been demonstrated to naturally produce and release high amounts of melatonin that exceed nocturnal human physiological levels in serum of up to five-fold. Inhibition of the melatonin biosynthetic pathways in lymphocytes led to the decrease in IL-2 production, which could be reversed by addition of exogenous melatonin in vitro (279). IL-2 is generally regarded as a pro-inflammatory cytokine (280).  There are indications that the production of IL-2 during viral infections can exacerbate the inflammatory environment in the lung (281). The production of IL-2 by lymphocytes may serve to promote effector CD4 T cell long-lived memory generation following pathogenic challenges. Deficient production of IL-2 by effector CD4 T cells initiates default apoptosis, while the addition of exogenous IL-2 can directly rescue some CD4⁺ from acute apoptotic death (282, 283). Melatonin is a multi-tasking pleiotropic immune modulator with known pro- and anti-inflammatory regulatory effects, with the capacity to reduce IL-2 expression and release during severe inflammatory conditions (284). Melatonin synthesized by lymphocytes not only regulates the IL-2/IL-2 receptor system (285), but could be essential in ensuring survival and functioning by enhancing G6PD enzyme activity, protecting glutathione levels (272).

7.3. Melatonin attenuates thrombocytopenia from oxidative hemolysis.

     The CD147CypA signaling pathway activates platelets leading to enhanced adhesion and thrombosis (57). Plasma levels of soluble CD147 (sCD147) in healthy donors and patients with or without coronary artery disease were found to be significantly correlated with standard platelet biomarker soluble GPVI (sGPVI, r=0.46, p=.004). sCD147 levels can be predicted using linear regression analysis of sGPVI levels (β = .445, p = 0.003) and age (β = 0.304, p = 0.038) (157). The effects of CD147/SP interactions on platelets needs to be further clarified as 20.45% +/- 1.63% of circulating platelets express CD147 in humans (56). Platelets contain whole, functional, respiratory competent mitochondria in normal physiological state (286). During oxidative hemolysis, oxidized cell-free MetHb can rapidly deplete platelets via the activation of ROS-mediated mitochondrial apoptotic pathways (287). Antioxidant protection from oxidative stress is essential for the survival and functionality of platelets. Patients with known G6PD deficiency showed enzyme activity reduced to 15% of control subjects, together with greatly diminished NADPH and glutathione levels, and elevated platelet aggregation measurements (288). In an irradiated mouse model (4 Gy), mice receiving 10mg/kg intraperitoneal injection of melatonin for 21 consecutive days after irradiation displayed enhanced recovery rate of platelets and white blood cell counts compared to controls without melatonin treatment (289). In human subjects, 200 patients with persistent thrombocytopenia due to different causes were randomized to receive 20mg/day melatonin orally in the evening for one month. 71/98 (72%) of patients achieved normalization of platelet counts where mean platelet number was rapidly and significantly increased. Among the patients, thrombocytopenia from disseminated intravascular coagulation (DIC) had the lowest response rate to melatonin supplementation (290). At present, the full implications of SARS-CoV-2 spike protein binding with CD147 and the subsequent effects on CD147-CypA signaling are not completely understood. Cyclophilin A has been associated with the pathogenesis of viral infection and replication including SARS-CoV (291, 292).  The fact that CyPA is also identified as a target for antiviral therapy (293) warrants further exploration into its role during COVID-19 infections.

     For the best interests of readers, the potential mechanisms of melatonin targeting CD147 SARS-CoV-2 spike protein are summarized in Figure 1.

Fig. 1. The potential mechanisms of melatonin in the attenuation of CD147 SARS-CoV-2 spike protein mediated damages during infection.

     *COVID-19 patients may show elevated von Willebrand factor and factor VIII, fibrinogen and fibrinogen degradation products D-dimer, while platelet counts are depleted, normal or elevated. GSH: glutathione; NO: nitric oxide; Ca2+: calcium ions; G6PD: glucose-6-phosphate dehydrogenase; eNOS: endothelial nitric oxide synthase; arrows: stimulations; red bars: attenuation.

8. CONCLUSION

     Melatonin is a well-established, potent, free radical scavenger with pleiotropic functions that includes epigenetic regulation (294) affecting circadian biology and maintenance of systemic homeostasis of essential biological processes. The ability of melatonin to preserve the survival and functioning of erythrocytes, platelets, lymphocytes, leukocytes, and mitochondria during SARS-CoV-2 infections renders this ancient molecule to be an indispensable component in the arsenal of pharmacological and natural remedies in the attenuation of symptoms in COVID-19 patients suffering from hypoxemia, myocardial injury, and other hemoglobinopathy related pathologies caused by oxidative hemolysis induced by CD147/SP interactions during receptor binding and subsequent viral fusion and invasion. Furthermore, melatonin can exert a formidable range of influences over the human immune response system, as discussed in great detail by Tan and Hardeland (295), as well as Anderson and Reiter (296). The need to identify optimal dosages for melatonin as effective treatment for COVID-19 infection is an urgent priority (297). While further clarification of CD147/SP interactions and effects is urgently needed, this review has demonstrated the efficacies in the use of melatonin as a safe and effective treatment for the prevention and attenuation of unique pathologies that may be related to CD147/SP interactions during COVID-19 infections. 

ACKNOWLEDGEMENTS 

     The author sincerely thanks DX Tan and reviewers for their insightful comments on this manuscript. Special appreciation to Elizabeth Martorana for her help in editing the manuscript, and to Allan Lenon Cura for the preparation and design of the graphic illustration.

AUTHORSHIP

     DL wrote and edited this article. 

CONFLICT OF INTEREST

     The author declares no conflict of interest.

REFERENCES

1. Lu R, Zhao X, Li J, et al. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet (London, England) 395 (10224): 565–574. https://doi.org/10.1016/S0140-6736(20)30251-8.

2. Ortega JT, Serrano ML, Pujol FH (2020) Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis. EXCLI J. 19: 410–417. doi:10.17179/excli2020-1167.

3. Beniac DR, deVarennes SL, Andonov A, He R, Booth TF (2007) Conformational reorganization of the SARS coronavirus spike following receptor binding: implications for membrane fusion. PloS one 2 (10): e1082. https://doi.org/10.1371/journal.pone.0001082.

4. Hoffmann M, Kleine-Weber H, Schroeder S, et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is blocked by a clinically proven protease inhibitor. Cell 181 (2): 271–280.e8. doi:10.1016/j.cell.2020.02.052.

5. Kuba K, Imai Y, Rao S, et al. (2005) A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat. Med. 11 (8): 875–879. doi:10.1038/nm1267.

6. Zheng YY, Ma YT, Zhang JY, et al. (2020) COVID-19 and the cardiovascular system. Nat. Rev. Cardiol. 17 (5): 259–260. doi:10.1038/s41569-020-0360-5.

7. Guo J, Huang Z, Lin L, et al. (2020) Coronavirus Disease 2019 (COVID-19) and cardiovascular disease: A viewpoint on the potential influence of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on onset and severity of severe acute respiratory syndrome coronavirus 2 infection. J. Am. Heart Assoc. 9 (7): e016219. doi:10.1161/JAHA.120.016219.

8. Patel VB, Zhong JC, Grant MB, Oudit GY (2016) Role of the ACE2/angiotensin 1-7 axis of the renin-angiotensin system in heart failure. Circ Res. 118 (8): 1313–1326. doi:10.1161/CIRCRESAHA.116.307708.

9. Clerkin KJ, Fried JA, Raikhelkar J, et al. (2020) Coronavirus disease 2019 (COVID-19) and cardiovascular disease. circulation. Circulation 141 (20):1 648‐1655. doi:10.1161/CIRCULATIONAHA.120.046941.

10. Chen J, Jiang Q, Xia X, et al. (2020) Individual variation of the SARS-CoV2 receptor ACE2 gene expression and regulation. Preprints 2020: 2020030191.

11. Jia HP, Look DC, Shi L, et al. (2005) ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia. J. Virol. 79 (23): 14614–14621. doi:10.1128/JVI.79.23.14614-14621.2005.

12. Leung JM, Yang CX, Tam A, et al. (2020) ACE-2 expression in the small airway epithelia of smokers and COPD patients: implications for COVID-19. Eur. Respir. J. 55 (5): 2000688. doi:10.1183/13993003.00688-2020.

13. Gattinoni L, Coppola S, Cressoni M, et al. (2020) Covid-19 does not lead to a "typical" acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 10: 1164/rccm.202003-0817LE. doi:10.1164/rccm.202003-0817LE.

14. Cascella M, Rajnik M, Cuomo A, et al. (2020) Features, evaluation and treatment coronavirus (COVID-19). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing.  Available from: https://www.ncbi.nlm.nih.gov/books/NBK554776/.

15. Arentz M, Yim E, Klaff L, et al. (2020) Characteristics and outcomes of 21 critically Ill patients with COVID-19 in Washington State. JAMA. e204326. doi:10.1001/jama.2020.4326.

16. Shi S, Qin M, Shen B, et al. (2020) Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China. JAMA Cardiol. 2020: e200950. doi:10.1001/jamacardio.2020.0950.

17. Guo T, Fan Y, Chen M, et al. (2019) Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19). JAMA Cardiol. 2020: e201017. doi:10.1001/jamacardio.2020.1017. 

18. Liu W, Li H (2020) COVID-19: Attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism. ChemRxiv https://doi.org/10.26434/chemrxiv.11938173.v5.

19. Wang K, Chen Q, Zhou YS, et al. (2020) SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. (preprint) bioRxiv 2020: 03.14.988345; doi: https://doi.org/10.1101/2020.03.14.988345.

20. Coste I, Gauchat JF, Wilson A, et al. (2001) Unavailability of CD147 leads to selective erythrocyte trapping in the spleen. Blood  97 (12): 3984–3988. doi:10.1182/blood.v97.12.3984.

21. Venkatesan B, Valente AJ, Prabhu SD, et al. (2010) EMMPRIN activates multiple transcription factors in cardiomyocytes, and induces interleukin-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-kappaB andMKK7/JNK/AP-1 signaling. J. Mol. Cell Cardiol. 49 (4): 655–663. doi:10.1016/j.yjmcc.2010.05.007.

22. Bian H, Zheng ZH, Wei D, et al. (2020) Meplazumab treats COVID-19 pneumonia: an open-labelled, concurrent controlled add-on clinical trial. medRxiv 2020: 03.21.20040691; doi: https://doi.org/10.1101/2020.03.21.20040691.

23. Takashi Muramatsu (2016) Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners. J. Biochem. 159 (5): 481–490. https://doi.org/10.1093/jb/mvv127.

24. Guindolet D, Gabison EE (2020) Role of CD147 (EMMPRIN/Basigin) in tissue remodeling. Anat. Rec. 303: 1584-1589. doi:10.1002/ar.24089.

25. Ge H, Yuan W, Liu J, et al. (2015) Functional relevance of protein glycosylation to the pro‐inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages. PloS One 10: e0117463.

26. Pennings GJ, Kritharides L (2014) CD147 in cardiovascular disease and thrombosis. Semin. Thromb. Hemost. 40 (7): 747–755. doi:10.1055/s-0034-1390001.

27. Crosnier C, Bustamante L, Bartholdson S, et al. (2011) Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature 480: 534–537. doi: 10.1038/nature106.

28. Poole RC, Sansom CE, Halestrap AP (1996) Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1). Biochem. J. 320: 817–824.  

29. Wilson MC, Meredith D, Halestrap AP (2002) Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ. J. Biol. Chem. 277 (5): 3666–3672. doi:10.1074/jbc.M109658200.

30. Sun S, Li H, Chen J, Qian Q (2017) Lactic acid: No longer an inert and end-product of glycolysis. Physiology (Bethesda) 32 (6): 453–463. doi:10.1152/physiol.00016.2017. 

31. Tilton WM, Seaman C, Carriero D, Piomelli S (1991) Regulation of glycolysis in the erythrocyte: role of the lactate/pyruvate and NAD/NADH ratios. J. Lab. Clin. Med.  118 (2): 146–152. 

32. Pivkin IV, Peng Z, Karniadakis GE, et al. (2017) Biomechanics of red blood cells in human spleen and consequences for physiology and disease. Proc. Natl. Acad. Sci.113 (28): 7804–7809. doi:10.1073/pnas.1606751113.

33. Li L, Duan M, Chen W, et al. (2017) The spleen in liver cirrhosis: revisiting an old enemy with novel targets. J. Transl. Med. 15 (1): 111. doi:10.1186/s12967-017-1214-8.

34. Li Y, Wu J, Xu L, et al. (2017) Regulation of leukocyte recruitment to the spleen and peritoneal cavity during pristane-induced inflammation. J. Immunol. Res. 2017: 9891348. doi:10.1155/2017/9891348

35. Mitra A, Dwyre DM, Schivo M, et al. (2020) Leukoerythroblastic reaction in a patient with COVID‐19 infection. Am. J. Hematol. 10.1002/ajh.25793. doi:10.1002/ajh.25793.

36. Arber N, Berliner S, Pras E, et al. (1991) Heterotypic leukocyte aggregation in the peripheral blood of patients with leukemia, inflammation and stress. Nouv. Rev. Fr. Hematol. 33 (3): 251–255.

37. Yee C, Main NM, Terry A, et al. (2019) CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury. PLoS One 14 (7): e0215557. doi:10.1371/journal.pone.0215557.

38. Zhang C, Shi L, Wang FS (2020) Liver injury in COVID-19: management and challenges. Lancet Gastroenterol. Hepatol. 5 (5): 428–430. doi:10.1016/S2468-1253(20)30057-1.

39. Tan L, Wang Q, Zhang D, et al. (2020) Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study. Signal Transduct Target Ther. 5: 33. doi:10.1038/s41392-020-0148-4.

40. Ruan Q, Yang K, Wang W, et al. (2020) Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med. 46 (5): 846‐848. doi:10.1007/s00134-020-05991-x.

41. Zheng M, Gao Y, Wang G, et al. (2020) Functional exhaustion of antiviral lymphocytes in COVID-19 patients. Cell Mol. Immunol. 17 (5): 533‐535. doi:10.1038/s41423-020-0402-2.

42. Qin C, Zhou L, Hu Z, et al. (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin Infect. Dis. ciaa248. doi:10.1093/cid/ciaa248.

43. Wang, X., Xu, W., Hu, G. et al. (2020) SARS-CoV-2 infects T lymphocytes through its spike protein-mediated membrane fusion. Cell Mol. Immunol. 1‐3. doi:10.1038/s41423-020-0424-9.

44. Lv M, Miao J, Zhao P, et al. (2018) CD147-mediated chemotaxis of CD4(+)CD161(+) T cells may contribute to local inflammation in rheumatoid arthritis. Clin. Rheumatol. 37 (1): 59–66.

45. Qu R, Ling Y, Zhang YH, et al. (2020) Platelet‐to‐lymphocyte ratio is associated with prognosis in patients with coronavirus disease‐19. J. Med. Virol. 10.1002/jmv.25767. doi:10.1002/jmv.25767.

46. Semeraro N, Ammollo CT, Semeraro F, et al. (2015) Coagulopathy of acute sepsis. Semin Thromb Hemost. 41 (6):650–658. doi:10.1055/s-0035-1556730.

47. Lippi G, Plebani M, Henry BM (2020) Thrombocytopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: A meta-analysis. Clin. Chim. Acta. 506: 145–148. doi:10.1016/j.cca.2020.03.022.

48. Yang X, Yang Q, Wang Y, et al. (2020) Thrombocytopenia and its association with mortality in patients with COVID‐19. J. Thromb. Haemost. 18: 1469–1472. https://doi.org/10.1111/jth.14848

49. Gralnick HR, Williams SB, McKeown LP, et al. (1991) Platelet von Willebrand factor. Mayo Clin. Proc. 66 (6): 634‐640. doi:10.1016/s0025-6196(12)60524-2.

50. Turner NA, Moake JL (2015) Factor VIII is synthesized in human endothelial cells, packaged in weibel-palade bodies and secreted bound to ULVWF strings. PLoS ONE 10 (10): e0140740. https://doi.org/10.1371/journal.pone.0140740.

51. Panigada, M, BottinoN, Tagliabue P, et al. (2020) Hypercoagulability of COVID‐19 patients in intensive care unit. a report of thromboelastography findings and other parameters of hemostasis.  Thromb. Haemost.10.1111/jth.14850. doi:10.1111/jth.14850.

52. Zachariah U, Nair SC, Goel A, et al. (2020) Targeting raised von Willebrand factor levels and macrophage activation in severe COVID-19: Consider low volume plasma exchange and low dose steroid. Thromb. Res. 192: 2. doi:10.1016/j.thromres.2020.05.001.

53. Sadler JE (2008) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood 112 (1): 11–18. doi:10.1182/blood-2008-02-078170.

54. Dai L, Trillo-Tinoco J, Chen Y, et al. (2016) CD147 and downstream ADAMTSs promote the tumorigenicity of Kaposi's sarcoma-associated herpesvirus infected endothelial cells. Oncotarget 7 (4): 3806–3818. doi:10.18632/oncotarget.6584. 

55. Schmidt R, Bültmann A, Fischel S, et al. (2008) Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates platelets, and augments nuclear factor kappaB-dependent inflammation in monocytes. Circ. Res. 102: 302–309. doi: 10.1161/CIRCRESAHA.107.157990.

56. Pennings GJ, Yong AS, Kritharides L (2010) Expression of EMMPRIN (CD147) on circulating platelets in vivo. J. Thromb. Haemost. 8 (3): 472–481. doi:10.1111/j.1538-7836.2009.03716.x.

57. Seizer P, Ungern-Sternberg SN, Schönberger T, et al. (2015) Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 35 (3): 655–663. doi:10.1161/ATVBAHA.114.305112.

58. Zhou F, Yu T, Du R, et al. (2020) Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 395 (10229): 1054–1062. https://doi.org/10.1016/S0140-6736(20)30566-3.

59. Liu XY, Li Z, Liu S, et al. (2020) Therapeutic effects of dipyridamole on COVID-19 patients with coagulation dysfunction. medRxiv 2020: 02.27.20027557; doi: https://doi.org/10.1101/2020.02.27.20027557.

60. Helms CC, Marvel M, Zhao W, et al. (2013) Mechanisms of hemolysis-associated platelet activation. J. Thromb. Haemost. 11 (12): 2148–2154. doi:10.1111/jth.12422.

61. Schanze N, Bode C, Duerschmied D (2019) Platelet contributions to myocardial ischemia/reperfusion injury. Front Immunol. 10: 1260. doi:10.3389/fimmu.2019.01260.

62. Schmidt R, Bültmann A, Fischel S, et al. (2008) Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates platelets, and augments nuclear factor kappaB-dependent inflammation in monocytes. Circ. Res. 102 (3): 302–309. doi:10.1161/CIRCRESAHA.107.157990.

63. Suzuki K, Satoh K, Ikeda S, et al. (2016) Basigin promotes cardiac fibrosis and failure in response to chronic pressure overload in mice. Arterioscler. Thromb. Vasc. Biol. 36 (4): 636–646. doi:10.1161/ATVBAHA.115.306686.

64. Qu R, Ling Y, Zhang YHZ, et al. (2020) Platelet‐to‐lymphocyte ratio is associated with prognosis in patients with coronavirus disease‐19. J. Med. Virol. doi:10.1002/jmv.25767.

65. Hao Zhou H, Wanxin Chen W, Ziping Li Z, et al. (2020) Delayed-phase thrombocytopenia in patients of coronavirus disease 2019 (COVID-19). medRxiv doi: https://doi.org/10.1101/2020.04.11.20059170.

66. Karsten E, Breen E, Herbert BR, (2018) Red blood cells are dynamic reservoirs of cytokines. Sci. Rep. 8 (1): 3101. doi:10.1038/s41598-018-21387-w.

67. Beaulieu LM, Lin E, Mick E, et al. (2014) Interleukin 1 receptor 1 and interleukin 1β regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans. Arterioscler. Thromb. Vasc. Biol. 34 (3): 552‐564. doi:10.1161/ATVBAHA.113.302700.

68. Erdei J, Tóth A, Balogh E, et al. (2018) Induction of NLRP3 inflammasome activation by heme in human endothelial cells. Oxid. Med. Cell Longev. 2018: 4310816. doi:10.1155/2018/4310816.

69. Nyakundi BB, Tóth A, Balogh E, et al. (2019) Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1β production upon intravascular hemolysis. Biochim. Biophys. Acta Mol. Basis Dis. 1865 (2): 464‐475. doi:10.1016/j.bbadis.2018.10.030.

70. Silveira AAA, Mahon OR, Cunningham CC, et al. (2019) S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation. J. Leukoc. Biol. 106 (1): 35‐43. doi:10.1002/JLB.3MIA1118-418RR.

71. Dutra FF, Alves LS, Rodrigues D, et al. (2014) Hemolysis-induced lethality involves inflammasome activation by heme. Proc. Natl. Acad. Sci. 111 (39): E4110‐E4118. doi:10.1073/pnas.1405023111.

72. Qu R, Ling Y, Zhang YH, et al. (2020) Platelet-to-lymphocyte ratio is associated with prognosis in patients with coronavirus disease-19. J. Med. Virol. 10.1002/jmv.25767. doi:10.1002/jmv.25767.

73. Chapin JC, Hajjar KA (2015) Fibrinolysis and the control of blood coagulation. Blood Rev. 29 (1): 17‐24. doi:10.1016/j.blre.2014.09.003.

74. Wright FL, Vogler TO, Moore EE, et al. (2020) Fibrinolysis shutdown correlates to thromboembolic events in severe COVID-19 infection. J. Am. Coll Surg. S1072-7515(20) 30400-2. doi:10.1016/j.jamcollsurg.2020.05.007.

75. Da Q, Teruya M, Guchhait P, et al. (2015) Free hemoglobin increases von Willebrand factor-mediated platelet adhesion in vitro: implications for circulatory devices. Blood 126 (20): 2338‐2341. doi:10.1182/blood-2015-05-648030.

76. Poon TC, Pang RT, Chan KC, et al. (2012) Proteomic analysis reveals platelet factor 4 and beta-thromboglobulin as prognostic markers in severe acute respiratory syndrome. Electrophoresis 33 (12): 1894‐1900. doi:10.1002/elps.201200002.

77. Lefrançais E, Ortiz-Muñoz G, Caudrillier A, et al. (2017) The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature 544 (7648): 105‐109. doi:10.1038/nature21706.

78. Nieswandt B, Stritt S (2015) Megakaryocyte rupture for acute platelet needs. J. Cell Biol. 209 (3): 327‐328. doi:10.1083/jcb.201504026.

79. Xu P, Zhou Q, Xu J (2020) Mechanism of thrombocytopenia in COVID-19 patients. Ann. Hematol. 99 (6): 1205‐1208. doi:10.1007/s00277-020-04019-0.

80. Foda HD, Rollo EE, Drews M, et al. (2001) Ventilator-induced lung injury upregulates and activates gelatinases and EMMPRIN: attenuation by the synthetic matrix metalloproteinase inhibitor, Prinomastat (AG3340). Am. J. Respir. Cell Mol. Biol. 25 (6): 717‐724. doi:10.1165/ajrcmb.25.6.4558f.

81. Wang C, Jin R, Zhu X, et al. (2015) Function of CD147 in atherosclerosis and atherothrombosis. J. Cardiovasc. Transl. Res. 8 (1): 59‐66. doi:10.1007/s12265-015-9608-6.

82. Lu G, Wang Q, Gao GF (2015) Bat-to-human: spike features determining 'host jump' of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 23 (8): 468–478. doi:10.1016/j.tim.2015.06.003.

83. Reiter CD, Wang X, Tanus-Santos JE, et al. (2002) Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8 (12): 1383–1389. doi:10.1038/nm1202-799.

84. Kuhn V, Diederich L, Keller TCS 4th, et al. (2017) Red blood cell function and dysfunction: redox regulation, nitric oxide metabolism, anemia. Antioxid. Redox Signal. 26 (13): 718–742. doi:10.1089/ars.2016.6954.

85. Franco R, Navarro G, Martínez-Pinilla E (2019) Antioxidant defense mechanisms in erythrocytes and in the central nervous system. Antioxidants (Basel) 8 (2):46. doi:10.3390/antiox8020046

86. Schaer DJ, Vinchi F, Ingoglia G (2014) Haptoglobin, hemopexin, and related defense pathways-basic science, clinical perspectives, and drug development. Front Physiol. 5: 415. doi:10.3389/fphys.2014.00415

87. Kassa T, Jana S, Meng F (2016) Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1. FEBS Open Bio. 6 (9):876–884. doi:10.1002/2211-5463.12103.

88. Sadrzadeh SM, Graf E, Panter SS, et al. (1984) Hemoglobin. A biologic fenton reagent. J. Biol. Chem. 259 (23): 14354‐14356.

89. Brabec V, Cermák J, Sebestík V (1990) Serum ferritin in patients with various haemolytic disorders. Folia Haematol. Int. Mag. Klin. Morphol. Blutforsch. 117 (2): 219–227.

90. Youssef L.A., Spitalnik S.L. (2019) Ferroptosis in hemolytic disorders. In: Tang D. (eds) Ferroptosis in Health and Disease. Springer, Cham.

91. Lippi G, Mattiuzzi C (2020) Hemoglobin value may be decreased in patients with severe coronavirus disease 2019 Hematol. Transfu.s Cell Ther. S2531-1379(20)30029-8. doi:10.1016/j.htct.2020.03.001.

92. Schaer DJ, Buehler PW, Alayash AI (2013) Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 121 (8): 1276–1284. doi:10.1182/blood-2012-11-451229.

93. Zhou Z, Yee DL, Guchhait P (2012) Molecular link between intravascular hemolysis and vascular occlusion in sickle cell disease. Curr. Vasc. Pharmacol. 10 (6): 756–761.

94. Goldhaber SZ, Bounameaux H (2012) Pulmonary embolism and deep vein thrombosis. Lancet 379 (9828):1835-46.

95. Tapson VF, Platt DM, Xia F, et al. (2016) Monitoring for Pulmonary Hypertension Following Pulmonary Embolism: The INFORM Study. Am. J. Med. 129 (9): 978–985.e2. doi:10.1016/j.amjmed.2016.03.006.

96. Ataga KI (2009) Hypercoagulability and thrombotic complications in hemolytic anemias. Haematologica 94 (11): 1481–1484. doi:10.3324/haematol.2009.013672.

97. Cui S, Chen S, Li X, et al. (2020) Prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia. J. Thromb. Haemost. 18 (6): 1421‐1424. doi:10.1111/jth.14830.

98. Huerta C, et al. (2008) Risk of myocardial infarction and overall mortality in survivors of venous thromboembolism. Thrombosis J. 6: 10. https://doi.org/10.1186/1477-9560-6-10.

99. Han H, Yang L, Liu R, et al. (2020) Prominent changes in blood coagulation of patients with SARS-CoV-2 infection. Clinical chemistry and laboratory medicine. DOI: 10.1515/cclm-2020-0188.

100. Casey K, Iteen A, Nicolini R, et al. (2020) COVID-19 pneumonia with hemoptysis: Acute segmental pulmonary emboli associated with novel coronavirus infection. Am. J. Emerg. Med. S0735-6757(20)30239-4. doi:10.1016/j.ajem.2020.04.011.

101. Huet Y, Lemaire F, Brun-Buisson C, et al. (1985) Hypoxemia in acute pulmonary embolism. Chest 88 (6): 829–836. doi:10.1378/chest.88.6.829.

102. Mehta C, Mehta Y (2016) Management of refractory hypoxemia. Ann. Card. Anaesth. 19 (1): 89–96. doi:10.4103/0971-9784.173030.

103. Aoki N, Yanagisawa A, Shimoyama K, et al. (1998) Clinical significance of hypoxemia without congestive heart failure in patients presenting with acute myocardial infarction. Cardiology 89 (1): 40–45. doi:10.1159/000006742.

104. Carreau A, El Hafny-Rahbi B, Matejuk A, et al. (2011) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J. Cell Mol. Med. 15 (6): 1239–1253. doi:10.1111/j.1582-4934.2011.01258.x.

105. Haase VH (2013) Regulation of erythropoiesis by hypoxia-inducible factors. Blood Rev. 27 (1): 41–53. doi:10.1016/j.blre.2012.12.003.

106. Spinello I, Saulle E, Quaranta MT, et al. (2019) The small-molecule compound AC-73 targeting CD147 inhibits leukemic cell proliferation, induces autophagy and increases the chemotherapeutic sensitivity of acute myeloid leukemia cells. Haematologica 104 (5): 973–985. doi:10.3324/haematol.2018.199661.

107. Amati E, Perbellini O, Rotta G, et al. (2018) High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources. Stem Cell Res. Ther.  9 (1): 10, https://doi.org/10.1186/s13287-017-0755-3.

108. Ulrich H, Pillat MM (2020) CD147 as a target for COVID-19 treatment: suggested effects of azithromycin and stem cell engagement. Stem Cell Rev. Rep. 16 (3): 434‐440. doi:10.1007/s12015-020-09976-7.

109. Falanga V (2012) Stem cells in tissue repair and regeneration. J. Invest. Dermatol. 132 (6): 1538‐1541. doi:10.1038/jid.2012.77.

110. Bortner CD, Gomez-Angelats M, Cidlowski JA (2001) Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis. J. Biol. Chem. 276 (6): 4304–4314. doi:10.1074/jbc.M005171200.

111. Varga Z, Flammer AJ, Steige P, et al. (2020) Endothelial cell infection and endotheliitis in COVID-19. Lancet 395 (10234): 1417‐1418. doi:10.1016/S0140-6736(20)30937-5.

112. Chan JF, Kok KH, Zhu Z, et al. (2020) Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg. Microbes Infect. 9 (1): 221–236. doi:10.1080/22221751.2020.1719902.

113. Schoeman D, Fielding BC (2019) Coronavirus envelope protein: current knowledge. Virol. J. 16 (1): 69. doi:10.1186/s12985-019-1182-0.

114. Lu W, Zheng BJ, Xu K, et al. (2006) Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc. Natl. Acad. Sci. 103 (33): 12540–12545. doi:10.1073/pnas.0605402103.

115. Verdiá-Báguena C, Nieto-Torres JL, Alcaraz A, et al. (2010) Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids. Virology 432 (2): 485‐494. doi:10.1016/j.virol.2012.07.005.

116. Liao Y, Tam JP, Liu DX (2006) Viroporin activity of SARS-CoV E protein. Adv. Exp. Med. Biol. 581: 199‐202. doi:10.1007/978-0-387-33012-9_34.

117. Nieto-Torres JL, Verdiá-Báguena C, Jimenez-Guardeño JM, et al. (2015) Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome. Virology 485: 330–339. doi:10.1016/j.virol.2015.08.010.

118. Castaño-Rodriguez C, Honrubia JM, Gutiérrez-Álvarez J, et al. (2018) Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis. mBio. 9 (3): e02325-17. doi:10.1128/mBio.02325-17.

119. Latz E, Xiao TS, Stutz A (2013) Activation and regulation of the inflammasomes. Nat. Rev. Immunol. 13 (6): 397–411. doi:10.1038/nri3452.

120. Murakami T, Ockinger J, Yu J, et al. (2012) Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc. Natl. Acad. Sci. 109 (28): 11282‐11287. doi:10.1073/pnas.1117765109.

121. Nieto-Torres JL, DeDiego ML, Verdiá-Báguena C, et al. (2014) Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog. 10 (5): e1004077. doi:10.1371/journal.ppat.1004077.

122. Alam I, Kamau A, Kulmanov M, et al. (2020) Functional pangenome analysis suggests inhibition of the protein E as a readily available therapy for COVID-2019. bioRxiv  02.17.952895. doi: https://doi.org/10.1101/2020.02.17.952895.

123. Gupta MK, Vemula S, Donde R, et al. (2020). In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel J. Biomol. Struct. Dyn. 1‐11 DOI:10.1080/07391102.2020.1751300.

124. Chen IY, Moriyama M, Chang MF, Ichinohe T (2019) Severe acute respiratory syndrome coronavirus viroporin 3a activates the NLRP3 inflammasome. Front Microbiol. 10: 50. doi:10.3389/fmicb.2019.00050.

125. Issa E, Merhi G, Panossian B, et al. (2020) SARS-CoV-2 and ORF3a: Nonsynonymous mutations, functional domains, and viral pathogenesis. mSystems 5 (3): e00266-20. DOI: 10.1128/mSystems.00266-20. 

126. Chan CM, Tsoi H, Chan WM, et al. (2009) The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function. Int. J. Biochem. Cell Biol. 41 (11): 2232‐2239. doi:10.1016/j.biocel.2009.04.019.

127. Chen D Jr., Li X, Song Q Sr., et al. (2020) Hypokalemia and clinical implications in patients with coronavirus disease 2019 (COVID-19) medRxiv 02.27.20028530. doi:https://doi.org/10.1101/2020.02.27.20028530.

128. Yaron JR, Gangaraju S, Rao MY, et al. (2015) K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells. Cell Death Dis. 6 (10): e1954. Published 2015 Oct 29. doi:10.1038/cddis.2015.277.

129. Minakshi R, Padhan K, Rehman S, et al. (2014) The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain. Virus Res. 191: 180‐183. doi:10.1016/j.virusres.2014.08.001.

130. Caulier A, Rapetti‐Mauss R, Guizouarn H, et al. (2018) Primary red cell hydration disorders: Pathogenesis and diagnosis. Int. J. Lab. Hem. 40 (Suppl. 1): 68‐73.

131. George Taiaroa, Daniel Rawlinson, Leo Featherstone, et al. (2020) Direct RNA sequencing and early evolution of SARS-CoV-2. bioRxiv 03.05.976167. doi: https://doi.org/10.1101/2020.03.05.976167. 

132. Liao Y, Lescar J, Tam JP, et al. (2004) Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability. Biochem. Biophys. Res. Commun. 325 (1): 374–380. doi:10.1016/j.bbrc.2004.10.050.

133. Tiruppathi C, et al. (2002) Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul. Pharmacol. 39 (4-5): 173–185. doi:10.1016/s1537-1891(03)00007-7.

134. He P, Curry FE (1991) Depolarization modulates endothelial cell calcium influx and microvessel permeability. Am. J. Physiol. 261 (4 Pt 2): H1246–H1254. doi:10.1152/ajpheart.1991.261.4.H1246.

135. Hertz L, Huisjes R, Llaudet-Planas E, et al. (2017) Is increased intracellular calcium in red blood cells a common component in the molecular mechanism causing anemia? Front Physiol. 8: 673. doi:10.3389/fphys.2017.00673.

136. Chaves-Moreira D, Souza FN, Fogaça RT, et al. (2011) The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin. J. Cell Biochem. 112 (9): 2529–2540. doi:10.1002/jcb.23177.

137. Smeets MW, Bierings R, Meems H, et al. (2017) Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor PLoS One 12 (3): e0173077.  doi:10.1371/journal.pone.0173077.

138. Ahn YS, Jy W, Harrington WJ, et al. (1987) Increased platelet calcium in thrombosis and related disorders and its correction by nifedipine. Thromb. Res. 45 (2): 135–143. doi:10.1016/0049-3848(87)90167-8.

139. Lissoni P, Mandala M, Rossini F, et al. (1999) Thrombopoietic property of the pineal hormone melatonin. Hematology 4: 4, 335-343, DOI: 10.1080/10245332.1999.11746457.

140. Squecco R, Tani A, Zecchi-Orlandini S, et al. (2015) Melatonin affects voltage-dependent calcium and potassium currents in MCF-7 cell line cultured either in growth or differentiation medium. Eur. J. Pharmacol. 758: 40–52. doi:10.1016/j.ejphar.2015.03.068.

141. León J, Acuña-Castroviejo D, Escames G, Tan DX, Reiter RJ. (2005) Melatonin mitigates mitochondrial malfunction. J. Pineal Res. 38 (1): 1–9. doi:10.1111/j.1600-079X.2004.00181.x.

142. Reiter RJ, Rosales-Corral S, Tan DX, et al. (2017) Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas. Cell Mol. Life Sci. 74 (21): 3863–3881. doi:10.1007/s00018-017-2609-7.

143. Suofu Y, Li W, Jean-Alphonse FG, et al. (2017) Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc. Natl. Acad. Sci. 114 (38): E7997–E8006. doi:10.1073/pnas.1705768114.

144. Zhao D, Yu Y, Shen Y, et al. (2019) Melatonin Synthesis and Function: Evolutionary History in Animals and Plants. Front Endocrinol. (Lausanne) 10: 249. doi:10.3389/fendo.2019.00249.

145. Hardeland R, Cardinali DP, Srinivasan V (2011) Melatonin--a pleiotropic, orchestrating regulator molecule. Prog. Neurobiol. 93 (3): 350–384. doi:10.1016/j.pneurobio.2010.12.004.

146. Reiter RJ (2000) Melatonin: Lowering the high price of free radicals. News Physiol. Sci. 15: 246–250. doi:10.1152/physiologyonline.2000.15.5.246.

147. Reiter RJ, Tan DX, Terron MP, et al. (2007) Melatonin and its metabolites: new findings regarding their production and their radical scavenging actions. Acta Biochim. Pol. 54 (1): 1–9.

148. Tan DX, Manchester LC, Esteban-Zubero E, et al. (2015) Melatonin as a potent and inducible endogenous antioxidant: synthesis and metabolism. Molecules 20 (10): 18886–18906. doi:10.3390/molecules201018886.

149. Jou M‐J, Peng T‐I, Reiter RJ (2019) Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3‐OHM and AFMK in RBA1 astrocytes. J. Pineal Res. 66: e12538. https://doi.org/10.1111/jpi.12538.

150. Reddy VS, Prabhu SD, Mummidi S, et al. (2010) Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-kappaB activation. Am. J. Physiol. Heart Circ. Physiol. 299 (4): H1242–H1254. doi:10.1152/ajpheart.00451.2010.

151. Han P, Trinidad BJ, Shi J (2015) Hypocalcemia-induced seizure: demystifying the calcium paradox. ASN Neuro. 7 (2): 1759091415578050. doi:10.1177/1759091415578050.

152. Yarmohammadi H, Uy-Evanado A, Reinier K, et al. (2017) Serum calcium and risk of sudden cardiac arrest in the general population. Mayo Clin Proc. 92 (10): 1479–1485. doi:10.1016/j.mayocp.2017.05.028.

153. Sun JK, Zhang WH, Lei Zou L, et al. (2020) Serum calcium as a biomarker of clinical severity and prognosis in patients with coronavirus disease 2019: a retrospective cross-sectional study. Crit. Care Emerg. Med. doi:10.21203/rs.3.rs-17575/v1.

154. Spinale FG, Coker ML, Heung LJ, et al. (2000) A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 102 (16):1944–1949. doi:10.1161/01.cir.102.16.1944.

155. Cao M, Yuan W, Peng M, et al. (2019) Role of CyPA in cardiac hypertrophy and remodeling. Biosci Rep. 39 (12): BSR20193190. doi:10.1042/BSR20193190.

156. Su H, Li J, Chen T, et al. (2016) Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CD147-CyPA signaling pathway. Mol. Cell Biochem. 422 (1-2): 85–95. doi:10.1007/s11010-016-2808-9.

157. Pennings GJ, Yong ASC, Wong C, et al. (2014) Circulating levels of soluble EMMPRIN (CD147) correlate with levels of soluble glycoprotein VI in human plasma. Platelets 25: 8, 639-642, DOI: 10.3109/09537104.2013.852660.

158. Satoh K, Satoh T, Kikuchi N, et al. (2014) Basigin mediates pulmonary hypertension by promoting inflammation and vascular smooth muscle cell proliferation. Circ. Res. 115 (8): 738–750. doi:10.1161/CIRCRESAHA.115.304563.

159. Garcia-Dorado D, Ruiz-Meana M, Inserte J, et al. (2012) Calcium-mediated cell death during myocardial reperfusion. Cardiovasc. Res. 94 (2): 168-180. DOI: 10.1093/cvr/cvs116.

160. Hu S, Zhu P, Zhou H, Zhang Y, et al. (2018) Melatonin-induced protective effects on cardiomyocytes against reperfusion injury partly through modulation of IP3R and SERCA2a via activation of ERK1. Arquivos Brasileiros de Cardiologia 110 (1): 44-51. DOI: 10.5935/abc.20180008.

161. Davidson SM, Duchen MR (2007) Endothelial mitochondria contributing to vascular function and disease. Circ. Res. 100 (8): 1128–1141. Doi: 10.1161/01.RES.0000261970.18328.1d.

162. Zhu H, Jin Q, Li Y, et al. (2018) Melatonin protected cardiac microvascular endothelial cells against oxidative stress injury via suppression of IP3R-[Ca2+]c/VDAC-[Ca2+]m axis by activation of MAPK/ERK signaling pathway. Cell Stress Chaperones 23 (1): 101–113. doi:10.1007/s12192-017-0827-4.

163. Imai Y, Kuba K, Rao S, et al. (2005) Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 436 (7047): 112–116. doi:10.1038/nature03712.

164. Rabelo LA, Todiras M, Nunes-Souza V, et al. (2016) Genetic deletion of ACE2 induces vascular dysfunction in C57BL/6 mice: role of nitric oxide imbalance and oxidative stress. PLoS One, 11 (4): e0150255. doi:10.1371/journal.pone.0150255.

165. Ferrini MG, Vernet D, Magee TR,  et al. (2002) Antifibrotic role of inducible nitric oxide synthase. Nitric Oxide 6 (3): 283-294. doi: 10.1006/niox.2001.0421.

166. Jones SP, Bolli R (2006) The ubiquitous role of nitric oxide in cardioprotection. J. Mo.l Cell Cardiol. 40 (1): 16–23. doi:10.1016/j.yjmcc.2005.09.011.

167. Simko F (2012) Chronobiology of blood pressure: emerging implications of melatonin. Eur. J. Clin. Invest. 42 (11): 1252–1254. doi:10.1111/j.1365-2362.2012.02705.x.

168. Pechanova O, Paulis L, Simko F (2014) Peripheral and central effects of melatonin on blood pressure regulation. Int. J. Mol. Sci. 15 (10): 17920–17937. doi:10.3390/ijms151017920.

169. Koh PO (2008) Melatonin regulates nitric oxide synthase expression in ischemic brain injury. J. Vet. Med. Sci. 70 (7): 747–750. doi:10.1292/jvms.70.747.

170. Simko F, Baka T, Krajcirovicova K, et al. (2018) Effect of melatonin on the renin-angiotensin-aldosterone system in l-NAME-induced hypertension. Molecules 23 (2): 265. Published 2018 Jan 29. doi:10.3390/molecules23020265.

171. Kosonen O, Kankaanranta H, Uotila J, et al. (2000) Inhibition by nitric oxide-releasing compounds of E-selectin expression in and neutrophil adhesion to human endothelial cells. Eur. J. Pharmacol. 394 (1): 149–156. doi:10.1016/s0014-2999(00)00141-2.

172. Sonar SA, Lal G (2019) The iNOS activity during an immune response controls the cns pathology in experimental autoimmune encephalomyelitis. Front Immunol. 10: 710. doi:10.3389/fimmu.2019.00710.

173. Donnelly LE, Barnes PJ (2002) Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells. Am. J. Respir Cell Mol. Biol. 26 (1): 144‐151. doi:10.1165/ajrcmb.26.1.4477.

174. Zamora R, Vodovotz Y, Billiar TR (2000) Inducible nitric oxide synthase and inflammatory diseases. Mol. Med. 6 (5): 347‐373.

175. Prado CM, et al. (2019) iNOS inhibition reduces lung mechanical alterations and remodeling induced by particulate matter in mice. Pulm. Med. 2019: 4781528. doi:10.1155/2019/4781528.

176. Hardeland R (2020) Melatonin and inflammation—Story of a double‐edged blade. J. Pineal Res. 65: e12525. https://doi.org/10.1111/jpi.12525.

177. Wu, G., Peng, C., Liao, W. et al. (2020) Melatonin receptor agonist protects against acute lung injury induced by ventilator through up-regulation of IL-10 production. Respir. Res. 21 (1): 65. https://doi.org/10.1186/s12931-020-1325-2.

178. Barnes BJ, Adrover JM, Baxter-Stoltzfus A, et al. (2020) Targeting potential drivers of COVID-19: Neutrophil extracellular traps. J. Exp. Med. 217 (6): e20200652. doi:10.1084/jem.20200652.

179. Ma Y, Yang X, Chatterjee V, et al. (2019) Role of neutrophil extracellular traps and vesicles in regulating vascular endothelial permeability. Front Immunol. 10: 1037. doi:10.3389/fimmu.2019.01037.

180. Hernández-Reséndiz S, Muñoz-Vega M, Contreras WE, et al. (2018) Responses of endothelial cells towards ischemic conditioning following acute myocardial infarction. Cond Med. 1(5):247–258.

181. Lotufo CM, Yamashita CE, Farsky SH, et al. (2006) Melatonin effect on endothelial cells reduces vascular permeability increase induced by leukotriene B4. Eur. J. Pharmacol. 534 (1-3): 258–263. doi:10.1016/j.ejphar.2006.01.050.

182. Wirtz PH, Spillmann M, Bärtschi C, et al. (2008) Oral melatonin reduces blood coagulation activity: a placebo-controlled study in healthy young men. J. Pineal Res. 44 (2): 127–133. doi:10.1111/j.1600-079X.2007.00499.x.

183. Barcellini W, Fattizzo B (2015) Clinical applications of hemolytic markers in the differential diagnosis and management of hemolytic anemia. Dis. Markers. 2015: 635670. doi:10.1155/2015/635670

184. Schaer CA, Deuel JW, Schildknecht D, et al. (2016) Haptoglobin preserves vascular nitric oxide signaling during hemolysis. Am. J. Respir. Crit. Care Med. 193 (10): 1111–1122. doi:10.1164/rccm.201510-2058OC.

185. Reiter CD, Wang X, et al. (2002) Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8 (12): 1383–1389. doi:10.1038/nm1202-799.

186. Helms CC, Gladwin MT, Kim-Shapiro DB (2018) Erythrocytes and vascular function: Oxygen and nitric oxide. Front Physiol. 9: 125. doi:10.3389/fphys.2018.00125.

187. Loscalzo J (2001) Nitric oxide insufficiency, platelet activation, and arterial thrombosis. Circ. Res. 88 (8): 756–762. doi:10.1161/hh0801.089861.

188. Nitric oxide gas inhalation for severe acute respiratory syndrome in COVID-19. (NOSARSCOVID) ClinicalTrials.gov Identifier: NCT04290871 https://clinicaltrials.gov/ct2/show/NCT04290871.

189. Holcomb C, Erdmann W, Corssen G (1976) The significance of diffusion hypoxemia. South Med. J. 69 (10): 1282–1284.

190. Yaron Ogen (2020) Assessing nitrogen dioxide (NO2) levels as a contributing factor to coronavirus (COVID-19) fatality. Sci. Total Environ. 726: 138605. doi:10.1016/j.scitotenv.2020.138605.

191. Raut MS, Maheshwari A (2017) Inhaled nitric oxide, methemoglobinemia, and route of delivery. Saudi. J. Anaesth. 11 (3): 364. doi:10.4103/sja.SJA_82_17.

192. Kon K, Maeda N, Shiga T (1977) Effect of nitric oxide on the oxygen transport of human erythrocytes. J. Toxicol. Environ. Health 2 (5): 1109–1113. doi:10.1080/15287397709529508.

193. Oda H, et al. (1980) Reaction of hemoglobin with nitric oxide and nitrogen dioxide in mice. J. Toxicol. Environ. Health 6 (3): 673–678. doi:10.1080/15287398009529884.

194. Stepuro TL, Zinchuk VV (2006) Nitric oxide effect on the hemoglobin-oxygen affinity. J Physiol Pharmacol. 57 (1): 29–38.

195. Dei Zotti F, Lobysheva II, Balligand J-L (2018) Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo. PLoS ONE 13 (7): e0200352.

196. Shao G, Zhang S, Nie J, et al. (2017) Effects of melatonin on mechanisms involved in hypertension using human umbilical vein endothelial cells. J. Toxicol. Environ. Health A. 80 (23-24): 1342–1348. doi:10.1080/15287394.2017.1384171.

197. Klimentova J, Cebova M, Barta A, et al. (2016) Effect of melatonin on blood pressure and nitric oxide generation in rats with metabolic syndrome. Physiol. Res. 65 (Suppl 3): S373–S380. doi:10.33549/physiolres.933436.

198. Huang CC, Lai CJ, Tsai MH, et al. (2015) Effects of melatonin on the nitric oxide system and protein nitration in the hypobaric hypoxic rat hippocampus. BMC Neurosci. 16: 61. doi:10.1186/s12868-015-0199-6.

199. Klok FA, Kruip MJHA, van der Meer NJM, et al. (2020) Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb. Res. S0049-3848(20)30120-1. doi:10.1016/j.thromres.2020.04.013.

200. Hung MW, Yeung HM, Lau CF, et al. (2017) Melatonin attenuates pulmonary hypertension in chronically hypoxic rats. Int. J. Mo.l Sci. 18 (6): 1125. doi:10.3390/ijms18061125.

201. Jensen FB (2009) The role of nitrite in nitric oxide homeostasis: a comparative perspective. Biochim. Biophys. Acta. 1787 (7): 841–848. doi:10.1016/j.bbabio.2009.02.010.

202. Grygorczyk R, Orlov SN (2017) Effects of hypoxia on erythrocyte membrane properties-implications for intravascular hemolysis and purinergic control of blood flow. Front Physiol. 8: 1110. Published 2017 Dec 22. doi:10.3389/fphys.2017.01110.

203. Kanias T, Acker JP (2010) Biopreservation of red blood cells – the struggle with hemoglobin oxidation. FEBS J. 277 (2): 343–356. doi:10.1111/j.1742-4658.2009.07472.x.

204. Nagababu E, Mohanty JG, Bhamidipaty S, et al. (2010) Role of the membrane in the formation of heme degradation products in red blood cells. Life Sci. 86 (3-4): 133–138. doi:10.1016/j.lfs.2009.11.015.

205. Chiu D, Lubin B (1989) Oxidative hemoglobin denaturation and RBC destruction: the effect of heme on red cell membranes. Semin. Hematol. 26 (2): 128–135.

206. Tesoriere L, D'Arpa D, Conti S, et al. (1999), Melatonin protects human red blood cells from oxidative hemolysis: New insights into the radical‐scavenging activity. J. Pineal Res. 27: 95-105. doi:10.1111/j.1600-079X.1999.tb00602.x.

207. Maitra D, Abdulhamid I, Diamond MP,  et al. (2012) Melatonin attenuates hypochlorous acid-mediated heme destruction, free iron release, and protein aggregation in hemoglobin. J. Pineal Res. 53 (2): 198–205. doi:10.1111/j.1600-079X.2012.00988.x.

208. Tesoriere L, et al. (2001) Reaction of melatonin with hemoglobin-derived oxoferryl radicals and inhibition of the hydroperoxide-induced hemoglobin denaturation in red blood cells. J. Pineal Res. 1 (2): 114–119. doi:10.1034/j.1600-079x.2001.310204.x.

209. Reeder BJ, Wilson MT (2005) Hemoglobin and myoglobin associated oxidative stress: from molecular mechanisms to disease States. Curr. Med. Chem. 12 (23): 2741–2751. doi:10.2174/092986705774463021.

210. Borgese N, Pietrini G, Gaetani S (1987) Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay. J. Clin. Invest. 80 (5): 1296‐1302. doi:10.1172/JCI113205.

211. Choury D, Leroux A, Kaplan JC (1987) Membrane-bound cytochrome b5 reductase (methemoglobin reductase) in human erythrocytes. Study in normal and methemoglobinemic subjects. J. Clin. Invest. 67 (1): 149‐155. doi:10.1172/JCI110007.

212. Bulbarelli A, Valentini A, DeSilvestris M, et al. (1998) An erythroid-specific transcript generates the soluble form of NADH-cytochrome b5 reductase in humans. Blood 92 (1): 310‐319.

213. Percy MJ, Lappin TR (2008) Recessive congenital methaemoglobinaemia: cytochrome b5 reductase deficiency. Br. J. Haematol. 141: 298-308. doi:10.1111/j.1365-2141.2008.07017.x.

214. Martin-Montalvo A, Sun Y, Diaz-Ruiz A, et al. (2016) Cytochrome b5 reductase and the control of lipid metabolism and healthspan. NPJ Aging Mech. Dis. 2: 16006. https://doi.org/10.1038/npjamd.2016.6. 

215. Martinez GR, Almeida EA, Klitzke CF, et al. (2005) Measurement of melatonin and its metabolites: importance for the evaluation of their biological roles. Endocrine. 27 (2): 111‐118. doi:10.1385/endo:27:2:111.

216. Tan DX, Manchester LC, Sainz RM, et al. (2005) Interactions between melatonin and nicotinamide nucleotide: NADH preservation in cells and in cell-free systems by melatonin. J. Pineal Res. 39 (2): 185‐194. doi:10.1111/j.1600-079X.2005.00234.x.

217. Reiter RJ, Tan DX, Terron MP, et al. (2007) Melatonin and its metabolites: new findings regarding their production and their radical scavenging actions. Acta Biochim. Pol. 54 (1): 1‐9.

218. Ross D, Siegel D (2017) Functions of NQO1 in cellular protection and CoQ10 metabolism and its potential role as a redox sensitive molecular switch. Front Physiol. 8: 595. doi:10.3389/fphys.2017.00595.

219. Yan C, Shieh B, David Siegel D, et al. (2007) Overexpression of NQO1 protects Jurkat leukemia cells from apoptosis. Cancer Res. 67 (9 Supplement): 4862.

220. Graves PR, Kwiek JJ, Fadden P, et al. (2002) Discovery of novel targets of quinoline drugs in the human purine binding proteome. Mol. Pharmacol. 62 (6): 1364‐1372. doi:10.1124/mol.62.6.1364.

221. Vasiliou V, Ross D, Nebert DW (2006) Update of the NAD(P)H:quinone oxidoreductase (NQO) gene family. Hum Genomics. 2 (5): 329–335. doi:10.1186/1479-7364-2-5-329.

222. Mailliet F, Ferry G, Vella F, et al. (2005) Characterization of the melatoninergic MT3 binding site on the NRH:quinone oxidoreductase 2 enzyme. Biochem. Pharmacol. 71 (1-2): 74–88. doi:10.1016/j.bcp.2005.09.030.

223. Calamini B, Santarsiero BD, Boutin JA, et al. (2008) Kinetic, thermodynamic and X-ray structural insights into the interaction of melatonin and analogues with quinone reductase 2. Biochem. J. 413 (1): 81–91. doi:10.1042/BJ20071373.

224. Boutin JA, Ferry G, (2018) Is there sufficient evidence that the melatonin binding site MT3 is quinone reductase 2? J. Pharmacol. Exp. Ther. 368 (1): 59‐65. doi:10.1124/jpet.118.253260.

225. Wu K, Knox R, Sun XZ, et al. (1997) Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase. Arch. Biochem. Biophys. 347 (2): 221‐228. doi:10.1006/abbi.1997.0344.

226. Reybier K, Perio P, Ferry G, et al. (2011) Insights into the redox cycle of human quinone reductase 2. Free Radic. Res. 45 (10): 1184‐1195. doi:10.3109/10715762.2011.605788.

227. Chen D, Li X, Liu X, et al. (2017) NQO2 inhibition relieves reactive oxygen species effects on mouse oocyte meiotic maturation and embryo development. Biol. Reprod. 97 (4): 598‐611. doi:10.1093/biolre/iox098.

228. Cassagnes LE, Rakotoarivelo N, Sirigu S, et al. (2017) Role of quinone reductase 2 in the antimalarial properties of indolone-type derivatives. Molecules 22 (2): 210. doi:10.3390/molecules22020210.

229. Tan DX, Manchester LC, Terron MP, et al. (2007) Melatonin as a naturally occurring co-substrate of quinone reductase-2, the putative MT3 melatonin membrane receptor: hypothesis and significance. J. Pineal Res. 43 (4): 317‐320. doi:10.1111/j.1600-079X.2007.00513.x.

230. Boutin JA, Marcheteau E, Hennig P, et al. (2008) MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate. J. Pineal Res 45 (4): 524-531. DOI: 10.1111/j.1600-079x.2008.00631.x.

231. Reithmeier RA, Casey JR, Kalli AC, et al. (2016) Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context. Biochim. Biophys. Acta. 1858 (7 Pt A): 1507–1532. doi:10.1016/j.bbamem.2016.03.030. 

232. Hamasaki N (1999) The role of band 3 protein in oxygen delivery by red blood cells. Indian J. Clin. Biochem. 14 (1): 49–58. doi:10.1007/BF02869151.

233. Wang, Da Neng (1994), Band 3 protein: Structure, flexibility and function, FEBS Lett. 346 (1): 26‐31. doi:10.1016/0014-5793(94)00468-4.

234. Ke X, Fei F, Chen Y, et al. (2012) Hypoxia upregulates CD147 through a combined effect of HIF-1α and Sp1 to promote glycolysis and tumor progression in epithelial solid tumors. Carcinogenesis 33 (8): 1598–1607. doi:10.1093/carcin/bgs196.

235. Zipser Y, Piade A, Barbul A, et al. (2002) Ca2+ promotes erythrocyte band 3 tyrosine phosphorylation via dissociation of phosphotyrosine phosphatase from band 3. Biochem. J. 368 (Pt 1): 137–144. doi:10.1042/BJ20020359.

236. Pantaleo A, Ferru E, Pau MC, et al. (2016) Band 3 erythrocyte membrane protein acts as redox stress sensor leading to its phosphorylation by p (72) syk. Oxid. Med. Cell Longev. 2016: 6051093. doi:10.1155/2016/6051093.

237. Shimo H, Arjunan SN, Machiyama H, et al. (2015) Particle simulation of oxidation induced band 3 clustering in human erythrocytes. PLoS Comput. Biol. 11 (6): e1004210. doi:10.1371/journal.pcbi.1004210.

238. Morabito R, Remigante A, Marino A. (2019) Melatonin protects band 3 protein in human erythrocytes against H2O2-induced oxidative stress. Molecules 24 (15): 2741. doi:10.3390/molecules24152741.

239. Huisjes R, Bogdanova A, van Solinge WW, et al. (2018) Squeezing for life - properties of red blood cell deformability. Front Physiol. 9: 656. doi:10.3389/fphys.2018.00656.

240. Richardson SL, Swietach P (2016) Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm. Sci. Rep. 6: 36018. doi:10.1038/srep36018.

241. Jeney V, Balla J, Yachie A et al. (2002) Pro-oxidant and cytotoxic effects of circulating heme. Blood 100 (3): 879–887. doi:10.1182/blood.v100.3.879.

242. Balla G, Jacob HS, Eaton JW, et al. (1991) Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury. Arterioscler. Thromb. 11 (6): 1700‐1711. doi:10.1161/01.atv.11.6.1700.

243. Rother RP, Bell L, Hillmen P, et al. (2005) The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA 293 (13): 1653‐1662, doi:10.1001/jama.293.13.1653.

244. Immenschuh S, Vijayan V, Janciauskiene S, et al. (2017) Heme as a target for therapeutic interventions. Front Pharmacol. 8: 146. doi:10.3389/fphar.2017.00146

245. Higdon AN, Benavides GA, Chacko BK, et al. (2012) Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy. Am. J. Physiol. Heart Circ. Physiol. 302 (7): H1394‐H1409. doi:10.1152/ajpheart.00584.2011.

246. Frimat M, Boudhabhay I, Roumenina LT (2019) Hemolysis derived products toxicity and endothelium: model of the second hit. Toxins (Basel). 11 (11): 660 doi:10.3390/toxins11110660.

247. Riphagen S, Gomez X, Gonzalez-Martinez C, et al. (2020) Hyperinflammatory shock in children during COVID-19 pandemic. Lancet 395 (10237): 1607‐1608. doi:10.1016/S0140-6736(20)31094-1.

248. Nakatani K, Takeshita S, Tsujimoto H, et al. (2003) Circulating endothelial cells in Kawasaki disease. Clin. Exp. Immunol. 131 (3): 536‐540. doi:10.1046/j.1365-2249.2003.02091.x.

249. Ishikawa T, Seki K (2018) The association between oxidative stress and endothelial dysfunction in early childhood patients with Kawasaki disease. BMC Cardiovasc. Disord. 18 (1): 30. doi:10.1186/s12872-018-0765-9.

250. Ueno K, Ninomiya Y, Hazeki D, et al. (2017) Disruption of endothelial cell homeostasis plays a key role in the early pathogenesis of coronary artery abnormalities in Kawasaki disease. Sci. Rep. 7, 43719. https://doi.org/10.1038/srep43719.

251. Dhillon R, Clarkson P, Donald AE, et al. (1996) Endothelial dysfunction late after Kawasaki disease. Circulation 94 (9): 2103‐2106. doi:10.1161/01.cir.94.9.2103.

252. Paul, S, Naaz, S, Ghosh, A, et al. (2018) Melatonin chelates iron and binds directly with phenylhydrazine to provide protection against phenylhydrazine induced oxidative damage in red blood cells along with its antioxidant mechanisms: an in vitro study. Melatonin Res. 1 (1): 1-20. DOI:https://doi.org/https://doi.org/10.32794/mr11250001.

253. Elkader MAE, Aly H (2015) Protective effect of melatonin against iron overload-induced toxicity in rats. Int. J. Pharmac. Pharmaceutic. Sci. 7: (9): 116-121. https://innovareacademics.in/journals/index.php/ijpps/article/view/7317.

254. Arnould T, Michiels C, Alexandre I, et al. (1992) Effect of hypoxia upon intracellular calcium concentration of human endothelial cells. J. Cell Physiol. 152 (1): 215–221. doi:10.1002/jcp.1041520127.

255. Yeung HM, Hung MW, Fung ML (2008) Melatonin ameliorates calcium homeostasis in myocardial and ischemia-reperfusion injury in chronically hypoxic rats. J. Pineal Res. 45 (4): 373–382. doi:10.1111/j.1600-079X.2008.00601.x. 

256. Yugo Tabata, Daisuke Yoshino, Kiyoe Funamoto, et al. (2019) Migration of vascular endothelial cells in monolayers under hypoxic exposure. Integr. Biol. (Camb). 11 (1): 26‐35. doi:10.1093/intbio/zyz002.

257. Yang L, Zheng J, Xu R, et al. (2014) Melatonin suppresses hypoxia-induced migration of HUVECs via inhibition of ERK/Rac1 activation. Int. J. Mol. Sci. 15 (8): 14102‐14121. doi:10.3390/ijms150814102.

258. Cheng J, Yang HL, Gu CJ, et al. (2019) Melatonin restricts the viability and angiogenesis of vascular endothelial cells by suppressing HIF-1α/ROS/VEGF. Int. J. Mol. Med. 43 (2): 945–955. doi:10.3892/ijmm.2018.4021.

259. Favero G, Franceschetti L, Bonomini F, et al. (2017) Melatonin as an anti-inflammatory agent modulating inflammasome activation. Int. J. Endocrinol. 2017: 1835195. doi:10.1155/2017/1835195 1835195.

260. Bonomini F, Dos Santos M, Veronese FV, et al. (2019) NLRP3 inflammasome modulation by melatonin supplementation in chronic pristane-induced lupus nephritis. Int. J. Mol. Sci. 20 (14): 3466. doi:10.3390/ijms20143466.

261. Reiter RJ, Sharma R, Ma Q, et al. (2020) Melatonin inhibits COVID-19-induced cytokine storm by reversing aerobic glycolysis in immune cells: A mechanistic analysis Med. Drug Discov. 6: 100044. doi:10.1016/j.medidd.2020.100044.

262. Hevia D, González-Menéndez P, Quiros-González I, et al. (2015) Melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer. J. Pineal Res. 58 (2): 234–250. doi:10.1111/jpi.12210.

263. Berg JM, Tymoczko JL, Stryer L (2002) The pentose phosphate pathway generates NADPH and synthesizes five-carbon sugars. biochemistry 5th edition. New York: W H Freeman; 20.3 Available from: https://www.ncbi.nlm.nih.gov/books/NBK22416/.

264. Prchal JT, Gregg XT (2005) Red cell enzymes. Hematol. Am. Soc. Hematol. Educ. Program. 2005 (1): 19–23. doi: https://doi.org/10.1182/asheducation-2005.1.19.

265. Doshi B, Kamdar A, Smith-Whitley K, et al. (2016) Incidence of hemolytic events after exposure to triggering medications in pediatric patients with G6PD deficiency. Blood 128 (22): 4810. doi: https://doi.org/10.1182/blood.V128.22.4810.4810.

266. Francis RO, Jhang JS, Pham HP, et al. (2013) Glucose-6-phosphate dehydrogenase deficiency in transfusion medicine: the unknown risks. Vox Sang. 105 (4): 271–282. doi:10.1111/vox.12068.

267. Horowitz RI, Freeman PR, Bruzzese J (2020) Efficacy of glutathione therapy in relieving dyspnea associated with COVID-19 pneumonia: A report of 2 cases, Respir. Med. Case Rep. 30: 101063. doi:10.1016/j.rmcr.2020.101063.

268. Rovira A, Angioletti MD, Camacho-Vanegas O, et al. (2000) Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer. Blood 96 (13): 4111–4117. doi: https://doi.org/10.1182/blood.V96.13.4111.

269. Ma F, Ebihara Y, Umeda K, et al. (2008) Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proc. Natl. Aca. Sci. 105 (35): 13087-13092. DOI: 10.1073/pnas.0802220105

270. Qiu C, Olivier EN, Velho M, et al. (2008) Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood 111 (4): 2400-2408. DOI: 10.1182/blood-2007-07-10208.

271. Paglialunga F, Fico A, Iaccarino I, et al. (2004) G6PD is indispensable for erythropoiesis after the embryonic-adult hemoglobin switch. Blood 104 (10): 3148–3152. doi:10.1182/blood-2004-03-0835.

272. Ciftçi M, Bilici D, Küfrevioğlu OI (2001) Effects of melatonin on enzyme activities of glucose-6-phosphate dehydrogenase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacol. Res. 44 (1): 7–11. doi:10.1006/phrs.2001.0837.

273. El-Missiry MA (2000) Prophylactic effect of melatonin on lead-induced inhibition of heme biosynthesis and deterioration of antioxidant systems in male rats. J. Biochem. Mol. Toxicol. 14 (1): 57–62. doi:10.1002/(sici)1099-0461(2000)14:1<57::aid-jbt8>3.0.co;2-b.

274. Hara T, Mukai H, Nakashima T, et al. (2015) Factors contributing to erythropoietin hyporesponsiveness in patients on long-term continuous ambulatory peritoneal dialysis: A cross-sectional study. Nephron Extra. 5 (3): 79–86. doi:10.1159/000441154.

275. HF, Allami HCA (2019) Melatonin improves erythropoietin hyporesponsiveness via suppression of inflammation. Rev. Recent Clin. Trials. 14 (3): 203–208. doi:10.2174/1574887114666190528120357.

276. Walsh AC, Michaud SG, Malossi JA, et al. (1995) Glutathione depletion in human T lymphocytes: analysis of activation-associated gene expression and the stress response. Toxicol. Appl. Pharmacol. 133 (2): 249–261. doi:10.1006/taap.1995.1149.

277. Puskas F, Gergely P Jr, Banki K, et al. (2000) Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C. FASEB J. 14 (10): 1352–1361. doi:10.1096/fj.14.10.1352.

278. Chen G, Wang SH, Converse CA (1994) Glutathione increases interleukin-2 production in human lymphocytes. Int. J. Immunopharmacol. 16 (9): 755‐760. doi:10.1016/0192-0561(94)90095-7.

279. Carrillo-Vico A, Calvo JR, Abreu P, et al. (2004) Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance. FASEB J. 18 (3): 537–539. doi:10.1096/fj.03-0694fje.

280. Waters RS, Perry JSA, Han S, et al. (2018) The effects of interleukin-2 on immune response regulation. Math. Med. Biol. 35 (1): 79‐119. doi:10.1093/imammb/dqw021.

281. McKinstry KK, Alam F, Flores-Malavet V, et al. (2019) Memory CD4 T cell-derived IL-2 synergizes with viral infection to exacerbate lung inflammation. PLoS Pathog. 15 (8): e1007989. doi:10.1371/journal.ppat.1007989.

282. McKinstry KK, Strutt TM, Bautista B, et al. (2014) Effector CD4 T-cell transition to memory requires late cognate interactions that induce autocrine IL-2. Nat. Commun. 5: 5377. doi:10.1038/ncomms6377.

283. Abbas AK, Murphy KM, Sher A (1996) Functional diversity of helper T lymphocytes. Nature 383 (6603): 787‐793. doi:10.1038/383787a0.

284. Hardeland R (2018) Melatonin and inflammation—Story of a double‐edged blade. J. Pineal Res. 65: e12525. https://doi.org/10.1111/jpi.12525.

285. Carrillo-Vico A, Lardone PJ, José M, et al. (2005) Human lymphocyte-synthesized melatonin is involved in the regulation of the interleukin-2/interleukin-2 receptor system, J Clin Endocrinol Metab. 90 (2): 992‐1000. doi:10.1210/jc.2004-1429.

286. Al Amir Dache Z, et al. (2020) Blood contains circulating cell-free respiratory competent mitochondria. FASEB J. 34 (3): 3616–3630. doi:10.1096/fj.201901917RR.

287. NaveenKumar SK, et al. (2017) Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38 MAP kinase. Biochem. Biophys. Res. Commun. 491 (1): 183–191. doi:10.1016/j.bbrc.2017.07.073.

288. Hofmann J, Bosia A, Arese P, et al. (1981) Glucose-6-phosphate dehydrogenase deficiency in human platelets and its effect on platelet aggregation. Acta Biol. Med. Ger. 40 (12): 1707–1714.

289. Yang M, Zhou M, Ye JY, et al. (2008) The effect and underlying mechanism of melatonin on platelet formation and survival in a thrombocytopenic model. Blood 112 (11): 1241. doi: https://doi.org/10.1182/blood.V112.11.1241.1241.

290. Lissoni P, Mandala M, Rossini F (1999) Thrombopoietic property of the pineal hormone melatonin. Hematology 4: 4, 335-343. DOI: 10.1080/1.

291. Tanaka Y, Sato Y, Sasaki T (2013) Suppression of coronavirus replication by cyclophilin inhibitors. Viruses  5 (5):1250‐1260. doi:10.3390/v5051250.

292. Zhou D, Mei Q, Li J, He H (2012) Cyclophilin A and viral infections. Biochem. Biophys. Res. Commun. 424 (4): 647‐650. doi:10.1016/j.bbrc.2012.07.024.

293. Dawar FU, Tu J, Khattak MN, et al. (2017) Cyclophilin A: A key factor in virus replication and potential target for anti-viral therapy. Curr. Issues Mol. Biol. 21: 1‐20. doi:10.21775/cimb.021.001.

294. Hardeland, R. (2019) Melatonin and chromatin. Melatonin Res. 2 (1): 67-93. DOI:https://doi.org/https://doi.org/10.32794/mr11250012.

295. Tan DX, Hardeland R (2020) Potential utility of melatonin in deadly infectious diseases related to the overreaction of innate immune response and destructive inflammation: focus on COVID-19. Melatonin Res. 3 (1): 120-143. doi:https://doi.org/https://doi.org/10.32794/mr11250052.

296. Anderson G, Reiter RJ (2020) Melatonin: Roles in influenza, Covid‐19, and other viral infections. Rev Med Virol. 30 (3): e2109. doi:10.1002/rmv.2109.

297. Reiter RJ, Abreu-Gonzalez P, Marik PE, et al. (2020) Therapeutic algorithm for use of melatonin in patients with COVID-19. Front. Med. 7: 226. doi: 10.3389/fmed.2020.00226.

      

     This work is licensed under a Creative Commons Attribution 4.0 International License