Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase-1 or CD39) is expressed in the surface of several cells, including platelets, leukocytes and endothelial cells (1). In addition, a soluble catalytically active form of CD39 circulates in human blood (2). CD39 constitutes a major regulator of purinergic signaling, a form of extracellular signaling mediated by purine nucleotides and nucleosides such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine (ADO) (3) that exert multiple functions in inflammation and coagulation as autocrine and paracrine signaling molecules (4). For instance, extracellular ATP (eATP) is recognized by the purinergic receptor P2X₇ (P2X₇R) which triggers the activation of NLRP3 inflammasome which not only results in inflammation but also in increased release of tissue factor (TF) (5), the latter involved in coagulation cascade initiation (6). Furthermore, extracellular ADP (eADP) is a major activator of platelets through P2Y₁₂ receptor (P2Y₁₂R) (7). Under homeostatic condition, proinflammatory and pro-coagulatory eATP and eADP are hydrolyzed to anti-inflammatory adenosine (ADO) thanks to the consecutive action of CD39 and 5’nucleotidase (CD73). Thus, CD39/CD73 axis constitutes a mechanism to ensure tissue protection under acute stress, by contributing to resolution of inflammation (8). Furthermore, CD39 is well described as a principal modulatory player in inflammation and coagulation (9, 10) and its soluble form has been previously proposed as a prognosis biomarker in chronic obstructive pulmonary disease (11). Interestingly, a recent review by Franciosi et al. (12), highlights the need for investigation about the prognostic value of CD39 levels in COVID-19 patients.

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV-2) responsible for the current pandemic, that has caused more than five million of deaths worldwide (13). COVID-19 patients manifest a broad spectrum of symptoms ranging from mild or no symptoms to severe pneumonia that might require ICU admission and can evolve to respiratory failure, multiorgan dysfunction and death (14). Some studies point to thromboinflammation (the coordinated activation of the inflammatory and thrombotic responses) as a major cause of disease severity in patients with COVID-19 (15). In fact, the inflammatory response in patients with severe COVID-19 is particularly striking: elevated levels of inflammatory markers (such as C-reactive protein, ferritin and various cytokines), which are associated with poor outcomes (16, 17). An intriguing finding is the correlation between elevated circulating levels of inflammatory cytokines and abnormal coagulation parameters (18). Indeed, levels of prothrombotic acute phase reactants, such as fibrinogen, and D‐dimer are increased in patients with COVID-19 (19, 20), suggesting that COVID-19 can be associated with a massive inflammatory response combined with a hypercoagulable state. Although the pathophysiology of these disturbances has not yet been defined; the role of CD39 in COVID-19 severity has been suggested in this context (9). Hence, we considered an interesting priority to unravel the role of CD39 in the pathophysiology of COVID-19, since CD39 might represent a useful predictive marker for COVID-19 severity and target for therapeutic interventions. Therefore, the aim of this study was to investigate the expression of CD39 and its involvement in clinical prognosis, as well as the effects of purinergic signaling on thromboinflammation in severe COVID-19 patients.

The present study demonstrates an upregulation of CD39 in severe COVID-19 patients. Although further research is needed, our data suggest that probably CD39 expression increases in severe COVID-19 patients due to the activation of HIF-1α and RIG-I pathways. In fact, previous studies suggest that CD39/CD73 axis can be transcriptionally regulated by hypoxia through SP1 and HIF-1α (25). Furthermore, it is known that the SARS-CoV-2 nucleocapsid protein interacts with RIG-I (26) which is involved in antiviral immune response (27). Also, antiviral immune response has been associated with CD39 expression (28). Therefore, we suggest that synergic action of hypoxia and antiviral immune response in COVID-19 patients is a possible cause of CD39 upregulation.

Moreover, we identified sCD39 as a potential biomarker of COVID-19 severity. In contrast, CD39 has been reported to exert a protective role in ischemia reperfusion injury and transplantation, preventing coagulation (29). Indeed, the use of recombinant soluble CD39 has even been proposed as a therapeutic strategy for noninfectious acute lung injury among other diseases (30). Inquiringly, CD39 expression has been also reported to be increased in other respiratory diseases, such as COPD, where it has been proposed as a compensatory mechanism in response to cigarette smoke-induced lung damage (11). However, the prognostic relevance of CD39 in COVID-19 patients might be justified by its potential implication in the processes related to thromboinflammation, which are main determinants of the evolution of COVID-19 (15). In this line, the relevance of CD39 is encouraged by its relationship with several inflammatory markers such as ferritin and CRP as well as with some coagulation markers, such as fibrinogen, CD40L, and TF. These three molecules have been described to be closely related with clot formation, and the upregulation of their soluble forms in plasma has been reported in a plethora of coagulation disorders, including COVID-19 (19, 20, 31, 32). Briefly, tissue factor initiates the generation of thrombin, which not only converts fibrinogen to fibrin, but also activates platelets. Activated platelets release sCD40L to the blood stream, which in turn stimulates endothelial cell activation and secretion of tissue factor, thereby amplifying thrombosis and coagulation (32). Interestingly, various reports have recently highlighted CD39 as a key player in the crosstalk between inflammation and thrombosis (10). In addition, CD39 upregulation in COVID-19 have been suggested. For instance, Shahbazi et al., reported high levels of CD39 in CD8 T-cells from COVID-19 patients (33). Moreover, in a recent study Na Wang et al., reported high levels of CD39 mRNA in the PBMCs of COVID-19 patients, that, interestingly, were related to disease severity (34). Meanwhile, Ahmadi et al., report no significant difference in the CD39 expression on some immune subsets between COVID-19 and healthy donors (23). Therefore, additional efforts are needed in order to elucidate CD39 role in COVID-19 pathophysiology. Here we provide a more extensive study, analyzing CD39 expression in several immune subsets as well as in its soluble form. Moreover, we provide evidence relating CD39 with inflammation and coagulation markers.

According to our findings, we proposed that the molecular mechanisms underlying CD39 role on COVID-19 severity are related to impaired purinergic signaling, characterized by high levels of eATP and eADP, in combination with low levels of ADO. Interestingly, elevated eATP has been already reported in COVID-19 patients (35). eATP constitutes an important alarm for the immune system (36) as it is recognized by P2X₇R, the most relevant purinergic receptor involved in inflammatory processes (37), such as sepsis (38). In fact, this receptor is capable of activating NLRP3 inflammasome promoting the release of pro-inflammatory and pro-coagulatory mediators (5). Thereby, inflammasome activation may constitute one angular stone in COVID-19 severity, in according with our data and previous research (39). Besides, we propose that accumulated eADP in the plasma of COVID-19 patients can activate P2Y₁₂R in platelets possibly leading to the reported procoagulant disorder. Strikingly, a reversible antagonist of P2Y₁₂R, ticagrelor, reduced platelet activation caused by COVID‐19 plasma. Indeed, ticagrelor can be useful to prevent sepsis-induced coagulopathy in COVID‐19 patients (40). In particular, ticagrelor not only acts on platelets by limiting its ADP‐dependent activation but can also reduce platelet-leucocyte interaction and decrease proinflammatory cytokines production (41), reducing mortality risk in patients suffering pulmonary adverse events as bacterial infection (42). In line, a retrospective observational clinical trial enrolling 1700 COVID‐19 patients concerning P2Y₁₂R inhibitors (ticagrelor, clopidogrel) has been recently completed (NCT04518735). Moreover, two phase-4 trials involving clopidogrel and/or ticagrelor to treat COVID‐19 associated thrombotic events are under patients’ recruitment. First, COVID‐PACT (NCT04409834) is a multicenter, randomized-controlled trial in critically ill COVID‐19 patients involving the use of anticoagulants (including clopidogrel). Second, ACTIV‐4 study (NCT04505774) is a randomized, adaptative platform trial to compare the effectiveness of antithrombotic strategies (including ticagrelor and clopidogrel). Altogether, these proposals highlight the rising interest in P2Y₁₂R inhibitors to prevent COVID‐19 associated coagulopathies.

However, our study has several limitations, which we acknowledge. First, limited sample size and follow-up time restrained the potential identification of robust prognostic events. More follow-up time will be necessary to assess the relation with different thrombotic events. Second, the results obtained allow us to suspect an association between different pathogenic pathways but do not establish a mechanistic description, since the local conditions of the pandemic restricted the procedures with infected cells. Third, this is an observational study carried out in patients with severe COVID-19 pneumonia treated according to conventional clinical practice, so the non-randomization does not allow us to infer the efficacy of different clinical approaches. And fourth, H1N1 infected patients’ biomaterial was restricted to plasma samples, preventing the realization of experiments involving immune cells.

In summary, this study reveals CD39 upregulation in severe COVID-19 patients. Moreover, the determination of its soluble form in plasma shows a certain discriminative capacity on the short-to-medium-term prognosis of these patients, so far, its usefulness as a potential biomarker could be evaluated. Moreover, this study reports impaired purinergic signaling in COVID-19, characterized by high levels of eATP and eADP in combination with low levels of ADO. The purinergic dysregulation might play an important role in modulating vascular homeostasis, inflammation, and thrombosis of patients with severe COVID-19 pneumonia. On the one hand, eATP contribute to hyperinflammation and TF release secondary to NLRP3 activation. On the other hand, eADP overproduction is linked to increased platelet activation ( Figure 5 ). This observation might be exclusive for COVID-19 patients, because patients with other viral infection as influenza A, although presenting elevated proinflammatory nucleotides, maintain normal production of anti‐inflammatory ADO, and do not show elevated levels of CD39. Additional work is needed to corroborate sCD39 usefulness as a potential biomarker and to elucidate whether CD39 could be implicated in the pathogenesis of COVID-19 or whether it plays a compensatory role.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by PI-4087. The patients/participants provided their written informed consent to participate in this study.

FG-R and CC-Z conceptualized the study. ED-G, EZ, AM, FG-R, and CC-Z advised on the study design and endpoints. ED-G, SG-T, EA, AV-D-R, RP-d-D, KN-N, EL-C, FG-R, and CC-Z performed designed experiments. EZ, AM, and RG. recruited COVID-19 patients and collect samples. JR-H, JS-V, and CR-G recruited influenza A patients and collect samples. ED-G, FG-R, and CC-Z analyzed data and performed statistical data. FG-R and CC-Z were responsible for the study management and coordination. FG-R and CC-Z drafted the paper. All authors have read and approved the final manuscript.

This work was supported by the following fundings: Fondo de Investigación Sanitario (FIS)‐Fondos FEDER, Spain: PI19/01612 (FG-R) and COV20/00207, CP18/00028 and PI19-01363 (CC-Z).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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