Involvement of P2 receptors in hematopoiesis and hematopoietic disorders, and as pharmacological targets

Abstract

Several reports have shown the presence of P2 receptors in hematopoietic stem cells (HSCs). These receptors are activated by extracellular nucleotides released from different sources. In the hematopoietic niche, the release of purines and pyrimidines in the milieu by lytic and nonlytic mechanisms has been described. The expression of P2 receptors from HSCs until maturity is still intriguing scientists. Several reports have shown the participation of P2 receptors in events associated with modulation of the immune system, but their participation in other physiological processes is under investigation. The presence of P2 receptors in HSCs and their ability to modulate this population have awakened interest in exploring the involvement of P2 receptors in hematopoiesis and their participation in hematopoietic disorders. Among the P2 receptors, the receptor P2X7 is of particular interest, because of its different roles in hematopoietic cells (e.g., infection, inflammation, cell death and survival, leukemias and lymphomas), making the P2X7 receptor a promising pharmacological target. Additionally, the role of P2Y12 receptor in platelet activation has been well-documented and is the main example of the importance of the pharmacological modulation of P2 receptor activity. In this review, we focus on the role of P2 receptors in the hematopoietic system, addressing these receptors as potential pharmacological targets.

Introduction

The regulation of quiescence, self-renewal, proliferation, differentiation, and cell death of the hematopoietic stem cell (HSC) is the core of the hematopoietic system. This process is known as hematopoiesis and is regulated by several signaling molecules, such as cytokines, which are the main regulators of hematopoiesis. However, other signaling molecules, such as ATP, are also relevant in this process. ATP is an important molecule linked to fundamental metabolic processes that also regulate the hematopoietic system by being the main agonist of ATP receptors (known as P2 receptors or purinergic receptors). These receptors are expressed in the most primitive hematopoietic cells until mature progeny. Although P2 receptors are mainly known to regulate the immune system, new physiological activities are being investigated.

ATP under normal conditions is at the millimolar range in the cytoplasm (3–10 mM) and at the nanomolar range in the extracellular side, providing a high chemical gradient [1]. ATP, in its ionized form (ATP⁴⁻) [2], can be released through the plasma membrane by lytic and nonlytic mechanisms in favor of a chemical gradient and due to the electrical potential difference across the plasma membrane, which is negative on the inside. A loss of plasma membrane integrity by stress mechanisms, or by cell death, leads to the release of intracellular ATP. Over the years, several nonlytic mechanisms by vesicles and conductive ATP release have been described [3]. For example, a cornerstone study published almost half a century ago demonstrated the release of ATP contained in vesicles with other neurotransmitters [4], which initiated the boom of investigations of purinergic receptors, while more recent ones showed the release of ATP under hypoxia by endothelial cells [5] and osteoblasts [6]. The activation of mechanoreceptors also promotes the release of ATP by osteoblasts [7, 8]. Another mechanism of ATP release was described by the autocrine activation of P2 receptors by ATP itself, with the participation of membrane channels formed by the pannexin-1 protein, which is highly expressed in various tissues [9–11]. Hemichannels formed by connexins are also another form of ATP release [12, 13]. Paracrine/autocrine ATP signaling on neutrophils has also been investigated either through the pannexin channels or by the vesicular nucleotide transporter [14, 15]. In addition to the cells described above, other cell types, such as erythrocytes [16], epithelial cells [17], and hepatocytes [18] can also release ATP. These reports have demonstrated the range of physiological situations in which P2 receptors can be activated.

Over the last decades, it has been shown that all hematopoietic cell types express P2X (ionotropic) and/or P2Y (metabotropic) P2 receptors that are activated by ATP and its analogs. The effects of ATP vary according to cell type, the degree of differentiation, agonist concentration, and the time of stimulus due to the large number of receptors and differences in the pharmacological characteristics of these receptors. In this review, we focus on the participation of P2 receptors in the hematopoietic system and how the knowledge of purinergic regulation can be used as potential pharmacological targets in blood disorders.

P2 receptors

Purinergic receptors are responsible for the transduction of signal triggers by extracellular nucleotides and nucleosides. P2X receptors are ligand-gated ion channels and assembled as homotrimers or heterotrimers [19]. To date, seven isoforms have been described (P2X1-7) (Fig. 1). These receptors have intracellular N- and C-subunits that bind to protein kinases and also have two transmembrane regions, the TM1, which is involved in the channel activation and the TM2, which coats the ionic pore [20, 21]. The homology among the P2X receptor genes is around 40–60% in mammals and the subtypes differ in their rates of desensitization; ion conductivity; and sensitivity to agonists, antagonists, and allosteric modulators [22, 23]. P2X1-7 receptors have three subunits that form a stretched trimer or a hexamer of conjugated trimers [24, 25], while transition metals such as Zn²⁺ and Cu²⁺ ions act as allosteric modulators [26]. Other metals, such as lanthanides, may also modulate P2X receptors, and ethanol has been shown to reduce the potency of ATP in several P2X receptors [27]. A comparative study shows 180 putative P2X genes in 32 animal species and four members in three species of lower plants exploring the evolutional relationship among different subtypes of P2X genes [28].

Fig. 1.

Roles of purinergic regulation in the hematopoiesis. ATP is an important molecule that also regulates the hematopoietic system by activation of receptors, known as P2 or purinergic receptors. Purinergic receptors are responsible for the signal transduction triggered by extracellular nucleotides and nucleosides. The release of ATP directly promotes the activation of P2X (ionotropic) and P2Y (metabotropic) receptors triggering signaling associated to Ca²⁺ mobilization, or by activation of enzymes linked to G proteins. The activation of these intracellular pathways affects the hematopoiesis, for instance modulating inflammation, HSC differentiation, and coagulation. The complexity of purinergic regulation involves not only the primary agonists and their receptors but also enzymes such as CD39 that hydrolyzes ATP to ADP and ADP to AMP, which can subsequently be hydrolyzed by CD73 to adenosine, which activates P1 receptors. The most emblematic clinical application of knowledge about the regulation of P2 receptors is the use of inhibitors of P2Y12 receptor such as clopidogrel, used as an antithrombotic drug

P2X1 receptor is widely expressed in smooth muscle and mediates the neurotransmitter actions of ATP released by the sympathetic and parasympathetic nerves [29, 30]. P2X1 receptor is also responsible for mediation of Ca²⁺ influx in platelet aggregation [31]. P2X2 receptors are widely expressed in central and peripheral neurons and have been implicated in neurotransmission [32, 33]. P2X3 receptors are expressed in sensory neurons, including nociceptive nerve endings [34, 35]. The agonist α,β-methylene-ATP distinguish the receptors (P2X1, P2X3) from the other homomeric forms (P2X2, P2X4, P2X6 and P2X7) [30]. However, the antagonist NF023, an analog of suramin, is about 20 times less effective in P2X3 compared to P2X1 receptors [36]. There are no selective agonists known for P2X2 receptors [19].

The receptors P2X4 and P2X6 are expressed in the central nervous system, endothelial cells, placenta, and thymus [37, 38]. Carbamazepine derivatives are potent agonists of the P2X4 receptor [39], and the allosteric modulator ivermectin increases ATP concentration and potentiates its effects [39]. The receptor P2X5 is highly expressed in skeletal muscle, skin, and epithelium [35]. ATP and 2-methylthio-ATP are full agonists for the P2X5 receptor, which in turn is inhibited by TNP-ATP, PPADS, and suramin [40, 41]. The P2X6 receptor was discovered in rat brain and by homology [42], being only functionally expressed as a heteromultimer [38, 43]. P2X7 receptor is expressed in immune cells, pancreas, skin, and microglia [35, 44]. Prolonged exposure of P2X7 receptor to high concentrations of ATP may lead to cell death [45–47]. BzATP is currently the most potent agonist at P2X7 receptors [42, 48], while oxidized ATP and KN-62 are selective antagonists [49, 50]. However, anti-inflammatory effects of oxidized ATP, independent of the expression or activation of P2 receptors, were described [51]. In addition, new antagonists for P2X7 receptor such as brilliant blue G, A740003, calmidazolium, AZ11645373, and ZINC58368839 have improved the pharmacological characterization of this receptor [52, 53]. Alternatively, a nanobody that specifically inhibits the P2X7 receptor was development [54]. This nanobody was 1000 times more potent in preventing IL-1b release than the P2X7 antagonists JNJ47065567 and AZ10606120 [54].

P2Y receptors are a family of G protein-coupled receptors, stimulated by nucleotides such as ATP, ADP, UTP, UDP, and UDP-glucose (Fig. 1). So far, there are eight P2Y receptors found in humans: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 [55]. Members of the P2Y1 receptor subgroup (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) are conjugated with Gαq/11 proteins that activate phospholipase C. P2Y11 also binds to Gαs via protein/adenylate cyclase [56]. Members of the P2Y12 receptor subgroup (P2Y12, P2Y13, P2Y14) are coupled to Gαi/o protein. P2Y1, P2Y12, and P2Y13 are activated by ATP and ADP [57–59], P2Y2 and P2Y4 are activated also by UTP [60, 61], and P2Y6 is activated by UTP [61], but P2Y14 receptor is only activated by UDP-glucose [62].

Presence and participation of P2 receptors in the immune system

Among the initial descriptions of the participation of purinergic receptors in cells of the hematopoietic system, we can highlight the work of Cockcroft & Gomperts in mast cells [63]. In this study, it was observed that ATP produced the release of histamine and the secretion of metabolites [63]. One particular receptor was described in macrophages in the murine J774 lineage, which was initially called the P2Z receptor [64] and is currently known as the P2X7 receptor [42]. Activation of the P2X7 receptor has been implicated in the formation of a cytolytic pore that allows the passage of molecules up to 900 Da [42, 64]. Subsequently, the P2X7 receptor was described in monocytes and macrophages of various origins [47, 65–67]. However, the P2X7 receptor is not the only receptor expressed in macrophages; in contrast, it is common that primary cells express more than one P2 receptor.

In addition to monocytes/macrophages, other myeloid cells also express P2 receptors. ATP and UTP promote an increase in the concentration of cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) in eosinophils, which was inhibited by the pertussis toxin, an inhibitor of G_αi/o protein, and also by U73122, an inhibitor of phospholipase C [68]. Different types of P2X and P2Y receptor agonists promote several effects in eosinophils, such as an increase in [Ca²⁺]_cyt, actin polymerization, reactive oxygen (ROS), and nitrogen (RNS) production increased CD11b expression and chemotaxis [69]. The P2Y2 receptor was shown to be involved in asthmatic airway inflammation in a mouse model caused by mediating ATP-triggered dendrocytes and eosinophils [70].

Macrophages stimulated with ATP were able to kill both bacteria Escherichia coli e Staphylococcus aureus, but macrophages isolated from P2X4^−/− animals were not able to induce Escherichia coli death, whereas no differences were observed in macrophages isolated from P2X7^−/− animals [71]. In addition, infected animals P2X4^−/− have a lower survival rate than their normal counterpart [71]. P2X7^−/− deficient mice were more susceptible to sepsis and the floxed P2X7 conditional knockout mouse model (P2X7^fl/fl-LysM-Cre), without expression of P2X7 receptor in myeloid cells, showed higher levels of cytokines and infection [72].

Studies performed in granulocytes have shown that P2X7 receptor activation generated superoxide anion and secretory granule exocytosis by increasing [Ca²⁺]_cyt and protein kinase C activation [73, 74]. It has also been reported that activation of the P2X7 receptor promotes apoptosis in murine granulocytic cells and that the ability to promote apoptosis is reduced by aging, with a concomitant decrease in P2X7 expression [46]. P2X1 receptor is involved in endotoxemia by its participation in the chemotaxis of neutrophils [75], P2X1^−/− mouse shows reduced inflammatory response with resistance to LPS-induced death [76]. Additionally, the evaluation of P2 receptor expression in murine granulocytes showed the presence of the P2Y1, P2Y2, and P2Y4 receptors, with heteromeric association of the P2Y1/P2Y2 and P2Y1/P2Y4 receptors [77].

Although there are several works on the release of ATP and osmotic regulation in erythrocytes and those related to the presence of P2 receptors and ectoATPases on the plasma membrane of erythrocytes [78–80], there are few works addressing the physiological mode of action regarding the signaling of P2 receptors in this cell type and its progenitor cells. The expression of P2X1, P2X4, P2X7, and P2Y1 receptors but not the P2Y2, P2Y4, or P2Y6 receptors was observed in erythroid precursor cells derived from human CD34⁺ cells [81]. Human erythrocytes were also responsive to ATP and BzATP by activation of the P2X7 receptor [82]. Murine erythroblasts express P2Y1, P2Y4, and P2Y12 receptors, and activation of the P2Y1 receptor in these cells promoted a biphasic increase in [Ca²⁺]_cyt. The initial transient phase was dependent on inositol 1,4,5-trisphosphate, and the sustained phase was dependent on the activation of protein kinase C and Ca²⁺ calmodulin kinase II [83].

Although the presence of P2 receptors in hematopoietic cells has been addressed in previous reviews [44, 84, 85], a complete molecular and pharmacological characterization of P2 receptors in different types of hematopoietic cells is still required.

One of the main functions of differentiated leukocytes (granulocytes, monocytes/macrophages, dendritic cells, and B and T lymphocytes) is their involvement in inflammation and the immune response (Fig. 1). Extracellular nucleotides can act as immunomodulators in the cellular response of these hematopoietic lineages [86–88].

Extracellular ATP may act as an endogenous adjuvant to initiate the inflammatory process by stimulating P2 receptors. The participation of the P2X7 receptor is the most studied in the immune system, since it participates in innate and adaptive immune responses. The activation of the P2X7 receptor by ATP is a signal for the activation of the inflammasome, inducing both maturation and release of proinflammatory cytokines such as IL-1β and IL-18 and the production of ROS and RNS [89, 90]. Moreover, during the adaptive immune response, the P2X7 receptor modulates the balance between the generation of T helper lymphocytes type 17 (T_h17) cells and T regulatory (T_reg) cells [89, 91].

The main function of ROS production during the inflammatory response is the destruction of microorganisms, and neutrophils as well as other leukocytes (lymphocytes and monocytes/macrophages), play a central role in this process [92]. After recruitment, neutrophils migrate to the inflammatory and/or infectious site, and together with macrophages, are able to eliminate the aggressor agent. There are primary and secondary granules inside neutrophils. The primary granule content enzymes, such as myeloperoxidase, are associated with the phagocytic process mainly in neutrophils (rather than macrophages). In the phagocytic phase, activation of the enzyme NADPH oxidase occurs, which together with the influx of ions, induces the myeloperoxidase activity. The activation of myeloperoxidase leads to the production of hypochlorous acid (HClO) that possesses microbicidal activity, which is produced from H₂O₂ and Cl⁻ ions, and may also cause oxidative damage [93].

There is evidence that activation of the neuronal P2X7 receptor leads to the production of ROS and subsequent nociceptive pain in mice [94]. ROS produced by activation of the P2X7 receptor play a critical role in the P2X7 signaling pathway of macrophages and microglia in the central nervous system [94]. It has been shown that pretreatment with a P2X7 receptor antagonist or an antioxidant efficiently protected cells from caspase-dependent cell death triggered by ultraviolet irradiation [95]. These results showed that autocrine signaling by the release of ATP and activation of the P2X7 receptor are required for the stimulation of oxidative stress in monocytes.

It should be noted that numerous intracellular pathways related to inflammation and activation by the P2X7 receptor have been described, including the inflammasome NLRP3, NF-kB, NFAT, GSK3β, and VEGF [96–98].

Inflammasome NLRP3 is an intracellular sensor that detects a broad range of viral infection, bacterial DNA, bacterial toxins, and protozoaries, and by P2X7 activation, resulting in the formation and activation of the NLRP3 inflammasome [99–101]. Activation of Toll-like receptor, tumor necrosis factor receptor, or IL-1 receptor leading to the transcriptional upregulation inflammasome components by NF-kB [99]. Initially, it was described that secreted ATP through pannexin-1 in bone marrow cells triggers activation of the complement cascade activation, initiating the mobilization of HSC from bone marrow to peripheral blood, whereas P2X7^−/− mice were poor mobilizers [102]. Then, the same group showed that HSC mobilization to peripheral blood by the ATP-driven NLRP3 inflammasome leads to the release of IL-1β and IL-18, as well as several danger-associated molecular pattern molecules [103]. In addition, adenosine exhibited an inhibitory function in this process, working as a negative feedback molecule [104]. These reports showed the participation of NLRP3 inflammasome/P2X7 in HSC mobilization.

P2X7 activation was usually associated with cell death. An unexpected role of P2X7 receptor in the immune system is its participation in the maintenance of immune cells. For instance, the P2X7 receptor is required for the establishment, maintenance, and functionality of long-lived central and tissue-resident memory CD8⁺ T cell populations [105]. This capability of P2X7 receptor provides a common currency that both alerts the nervous and immune systems to tissue damage, promoting a durable adaptive immunological memory related to the immune response against reinfection, which is the basis of vaccines [105].

The P2X7 receptor may be involved in the pathophysiology of chronic inflammatory diseases, and this receptor could be a therapeutic target by the use of antagonists [96, 106].

The development of a tumor generated by injection of murine B16 melanoma was compared in a P2X7^−/− deficiency mouse model versus the use of P2X7 antagonist, A740003. P2X7 pharmacological blockade does not replicate the immunosuppressive effects due to genetic ablation, rather it enhances tumor infiltration by CD4⁺ T effector cells and decreases CD39 and CD73 expression, thus reducing immunosuppression in the tumor microenvironment [53].

The P2X7 receptor expressed in macrophages may be implicated in several pathophysiological mechanisms in infections, such as in the spread of human immunodeficiency virus infection since the stimulation of ATP promoted the release of microvesicles loaded with the human immunodeficiency virus [107]. In addition, the P2X7 receptor may be involved in inflammation and coagulation. The stimulation of P2X7 receptors in macrophages increased the expression and release of microvesicles containing tissue factor, producing a strong prothrombotic response [108]. It is important to emphasize that the activation of the P2X7 receptor induces the release of proinflammatory factors, such as IL-6 (fibroblasts, microglia, mast cells, and dendritic cells), and may also release anti-inflammatory factors, such as IL-10 and TGF-β, in macrophages [109–112].

P2 receptors and their involvement in hematopoiesis

Although hematopoietic cells are known to express P2 receptors, their involvement in hematopoiesis has not yet been fully elucidated. To date, the presence of these receptors, even in the most undifferentiated cells, and the variation in their expression with differentiation, indicate their probable participation in hematopoiesis. Endothelial cells and osteoblasts, which can release ATP [5, 6, 8], are cells present in the different bone marrow niches where HSCs are present; ATP and other signaling molecules released by these cells or by another mechanism can regulate the activity of HSCs [113, 114] (Fig. 2).

Fig. 2.

Regulation of HSC by purinergic regulation. The bone marrow is the hematopoietic environment where HSC resides. HSC is mainly regulated by cytokines, but other signaling molecules such as ATP also can modulate hematopoiesis. ATP can be released by lytic and non-lytic mechanisms by cells of the hematopoietic environment, such as endothelial cells and osteoblasts. Recent reports have shown that ATP and analogs promote differentiation and migration of HSC. But the real participation of P2 receptors and whether the purinergic signaling foments or avoids the growth of leukemias, is not clear. The presence of several P2 and P1 receptors and ectonucleotidases, which hydrolysis of ATP/UTP, evince the complexity of purinergic regulation in the hematopoietic microenvironment

Synergism between ATP and cytokines has been shown by increasing the primitive human hematopoietic CD34⁺ population in methylcellulose [115]. This primitive human hematopoietic CD34⁺ subset expresses several P2 receptors, including P2Y1, P2Y2, P2Y12, and P2X1-7 [115, 116]. Furthermore, UTP stimulation increased the migration of HSCs, inhibited the downregulation of the CXC type 4 chemokine receptor and increased the fibronectin adhesion of human CD34⁺, which were observed in vitro [117]. Using an in vivo assay, immunodeficient animals showed that the stimulation of human CD34⁺ cells with UTP increased the homing efficiency [117]. Differentiation by the direct modulation of primitive hematopoietic cells by ATP has also been reported. ATP and its analogs produce a large increase in [Ca²⁺]_cyt, leading to the myeloid differentiation of hematopoietic primitive cells in bone marrow cultures, unlike cytokines, which produce a small [Ca²⁺]_cyt increase [118]. Subsequently, it was shown that the differentiation promoted by ATP mainly affected HSCs and granulocyte-macrophage progenitor populations [119]. Moreover, it has been reported that ATP reduced the number of hematopoietic primitive populations, increased the myeloid population Gr-1⁺Mac-1⁺, decreased Notch-1 receptor expression, and reduced both quiescence and the engraftment ability of HSCs [119]. Another interesting aspect observed was the increase in [Ca²⁺]_cyt by ATP, which was reduced in the presence of cytokines such as interleukin-3 and stem cell factor in HSCs, reducing the differentiation effect of ATP [119].

The P2Y14 receptor was described as a hematopoietic modulator. Under normal conditions, the absence of the receptor in knockout animals (P2Y14^−/−) did not affect the biology of the cells of the hematopoietic system, but in situations such as radiation stress, aging, chemotherapy, and transplantation, the senescence pattern and cell death of HSCs were modified by increasing the levels of ROS production, the expression of p16^INK4a, and the hypophosphorylation of retinoblastoma protein [120].

The participation of the P2X7 receptor in hematopoiesis has also been demonstrated. The P2X7 receptor is expressed weakly in murine HSCs, but overexpression of the P2X7 receptor in this population by viral transfection reduced both the ability to form colonies in vitro (mainly affecting the most primitive colony-forming unit of granulocytes/erythrocytes/macrophages/megakaryocytes), as well as its engraftment potential in vivo [121]. The mobilization of HSCs is important in processes of the immune response when damage to cells or tissues occurs or in response to drugs that mobilize HSCs, such as granulocytic colony-stimulating factor, a physiological cytokine. The mobilization of HSCs was affected by the release of ATP via pannexin-1 and the formation of its metabolite adenosine [102], whereas P2X7^−/− knockout animals showed a low mobilization ability [102].

P2X7 polymorphism induces a function gain of P2X7, as in the presence of SNP Gln460Arg which is co-inherited with Ala348Thr to form the gain-of-function haplotype 4, then the mobilization ability increases [122].

The role of ectonucleotidases such as NTPDase (CD39) and ecto-5′-nucleotidase (CD73) may also be associated with regulation of the undifferentiated state of HSCs. In addition to the ATP-dependent differentiation of murine HSCs, HSCs exhibit higher expression of CD39 and CD73 than do mature hematopoietic cells [119]. Moreover, primitive c-Kit⁺ cells exhibited higher ectonucleotidase activity than did mature hematopoietic cells or CD45⁻ Ter119⁻ populations [119]. A previous report described the presence of a T_reg CD150^high population located in the niche of bone marrow HSCs [123]. A reduction in the T_reg CD150^high population from bone marrow, obtained by the conditional deletion of C-X-C chemokine receptor type 4, increased the number of HSCs [123]. Experiments that used animals with the conditional expression of CD39 on T_reg CD150^high cells showed that extracellular adenosine generated by the activity of the CD39 enzyme protects against oxidative stress and maintains HSCs in a quiescent state [123]. Moreover, T_reg cells modulated the HSC population via activation of the CD73 enzyme [80]. Deletion of the CD73 enzyme increased the number, cell cycle frequency, and ROS production of HSCs [123, 124]. The use of antioxidants inhibited the increase in HSCs in CD73^−/− knockout mice and maintained HSCs in a quiescent state [80]. Thus, the activity of CD39 and CD73 enzymes could prevent HSC differentiation promoted by ATP through its degradation by the production of adenosine, which maintains the quiescent state of HSCs (Fig. 2).

Purinergic receptors as likely therapeutic targets in leukemia

The importance of P2 receptors in cells from the hematopoietic system has been demonstrated by several studies; therefore, the investigation of the role of P2 receptors in related disorders has great relevance. Leukemias as well the other main hematological cancers arise from problems in the maturation and proliferation of primitive hematological cells. The alteration in purinergic regulation can currently be well-evaluated, and the investigation of basic mechanisms and alterations can provide novel diagnoses, prognoses, and treatments.

Initial studies have shown the presence of P2 receptors in leukemic lineages, such as HL-60 (acute promyelocytic leukemia) and CB1 (acute lymphoblastic leukemia) [125, 126]. Variation in P2 receptor expression and their functional responses has been reported during myeloid differentiation in leukemia lineages, particularly in the HL-60 lineage [125, 127, 128].

The expression and function of the P2X7 receptor are particularly evaluated for its unique pharmacological characteristics. Activation of the P2X7 receptor was initially correlated with cell death by its ability to form pores in the membrane [42, 45, 64, 129], but its activation was also related to cellular proliferation [130]. In leukemias, the participation of the P2X7 receptor in proliferation and cell death by apoptosis and necrosis was described. Initially, it was found that the P2X7 receptor was highly expressed in U-937 (histiocytic lymphoma) and KG-1 (acute myeloid leukemia-AML) lineages. In HL-60 and J6-1 (myeloid leukemia) lineages, the P2X7 receptor had moderate expression, while in K562 (chronic myeloid leukemia) and Burkitt lymphoma (Raji and Ramos) lineages, the expression of this receptor was not detected [131]. Another study reported functional expression of the P2X7 receptor in KG-1 cells [132]. In the murine erythroleukemia cell line, the P2X7 receptor was responsible for inducing cell death and the release of microparticles [133].

Some studies have identified alterations in the gene expression of P2 receptors in myeloid leukemias. Cells extracted from patients with AML exhibited higher expression of P2X1, P2X4, P2X5, and P2X7 receptors than those extracted from normal cells, whereas P2X2, P2X3, and P2X6 were not detected [131].

Among the positively regulated receptors in AML, the P2X7 receptor stands out. Studies have shown that the remission rate of AML is higher in patients with lower P2X7 receptor expression after chemotherapy and that patients with relapse have the highest levels of P2X7 expression [131, 134]. The morphological and immunophenotypic variations that differentiate myeloid leukemias are also reflected in the expression of the P2X7 receptor. High levels of the P2X7 receptor were observed in some AML subtypes, such as M4, M5, and M6, and low expression of this receptor was observed in the M2 and M3 subtypes, suggesting that its expression is higher due to the greater number of immature monocytes [131]. Antiproliferative effects were observed in blast cells and in leukemic stem cells of AML patients by stimulation with ATP, but the same was not observed in normal cells, generating expectations for innovative therapies [135, 136].

The effect of ATP release from chemotherapy-treated dying leukemia cells was investigated. The immunosuppressor effect of ATP release by the activation of P2X7 receptors induces the increase of T_reg and dendritic cells [137].

A relationship between P2X7 receptor expression and apoptosis induction has been related in myeloid leukemias. The apoptosis elicited by aminopyridine 4, a proapoptotic agent, was Ca²⁺-dependent and blocked by KN-62, an antagonist of the P2X7 receptor [138]. The same results occur when cells of myeloid leukemia lineages are treated with ATP (enhanced Ca²⁺ influx, alteration of the cell cycle, the activation of intracellular caspases and consequent cellular apoptosis), suggesting a possible therapeutic ability of ATP [139]. However, some authors suggest that this cytotoxic effect mediated by P2X7 receptors may occur independently of the influx of Ca²⁺ [140]. Activation of the P2X7 receptor induced ROS formation in murine erythroleukemia, which may be involved with apoptosis [141].

A comparative study observed that the lineage of acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts promoting autocrine purinergic signaling and uncontrolled proliferation of leukemia cells [142]. The inhibition of P2X1 and P2X7 receptors reduced the decrease in mitochondrial activity and proliferation of leukemia cells [142].

B-cell chronic lymphocytic leukemia (B-CLL) is the most frequent leukemia among adults in the Western World. It is caused by the clonal expansion of B lymphocytes and is characterized by a loss of apoptotic capacity in CD5⁺ leukemic lymphocytes that accumulate in the blood [143, 144]. P2X7 receptor expression was correlated with disease severity in patients with B-CLL [145]. The expression of the P2X7 receptor was higher in patients with the most aggressive form of B-CLL [145].

Single nucleotide polymorphisms were observed in the gene encoding the P2X7 receptor in the KG1 lineage; polymorphisms such as H155Y and A348T were related to gain of function, and T357S and Q460R were related to loss of function [132]. Among the single nucleotide polymorphisms in the P2X7 receptor, 1513A/C is characterized by the substitution of adenine for cytosine at position 1513; this alteration converts glutamic acid to alanine at residue 496 and causes an almost complete loss of function of the P2X7 receptor [132]. Some studies investigated a possible relationship between this polymorphism and B-LLC. It was proposed that the loss of receptor function caused by this mutation could favor antiapoptotic effects and, consequently increase the pathogenesis [144]. The correlation between this polymorphism and the improvement of clinical conditions in patients with B-LLC was already described [146]. However, further studies did not support the role of the P2X7 genotype as a prognostic marker in B-cell CLL [147–150].

B cells from CLL patients have P2X7 receptor and ASC (apoptosis-associated speck-like protein containing a CARD) overexpressed while NLRP3 is down-modulated and promotes growth. On the other hand, NLRP3 overexpression stimulates cell death by apoptosis [151].

Although previous reports use the term apoptosis or necrosis-like to describe the P2X7 receptor-dependent cell death the revision of this term is necessary. The cell death elicited by P2X7 receptor activation is usually associated with membrane permeabilization, loss of cell and mitochondrial potential membrane, caspase activation, and release of interleukins [44–46, 67, 135, 140]. Pyroptosis is a programmed cell death initially described in monocytes/macrophages by caspase 1 activation [152]. Recent findings indicate that pyroptosis can be also driven by several other caspases including caspase 3 and murine caspase 11, human caspase 4 and 5 and can also occur in cell types other than cells from the immune system [153]. Sometimes it is associated with IL-1β and IL18 secretion, promoting the inflammation process, culminating with chromatin condensation as well as cellular swelling of plasma membrane permeabilization [152, 153]. Current studies are associating the cell death elicited by pannexin-1, caspases, and P2X7 with pyroptosis [154–157].

Taken together, these studies related to normal and altered hematopoiesis, illustrate an important role of P2 receptors, and among them, the P2X7 receptor arises as an important target to investigate against leukemias. The involvement of P2X7 receptor for or against tumor progression is still unclear. However, participation and function of other P2 receptors in leukemias and other disorders deserve more attention (Fig. 2).

Therapeutic application of a purinergic antagonist as an antithrombotic

Other hematopoietic populations such as platelets also respond to the ATP- and αβMeATP-induced increase in [Ca²⁺]_cyt by activating the P2X1 receptor [158]. Among the P2Y receptors, the P2Y1 receptor initiates platelet aggregation by increasing [Ca²⁺]_cyt, and the P2Y12 receptor modulates platelet aggregation by inhibiting adenylate cyclase [59, 159, 160]. Both receptors, P2Y1 and P2Y12, are mainly activated by ADP and analogs such as 2-methylthio-ADP, whereas ATP acts as a partial agonist or can even act as a competitive antagonist of P2Y1 and P2Y2 receptors [161–163]; the P2Y1 receptor is mainly coupled to the G_αq protein, while the P2Y12 receptor is coupled to protein G_αi [164]. The knowledge of purinergic signaling has been very promising in the development of antithrombotic drugs in platelet aggregation (Fig. 1).

There are already some commercial drugs that act as inhibitors of purinergic receptors blocking platelet aggregation. The first commercial drug that inhibited a P2 receptor as medicine was ticlopidine, which was discovered in 1972 and commercialized in 1978; used as a selective inhibitor of ADP-dependent platelet aggregation [165]. This investigation contributed to elucidating the role of ADP in arterial thrombosis. Clopidogrel was developed in 1986 and marketed in 1997 [166]. Ticlopinide and clopidogrel reduce the risk of stroke and heart attacks, especially when combined with aspirin [167, 168]. Ticlopinide and clopidogrel are inhibitors of the P2Y12 receptor and act as prodrugs [169]. Owing to the increased safety attributed to treatment with clopidogrel, it has become the most widely used inhibitor of the P2Y12 receptor, both clinically and experimentally [170].

Two new P2Y12 receptor inhibitors were approved for clinical use by the FDA in 2009 and 2011: prasugrel and ticagrelor [171]. They are more potent and have a faster action and a more predictable effect than clopidogrel, representing an advance in the treatment of acute coronary syndrome but with an increased risk of hemorrhage [171]. Prasugrel, similar to clopidogrel, is also a prodrug. Their active metabolites have the same affinity for the P2Y12 receptor, but the metabolism of prasugrel results in the most availability of the active component in vivo [172], in contrast to ticagrelor, which does not require metabolic activation [173].

The choice of drug in patients should be based on individual characteristics. Clopidogrel can be used when prasugrel and ticagrelor were not indicated [174]. However, there may be cases in which none of these oral inhibitors of the P2Y12 receptor can be indicated, since platelets may remain activated by other routes and are not inhibited by these agents [171]. Owing to the limitations of oral application, intravenous P2Y12 receptor antagonist drugs have been developed.

Cangrelor, an inhibitor of the P2Y12 receptor that does not require metabolic activation, was recently approved for use by the FDA [175]. This inhibitor has a similar structure to ATP, and ticagrelor binds directly and reversibly to the P2Y12 receptor [176, 177].

Elinogrel is also an intravenous drug that acts on the P2Y12 receptor but is still under clinical testing [171, 175]. It is also available in oral form and binds reversibly and directly to the P2Y12 receptor [171]. Elinogrel presents better levels of platelet aggregation than does clopidogrel but is associated with increased levels of liver enzymes and dyspnea [178].

Studies using P2Y1-knockout mice, thrombosis models, and P2Y1 receptor antagonists have shown that this receptor can also be an important potential target for novel antithrombotic drugs [159]. Some antagonists of the P2Y1 receptor are under investigation, such as MRS2179 and MRS2500 [171, 179]. In addition, the synergistic effect of the use of P2Y1 receptor antagonists in the presence of clopidogrel has been demonstrated in the prevention of thromboembolism [180].

Antagonists of P2 receptors are being tested in pathologies not related to platelet aggregation. Suramin, the oldest P2 antagonists, was synthesized in 1916 to treat protozoan infections. Its mechanism of action is related to the nonspecific and reversible inhibition of P2 receptors [181, 182]. Diquafosol acts on the P2Y2 receptors as an agonist, increasing the transport of liquid via chloride channels [183] and facilitating the secretion of mucin by conjunctival epithelial cells [184].

As ATP is degraded to adenosine by ectonucleotidases, it was observed that adenosine exerts regulatory effects by activating P1 receptors; therefore, ectonucleotidases have also become a target of research. Preclinical studies have shown that it is possible to reduce tumor growth by inhibiting CD73 [185]. Phase I clinical studies have been conducted to inhibit this ectoenzyme through monoclonal antibodies (nivolumab and durvalumab) and small molecules as substituted derivatives of benzothiadiazine, osimertinib, and CPI-444 [186].

Conclusions

The presence of these P2 receptors, even in the most undifferentiated cells of the hematopoietic system, and the variation in their expression in the differentiation process, suggests the participation of P2 receptors in several aspects of hematopoiesis. Important reports in this area have shown the participation of P2 receptors in the modulation of normal and leukemic HSCs. This complex system is controlled by P2 receptors with the participation of ectonucleotidases and the activation of P1 receptors by adenosine. These studies demonstrate complex purinergic regulation that can be used for clinical purposes. The perspective of developing new drugs for hematological and inflammatory diseases by the modulation of purinergic receptors is very promising. Many drugs that are under preclinical testing are also very promising and expected to revolutionize the treatment of these diseases, thereby improving the lives of patients.

AcknowledgmentsThe authors would like to thank Professor Danilo Wilhelm Filho and Professor Ed Yates for constructive criticism of the manuscript.

Funding information

This publication and the previous related results were supported by “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP. Proc. 2018/23870-4) and “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq 425965/2018-0).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.