Purinergic Signaling in Covid-19 Disease

Abstract

SARS-CoV-2 virus infection causes the Covid-19 disease pandemic. Purinergic signaling is a form of extracellular signaling. Purinergic signaling plays significant role in the pathology of Covid-19. Purinergic system includes extracellular nucleotides, nucleosides, ectonucleotidases, and purinergic receptors. ATP, ADP, and adenosine are the main nucleotides, nucleosides. CD39 and CD73 are the main ectonucleotidases. There are two classes of purinergic receptors, P1 and P2. Each of them can be further divided, P1 into A1, A2A, A2B, and A3, P2 into P2X, and P2Y. In Covid-19, the purinergic system is disordered. SARS-CoV-2 viruses invading leads to extracellular ATP and ADP accumulation, purinergic receptor abnormally activation, tissue homeostasis balance is broken, which lead to inflammation even hyperinflammation with cytokine storm and thrombosis et al. symptoms. Currently, Covid-19 therapeutic medicine is still in shortage. Target purinergic system components is a promising way to treat Covid-19, which will help inhibit inflammation and prevent thrombosis. Currently, many relevant preclinical and clinical trials are ongoing. Some are very promising.

1. Introduction

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. The first Covid-19 case was reported at the end of December 2019 in Wuhan, China [2]. From then on, the virus transmission was swiftly spread leading to the Covid-19 pandemic. So far, more than 6 billion people have infected SARS-CoV-2 virus worldwide, and more than 6million people died in the pandemic. Although now there are several kinds of vaccines have been put into use and been proven to be safe, effective and life-saving, the pandemic does not stop. There are several reasons may be responsible for this. The first one, not 100 percent people inoculate with vaccines. Second, like all the other vaccines, the Covid-19 vaccines do not fully protect everyone who is vaccinated, namely the efficacy rates of the vaccines are less than 100%. The SARS-CoV-2 is enveloped positive-chain RNA virus, which is prone to mutate. New mutation strains more easily escape from the vaccine defense. Third, vaccine protection is time-dependent, not lifelong. The SARS-CoV-2 virus transmission is through hand-mouth-eye contact and infected droplets released by coughing and sneezing. After the virus enters the body, it will combine with the cell surface angiotensin-converting enzyme 2 (ACE2) receptor through its envelope spike protein [3]. With the help of transmembrane serine protease2 (TMPRSS2), the virus will enter successfully into the cell [4]. There are some other cell-surface proteins, like Eph receptors, Neuropilin 1, and CD147 et al. which can also act as SARS-CoV-2 virus cell entry helpers [5, 6]. From asymptomatic to life-threatening acute respiratory distress syndrome (ARDS), the manifestations of the SARS-CoV-2 virus-infected people are quite variant. The most common symptoms found in the clinical presentation are fatigue, anosmia, ageusia, dizziness, headache, obtundation, myalgia, diarrhea, anorexia, fever, cough, pneumonia, and dyspnea [7, 8]. In severe cases, the virus infection will cause hyper inflammation with cytokine storm and also thrombogenesis can occur [9].

Purinergic signaling plays a pivotal role in SARS-CoV-2 virus infection, participates in the regulation of the innate immune system and platelet function, which are highly relevant for hemostasis, inflammatory, and thrombosis processes [10]. The purinergic system may be a possible target for SARS-CoV-2 treatments [11]. In this chapter, the purinergic signaling in Covid-19 disease will be introduced.

2. Purinergic system

Purinergic signaling is a form of extracellular signaling. In 1972, Geoffrey Burnstock proposed that adenosine triphosphate (ATP) can act as a neurotransmitter, which opened up a new area [12]. Purinergic system is composed of extracellular nucleotides, nucleosides, ectonucleartides, and purinergic receptors.

2.1 Nucleotides and nucleosides

The nucleotides and nucleosides are mainly ATP, adenosine diphosphate (ADP), and adenosine (Ado). In addition, Uridine diphosphate (UDP), uracil-diphosphate-sugar (UDP -sugar), and Nicotinamide adenine dinucleotide (NAD+) can also act as purinergic signal molecules [13]. Besides as a purinergic signal molecule, ATP is the universal currency of energy metabolism. 10% ATP is produced in cytoplasm by glycolysis. In this way, 1 molecular glucose lysis can generate 2 molecular ATPs. While the 90% ATP is synthesized in mitochondria through Krebs cycle. 1 molecular glucose oxidative-phosphorylation can create 30 ATPs. The Krebs cycle needs oxygen. As ACE2 receptors are highly expressed in lung, SARS-CoV-2 virus very easily invades this tissue leading to lower ventilation rates in Covid-19 patients. Hypoxia will cause ATP production inefficiency to lead to low intracellular ATP [14, 15]. The situation will be further intensified by ATP going outside the cell through Pannexin-1 channel (PANX1). PANX1 expression is increasing in Covid 19. Normally, extracellular ATP concentration is very low, commonly the concentration is less than 3 nanomolar, while intracellular one can be high at millimolar (2–8 mM). So, Covid-19 patients’ extracellular ATP level is dramatically increased, along with extracellular ADP accumulation. SARS-CoV-2 virus-infected cell lysis is another important reason for high extracellular ATP. Extracellular ATP catalyzed by CD39 (ectonucleoside triphosphate diphosphohydrolase-1, ENTPD1) to dephosphorylated into ADP, further into AMP which is still by CD39. Then CD73 (ecto-5′-nucleotidase, NT5E) will fully convert AMP to Ado. From ATP to Ado, this pathway is called canonical adenosinergic pathway, which was firstly illustrated by Yegutkin et al. Ado can be produced through a non-canonical alternative pathway, which starts from NAD+. NAD+ first is metabolized into ADP-ribose (ADPR) by CD38, then into AMP by CD203a, and further into Ado by CD73. So, two pathways converge on CD73. The life of Ado is very short. It will soon be deaminized into inosine by adenosine deaminase (ADA). Inosine is much more stable. Ado can also be taken into the cell through equilibrative nucleoside transporter-1 (ENT1) [16].

2.2 Ectonucleartidases

Ectonucleotidases are nucleotide metabolizing enzymes, which located on cytoplasmic membrane. The main function of Ectonucleotidases is to catalyze nucleotides hydrolyzation to balance nucleotides and neucleosides. Ectonucleotidases can be classed into 4 families: the ectonucleoside triphosphate diphosphohydrolases (NTPDase1-4,8), the ectonucleotide pyrophosphatase phosphodiesterases (NPP1-3), ecto-5′-nucleotidase and alkaline phosphatase (Table 1). CD39 is type 1 ectonucleoside triphosphate diphosphohydrolase, while CD73 is ecto-5′-nucleotidase. CD39 and CD73 not only regulate AMP existing, but also GMP state [17]. CD39 was overexpressed in COVID-19 patients’ plasma and some immune cell subsets and related to hypoxemia [18]. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission. Soluble Plasma CD39 may be used to predict covid-19 patients’ clinical prognosis, which is suggested as a promising biomarker for COVID-19 severity. CD39 is a defined marker of exhausted T cell [19]. T cell exhaustion and dysfunction are hallmarks of severe COVID-19. Both CD4⁺ Tim-3⁺ CD39⁺ T cell and CD8⁺ Tim-3⁺ CD39⁺ T cell significant increase in multiple tissues, like lung, liver, spleen and PBMCs, of critical covid 19 patients [20]. CD39 expression was also found up-regulated in plasmablasts. CD39 higher expression was also reported in the placenta of a 23 year old woman of pregnancy complicated by SARS-CoV-2 virus infection and the accompanying placental complement C4d deposition [21]. Regulatory T (Treg) cells have been shown to play an essential role in immune homeostasis in many diseases and pathological conditions [22]. Some studies have reported that CD4⁺CD25⁺CD39⁺ Tregs have more immunosuppressive effects than CD4⁺CD25⁺CD39⁻ Tregs [23]. In covid-19, CD39⁺ Tregs are decreased in juvenile patients in an age-dependent manner while in adult patients,CD39⁺ Tregs increased with disease severity [24]. However, CD73 expression is down-regulated in plasmablasts, CD8⁺ T cells and natural killer T cells (NKT)[25]. But there is one paper shows that moderate and severe cases have increased expression of CD39 and CD73 in total leukocytes. CD38, a catalytic case of non-canonical adenosinergic pathway mentioned above, is upregulated.

Family	Members	Hydrolysis pathways	—	—
NTPDases	NTPDase 1	ATP → ADP + Pi	ADP → AMP + Pi	—
—	NTPDase 2	ATP → ADP + Pi	ADP → AMP + Pi	—
—	NTPDase 3	ATP → ADP + Pi	ADP → AMP + Pi	—
—	NTPDase 4	UDP → UMP + Pi	UTP → UDP + Pi	—
—	NTPDase 5*	UDP → UMP + Pi	GDP → GMP + Pi	—
—	NTPDase 6*	GDP → GMP + Pi		—
—	NTPDase 7*	UTP → UDP + Pi	GTP → GDP + Pi	—
—	NTPDase 8	ATP → ADP + Pi	ADP → AMP + Pi	—
NPPs	NPP1	ATP → AMP + 2Pi	ADP → AMP + Pi	3′,5′-cAMP → AMP
—	NPP2	ATP → AMP + 2Pi	ATP → ADP + Pi	ADP → AMP + Pi
—		GTP → GDP + Pi	3′,5′-cAMP → AMP	AMP → adenosine + Pi
—	NPP3	ATP → AMP + 2Pi	ADP → AMP + Pi	3′,5′-cAMP → AMP
Ecto-5′-nucleotidase		AMP → adenosine + Pi		—
Alkaline phosphatase	—	ATP → ADP + Pi	ADP → AMP + Pi	AMP → adenosine + Pi

Table 1.

Major hydrolysis pathways of Ectonucleotidases.

^*

Intracellular enzymes.

Adapt from Seldin and Giebisch, The Kidney Physiological &Pathophysiological, 4th Edition, Oct1, 2007.

2.3 Purinergic receptors

Purinergic receptors can be separated into P1 and P2 [26]. P1 receptor can be further subclassed into A1, A2A, A2B and A3, P2 receptor further into P2X and P2Y. P1 and P2Y are G protein-coupled metabolic receptors, while P2X receptors are fast ligand gated ion channel.

P1 receptor is also known as adenosine receptor as its endogenous ligand is Ado. Caffeine and theophylline are two best known P1 receptor antagonists [27, 28]. Once Ado binding to P1 receptor, the conformation of the receptor will change to activate the coupled G protein. Activated G protein will cause intracellular cyclic adenosine monophosphate (cAMP) level change through acting on adenylate-cyclase. 4 subtype P1 receptors in human, encoded by different genes, interact with different G protein subunits. A1 and A3 receptors preferentially bind to inhibitory regulative Gi/o proteins, inhibiting adenylate-cyclase and cyclic AMP production, whereas the receptors of A2 family are generally coupled to stimulative regulative Gs protein that trigger intracellular cAMP accumulation. A1 and A2A are high affinity receptors, while A2B and A3 are low affinity receptors. Mitogen-activated protein kinase signaling pathway has also been reported to be another P1 receptor downstream pathway. In immune system, A1 receptor is mainly expressed on Neutrophils and immature dendritic cells, A2A on most immune cells, A2B on macrophages and dendritic cells, while A3 on neutrophils and mast cells. Ado binding to A1 receptor will produce chemotaxis function. On the contrary, A3 receptor activation will reduce neutrophil and stimulate mast cells degranulation. A2A and A2B receptors evoke immune suppress. The well-known anti-inflammatory effects of Ado are mediated by these two receptors [29]. A2B expression is upregulated in Covid 19.

P2 receptor can be sub-grouped into P2X and P2Y. P2X receptors belong to a larger family of receptors known as the ENaC/P2X superfamily. They are homologs. Structurally, P2X receptors and ENaC are very similar. PX2 receptors have a wide tissue distribution, which being expressed in nervous system, the pulmonary and digestive systems, muscle, bone, and immune system et al. Blood cells, like red cells, lymphocytes and macrophages and platelets, can be traced to have P2X receptors’ expression. P2X receptor family contains 7 members, P2X1 to P2X7 respectively, which are heterotrimers or homotrimers. Another name of P2X7 receptor is P2Z. ATP is the full agonist of P2X receptors, which can activate all P2X family receptors. NAD+ is also an activator of P2X receptors. However, there are some nucleotide-specific variations between these two ligands. For example, among the P2X receptor subtypes, the P2X7 receptor is unique in facilitating the induction of nonselective pores that allow entry of organic cations and dye molecules. Upon stimulation with ATP. As little as 100 μM ATP was sufficient to activate the nonselective pore, whereas NAD+ at concentrations up to 2 mM had no effect. The affinities between ATP and different P2X subgroups are also different. ADP and AMP, when purified, are inactive at P2X receptors. Activation of P2X receptors leads to influx of cations such as sodium and calcium, and further to depolarize the excitable cells. Among the 7 P2X receptors, P2X7 which mainly expressed on Macrophages, mast cells, microglia, pancreas, skin, and endocrine organs, is most studied and plays a pivotal role in SARS-CoV-2 virus infection associated inflammation, which is a promising target to treat Covid-19 disease [30]. Beside to P2X7, P2X1, P2X4, and P2X5 have been detected increasing expression in Covid 19 patients either.

P2Y receptors are seven-transmembrane proteins belonging to the class A family of G protein-coupled receptors (GPCRs), which are the δ group of rhodopsin-like GPCRs [31]. Structurally, P2Y receptors are characterized by extracellular N-terminal, which followed by seven hydrophobic transmembrane (7-TM) α-helices (TM-1 to TM-7) connected by three extracellular loops (ECL) and three intracellular loops, and ending in an intracellular C-terminus. An ECL serves to bind the receptor ligand(s), while intracellular regions mediate G protein activation and participate in P2Y receptor regulation. P2Y receptors can form both homodimers and heterodimers to further increase the biochemical and pharmacological spectrum of P2YRs. P2Y receptor’s family consists of 8 subunits, P2Y [4, 5, 14, 18, 28, 30, 32, 33]. The gaps of the subunit numbers are because of the fact that the assignment of numbers to certain putative P2Y receptors was later shown to be premature, with some of the previously designated sequences being P2Y species homologs and others being other types of receptors. P2Y receptors are present in almost all human tissues, where they exert various biological functions based on their G-protein coupling. Different from P2X receptors which have only ATP and NAD⁺ two native nucleotide agonists, P2Y receptors respond not only to nucleotides (ATP, ADP, UTP, UDP, NAD⁺) but also to nucleotide sugars such as UDP-glucose. According to the G-protein coupling difference, P2Y can be classed into Gq-coupled, P2Y1-like receptors (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) and Gi -coupled, P2Y12-like receptors (P2Y12, P2Y13 and P2Y14) (Table 2). As P1 receptors, when ligands combine to P2Y receptors, the conformations of the receptors will change to transfer the signal to the coupled G-proteins. Heteromeric G-proteins (Gαβγ) will dissociate into Gα subunits and Gβγ complexes, which activate or regulate downstream effector pathways. P2Y11 is the only P2Y member which can activate cAMP pathway. P2Y receptors also play very important roles in in SARS-CoV-2 virus infection related inflammation and can be served as therapeutic targets. P2RY1 and P2RY12 have been shown to be elevated in Covid-19.

groups	Receptors	Tissue distribution	Agonist(s)	G Protein coupling
P2Y1-like	P2Y1	Brain, epithelial and endothelial cells, platelets, immune cells, osteoclasts	ADP, NAD^+	Gq
—	P2Y2	Immune cells, epithelial and endothelial cells, kidney tubules, osteoblasts	ATP ≈ UTP	Gq-Gi
—	P2Y4	endothelial cells, placenta	UTP	Gq-Gi
—	P2Y6	airway and intestinal epithelial cells, spleen, placenta, T-cells, thymus,	UDP	Gq
—	P2Y11	spleen, intestine, granulocytes, macrophage	ATP, NAD^+	Gq-Gs
P2Y12-like	P2Y12	Platelets, brain (glial cells), microglial cells	ADP	Gi
—	P2Y13	Spleen, brain, lymph nodes, bone marrow	UDP-Sugar	Gi
—	P2Y14	Placenta, mast cells, adipose tissue, stomach, intestine, discrete brain regions	UDP	Gi

Table 2.

Human P2Y receptors.

2.4 Roles of purinergic signaling in the inflammation of Covid-19

The inflammation of Covid-19 is the most important biological response of the body tissue to SARS-CoV-2 invasion [34]. Usually inflammation is the innate immune protective response involving immune cells, blood vessels, and molecular mediators. However, in severe covid-19 patients, it can develop into hyperinflammation, which can be life-threatening. Hyperinflammation is thought to be the base to develop into severe Covid 19. About hyperinflammation, currently there is no clear-cut definition. The criteria of hyperinflammation are not consistent. Most people think the condition of hyperinflammation as a form of very severe inflammation with cytokine storm which is out of tissue homeostatic control to lead to ARDS or other organs failure. Purinergic signal system is in the pivotal position of pro-inflammation and anti-inflammation axis. Once the balance is broken, pro-inflammation factors being far more than the ones of anti-inflammation, hyperinflammation will happen.

SARS-CoV-2 invasion leads to extracellular ATP and ADP accumulation. ATP will bind to P2X7 receptor. Though P2X7 receptor expressed on almost all type human and mouse cells, the levels of the ones on monocyte and macrophage are much higher. ATP binding to P2X7 receptor leads to pore forming on the cell surface to cause K⁺ efflux. Intracellular K⁺ depletion and extracellular K⁺ concentration increase is necessary and sufficient to activate and assembly the NLR family pyrin domain containing 3 (NLRP3) inflammasome to promote proteolytic cleavage, maturation and secretion of pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18)[30]. So far there is no evidence to show that SARS-CoV-2 can directly activate NLRP3 inflammasome. Flowing P2X7 receptor activation other cytokines and chemokines, for example, IL-6, TNF-α, CCL2, IL-8, CCL3 and CXCL2, of pro-fibrotic factors such as TGF-β, and extracellular matrix remodeling factors, for example, metalloproteinase-9 and tissue inhibitor of metalloproteinase (TIMP)-1 will also be released. In mild Covid 19, the extracellular ATP concentration lower than 100uM, after proinflammation process starts the anti-inflammation response will be triggered either. First reaction is CD39 / CD73 will convert ATP into Ado. Ado will activate P1 receptor. As above mentioned, A2A and A2B receptors activation will launch immune suppress. At the same time, activation of A2A receptor will promote the differentiation of naïve T-cells towards regulatory T-cells (Tregs). Treg will secrete immune suppressive factors, like IL-10 and TGF-β, to restrict immune reaction. However, when extracellular ATP concentration over 100uM, the situation will become worse, dramatic immune response will lead to sever inflammation. If extracellular ATP concentration is over 1 mM, hyperinflammation will be inevitable in most cases. High amount extracellular ATP accumulation leads to prolong P2X7 receptor activation. P2x7 receptor overactivation leads to macropore formation and cytolysis with uncontrolled ATP outgoing and cytokines release. What making the situation even worse is the anti-inflammation process being out of control. P2X7 receptor activation inhibits he suppressive potential and stability of Tregs. Tregs clonal proliferation and mature are suppressed, Treg death increasing. Treg depletion leads to IL10 et al. immune suppressive factors drop. In severe covid-19 patient, the expression of forkhead box protein P3 (FoxP3), a marker of Treg, was monitored lower than that in health control. On the other side, CD73 express is down-regulated, which blocks the production of Ado, which cause P1 receptors desensitization [35]. So, though A2B is detected to have higher expression in Covid-19, it is less activated. The homeostatic out-control at last results in the hyperinflammation exploding. Not only P2X7 receptor play a role in Covid-19 inflammation, other P2 receptors also have functions in the proinflammation. For ex, ATP-P2X4, ADP-P2Y6, ADP-P2Y12, and UDP-sugure-P2Y14 et al. mediated signaling all can stimulate inflammation via actions on innate immune cells, especially dendritic cells and macrophages.

2.5 Roles of purinergic signaling in the thrombosis of Covid-19

In addition to inflammation, many Covid-19 patients also have microvascular thrombosis, which have been confirmed by autopsy. Clinical detection has also provided very solid evidence. Covid-19 patients have high level of circulating D-dimeris (a fibrin/fibrinogen degradation product), prolonged prothrombin time, upregulated expression of tissue factor (TF, encoded by F3 gene) et al [32]. The Covid-19 related stroke incidence was reported increase, either. Purinergic system not only participate in inflammation but also involved in thrombosis, it is like a bridge to connect the two processes. The complex interplay between the two processes is described as thromoinflammation [36, 37].

Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. In Covid-19 disease the balance of coagulation and fibrinolysis is broken, which leads to thrombosis happen [38]. Blood coagulation can be divided into two pathways: intrinsic pathway and extrinsic pathway. Intrinsic cascade starts from blood contacts the damaged blood vessel surface or other high molecular surface with negative charges to induce factor XII activation, which following by factor XI and activation. On the phospholipid surface of the activated platelet, factor IX together with factor VIII (vW factor) and Ca²⁺ will activate factor X. Extrinsic pathway, which also called tissue factor pathway, is beginning from factor VII being activated by tissue factor. Activated factor VII can directly activate factor X. So, two coagulation pathways converge on factor X activation. Activated factor collaborated with factor V and Ca²⁺catalyzes prothrombin to become thrombin. Also, the phospholipid surface of the activated platelet is necessary for this reaction. Thrombin will continue to catalyze fibrinogen to convert into fibrin. Purinergic system can promote coagulation from several aspects [33]. Activated platelet plays very important roles in blood coagulation. ADP can directly activate platelet. As above mentioned, Covid-19 patients have extracellular ADP accumulation. The accumulated ADP can bind to P2Y12 receptor located on the surface of the platelet to activate it. The activated platelet will secrete more ADP and vW factor et al. ADP can also activate platelet through combine to P2Y1 receptor. ATP is also a platelet activator. ATP can interact with platelet P2X1 receptor. ATP binds to macrophage P2X7 receptor enhance tissue factor expression and release to trigger extrinsic pathway [39]. The third way is that purinergic signal can stimulate neutrophils activation to produce reactive oxygen (ROS). The overwhelming production of ROS can result the release of neutrophil extracellular traps (NETs), which is web-like structures composed of chromatin containing neutrophil granule proteins [40]. NETs can further activate factor XII to activate the intrinsic pathway [19].

3. Therapeutic targets

Regarding the important roles purinergic signal plays in Covid-19 disease, the members of purinergic system have been used as therapeutic targets to reduce morbidity and mortality.

3.1 Adenosine and Ado metabolism enzymes

As above described, Ado can exert anti-inflammation effects through active P1 receptor [41, 42]. Clinically, Ado is used in cardiac diseases diagnosis and treatment. Preclinically, Ado administration was demonstrated to be able to attenuate lung injury [43]. Ado being reported can also be applied in Covid-19 patient treatment. A patient suffering from SARS-CoV-2-related ARDS on routine therapies who did not show clinical improvements, inhaled adenosine in a mixture of 21% oxygen was applied. After 5 days, the SARS-CoV-2 test was negative and a rapid improvement in clinical condition as well as radiological pictures were shown. The main concern about Ado used in disease treatment is its short half-life in vivo. In the future more stable Ado analogs may be developed. Ado metabolism enzymes blocking methods is another way to elevate extracellular Ado. Pentostatin (2′ deoxycoformycin) and EHNA (erytho-9-(2-hydroxy-3-nonyl) adenine hydrochloride) are two ADA inhibitors. Clinically, pentostatin is used in Hairy Cell Leukemia treatment. It is suggested pentostatin might be beneficial in late-stage ARDS. Not like pentostatin which only inhibits ADA enzyme activity, EHNA can also bind to P1 receptors and adenosine deaminase complexing protein 2(CD26). EHNA potentially has anticancer effects, but so far has been used clinically. EHNA is also suggested to be potentially used in Covid-19 therapy. Dipyridamole (DIP) is a ENT1 inhibitor, which can prevent extracellular Ado uptake [44]. DIP is an approved antiplatelet drug, clinically being used to prevent stroke, and being proved to have high safety [45]. The bleeding risk of DIP is similar to that of aspirin. Currently, three clinical trials evaluating efficacy of dipyridamole for the treatment of COVID-19 have been registered (identifiers: NCT04424901; NCT04391179; NCT04410328) [46, 47]. Apart from anticoagulant and anti-inflammatory effects, it is speculated that DIP can also blunt SARS-CoV-2 replication.

3.2 CD39

As CD39 plays very important roles in extracellular ATP and ADP hydrolysis, its expression and activity closely related to inflammation and thrombosis [48]. Several approaches have been attempted to target CD39. One of them is using soluble CD39 to antithrombosis [48]. However, this method easily causes bleeding. To overcome this side effect, new strategies is worked out. The core of these new strategies is to link the recombinant soluble CD39 to other molecules, like PSGL-1, the receptor for P selectin on leukocyte surface, and single chain antibody (scFV) specific against GPIIb/IIIa, the platelet fibrinogen receptor, and glycoprotein VI (GPVI) Fc fusion protein et al.

3.3 P1 receptors

P1 receptor family contain1 A1, A2A, A2B, A3. These 4 receptors have different functions [49]. Activated A2A and A2B can suppress immune response. As mentioned above that caffeine and theophylline are two best known P1 receptors antagonists. Both are non-selective antagonists except for A3 receptor, they can inhibit the other 3 P1 receptors, namely A1, A2A, and A2B, at therapeutic concentrations. Theophylline is more potent. As these receptors have different functions in inflammation, inhibit these three receptors will have different effects. For example, theophylline has been shown to have both proinflammatory and anti-inflammatory effects [50]. The latter one might be stronger. Recently, shown by preclinical data that theophylline can potentially amplify the anti-inflammatory effect of corticosteroids and reduce corticosteroid resistance. Now one clinical trial, which theophylline is designed to be nasally administrated to treat the Covid-19 patients who have been received intranasal and oral corticosteroids, is on-going (Identifier: NCT04789499). However, there is one report sharing that theophylline treatment induced sinus bradycardia in two cases of Covid-19 patients [51]. Pentoxifylline (PTX) can active A2A receptor. When PTX binds to A2A receptor, it will stimulate to secrete IL-10 et al. immune suppressive molecules to inhibit inflammation. PTX treatment is also shown to help reduce IL-6 serum concentration, as well as diminish IL-1b level. PTX has been recommended to be applied in Covid-19 therapy.

3.4 X2P7 receptor

X2P7 receptor is the most important pro- inflammation purinergic receptor. X2P7 receptor block is predicated to be able to ameliorate inflammation. X2P7 receptor activation can also potentially induce of VEGF release. P2X7 receptor blockade can inhibit VEGF-dependent increase in vascular permeability, and therefor prevent lung oedema. Several X2P7 receptor antagonists are suggested to be used in Covid-19 therapy [27, 52]. Colchicine is one of such inhibitors. Colchicine is a tricyclic lipid-soluble alkaloid extracted from Colchicum autumnalle and gloriosa superba. Colchicine is a well-known of microtubule polymerization inhibitor, which in the early time was found to be able to block cell mitosis. Hereafter, its anti-inflammation effects was revealed. Colchicine has been clinically used as an anti-inflammatory agent for long-term treatment of Behçet's disease and also used to treat many other diseases, like pericarditis, pulmonary fibrosis, biliary cirrhosis, various vasculitides, pseudogout, spondyloarthropathies, calcinosis, scleroderma, and amyloidosis et al. Colchicine not only can inhibit X2P7 receptor, but also can block X2P2 receptor pathway. Several clinical trials have shown that colchicine can limit the production of some cytokines, like IL-1b, IL-18, and IL-6 et al., of Covid-19 patients. NIH has included colchicine in Covid-19 treatment guideline.

Lidocaine is another P2X7 receptor antagonist, which routinely used as local anesthesia in clinic. It’s readily available and affordable. Recently, a clinical trial (Identifier:NCT04609865) is carrying out in a French group, in which lidocaine is intravenously administrated to treat Covid-19 disease [6]. However, the half-maximal effective concentration (IC50) for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. A Peru group modified the protocol. 28 (three mild, 21 moderate and four severe) COVID-19 patients were treated with 0.5% lidocaine HCL solution with an intravenous dose of 1 mg/kg once a day for 2 days and 2% lidocaine HCL solution with a subcutaneous dose of 1 mg/kg once a day for 2 days, which results in the improvement in pain, cough, respiratory rate and oxygen saturation. Another group directly carried out subdermal administration of lidocaine in 6 critical ill Covid-19 induced ARDS patients. The author claimed that although all six patients appeared to respond positively to the treatment and no severe adverse effects were observed, no final conclusions could be made on the efficacy of lidocaine in critically ill COVID-19 patients.

3.5 P2Y12 receptor

P2Y12 receptor is the main purinergic receptor responsible for SARS-CoV-2 virus related thrombosis, Theoretically, P2Y12 receptor blocking can confine thrombosis of Covid-19, and also can curb the inflammation. Several P2Y12 antagonists (clopidorgel, prasugrel, ticagrelor and cangrelor) have been clinically used to prevent thrombosis in patients at risk of heart attack for about 20 years [27, 53]. Recently, these antagonists have been reevaluated for its effects in Covid-19 related thrombosis treatment. Several clinical trials (Identifiers are NCT04505774; NCT04409834, and NCT04333407.) are on the way. One of them, titled “accelerating Covid-19 therapeutic and vaccines 4 acute (ACTIV-4A, Identifier: NCT04505774)” has been finished and reported [54]. This is a randomized clinical trial, which aims to test if P2Y12receptor antagonists can enhance heparin therapeutic effects in mild Covid-19 patients. The answer is no. The results demonstrated that among non- critical ill hospitalized Covid-19 patients, the use of a P2Y12 receptor antagonist in addition to a therapeutic dose of heparin, compared with a therapeutic dose of heparin only, did not result in an increased odds of improvement in organ support– free days within 21 days during hospitalization. However, as the author mentioned that this trial tested only the combination of a P2Y12 inhibitor with anticoagulant therapy, it remains possible that use of a P2Y12 inhibitor as a sole antithrombotic agent may improve outcomes in patients with COVID-19. In addition, the potential for benefit with a longer treatment duration or at an earlier stage of illness (before hospitalization) cannot be ruled out.

4. Conclusion

Purinergic signal is involved in SARS-CoV-2 viruses causing Covid-19, which plays a pivotal role in the pathology of Covid-19 disease. Purinergic signal participates in the regulation of the innate immune system and platelet function et al., which are highly relevant for hemostasis, inflammatory and thrombosis processes. SARS-CoV-2 virus infection will lead to the abnormality of purinergic system to break the body homeostasis further to inflammation and thrombosis. Purinergic system components have been suggested to be Covid-19 therapeutic targets. Currently many preclinical and clinical trials have been in progress to test this hypothesis. Promising data have brought new hope to the patients.