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Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets

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Abstract

Extracellular nucleotide P2-receptor-mediated effects on platelets, leukocytes and endothelium are modulated by ecto-nucleotidases. These ecto-enzymes hydrolyze extracellular nucleotides to the respective nucleosides. The dominant ecto-nucleotidase expressed by the endothelium, by monocytes and vascular smooth muscle cells is CD39/NTPDase1. Ecto-nucleotidase biochemical activity of CD39 is lost at sites of acute vascular injury, such as in ischemia reperfusion and immune graft rejection. CD39L(Like)1/NTPDase2, a related protein, is associated with the basolateral surface of endothelium, the adventitia of vessels and microvascular pericytes. CD39/NTPDase1 hydrolyzes both tri- and diphosphonucleosides and blocks platelet aggregation responses to ADP. In contrast, CD39L1/NTPDase2, a preferential nucleoside triphosphatase, activates platelets by preferentially converting ATP to ADP, the major agonist of platelet P2 receptors. Spatial and temporal expression of NTPDases in the vasculature appears to control platelet activation, thrombus size and stability by regulating phosphohydrolytic activity and consequent P2 receptor signaling. Constitutively circulating microparticles appear to be associated with functional NTPDases, and accumulation of these at sites of vascular injury might influence local thrombus formation and evolution. The phenotype of the cd39-null mouse is in keeping with disordered thromboregulation with heightened susceptibility to inflammatory vasculary reactions, increased permeability and high levels of tissue fibrin. Paradoxically, these mutant mice also exhibit a bleeding phenotype with differential platelet P2Y1 desensitization. Over-expression of CD39 at sites of vascular injury and inflammation by adenoviral vectors, by transgenesis or by the use of pharmacological modalities with soluble derivatives has been shown to have major potential in several animal models tested to date. Future clinical applications will involve the development of new therapeutic strategies to various inflammatory vascular diseases and in transplantation.

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... Interestingly, certain portions of cellular AK and/or NDPK was shown to be released from the platelets and red blood cells [131], airway epithelial cells [114], and breast cancer cells [139], whereas the potential sources for secreted CD39 and CD73 might include nerve terminals [140], pancreatic acini [137], vascular endothelium [54,141], and other cell types. Alternatively, CD39 and CD73 can also be incorporated into exosomes (a subclass of membrane-derived extracellular vesicles secreted into the extracellular milieu by most cell types), where they impact endothelial activation and inhibit angiogenesis [142,143], mediate immunosuppressive effects in the tumor microenvironment [8,144], modulate vascular thrombosis by preventing platelet aggregations [145,146], and control pancreatic secretion [147], and gastrointestinal and hepatic functions [77]. Further studies are required to understand the relevance of soluble and exosome-wrapped purinergic activities contributing, in conjunction with the larger framework of membrane-bound ectoenzymes, to the spatial propagation or termination of purinergic signaling responses in the vascular system. ...
... In contrast to the enhanced nucleotide phosphohydrolysis following the exposure of endothelial cells to short-term hypoxia [40,72,148], CD39 and CD73 activities may be down-regulated or even lost on vascular and microvascular endothelium and other cell types at sites of vascular injury and atherogenesis [145,157], during chronic oxygen deprivation [115], organ transplantation [152], and also arterial [185] and pulmonary [163] hypertension. Significant down-regulation of CD39 activity has also been demonstrated in aortas from apolipoprotein E-deficient (ApoE -/-) mice prone to atherosclerosis [157,184], and also in blood vessels from abdominal aortas of patients with atherosclerosis and atherosclerosis-like diseases [186]. ...
... In turn, rapid inactivation of ATP and other circulating nucleotides via transiently up-regulated serum ectonucleotidase activities may serve as another important auxiliary effector system counteracting acute pro-thrombotic state during vigorous exercise in humans [136], and contributing to the maintenance of adenosinergic signaling pathway in human and mouse blood during adaptation to high altitude and hypoxia [129]. It is pertinent to note that soluble NTPDase (apyrase) is considered a promising drug, which prevents sustained ADP-induced activation of the major platelet adhesion receptor GPIIb/IIIa and inhibits thrombus formation during sepsis, vascular injury, ischemia-reperfusion and other pro-thrombotic conditions [145,191,192]. Other strategies to offset the reduced effectiveness of endothelial nucleotidases and prevent excessive vascular thrombosis include CD39 expression in the injured vessels [193,194], up-regulation of endogenous CD39 through the use of phosphodiesterase3-inhibiting drugs [195], and targeting of soluble CD39 (targ-CD39) recombinantly fused to activated platelet GPIIb/IIIa-specific single-chain antibody [164,196]. ...
Article
The concept of extracellular purinergic signaling was first proposed by Geoffrey Burnstock in the early 1970s. Since then, extracellular ATP and its metabolites ADP and adenosine have attracted an enormous amount of attention in terms of their involvement in a wide range of immunomodulatory, thromboregulatory, angiogenic, vasoactive and other pathophysiological activities in different organs and tissues, including the vascular system. In addition to significant progress in understanding the properties of nucleotide- and adenosine-selective receptors, recent studies have begun to uncover the complexity of regulatory mechanisms governing the duration and magnitude of the purinergic signaling cascade. This knowledge has led to the development of new paradigms in understanding the entire purinome by taking into account the multitude of signaling and metabolic pathways involved in biological effects of ATP and adenosine and compartmentalization of the adenosine system. Along with the “canonical route” of ATP breakdown to adenosine via sequential ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) and ecto-5’-nucleotidase/CD73 activities, it has now become clear that purine metabolism is the result of concerted effort between ATP release, its metabolism through redundant nucleotide-inactivating and counteracting ATP-regenerating ectoenzymatic pathways, as well as cellular nucleoside uptake and phosphorylation of adenosine to ATP through complex phosphotransfer reactions. In this review I provide an overview of key enzymes involved in adenosine metabolic network, with special emphasis on the emerging roles of purine-converting ectoenzymes as novel targets for cancer and vascular therapies. To whom correspondence should be addressed: Gennady G. Yegutkin: Medicity Research Laboratory, University of Turku, Tykistökatu 6A, 20520, Finland. Email: [email protected]; Tel: +358458775353
... Physical exercise is a well-known trigger for short and long-term molecular and tissue adaptations, and is a non-pharmacological tool for the prevention and treatment of many diseases (Heinonen et al., 2014;Pedersen et al., 2015). In turn, purinergic signalling is an elegant system of extracellular communication, which has been poorly explored in exercise science, in which nucleotides and nucleosides modulate immune, thrombotic, vascular and cardiac responses (Atkinson et al., 2006;Burnstock & Ralevic, 2014;Faas et al., 2017). ...
... NTPDase1 and 5′-NT are key regulators of immunological and coagulation processes (Atkinson et al., 2006;Faas et al., 2017). Over the last few years, it has been suggested that these enzymes have a protective role in cardiac and vascular tissues due, at least in part, to the control of extracellular levels of ATP, ADP and ADO (Eckle et al., 2007;Marcus et al., 2005;Schetinger et al., 2007). ...
... post-and 1 h post-exercise(Fragala Maren et al., 2017;Rudberg et al., 2000;Yegutkin, 2014). Furthermore, E-NTPDase1/CD39, E-NTPDase2/CD39L1 and E-5′-NT/CD73 are highly expressed at the membranes of platelets and endothelial cells, and participate in the metabolism of nucleotides in the bloodstream(Atkinson et al., 2006;Heber & Volf, 2015;Kanthi et al., 2014). The magnitude and duration of purinergic signalling includes ATP-generating and ATP-consuming or inactivation pathways, in addition to ADO-generating, ADOconsuming or inactivation and coexisting reuptake(Pastor-Anglada & Pérez-Torras, 2018;Yegutkin, 2008;Yegutkin et al., 2002). ...
Article
New finding: What is the central question of this study? How does moderate-intensity aerobic exercise affect the behavior of purinergic enzymes in sedentary, overweight and physically active subjects? What is the relationship between purinergic and inflammatory responses triggered by exercise? What is the main finding and its importance? Moderate-intensity aerobic exercise modifies the activity of purinergic enzymes and the levels of nucleotides and nucleosides. These results are similar in subjects with different biological characteristics. 5'-nucleotidase activity and adenosine levels are associated with inflammatory responses. This study suggests that a purinergic pathway is related to the inflammatory responses triggered by exercise. Abstract: Purinergic signaling is a mechanism of extracellular communication that modulate events related to exercise, such as inflammation and coagulation. Herein, we evaluated the effects of acute moderate-intensity exercise on the activities of purinergic enzymes and plasma levels of adenine nucleotides in individuals with distinct metabolic characteristics. We analyzed the relationship between purinergic parameters, inflammatory responses and cardiometabolic markers. Twenty-four healthy males were assigned to three groups: normal weight sedentary (n = 8), overweight sedentary (n = 8) and normal weight physically active (n = 8). The volunteers performed an acute session of moderate-intensity aerobic exercise on a treadmill at 70% of V̇O2peak ; blood samples were drawn at baseline, immediately post-exercise and at 1h post-exercise. Immediately post-exercise, all subjects showed increases in ATP, ADP, AMP and p-Nph-5'-TMP hydrolysis, while AMP hydrolysis remained increased at 1h after exercise. High-performance liquid chromatography analysis demonstrated lower levels of ATP and ADP at post- and 1h post-exercise in all groups. Conversely, adenosine and inosine levels increased at post-exercise, but only adenosine remained augmented at 1h after exercise in all groups. With regard to inflammatory responses, the exercise protocol increased TNF-α and IL-8 concentrations in all subjects, but only TNF-α remained elevated at 1h after exercise. Significant correlations were found between the activity of 5'-NT, adenosine levels, V̇O2peak , triglyceride, TNF-α and IL-8 levels. Our findings suggest a purinergic signaling pathway that participates, at least partially, in the inflammatory responses triggered by acute moderate-intensity exercise. The response of soluble nucleotidases to acute moderate exercise appears to be similar between subjects of different biological profiles. This article is protected by copyright. All rights reserved.
... In lymphocytes, ATP promotes activation followed by pro-inflammatory effects and adenosine is considered a potent anti-inflammatory molecule. On platelets, ATP and ADP are pro-aggregating molecules while adenosine inhibits platelet aggregation [7][8][9]. ...
... Platelets Lymphocytes Since NTPDase (ADP) activity is augmented, it means that more ADP is being hydrolyzed [9] and it could explain one of the training mechanisms on tromboregulation. Since less concentration of ADP is in the microenvironment and ADP is the most important molecule that pursues pro-aggregant action, the platelet aggregation occurs in low intensity [9]. ...
... Platelets Lymphocytes Since NTPDase (ADP) activity is augmented, it means that more ADP is being hydrolyzed [9] and it could explain one of the training mechanisms on tromboregulation. Since less concentration of ADP is in the microenvironment and ADP is the most important molecule that pursues pro-aggregant action, the platelet aggregation occurs in low intensity [9]. This training effect could be understood as one of the main protector actions of chronic exercise on cardiovascular health. ...
Article
The aim of this letter is to summarize the impact of ectonucleotidases activities on platelets and lymphocytes in a model of moderate physical exercise: rats submitted to swimming training.
... Extracellular adenine nucleotides-such as adenosine triphosphate (ATP), ADP, adenosine monophosphate (AMP), and their metabolite (adenosine)-can modulate multiple effects on the vascular system by interacting with specific purinergic receptors in platelets and endothelial cells [14]. Platelets express a multienzymatic complex on their surfaces that permit extracellular nucleotide hydrolysis. ...
... It plays an important role in controlling blood flow and the regulation of ADP [17]. High concentrations of ATP have also been shown in vitro to inhibit the process of platelet induction by ADP; ATP is considered a competitive antagonist of ADP by the P2Y platelet receptor [14]. When secreted into the extracellular medium of platelets, ATP is capable of mediating platelet reactivity [18]. ...
Article
Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p < 0.05) increases in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolyses, and in the deamination of adenosine in the CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p < 0.05) in the CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events.
... Entretanto, pouco é conhecido acerca da função das endo-apirases sendo sugerido envolvimento na glicosilação de proteínas (Gao et al. 1999;Uccelletti et al. 2007), e virulência do patógeno Legionella pneumophila (Sansom et al. 2007 (Maliszewski et al. 1994) com atividade enzimática (Wang e Guidotti 1996); é caracterizada como uma proteína tetramérica não covalente (Wang et al. 1998) que hidrolisa os dois substratos ATP e ADP com igual eficiência, enquanto a enzima ATPase somente hidrolisa o ATP (Atkinson et al. 2006). ...
... Estas características descritas podem indicar que as NTPDases podem interferir com a sinalização por receptores P2, o que se somaria à característica principal deste tipo de enzima, que seria a inibição da agregação plaquetária devido à atividade ADPásica que ambas as SmATPDases apresentam (Atkinson et al. 2006). ...
Thesis
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ATPDases or ATP-diphosphohydrolases are enzymes that cleave ATP and ADP to AMP and Pi and are involved in inhibition of platelet aggregation. In the parasite Schistosoma mansoni our group had identified and cloned the ATPDase1 gene and localized the protein on the tegument surface. Recently, we cloned the ATPDase2 gene using S. mansoni EST databank information and we immunolocalized it also in the tegument. ATPDase 2 was found on both the apical and basal tegument membranes together with ATPDase1, but only ATPDse2 was found in the syncytium space of the tegument. The presence of both enzymes on the tegumental outer surface suggests a major role in regulation of nucleotides abundance. Expression analysis of both genes was performed by Real Time RT- PCR using RNA from eggs, miracidia, cercariae, schistosomula and adult worms. The results showed that ATPDase1 gene was more expressed in eggs (7-fold), adults (6-fold), cercariae (3.5-fold) and schistosomula (1.5-fold) when compared to miracidia, which was taken as the reference. ATPDase2 gene was more expressed in eggs (16-fold), cercariae (11-fold), miracidia (7-fold) and adult worms (2-fold) when compared to schistosomula, showing that both genes are modulated along the life cycle stages. For further characterization of these enzymes, they were expressed heterologously in the yeast Pichia pastoris as fusion proteins with hexa-histidine tags and the recombinant proteins were purified by Ni-NTA affinity chromatography. The recombinant ATPDases were obtained in active form and activity measurements were performed. ATPDase1 did show ATPase and ADPase activities about 650 and 160 nmoles Pi.min-1.mg-1 , respectively. ATPDase2 had ATPase and ADPase activities in the range of 1050 and 250 nmoles Pi.min-1.mg-1 , respectively. These results were obtained in the presence of calcium as cofactor. Additionally, UTPase and UDPase activities were found for both enzymes. Circular dichroism studies with these enzymes elucidated their secondary structures; ATPDase1 (S66 to Q507 ) has alpha helix (7%), beta sheet (45%) and random coil (48%), whereas ATPDase2 (N83 a K564 ) showed alpha helix (14%), beta sheet (33%) and random coil (53%). We found that ATPDase2 was secreted by the parasite to the medium, similar to what has been described for human CD39-L2 and CD39-L4 ATP-diphosphohydrolases. Additionally, a penetration assay (cercaria to mice) using antibody anti-ATPDase1 did show a decrease of 20% in the penetration capacity through mice skin of cercaria previously incubated with this antiserum. Because ATPDase2 gene expression was increased in miracidia and cercariae, the stages that infect snail and human, respectively, we postulate that ATPDase2 may help the parasite's invasion process. In the egg stage both genes were highly expressed suggesting a possible involvement of the ATPDases in the protection response of eggs against the human immune system. Assays of protection against S. mansoni in mice, using recombinant ATPDase1 and 2 as antigens, resulted in low protection obtaining no more than 20% of parasite burden reduction.
... 19) Die Anwesenheit von CD39 im Versuchsaufbau begründete sich also mindestens auf den Thrombozyten. Weiterhin könnte CD39 beispielsweise durch Thrombozyten auf MSC induziert werden und es besteht die Annahme, dass auch lösliches CD39 aus dem Serum einen Einfluss auf den extrazellulären Purinmetabolismus besitzt (Atkinson et al., 2006;Yegutkin et al., 2012). Da durch die Hemmung von CD39 der ATP und ADP Abbau zu AMP blockiert werden sollte, wäresollte dieser Mechanismus mitverantwortlich sein -eine Aufhebung der ausgelösten Inhibition aller Zellarten zu erwarten gewesen. ...
... 1.3.5), von einem CD39 Verlust berichtet (Atkinson et al., 2006;Robson et al., 2005). Dies könnte ein wichtiger Kofaktor für die Sicherheit der MSC Applikation sein. ...
Thesis
Wegen ihrer vielfältigen Eigenschaften ist die therapeutische Anwendung mesenchymaler Stromazellen (MSC) von großem wissenschaftlichem und klinischem Interesse. In den bisherigen klinischen Studien erwies sich die Applikation als sicher und weitgehend nebenwirkungsfrei. Es gibt jedoch einige Berichte über thromboembolische Ereignisse nach der Infusion von MSC. In vitro Studien untersuchten in diesem Zusammenhang bisher hauptsächlich die plasmatische Gerinnung und zeigten sowohl pro- als auch anti-koagulatorische Einflüsse der MSC. Um die Sicherheit der MSC Anwendung weiterhin zu untersuchen, wurde in dieser Arbeit der Einfluss von MSC auf die zelluläre Gerinnung untersucht. Dazu wurde die Thrombozytenaktivierung in Anwesenheit von MSC impedanzaggregometrisch und durchflusszytometrisch quantifiziert. Es wurden MSC aus Lipoaspirat, Nabelschnurblut und Knochenmark eingesetzt. Die verschiedenen MSC wurden außerdem mit menschlichen Endothelzellen der Nabelschnurvene (HUVEC) und Zellen der Tumorlinie HeLa als thrombozyteninhibierende, bzw. -aktivierende Zellpopulation verglichen. In der Durchflusszytometrie führten HeLa Zellen wie erwartet zu einer Aktivierung der Thrombozyten. Interessanterweise führten Nabelschnurblut MSC zu einem ähnlichen Ergebnis. Im Gegensatz dazu reduzierten MSC aus Knochenmark und Lipoaspirat sowie HUVEC die induzierte Thrombozytenstimulation unabhängig von den eingesetzten Agonisten. Die Impedanzaggregometrie konnte die beschriebenen Ergebnisse nicht nachweisen. Durchflusszytometrisch wurden weiterhin cyclooxygenase- und CD62P abhängige Interaktionen von MSC und Thrombozyten als mögliche Wirkmechanismen untersucht. Hier zeigte sich kein Zusammenhang mit den beobachteten Effekten. Durch die Blockade der Ectonucleotidasen CD39 und CD73 konnte dagegen gezeigt werden, dass durch CD73 konvertiertes Adenosin für die Thrombozyteninhibition durch MSC verantwortlich ist. Die Daten belegen, dass die durch MSC aus Lipoaspirat und Knochenmark verursachte Hemmung der Thrombozytenaktivierung durch Adenosin, welches durch MSC exprimiertes CD73 gebildet wird, vermittelt wird. Dadurch werden die klinischen Daten, die der MSC Applikation eine hohe Sicherheit bescheinigen, weiter unterstützt. Interessanterweise scheinen sich MSC aus Nabelschnurblut in diesem Aspekt von MSC anderer Quellen zu unterscheiden. Der extrazelluläre Adenosinstoffwechsel gewinnt durch diese Ergebnisse weiter an Relevanz für die Wirkmechanismen von MSC.
... E-mail: sudakk@tu.ac.th activated B cells, natural-killer cells, macrophages, dendritic cells, neurons, glial cells and astrocytes. CD39 has roles on inflammatory process and regulation of platelet activation [10][11][12]. Consequently, CD39 has been proposed as a potential target for treatment of patients with complications caused by platelet activation [11][12][13]. CD39 is an ecto-enzyme expressed on VECs and is an important player in the regulation of inflammation and platelet activation, associated with DM complications [14,15]. ...
... CD39 has roles on inflammatory process and regulation of platelet activation [10][11][12]. Consequently, CD39 has been proposed as a potential target for treatment of patients with complications caused by platelet activation [11][12][13]. CD39 is an ecto-enzyme expressed on VECs and is an important player in the regulation of inflammation and platelet activation, associated with DM complications [14,15]. However, it has not been reported if hyperglycemia has effects of CD39 in VECs. ...
Article
Full-text available
Background: Diabetes mellitus (DM) patients lose their ability to control normal blood glucose levels, resulting in high blood glucose levels (hyperglycemia). Hyperglycemia causes DM complications. This involves responses of vascular endothelial cells (VECs) to hyperglycemia, affecting inflammatory process and platelet activity. Ecto-enzyme CD39 is expressed on VECs, catalyzing the hydrolysis of ATP and ADP to AMP and, consequently, regulating inflammatory process and platelet activation. Objective: We studied whether high glucose concentration has an effect on CD39 expression on VECs. Methods: Cultured human umbilical vein endothelial cells (HUVEC) were used as a model of study. HUVEC were cultured in different glucose conditions (4, 9, 24, and 34 mM) for 24 hours. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay and expression of CD39 was examined by using SDS-PAGE and western blot techniques. Results: HUVEC were cultured in normal (4 and 9 mM) or high (24 and 34 mM) glucose concentrations for short term (24 hours). The results showed that high glucose (24 and 34 mM) reduced cell viability to 89.5 ± 11.3 and 86.3 ± 13.5 (mean ± SD), compared with control (4 mM), respectively. High glucose also induced increases in CD39 expression in HUVEC. Conclusion: High glucose decreases cell viability and increases CD39 expression in HUVEC, suggesting involvement of CD39 in cell responses to high glucose.
... Also, other authors reported that ATP and activity of valvular ectonucleotidases engaged in its metabolism could play a substantial role in the development and progression of CAVD [19]. Moreover, extracellular ATP exerts pro-inflammatory and pro-aggregatory actions that promote pathological processes within valves and vessel wall [20,21]. In addition to ATP (which acts through P2 receptors), its breakdown product-adenosine, also displays many regulatory functions via P1 receptors, widely distributed in the cardiovascular system. ...
Article
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Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.
... Koziak et al. (1999) [35] reported that the attenuation of E-NTPDase/CD39 expression in platelets and endothelial cells of healthy individuals. However, the modulation of E-NTPDase/ CD39 expression was associated with platelet activation, thrombus formation and may be possible chemotherapeutic targets [36]. Reduced degradation of ADP by ectonucleotidases contributes to the amplification of ADP evoked aggregation [37]. ...
Article
Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5′-nucleotidase (E-5′-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P < 0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P < 0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients.
... CD39 (ATP-diphosphohydrolase) is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73 (ecto-5'-nucleotidase), it contributes to extracellular adenosine accumulation [3]. CD39 is considered a key modulator of thrombus formation, since it may inhibit platelet aggregation promoting ADP hydrolysis and, in combination with CD73, promotes adenosine accumulation that is a vasodilator and a platelet antiaggregating agent [4][5][6][7]. Loss of CD39 activity from the endothelium sustains platelet aggregation and thrombogenesis [8][9][10][11][12][13]. There is evidence that moderate red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats [14]; furthermore, flavonoids, which are major wine constituents and well known vasoprotective agents, also increase ATP -and ADPase activity in rat serum [15]. ...
Article
Full-text available
Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP - and ADPase activity in rat platelets.
... El sistema de señalización purinérgica cumple importantes funciones: regulador en la inflamación, la activación celular, el flujo sanguíneo y la trombosis vascular por biomoléculas extracelulares, tales como nucleótidos de adenina (ATP, ADP y AMP) y de adenosina (37). Como una consecuencia de la infección, los eritrocitos, leucocitos y las plaquetas secretan altas concentraciones de adenosina al medio extracelular (38). Estos nucleótidos ejercen sus funciones a través de tres purinoceptores P2 en plaquetas: P2X1 (receptor ionotrópico que provoca una rápida entrada de calcio hacia el citosol), P2Y1 (receptor metabotrópico que moviliza el calcio desde las cisternas internas) y P2Y12 (receptor acoplado a la proteína Gαi que estabiliza la agregación plaquetaria) (39)(40)(41). ...
Article
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La adenosin deaminasa representa un punto de control en la regulación de los niveles extracelulares de adenosina, desempeñando así un papel fundamental en la modulación de las respuestas purinérgicas a ciertos eventos patofisiológicos. Diversos estudios señalan que los niveles séricos y plasmáticos de la enzima se elevan en algunas enfermedades causadas por microorganismos, lo cual podría representar un mecanismo compensatorio como consecuencia de la elevación de las concentraciones de adenosina y la liberación de mediadores inflamatorios. Recientes investigaciones indican que la actividad de la adenosin deaminasa disminuye e influye en los parámetros hematológicos de animales infectados con Trypanosoma evansi, de manera que tales alteraciones podrían tener implicaciones en la patogénesis de la enfermedad. Adicionalmente, la enzima ha sido detectada en este parásito; lo que permite inferir que podría estar asociada a las funciones vitales del mismo, de manera similar a lo que ocurre en los mamíferos. Este conocimiento puede ser útil al asociar la quimioterapia con inhibidores específicos de la enzima en futuros estudios.
... Further, the NTPDase bound PET surface shows a significantly greater reduction in platelet adhesion compared to both PET and PET- [COOH]. This might be due to the inhibition of platelet adhesion by blocking ADP-dependent platelet activation [24]. In the host system, the AMP produced may also be hydrolyzed to adenosine by ecto-5'-nucleotidase, which can potentially inhibit platelet activation [25,26]. ...
Article
Poor haemocompatibility of material surfaces is a serious limitation that can lead to failure of blood-contact devices and implants. In this work, we have improved the haemocompatibility of polyethylene terephthalate (PET) surfaces by immobilizing apyrase/ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) on to the carboxylated PET. NTPDase immobilized PET surfaces scavenge the ADP released by activated platelets, which prevents further platelet activation and aggregation. The surface properties of the modified PET were characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDAX), and contact angle measurement. The enzyme attachment and stability on the modified PET surfaces were evaluated. The kinetics of free enzyme and immobilized enzyme were studied and fitted using the Michaelis-Menten kinetic model. Both free and immobilized NTPDase followed Michaelis-Menten kinetics with similar Michaelis-Menten constants (Km). This suggests that the activity of NTPDase was unchanged upon immobilization. Protein adsorption and %hemolysis was significantly reduced for carboxylated PET and NTPDase immobilized PET surfaces compared to unmodified PET. Lactate dehydrogenase assay showed that the number of adhered platelets reduced by more than an order of magnitude for the NTPDase immobilized PET surface compared to unmodified PET. These results clearly indicate that NTPDase immobilization significantly enhances the haemocompatibility of PET surfaces.
... Thus, using 2 human groups with a clotting phenotype, we elucidated an important genetic regulatory mechanism of NTPDase1/CD39 function that provides evidence for interindividual differences in activity of this enzyme. Such differences are important to the purinergic regulation of clotting and potentially relevant for the regulation of inflammation and regulatory T cell function (2,28,29). We suggest that the term "CD39 Denver" be used to more succinctly denote the gain-of-function phenotype of NTPDase1/CD39 due to the rs3814159 G allele and to recognize the region where this mechanism was described. ...
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Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) degrades the purines ATP and ADP that are key regulators of inflammation and clotting. We hypothesized that NTPDase1 polymorphisms exist and that they regulate this pathway. We sequenced the ENTPD1 gene (encoding NTPDase1) in 216 subjects then assessed genotypes in 2 cohorts comprising 2213 humans to identify ENTPD1 polymorphisms associated with venous thromboembolism (VTE). The G allele of the intron 1 polymorphism rs3176891 was more common in VTE vs. controls (odds ratio 1.26-1.9); it did not affect RNA splicing, but it was in strong linkage disequilibrium with the G allele of the promoter polymorphism rs3814159, which increased transcriptional activity by 8-fold. Oligonucleotides containing the G allele of this promoter region bound nuclear extracts more avidly. Carriers of rs3176891 G had endothelial cells with increased NTPDase1 activity and protein expression, and had platelets with enhanced aggregation. Thus, the G allele of rs3176891 marks a haplotype associated with increased clotting and platelet aggregation attributable to a promoter variant associated with increased transcription, expression, and activity of NTPDase1. We term this gain-of-function phenotype observed with rs3814159 G "CD39 Denver."-Maloney, J. P., Branchford, B. R., Brodsky, G. L., Cosmic, M. S., Calabrese, D. W., Aquilante, C. L., Maloney, K. W., Gonzalez, J. R., Zhang, W., Moreau, K. L., Wiggins, K. L., Smith, N. L., Broeckel, U., Di Paola, J. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.
... E5NT and ENTPD1 are ectonucleotidases that regulate the extracellular concentration and signaling of adenine nucleotides and adenosine [3]. Several reports showed the anti-thrombotic and anti-inflammatory effects of ENTPD1, mainly by catabolizing ATP and ADP [39,40]. On the other hand, E5NT has been demonstrated to exert anti-inflammatory and cytoprotective action through its reaction product, adenosine [39]. ...
Article
Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5'-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig-to-non-human primates xenotransplantation models.
... The dense granules store calcium, serotonin, histamine and nucleotides, some of which are involved in purinergic signalling, such as adenosine diphosphate and triphosphate [1, 2]. Once released, these nucleotides are regulated by the ectonucleoside triphosphate diphosphohydrolase-1 CD39 [56] and impact liver regeneration through P2Y 2 R purinergic signalling on LSEC [57, 58] as well as platelet aggregation and efficacy [59, 60] . Platelets also have the capacity to release surfacederived microvesicles and lysosomes containing a wide variety of proteins, which are thought to contribute to the clearance of thrombi [1, 61]. ...
Article
Platelets are involved in the early phases of liver regeneration. Moreover, platelet transfusion and thrombocytosis were recently shown to enhance hepatocyte proliferation. However, the precise mechanisms remain elusive. This review discusses the latest updates regarding the mechanisms by which platelets stimulate liver regeneration, focusing on their interactions with liver sinusoidal endothelial cells and on their fate within the liver. Following liver injury, platelets are recruited to and trapped within the liver, where they adhere to the endothelium. Subsequent platelet activation results in the release of platelet granules, which stimulate hepatocyte proliferation through activation of the Akt and ERK1/2 signalling pathways. Platelets activate liver sinusoidal endothelial cells, leading to the secretion of growth factors, such as interleukin-6. Finally, liver sinusoidal cells and hepatocytes can also internalize platelets, but the effects of this alternate process on liver regeneration remain to be explored. A better understanding of the mechanisms by which platelets stimulate liver regeneration could lead to improvement in post-operative organ function and allow hepatectomies of a greater extent to be performed. Copyright © 2015. Published by Elsevier B.V.
... The catalytic activity of NTPDases is divalent cation dependent, typically upon Ca 2+ or Mg 2+ ions, NTPDases are inactive in the absence of these cations. NTPDases are of further eight types NTPDase 1 (CD39), NTPDase 2, NTPDase 3, NTPDase 4, NTPDase 5, NTPDase 6, NTPDase 7, and NTPDase 8. Out of these, only four NTPDases, that is NTPDase 1, NTPDase 2, NTPDase 3, and NTPDase 8 are anchored to the cell-surface via transmembrane domains with extracellular active sites, the remaining are present intracellularly [20,21]. To date, 13 crystal structures of rat (Rattus norvegicus) NTPDases are available from the PDB, but so far no crystal structure of human NTPDase is available. ...
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Introduction: Ectonucleotidases are a broad family of metallo-ectoenzymes that are responsible for hydrolysing a variety of nucleotides to nucleosides, hence orchestrating the activation of P1 and P2 cell receptors via controlled release of nucleotides and nucleosides. Many disorders such as impaired calcification including aortic calcification, neurological and immunological disorders, platelet aggregation, cell proliferation and metastasis. are characterized by an increase in expression of these ectonucleotidases. Consequently, selective inhibitors of ectonucleotidases are required for therapeutic intervention. Area covered: Several classes of compounds such as purine, nucleotide derivatives (e.g., ARL67156) and monoclonal antibodies, have shown promising ectonucleotidase inhibitory potential. This review discusses chemistry and therapeutic applications of ectonucleotidase inhibitors patented from 2011 to 2016. Expert opinion: All eukaryotic cells express nucleotide and nucleoside receptors on their cell surface and are capable of releasing extracellular nucleotides. Ectonucleotidases are a broad family of metallo-ectoenzymes that hydrolyze a variety of nucleotides to nucleosides. These extracellular nucleotides and nucleosides are important cell signalling molecules and mediate a variety of (patho)physiological processes by acting upon their respective P1 and/or P2 receptors. Discovery of molecules that can selectively inhibit or activate ectonucleotidases is crucial from therapeutic point of view, since it allows human intervention into purinergic cell signalling, thereby allowing us to modulate related (patho)physiological processes as desired.
... Under physiological conditions, the endothelium exhibits an antithrombotic surface by separating blood from the subendothelial matrix and by secreting several anti-thrombotic mediators, including nitric oxide [1,2]. Moreover, endothelial cells express thrombomodulin, which inhibits platelet stimulation by thrombin and the ecto-ATPase CD39 [3]. ...
Article
Introduction: Thrombus formation is a complex process, which is characterized by the dynamic interaction of platelets, leukocytes and endothelial cells. The activation of these cells is strictly mediated by different phospho-regulated signaling pathways. Recently, it has been reported that inhibition of protein kinase CK2 affects platelet function by suppressing phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) signaling. Based on this finding, we herein analyzed whether CK2 acts as a crucial regulator of thrombus formation. Materials and methods: We examined the effect of CK2 inhibition on platelet activation and aggregation, the formation of platelet-leukocyte aggregates (PLA), the endothelial expression of von Willebrand factor (vWF), intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and the subcellular localization of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and phospho-p65 in human dermal microvascular endothelial cells (HDMEC). Dorsal skinfold chambers were prepared in BALB/c mice to analyze in vivo the effect of CK2 inhibition on photochemically induced thrombus formation using intravital fluorescence microscopy. Results: CK2 inhibition by CX-4945 suppressed adenosin diphosphate (ADP)- and proteinase-activated receptor-1-peptide (PAR-1-AP)-stimulated platelet aggregation, which was associated with down-regulation of P-selectin, GPIIb/IIIa and a reduced formation of PLA. Expression and secretion of vWF was diminished in CX-4945-treated HDMEC. Moreover, CK2 inhibition attenuated the endothelial expression of VCAM-1, whereas the expression of ICAM-1 was not affected. Finally, CX-4945-treated mice exhibited a significantly delayed photochemically induced thrombus formation when compared to vehicle-treated controls. Conclusion: These results indicate that CK2 is a pleiotropic regulator of thrombus formation, affecting multiple interactions of platelets, leukocytes and endothelial cells.
... Organs that overexpress CD39 have improved graft function in both kidney [71] and liver [76] mouse transplant models encompassing extended cold preservation. Moreover, several studies during the past two decades indicated that the activity of CD39 influences severity of inflammation and autoimmune response [108][109][110][111][112][113]. ...
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Extracellular ATP interacts with purinergic type 2 (P2) receptors and elicits many crucial biological functions. Extracellular ATP is sequentially hydrolyzed to ADP and AMP by the actions of defined nucleotidases, such as CD39, and AMP is converted to adenosine, largely by CD73, an ecto-5′-nucleotidase. Extracellular adenosine interacts with P1 receptors and often opposes the effects of P2 receptor activation. The balance between extracellular ATP and adenosine in the blood and extracellular fluid is regulated chiefly by the activities of CD39 and CD73, which constitute the CD39-adenosinergic axis. In recent years, several studies have shown this axis to play critical roles in transport of water/sodium, tubuloglomerular feedback, renin secretion, ischemia reperfusion injury, renal fibrosis, hypertension, diabetic nephropathy, transplantation, inflammation, and macrophage transformation. Important developments include global and targeted gene knockout and/or transgenic mouse models of CD39 or CD73, biological or small molecule inhibitors, and soluble engineered ectonucleotidases to directly impact the CD39-adenosinergic axis. This review presents a comprehensive picture of the multiple roles of CD39-adenosinergic axis in renal physiology, pathophysiology, and therapeutics. Scientific advances and greater understanding of the role of this axis in the kidney, in both health and illness, will direct development of innovative therapies for renal diseases.
... Nucleoside triphosphate dephosphorylase (CD39) and ecto-5'nucleotidase (CD73) are also classical cell surface ATP apyrases that hydrolyze ATP/ADP to AMP and adenosine, respectively. 27,36 However, in contrast to the expression of TNSALP, the expression of CD39 and CD73 did not significantly differ between the WT and Alpl +/-MSCs (Fig. 4f). Additionally, in contrast to the classical ATPapyrase, the enzymatic reaction experiments revealed that the hydrolysis of TNSALP occurred in a dose-and time-dependent manner (Fig. 4g, h). ...
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Mutations in the liver/bone/kidney alkaline phosphatase (Alpl) gene cause hypophosphatasia (HPP) and early-onset bone dysplasia, suggesting that this gene is a key factor in human bone development. However, how and where Alpl acts in bone ageing is largely unknown. Here, we determined that ablation of Alpl induces prototypical premature bone ageing characteristics, including bone mass loss and marrow fat gain coupled with elevated expression of p16INK4A (p16) and p53 due to senescence and impaired differentiation in mesenchymal stem cells (MSCs). Mechanistically, Alpl deficiency in MSCs enhances ATP release and reduces ATP hydrolysis. Then, the excessive extracellular ATP is, in turn, internalized by MSCs and causes an elevation in the intracellular ATP level, which consequently inactivates the AMPKα pathway and contributes to the cell fate switch of MSCs. Reactivating AMPKα by metformin treatment successfully prevents premature bone ageing in Alpl+/- mice by improving the function of endogenous MSCs. These results identify a previously unknown role of Alpl in the regulation of ATP-mediated AMPKα alterations that maintain MSC stemness and prevent bone ageing and show that metformin offers a potential therapeutic option.
... Oxygen deprivation stimulates ECs to release ATP that triggers vascular inflammation, endothelial permeability, and thrombosis via P2 receptor stimulation [9,44]. These pathological effects of soluble nucleotides can be suppressed by redirecting them to extracellular hydrolysis via the CD39-CD73 axis [45]. In the cardiovascular system, the endothelial ecto-nucleotidases play a key role in the balance between nucleotides and protective adenosine, maintaining and strictly regulating the homeostatic process and counteracting excessive vascular leakage, or clot formation [46,47]. ...
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Chronic hypoxia drives vascular dysfunction by various mechanisms, including changes in mitochondrial respiration. Although endothelial cells (ECs) rely predominantly on glycolysis, hypoxia is known to alter oxidative phosphorylation, promote oxidative stress and induce dysfunction in ECs. Our work aimed to analyze the effects of prolonged treatment with hypoxia-mimetic agent CoCl2 on intracellular nucleotide concentration, extracellular nucleotide breakdown, mitochondrial function, and nitric oxide (NO) production in microvascular ECs. Moreover, we investigated how nucleotide precursor supplementation and adenosine deaminase inhibition protected against CoCl2-mediated disturbances. Mouse (H5V) and human (HMEC-1) microvascular ECs were exposed to CoCl2-mimicked hypoxia for 24 h in the presence of nucleotide precursors: adenine and ribose, and adenosine deaminase inhibitor, 2′deoxycoformycin. CoCl2 treatment decreased NO production by ECs, depleted intracellular ATP concentration, and increased extracellular nucleotide and adenosine catabolism in both H5V and HMEC-1 cell lines. Diminished intracellular ATP level was the effect of disturbed mitochondrial phosphorylation, while nucleotide precursors effectively restored the ATP pool via the salvage pathway and improved endothelial function under CoCl2 treatment. Endothelial protective effects of adenine and ribose were further enhanced by adenosine deaminase inhibition, that increased adenosine concentration. This work points to a novel strategy for protection of hypoxic ECs by replenishing the adenine nucleotide pool and promoting adenosine signaling.
... Extracellular ATP, ADP, AMP, or adenosine may be involved in pathophysiological conditions such as platelet aggregation [5,6], cardiovascular disease, immune response, chronic obstructive pulmonary disease, or asthma [7][8][9][10][11]. Mechanisms mediating extracellular ATP effects in human asthma or animal models of asthma include neurogenic bronchoconstriction [12], enhanced mast cell response to allergic stimuli [13], and TXA 2 -mediated bronchoconstriction [10]. ...
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Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sensitized guinea pigs were used for determining the extracellular metabolism (as assessed by inorganic phosphate production) of ATP, ADP, AMP, or adenosine, and for detecting possible changes in the expression (qPCR and Western blot) of major ectonucleotidases (NTPDase1, NTPDase3, and NPP1) and purinoceptors (P2X1, P2X7, P2Y4, and P2Y6). Contrary to our hypothesis, we found that leukocytes from allergic animals produced higher amounts of inorganic phosphate after stimulation with ATP and ADP, as compared with leukocytes from non-sensitized animals. Although at first glance, this result suggested that sensitization caused higher efficiency of ectonucleotidases, their mRNA and protein expressions were unaffected. On the other hand, after sensitization, we found a significant increase in the protein expression of P2X7 and P2Y4, two purinoceptors known to be responsible for ATP release after activation. We concluded that allergic sensitization increased the amount of ATP hydrolyzed by ectonucleotidases, the latter probably not due to the enhanced efficiency of its enzymatic breakdown, but rather due to an increased release of endogenous ATP or other nucleotides, partly mediated by enhanced expression or P2X7 and P2Y4 receptors.
... According to the literature, the nucleotides ATP, ADP, and AMP as well as derivatives of adenosine nucleosides are important signaling molecules in the vasculature able to influence vasomotor responses, inflammation, cardiac physiology and pathophysiology including myocardial ischemia and reactivity of platelets [23]. Microparticles released at the site of cellular damage appear to be associated with functional NTPDases, and the accumulation of these at sites of vascular injury can induce platelet-aggregation and mediate thrombosis, inflammation, cell apoptosis and necrosis [24]. ...
Article
The hydrolysis of adenine nucleotide linked to the membrane of the platelets is changed in acute myocardial infarction (AMI) probably due to a greater arterial blockage and cell damage in patients with ST elevation (STEMI) than in those without ST segment elevation (NSTEM). This study aimed to compare the extracellular hydrolysis of adenine nucleotides on the platelet surface of STEMI and NSTEMI patients. This study was carried out with 50 patients with AMI (STEMI and NSTEMI). The extracellular hydrolysis of adenine nucleotides and nucleoside adenosine as well as the expression of NTPDase were verified in platelets. The results demonstrated that STEMI patients had significantly higher extracellular hydrolysis of adenine nucleotides (p < 0.001), ADA (adenosine deaminase) activity (p < 0.05), as well as troponin levels (p < 0.0001) when compared to NSTEMI patients. Findings suggest that the extracellular hydrolysis of adenine nucleotides and increase in the ADA activity are higher in patients with STEMI than in those with NSTEMI probably because there was a blockage in this major arterial with a large area of damaged tissue.
... Our results show that PhaHV1 NTPDase readily hydrolyzes ADP. Hydrolysis of ATP to only ADP promotes platelet aggregation, which is one role of NTPDase2 (53), whereas ongoing hydrolysis to AMP results in inhibition of platelet aggregation (54), which may be advantageous during viral infection. Further biochemical analysis is needed to determine if the VoHV1 V4 encodes a functional NTPDase or another biologically relevant active enzyme. ...
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The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae . Their genomes contain several new ORFs, including ORFs encoding a β-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.
... Despite the much lower concentration of ATP in extracellular space (nanomolar) than in cell (milimolar), its role as a signaling molecule seems to be important since it is known that nucleotides exist in the pericellular space at micromolar levels [12]. In addition to VIC differentiation, nucleotide receptor activation also affects such conditions as chronic inflammation by the stimulation of immune cell transmigration via endothelium or thromboregulation by platelets activation, which plays a significant role in CAVD initiation and progression [13][14][15][16][17]. ...
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Background Extracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors. Results Nucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm²) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm²) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm²). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = − 0.30, p < 0.05; vs. AMP hydrolysis: r = − 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = − 0.26, p < 0.05; vs. AMP hydrolysis: r = − 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = − 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = − 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD. Conclusions This work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy. Graphic abstract Open image in new window .
... Purinergic signaling is an important modulating pathway for a variety of physiological processes involved in many neuronal and non-neuronal mechanisms and in short-and long-term events, including immune responses, inflammation, endothelium-mediated vasodilation, proliferation and cell death [2,4]. NTPDases and 5'-nucleotidase are enzymes involved in purinergic signaling responsible for controlling purine levels, playing an important role in physiological processes, as well as in inflammatory diseases [20]. ...
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Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associated with inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are not known. B. ovis is generally asymptomatic in sheep. Hepatosplenomegaly has been described in B. ovis, a non-zoonotic species, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved with the modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigate the role of ectonucleotidases and cholinesterase’s in the brains of mice experimentally infected with B. ovis.Materials, Methods & Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24). In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brains were collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substances reactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP and ADP as substrate) and 5'-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathological analysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI. However,multifocal areas with moderate microgliosis and inflammatory infiltrates in the cerebral cortex were observed at day 60 PI in the infected animals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation in the brains of infected animals than in the control group at day 60PI. No significant results were observed at 7, 15 or day 30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than in the control group. NTPDase activity (using ATP and AMP as substrate) was lower at days 7 and 15 PI in infected animals than in control. However, during the course of infection there was an increase in NTPDase activity at day 60 PI in the infected group. The infected animals showed a decrease of 5´-nucleotidase (AMP as substrate) activity at days 7 and 30 PI. On the other hand, 5´-nucleotidase activity was greater on day 60 PI in the experimental group than in the control. The results suggest that nucleotide hydrolysis was low in the acute phase (up to day 30 PI) due to the decrease of NTPDase and 5´-nucleotidase activities. After day 60 PI, there was a reversal in enzyme activities, probably with concomitant increase of extracellular nucleotides. AChE activity in brain on days 30 and 60 PI compared to control.Discussion: Among the functions of NTPDase are inhibition of platelet aggregation, vascular homeostasis, modulation of inflammation and immune response, all via its regulation of extracellular concentrations of ATP, a pro-inflammatory molecule. E-NTPDase plays an important role in controlling lymphocyte function, including antigen recognition and activation of cytotoxic T cell effector functions, as well as the generation of signals. The enzyme E-5´-nucleotidase also exerts non-enzymatic functions, including induction of intracellular signaling and mediation of cell-cell adhesion and cell-matrix and migration. Levels of acetylcholine are regulated by cholinesterase enzymes that are present in cholinergic and noncholinergic tissues, as the acetylcholinesterase (AChE) is a membrane-bound enzyme, primarily found in the brain and cholinergic neurons, where it participates in the structural regulation of postsynaptic differentiation. The results demonstrated that the chronicity of infection by B. ovis causes oxidative damage and inflammation in the brain, as well as modulation of ectonucleotidases and AChE activities.
... This sophisticated cell-cell communication system orchestrates numerous cellular responses in the context of health and disease, displaying immunomodulatory capabilities and widely influencing cellular proliferation, differentiation, and death processes. Extracellular signaling molecules include nucleotides such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and the nucleoside adenosine (Ado), implicated in several pathophysiological events [13][14][15][16][17]. ...
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly impacted the world and has driven many researchers into the pathophysiology of COVID-19. In the findings, there is a close association between purinergic signaling and the immune response. Then, this study aimed to evaluate alterations in the purinergic signaling in COVID-19 patients according to range severity. We divided the COVID-19 patients into moderate and severe cases following the guideless of NIH and WHO, together with clinical characteristics. The blood samples were collected to obtain PBMCs and platelets. We analyzed the ectonucleotidase activities through ATP, ADP, AMP, Ado hydrolysis, E-NTPDase1 (CD39), and 5′-NT (CD73) expression by flow cytometry in total leukocytes. The extracellular ATP was measured by bioluminescence, and cytokines were analyzed by flow cytometry. We observed a decrease in ATP hydrolysis and increased AMP hydrolysis in PBMCs for both groups. In severe cases, ATP hydrolysis was raised for the platelets, while ADP and AMP hydrolysis have risen significantly in both groups. Additionally, there was a significant increase in ADP hydrolysis in severe cases compared to moderate cases. In addition, we observed an increase in the ADA activity in platelets of moderate patients. Moderate and severe cases showed increased expression of CD39 and CD73 in total leukocytes. To finalize the purinergic signaling, extracellular ATP was increased in both groups. Furthermore, there was an increase in IL-2, IL-6, IL-10, and IL-17 in moderate and severe groups. Thus, for the first time, our findings confirm the changes in purinergic signaling and immune response in COVID-19, in addition to making it more evident that the severity range directly impacts these changes. Therefore, the therapeutic potential of the purinergic system must be highlighted and studied as a possible target for the treatment of SARS-CoV-2 disease. Key messages COVID-19 patients exhibit alterations in purinergic system and immune response. High levels of extracellular ATP lead to different inflammatory responses. CD39 and CD73 expression were increased in COVID-19 patients. Cytokines IL-2, IL-6, IL-10, and IL-17 also were altered in these patients. The purinergic system may be a possibility target to SARS-CoV-2 treatments. Graphical abstract
... Additionally, ATP, ADP, and Ado can also modulate the vascular system through interactions with specific purinergic receptors. In this sense, it is known that the platelet membrane possesses the entire cascade of ectonucleotidases that are responsible for the hydrolysis of these molecules [41][42][43]. Thus, these enzymes play an important role in thromboregulation, and altered activities of these enzymes have been observed in various diseases. ...
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Glioblastoma (GB) is a highly aggressive and invasive brain tumor; its treatment remains palliative. Tannic acid (TA) is a polyphenol widely found in foods and possesses antitumor and neuroprotective activities. This study aimed to investigate the effect of TA on oxidative stress parameters and the activity of ectonucleotidases in the serum, platelets, and lymphocytes and/or in the brain of rats with preclinical GB. Rats with GB were treated intragastrically with TA (50 mg/kg/day) for 15 days or with a vehicle. In the platelets of the animals with glioma, the adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolysis and the catalase (CAT) activity decreased. Besides, the adenosine diphosphate (ADP) hydrolysis, adenosine (Ado) deamination, and the reactive oxygen species (ROS) and nitrite levels were increased in glioma animals; however, TA reversed ROS and nitrite levels and AMP hydrolysis alterations. In lymphocytes from animals with glioma, the ATP and ADP hydrolysis, as well as Ado deamination were increased; TA treatment countered this increase. In the brain of the animals with glioma, the ROS, nitrite, and thiobarbituric acid reactive substance (TBARS) levels increased and the thiol (SH) levels and CAT and superoxide dismutase (SOD) activities were decreased; TA treatment decreased the ROS and TBARS levels and restored the SOD activity. In the serum of the animals with glioma, the ATP hydrolysis decreased; TA treatment restored this parameter. Additionally, the ROS levels increased and the SH and SOD activity decreased by glioma implant; TA treatment enhanced nitrite levels and reversed SOD activity. Altogether, our results suggest that TA is an important target in the treatment of GB, as it modulates purinergic and redox systems.
... The purinergic system is a signaling cell pathway present in basically all body tissues, composed by extracell molecules of nucleotides and nucleosides, receptors and enzymes (ectoenzymes), which participate in physiologic and pathologic mechanisms of the human organism, such as cell proliferation, differentiation, and death, as well as immunomodulation [72][73][74][75]. A recent study hypothesized on the potential role of the purinergic system on therapy in cardiovascular diseases mediated by SARS-CoV-2 [76]. ...
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Cancer cases have increased significantly in Brazil and worldwide, with cutaneous melanoma (CM) being responsible for nearly 57,000 deaths in the world. Thus, this review article aims at exploring and proposed hypotheses with respect to the possibility that RA can be a promising and alternative compound to be used as an adjuvant in melanoma treatment, acting on purinergic signaling. The scarcity of articles evidencing the action of this compound in this signaling pathway requires further studies. Considering diverse evidence found in the literature, we hypothesize that RA can be an effective candidate for the treatment of CM acting as a modulating molecule of purinergic cellular pathway through P2X7 blocking, mitigating the Warburg effect, and as antagonic molecule of the P2Y12 receptor, reducing the formation of adhesive molecules that prevent adherence in tumor cells. In this way, our proposals for CM treatment based on targeting purinergic signaling permeate the integral practice, going from intracell to extracell. Undoubtedly, much is still to be discovered and elucidated about this promising compound, this paper being an interesting work baseline to support more research studies.
... In the vasculature, the increase in nucleotide bioavailability results in vascular effects due to P1 and P2 receptor activation, such as apoptosis, fibrosis, vasoconstriction or vasorelaxation, platelet aggregation, inflammation, and vascular permeability [33, [42][43][44][45]. The loss of CD39 on activated endothelium sustains platelet aggregation and thrombogenesis, resulting in enhanced platelet P2Y1 and P2Y12 activation [6,46]. Pathological conditions characterized by increased cardiovascular risk might be associated with a reduced CD39 expression and/or activity [47][48][49]. ...
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There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5′-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.
... The CD39/CD73 axis regulates leukocyte extravasation [24][25][26][27] and disruption results in increased vascular inflammation and thrombus formation. 45,46 In both cells types, ABC treatment resulted in lower CD39 + CD73 + populations and reduced phosphohydrolytic activity, including in the presence of TNAP inhibitors, suggesting a specific effect on ectonucleotidase activity. ...
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Rationale People living with HIV on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established. Objective To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function to better understand cardiovascular risk in people living with HIV. Methods and Results Human umbilical cord vein endothelial cells or human coronary artery endothelial cells were pretreated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate, or tenofovir alafenamide. Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM (intercellular adhesion molecule)-1 and TF expression following TNF (tumor necrosis factor)-α stimulation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide treatment gave rise to greater populations of CD39 ⁺ CD73 ⁺ cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, while tenofovir disoproxil fumarate- and tenofovir alafenamide-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release. Conclusions We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, prothrombotic endothelial phenotype that promoted platelet activation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in people living with HIV.
... Expression of CD39 is a key determinant of occlusive thrombus formation and therapies that promote CD39 expression are likely to increase antithrombotic efficacy. Global overexpression of human CD39 yields profound antithrombotic effects [94][95][96], though its effect may in part be due to the reduction of ADP-induced platelet activation [97,98]. CD39 is also important in leukocyte trafficking across the endothelium in response to chemokines and regulates immune cell adhesion to the endothelial layer where cell adhesion is promoted by the ATP rich environment and inhibited by adenosine [99]. ...
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The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood–brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain.
... In tumor environment, the expression of CD39 in vascular system, especially endothelial cells, can promote tumor growth by scavenging eATP [95] . The endothelial CD39/CD73 axis regulates hemostasis by transforming the local environment from a prethrombotic state rich in ATP and ADP into an antithrombotic environment rich in ADO [96][97][98] . CD39 plays the role of an endothelial thromboregulator by demonstrating that CD39transfected COS cells acquire the ability to inhibit ADP-induced aggregation in platelet-rich plasma [99][100][101] . ...
... In the central nervous system, adenosine homeostasis is controlled in a coordinated manner by a panel of proteins. 11,12 Extracellular adenosine can be produced by the degradation of ATP/ ADP by membrane-associated ecto-5¢-nucleotidases (i.e., CD39 and CD73; Fig. 1), [13][14][15][16] as well as by release through membrane equilibrative nucleoside transporters (ENTs; see below). Many pathological events (such as exocytosis, trauma-evoked membrane leakage or damage, and connexin hemichannels) may enhance the level of extracellular ATP, which results in an increase in adenosine tone in the brain. ...
... There is evidence that in an inflammatory environment, the loss of CD39 activity from activated endothelium sustains platelet aggregation and thrombogenesis (Atkinson et al., 2006). On the other hand, within a damaged tissue the increased expression of CD39 on inflammatory cells, working in tandem with CD73, might cause inhibition of platelet activation by increasing extracellular adenosine levels (Johnston-Cox et al., 2011). ...
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There is evidence that an imbalance of extracellular purine levels may be associated with increased cardiovascular risk. Platelets play a pivotal role in vascular homeostasis and thrombosis and are important source of purine nucleotides and nucleosides. Hydrolysis of nucleotides ATP and ADP is regulated by two ectonucleotidases, triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5’-nucleotidase (ecto-5’-NT/CD73). CD39 enzyme is expressed on the endothelium, circulating blood cells, and smooth muscle cells; there is evidence that changes in CD39 expression and activity affects the potential thrombogenic of a tissue. Gender difference in the cardiovascular risk has been extensively observed; however, while the age-dependent difference in the prevalence of cardiovascular events between men and women has been attributed to the loss of the protective effect of estrogens in the postmenopausal period, the physiological mechanism behind gender disparity is still unclear. Here, we evaluated comparatively male and female rat platelet reactivity and considered the possible role of CD39 at the basis of difference observed. We found a reduced in vitro response to ADP (1–30 µM) of female compared to male platelets, associated to increased platelet CD39 expression and activity. Platelet response to ADP was strongly increased by incubation (10 min) with the CD39 inhibitor, ARL67156 (100 µM), while male platelet response was unaffected. Rat treatment with clopidogrel (30 mg/kg, per os) inhibited ex vivo platelet aggregation. Bleeding time was prolonged in female compared to male. Taken together, our results suggest that platelet ATPase and ADPase activity might be a reliable predictor of platelet reactivity.
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Extracellular nucleotide catabolism contributes to immunomodulation, cell differentiation and tissue mineralization by controlling nucleotide and adenosine concentrations and its purinergic effects. Disturbances of purinergic signaling in valves may lead to its calcification. This study aimed to investigate the side-specific changes in extracellular nucleotide and adenosine metabolism in the aortic valve during calcific aortic valve disease (CAVD) and to identify the individual enzymes that are involved in these pathways as well as their cellular origin. Stenotic aortic valves were characterized by reduced levels of extracellular ATP removal and impaired production of adenosine. Respectively, already reduced levels of extracellular adenosine were immediately degraded further due to the elevated rate of adenosine deamination. For the first time, we revealed that this metabolic pattern was observed only on the fibrosa surface of stenotic valve that is consistent with the mineral deposition on the aortic side of the valve. Furthermore, we demonstrated that non-stenotic valves expressed mostly ecto-nucleoside triphosphate diphosphohydrolase 1 (eNTPD1) and ecto-5’nucleotidase (e5NT), while stenotic valves ecto-nucleotide pyrophosphatase/ phosphodiesterase 1, alkaline phosphatase and ecto-adenosine deaminase (eADA). On the surface of endothelial cells, isolated from non-stenotic valves, high activities of eNTPD1 and e5NT were found. Whereas, in valvular interstitial cells, eNPP1 activity was also detected. Stenotic valve immune infiltrate was an additional source of eADA. We demonstrated the presence of A1, A2a and A2b adenosine receptors in both, non-stenotic and stenotic valves with diminished expression of A2a and A2b in the former. Extracellular nucleotide and adenosine metabolism that involves complex ecto-enzyme pathways and adenosine receptor signaling were adversely modified in CAVD. In particular, diminished activities of eNTPD1 and e5NT with the increase in eADA that originated from valvular endothelial and interstitial cells as well as from immune inflitrate may affect aortic valve extracellular nucleotide concentrations to favor a pro-inflammatory milieu, highlighting a potential mechanism and target for CAVD therapy.
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Background: Although electroacupuncture is widely used in chronic pain management, it is quite controversial due to its unclear mechanism. We hypothesised that EA alleviates pain by inhibiting degradation of the ecto-nucleotidase prostatic acid phosphatase (PAP) and facilitating ATP dephosphorylation in dorsal root ganglions (DRGs). Methods: We applied EA in male C57 mice subjected to chronic constriction injury (CCI) and assessed extracellular ATP and 5'-nucleotidease expression in DRGs. Specifically, we used a luminescence assay, quantitative reverse transcriptase-polymerase chain reaction, western blotting, immunohistochemistry and nociceptive-related behavioural changes to gather data, and we tested for effects after PAP expression was inhibited with an adeno-associated virus (AAV). Moreover, membrane PAP degradation was investigated in cultured DRG neurons and the inhibitory effects of EA on this degradation were assessed using immunoprecipitation. Results: EA treatment alleviated CCI surgery induced mechanical pain hypersensitivity. Furthermore, extracellular ATP decreased significantly in both the DRGs and dorsal horn of EA-treated mice. PAP protein but not mRNA increased in L4-L5 DRGs, and inhibition of PAP expression via AAV microinjection reversed the analgesic effect of EA. Membrane PAP degradation occurred through a clathrin-mediated endocytosis pathway in cultured DRG neurons; EA treatment inhibited the phosphorylation of adaptor protein complex 2, which subsequently reduced the endocytosis of membrane PAP. Conclusions: EA treatment alleviated peripheral nerve injury-induced mechanical pain hypersensitivity in mice by inhibiting membrane PAP degradation via reduced endocytosis and subsequently promote ATP dephosphorylation in DRGs.
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Research on adenosine has a long history, and since the beginning more than 80 years ago there has been a link to energy metabolism. This is due to the fact that levels of adenosine increase under conditions of relative energy deficiency and that adenosine can have actions that limit this: increase respiration, increase blood flow, and reduce cellular work. There are also effects on intermediary metabolism and on overall energy homeostasis. This minireview provides a brief and personal historical background to these developments. © 2013 Springer Science+Business Media New York. All rights are reserved.
Article
Ecto-nucleotidases hydrolyze extracellular nucleotides. Nucleotides are amongst the most ubiquitous messenger substances in the vertebrate body. Receptors for nucleotides are expressed on the surface of essentially every cell and many cells carry several types of nucleotide receptors. Several families of ecto-nucleotidases have been identified that differ in tissue distribution and functional properties. They modulate ligand availability at nucleotide and adenosine receptors. Ecto-nucleotidases were first identified in the 1940ies. Work of the past two decades has unraveled molecular identities and important functional properties. Using targeted gene deletion clear examples highlighting the importance of ecto-nucleotidases in nucleotide and adenosine signaling have been elaborated. These reach from the control of blood flow and angiogenesis to the modulation of immune functions and neural development. Specific ecto-nucleotidases are associated with stem cells in the adult mammalian brain, implicating a role of nucleotides and nucleosides in the control of adult neurogenesis. Ecto-nucleotidases represent important therapeutic targets to interfere with P2 or P1 receptor-mediated receptor signaling pathways. The development of high throughput assays promises a considerable acceleration in the development of subtype-specific ecto-nucleotidase inhibitors.
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Background: Exercise-induced pulmonary hemorrhage (EIPH) is a common disorder of equine athletes. The role of polymorphisms in genes encoding hemostasis-regulatory proteins in horses with abnormal hemorrhage is unknown. Objectives: The goal of this study was to evaluate the genes encoding 2 ectonucleotidases, CD39/NTPDase-1 and CD39L1/NTPDase-2, and one ecto-5' nucleotidase, CD73, in horses with abnormal hemorrhage or pathologic changes consistent with EIPH. Methods: Twenty-three horses with histories of abnormal hemorrhage, 8 horses with gastrointestinal signs, and 45 healthy horses were evaluated using polymerase chain reaction-based techniques. Formalin-fixed tissues from 21 horses with pathologic changes consistent with EIPH were also evaluated. Results: Three single nucleotide polymorphisms (SNPs) were identified in the gene encoding CD39 and one SNP was identified in the gene encoding CD39L1. No SNPs were identified in the gene encoding CD73. CD39 SNPs were identified in 19 of 20 (95%) horses with unexplained hemorrhage and 20 of 21 (95%) horses with pathologic features consistent with EIPH. CD39L1 SNPs were identified in 6 of 20 (30%) horses with unexplained hemorrhage and 8 of 21 (38%) horses with pathologic features consistent with EIPH. CD39 and CD39L1 SNPs were identified in 5 of 8 (62.5%) and one of 8 (12.5%) horses, respectively, presenting with colic or weight loss. CD39 and CD39L1 SNPs were identified in 28 of 45 (62%) and 13 of 45 (28.8%) healthy horses, respectively. Conclusions: CD39 and CD39L1 are critically important in maintaining normal hemostasis and limiting inflammation. Further studies are needed to evaluate their role in the pathogenesis of equine EIPH.
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This review covers a broad spectrum of issues related to extracellular adenine compounds within the cardiovascular system. Topics covered include: the cellular sources and detection of extracellular adenine compounds; the ecto-enzymes that mediate their metabolism; protein phosphorylation and function, and only briefly; purinoceptors and their functions. Extracellular nucleotides are generally not synthesized in biological extracellular environments. Why do some cell types store metabolically inert levels of ATP in storage granules that are released upon specific signals into the extracellular environment? Why are there a cadre of enzymes in many extracellular environments that can hydrolyze ATP to other bioactive molecules, such as ADP and adenosine, and kinases that transfer the gamma-phosphate of ATP to ecto-membrane/soluble proteins and protein phosphatases that reverse these reactions? A great deal of investigative attention has been focused on the characterization and functions of the purinergic cell surface receptors while less attention has been paid to the extracellular enzymes that catalyze a host of reactions with extracellular adenine compounds, such as protein phosphorylation and the functional outcomes of these reactions. This review is an attempt to garner more scientific interest in the potential roles extracellular adenine compounds play in the regulation of cell and cell-cell functions other than via purinergic signaling pathways as many of these reactions may play a role in diseased states and may serve as targets for new drug development. Keywords: Extracellular adenine compounds, Extracellular protein phosphorylation, Extracellular nucleotidases, Extracellular kinases, Extracellular enzymes
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Extracellular adenosine triphosphate (eATP) mediates pro-inflammatory responses by recruiting and activating inflammatory cells. CD39 can hydrolyze eATP into adenosine monophosphate (AMP), while CD73 can convert AMP into the immunosuppressive nucleoside adenosine (ADO). CD39 is a rate-limiting enzyme in this cascade, which is regarded as an immunological switch shifting the ATP-mediated pro-inflammatory environment to the ADO- mediated anti-inflammatory status. The CD39 expression can be detected in a wide spectrum of immunocytes, which is under the influence of environmental and genetic factors. It is increasingly suggested that, CD39 participates in some pathophysiological processes, like inflammatory bowel disease (IBD), sepsis, multiple sclerosis (MS), allergic diseases, ischemia-reperfusion (I/R) injury, systemic lupus erythematosus (SLE), diabetes and cancer. Here, we focus on the current understanding of CD39 in immunity, and comprehensively illustrate the diverse CD39 functions within a variety of disorders.
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Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.
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The human immunodeficiency virus (HIV) infection results in biochemical and vascular dysfunctions. The highly active antiretroviral therapy (HAART) markedly reduces mortality and opportunistic diseases associated with acquired immunodeficiency syndrome (AIDS). This increased survival time predisposes the development of cardiovascular diseases. Platelets present purinergic system ectoenzymes such as E-NTPDase, E-5'-nucleotidase and E-ADA on its surface. In view of this, the aim of this study was to evaluate the activity of these ectoenzymes in platelets as well as the platelet aggregation and lipid profile of patients with HIV infection and also patients receiving HAART. The results showed an increase in the E-NTPDase activity for ATP hydrolysis in the HIV group compared with the control group and the HIV/HAART group. When assessing the activity E-NTPDase hydrolysis to ADP, the results revealed an increase in activity in the HIV group when compared to the control group, and a decrease in activity when in the HIV/HAART group when compared to the control and HIV groups. The activity of E-5'-nucleotidase revealed an increase in AMP hydrolysis in the HIV group, as the results from control and HIV/HAART groups showed no statistical difference. Regarding the E-ADA activity, the HIV and HIV/HAART groups revealed a decreased deamination of adenosine when compared with the control group. Furthermore, we observed an increased platelet aggregation of HIV/HAART group compared with the control group. Thus, our results suggest that antiretroviral treatment against HIV has a significant effect on the activity of purinergic system ectoenzymes demonstrating that thromboregulation is involved in the process.
Chapter
Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca2+ whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells. Much of the detailed information on the contribution of P2X1 to Ca2+ signalling and downstream functional events has been derived from the platelet. The underlying primary P2X1-generated signalling event in non-excitable cells is principally due to Ca2+ influx, although Na+ entry will also occur along with membrane depolarization. P2X1 receptor stimulation can lead to additional Ca2+ mobilization via a range of routes such as amplification of G-protein-coupled receptor-dependent Ca2+ responses. This chapter also considers the mechanism by which cells generate extracellular ATP for autocrine or paracrine activation of P2X1 receptors. For example cytosolic ATP efflux can result from opening of pannexin anion-permeable channels or following damage to the cell membrane. Alternatively, ATP stored in specialised secretory vesicles can undergo quantal release via the process of exocytosis. Examples of physiological or pathophysiological roles of P2X1-dependent signalling in non-excitable cells are also discussed, such as thrombosis and immune responses.
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Objectives: This study sought to evaluate the profile of pro and anti-inflammatory cytokines (IL-10, IL-6, IL-17, TNF, IL-4, IL-2 and IFN-gamma), cardiac markers (troponin, CK, CK MB, LDH, CRP) in HIV-positive patients and assess the in vitro effect of antiretroviral therapy on the activities of ectonucleotidases (E-NTPDase and E-5'-nucleotidase) in human platelets. Design and methods: Ten blood samples were obtained from ten HIV positive patients at the Infectious Disease Clinic in the University Hospital of Santa Maria, Brazil and ten HIV negative individuals (control group) for this study. Results: The results revealed that there were significant (P < 0.05) increases in serum levels of IL-6 and IFN-gamma with no significant (P > 0.05) changes in the serum levels of the cardiac markers investigated (CK, CK-MB, troponin, LDH and CRP). In addition, the ectonucleotidases (E-NTPDase and E-5'-nucleotidase) activities were not altered (P > 0.05) in human platelets when incubated with different antiretroviral drugs in vitro. Conclusions: The results of this study suggest that, despite successful treatment, HIV patients a proinflammatory state remains in HIV patients, and that antiretroviral therapy per se does not lead alter the purinergic profile.
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Extracellular di- and tri-phosphate nucleotides are released from activated or injured cells to trigger vascular and immune P 2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ecto-nucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the mono-phosphate nucleoside end-products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ecto-enzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ecto-enzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase (ALP-ECD) and acid phosphatase (HAP-ECD) fusion proteins were also generated, characterized and compared with these CD39-ECD, CD73-ECD and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ecto-enzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting tri- and di-phosphate nucleotides into nucleosides when compared to ALP-ECD and HAP-ECD. We also note a pH-sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ecto-enzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge pro-inflammatory ATP and also generate anti-inflammatory adenosine.
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The aim of this study was to evaluate nucleoside triphosphate diphosphohydrolase, 5′-nucleotidase, and adenosine deaminase activities in cattle fed on cuttings from pastures fertilized with urea compared to animals fed on cuttings from organically fertilized or from non-fertilized pastures. Blood samples were collected up to 9 h after feeding, and nitrite and enzyme activities were evaluated. The animals feeding on urea-fertilized pasture suffered subclinical intoxication. Serum nitrite levels increased by about 50% and adenosine deaminase activity increased almost sixfold 4 and 6 h after feeding on urea-fertilized pasture, and adenosine deaminase in platelets almost doubled 6 and 9 h after feeding. In platelets, nucleoside triphosphate diphosphohydrolase activity with adenosine triphosphate as substrate increased 2, 4, 6 and 9 h after feeding while nucleoside triphosphate diphosphohydrolase with adenosine diphosphate as substrate and 5′-nucleotidase activities increased only 9 h after feeding. Increased activities of these enzymes may be implicated in the pathophysiology of subclinical intoxication since they are involved in many biochemical functions.
Chapter
Cutaneous melanoma is an aggressive and difficult-to-treat disease that has rapidly grown worldwide. The pharmacotherapy available in so many cases results in low response and undesirable side effects, which impair the life quality of those affected. Several studies have been shown that the purinergic system is involved in cancer context, such as in cutaneous melanoma. With technological advances, several bioactive compounds from nature are studied and presented as promising adjuvant therapies against cancer, as phenolic compounds and related action by purinergic system modulations. Thus, phenolic compounds such as rosmarinic acid, resveratrol, tannic acid, as well as vitamin D may be promising substances in a therapeutic perspective to treat cutaneous melanoma via purinergic system pathway. More research needs to be done to open up new horizons in the treatment of melanoma by the purinergic signaling.
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Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.
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CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.
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A full-length E(ecto)-ATPase (Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ > Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.
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Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.
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Although the presence of nucleotidase activities on the extracellular surface of cells has been known for many years, the enzymes responsible for these activities in animal cells have only recently been identified. Here, we describe how we cloned the gene for potato apyrase and, thus, found the way to identify CD39 as the animal cell ectoapyrase. CD39 has two transmembrane domains, one at each end of the molecule, small cytoplasmic NH2- and COOH- terminal domains, and a large extracellular domain with the enzymatic activity. One of the characteristic features of this enzyme is the loss of activity caused by solubilization with detergents. Exploration of this phenomenon revealed that ectoapyrase (CD39) is a tetramer in the membrane; solubilization with Triton X-100 causes dissociation of the tetramer to monomers with concomitant loss of activity. On the other hand, the enzyme can be solubilized with digitonin without loss of activity and retention of the tetrameric state. The conclusion is that the transmembrane segments of the protein are involved in association of the monomers to tetramers.
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Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
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Endothelial cell activation with accompanying vascular inflammatory changes is considered central to the experimental manifestations of both hyperacute and delayed xenograft rejection responses. Natural xenoreactive antibodies directed at α-galactosyl residues of xenogeneic glycoproteins and glycolipids, with associated complement activation via the classical pathway, are considered major immediate mediators of graft endothelial cell injury in the clinically relevant discordant swine to primate combinations. In delayed xenograft rejection processes, where recipients are treated prophylactically to ameliorate these initial events, activation of infiltrating mononuclear phagocytes and natural killer cells are associated with ongoing endothelial cell activation processes, procoagulant generation and vascular thrombosis. Allograft hyperacute rejection is observed when vascularised organs are transplanted to sensitized individuals with high levels of cytotoxic antibodies. Less dramatic forms of humoral allograft rejection (termed accelerated or vascular rejection) and the more common cell-mediated endothelialitis are associated with significant graft damage. Endothelial cell activation is also linked with graft preservation injury, forms of chronic rejection and delayed graft loss. Experimental work is currently being directed at the control of hyperacute rejection, the close understanding of endothelial cell thromboregulation in both transplanted xeno- and allografts and the development of novel therapeutic agents including gene therapy and the possible use of organs from transgenic animals.Copyright © 1995 S. Karger AG, Basel
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Although the presence of nucleotidase activities on the extracellular surface of cells has been known for many years, the enzymes responsible for these activities in animal cells have only recently been identified. Here, we describe how we cloned the gene for potato apyrase and, thus, found the way to identify CD39 as the animal cell ectoapyrase. CD39 has two transmembrane domains, one at each end of the molecule, small cytoplasmic NH2- and COOH-terminal domains, and a large extracellular domain with the enzymatic activity. One of the characteristic features of this enzyme is the loss of activity caused by solubilization with detergents. Exploration of this phenomenon revealed that ectoapyrase (CD39) is a tetramer in the membrane; solubilization with Triton X-100 causes dissociation of the tetramer to monomers with concomitant loss of activity. On the other hand, the enzyme can be solubilized with digitonin without loss of activity and retention of the tetrameric state. The conclusion is that the transmembrane segments of the protein are involved in association of the monomers to tetramers. Drug Dev. Res. 45:245–252, 1998. © 1998 Wiley-Liss, Inc.
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Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases, Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.
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Hypoxia is a common denominator of ischemic microenvironments. Endothelium subjected to oxygen deprivation maintains cell viability and basic biosynthetic mechanisms, but displays multiple changes in properties relevant to vascular homeostasis, including suppression of the anticoagulant cofactor thrombomodulin, decreased barrier function, and generation of proinflammatory cytokines. Diminished intracellular cAMP during the period of hypoxia and lowered nitric oxide/cGMP in the subsequent reperfusion period are proposed as fundamental mechanisms driving vascular dysfunction impacting on coagulation, permeability, vasomotor tone and leukocyte adhesivity. The period of organ preservation for transplantation, recognized to be associated with hypoxia, primes mechanisms leading to subsequent vascular dysfunction which can be ameliorated by buttressing cAMP and nitric oxide/cGMP intra- and intercellular second messenger systems. A mechanism likely to contribute to hypoxia-mediated generation of cytokines, such as interleukin 6, is activation of the transcription factor NF-IL-6, which occurs in oxygen deprivation. These data indicate that study of cellular mechanisms of endothelial perturbation in hypoxia is likely to provide insights ultimately applicable to ischemia-induced vascular damage.
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CD73 or ecto-5′-nucleotidase (S-NT) is a widely expressed ecto-enzyme which catalyzes the dephosphorylation of AMP and other nucleoside monophosphates. CD 73 participates in purine salvage through this enzymatic activity, supplying cells with precursors for energy metabolism and nucleic acid biosynthesis. As an enzyme that produces adenosine, CD 73 can also regulate adenosine receptor engagement in many tissues. However, CD73 also has functions independent of its enzyme activity like many glycosyl phosphatidylinositol (GPI)-anchored molecules, it transmits potent activation signals in T cells when ligated by antibodies. Less compelling evidence suggests that CD 73 may function as a cell adhesion molecule. In the human immune system, CD73 is expressed on subsets of T and B cells, on germinal center follicular dendritic cells, and on thymic medullary reticular fibroblasts and epithelial cells. Many challenging areas remain to be explored before the role of CD73 in the immune system will be fully understood. These include an evaluation of the role of adenosine receptors in lymphoid development, the identification of physiological CD73 ligands, a functional assessment of the GPI anchor, and an analysis of the intricate cell-type-specific and developmental regulation of CD 73 expression.
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Rejection of xenografts is associated with vascular-based inflammation, thrombocytopenia and the consumption of coagulation factors that may evolve into disseminated intravascular coagulation (DIC). Similarly, bone marrow-derived cellular xenotransplantation procedures are associated with endothelial cell activation and thrombotic microangiopathic injury. These complications generally develop despite the best available measures for depletion of xenoreactive natural antibody, inhibition of complement activation and suppression of T- and B-cell mediated immune responses. The mechanisms underlying the DIC and thrombotic microangiopathy associated with xenotransplantation are unclear. A proposed primary biological dysfunction of xenografts with respect to regulation of clotting could amplify vascular injury, promote immunological responses and independently contribute to graft failure. Disordered thromboregulation could have deleterious effects, comparable to unregulated complement activation, in the pathogenesis of xenograft rejection and may therefore represent a substantive barrier to xenotransplantation.
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The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC50 = 9.1-21.2 M) than UTP (IC50 = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.
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The human CD38 molecule appears to mediate several diverse activities, including signal transduction, cell adhesion and cyclic ADP-ribose synthesis. In this article, the authors consolidate the information available on this highly interesting, multifunctional protein.
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Extracellular nucleotide stimulation of purinergic/pyrimidinergic type-2 (P2) receptors are components of platelet, endothelial cell (EC), and leukocyte activation that culminate in vascular thrombosis and inflammation in vivo. CD39, the prototype nucleoside triphosphate diphosphohydrolase (or NTPDase-1), is highly expressed on quiescent endothelium, monocytes, and activated lymphocytes and therefore could influence these pathways. The potential of NTPDase-1 to regulate P2-receptor function in the vasculature has been established by our generation of cd39-null mice. These mice exhibit a prothrombotic vascular phenotype ascribed to overexpression of tissue factor by endothelial cells following aberrant P2- (and potentially adenosine 2a/3) receptor activation. Mutant mice also show perturbations in hemostasis, secondary to platelet P2Y1-receptor desensitization. In addition, administration of soluble NTPDase and/or induction of CD39 overexpression by adenoviral vectors consistently result in amelioration of vascular injury in several animal models tested. CD39 is also the major NTPDase expressed by monocyte-macrophages (Mo). Upregulation of tissue factor expression by Mo in vitro and alterations in splenic populations in vivo have been observed in cd39-null mice. Paradoxical inhibition of integrin-mediated adhesion and transendothelial migration of cd39-null Mo are also related to aberrant P2-receptor activation and have also been observed in vitro and in vivo. Overexpression of CD39 following infection with recombinant adenoviral vectors also blocks LPS-induced ATP secretion and inhibits IL-1 release in vitro. These studies confirm a role for CD39 in the differential regulation of P2-receptor activity and function in platelets, vascular, and immune cells. Drug Dev. Res. 53:193–207, 2001. © 2001 Wiley-Liss, Inc.
Article
CD73 or ecto-5'-nucleotidase (5'-NT) is a widely expressed ecto-enzyme which catalyzes the dephosphorylation of AMP and other nucleoside monophosphates. CD73 participates in purine salvage through this enzymatic activity, supplying cells with precursors for energy metabolism and nucleic acid biosynthesis. As an enzyme that produces adenosine, CD73 can also regulate adenosine receptor engagement in many tissues. However, CD73 also has functions independent of its enzyme activity. Like many glycosyl phosphatidylinositol (GPI)-anchored molecules, it transmits potent activation signals in T cells when ligated by antibodies. Less compelling evidence suggests that CD73 may function as a cell adhesion molecule. In the human immune system, CD73 is expressed on subsets of T and B cells, on germinal center follicular dendritic cells, and on thymic medullary reticular fibroblasts and epithelial cells. Many challenging areas remain to be explored before the role of CD73 in the immune system will be fully understood. These include an evaluation of the role of adenosine receptors in lymphoid development, the identification of physiological CD73 ligands, a functional assessment of the GPI anchor, and an analysis of the intricate cell-type-specific and developmental regulation of CD73 expression.
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It is well known that the function of platelets decreases progressively during storage of platelet concentrates at room temperature. To investigate this phenomenon in more detail, we have resuspended platelets that had been stored for 24 h or 72 h in fresh plasma, and we have measured the aggregation response and the ATP secretion. Conversely, the effect of plasma in which platelet concentrates (PC) had been stored for 24 h or 72 h, was tested on fresh platelets. Both the aggregation response to collagen and ADP and the collagen-induced ATP secretion of stored platelets partially recovered after incubation with fresh plasma (p <0.05). The same parameters measured with fresh platelets incubated in stored PC-plasma were found to be significantly reduced in comparison with the response of fresh platelets in fresh plasma (p <0.05). Finally, platelets were stored in a plasma-free medium, suitable for platelet storage and the supernatant was tested. This supernatant inhibited the function of fresh platelets in a storage time-dependent fashion. Boiling of these supernatants did not change the inhibiting capacities, whereas filtration over active charcoal did. Analysis of this supernatant revealed AMP and diadenosine tetraphosphate, which both inhibit platelet function. These data show that stored platelets release nucleotides that inhibit platelet function in a reversible manner. This phenomenon may contribute to the decrease of platelet function during storage and the recovery of platelet function after transfusion.
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5'-Nucleotidase activity in vertebrate tissues reflects the activity of four different forms of enzymes, a surface-located GPI-anchored ecto-form (e-N), a soluble form derived from it by cleavage of the membrane anchor (e-N(s)), and two cytosolic enzymes (c-N-I and c-N-II). Only the surface-located ecto-5'nucleotidase (e-N) has been characterized in molecular terms. It is expected that future sequence work will reveal that the closely related cytoplasmic forms c-N-I and c-N-II represent different genomic products. As yet the possibility of tissue-specific forms of 5'-nucleotidase as well as of splicing variants cannot be excluded. The e-N form reveals significant sequence identities with multispecific nucleotidases in bacteria, indicating common origin. Multispecific 5'-nucleotidases also occur in yeast but plant enzymes generally display marked substrate specificity. A characterization of the three-dimensional structure and of the active site would provide a basis for the interpretation of substrate specificities. The production of nucleosides is the general result of 5'nucleotidase activity. But the physiological implication of this catalytic process at thc cellular or tissue level varies. The intracellular enzymes c-N-I and c-N-II are involved in the catabolism of AMP and IMP and also of other mononucleotides and correspondingly produce the nucleoside. They are thought to be of particular importance for the hydrolysis of AMP and IMP in situations of low energy charge. The stimulatory effect of ATP and/or ADP on both affinity and rate seems to be of major physiological significance. A detailed analysis of the general biochemical properties with highly purified enzyme preparations is essential for setting the functional interpretations on a reliable basis. Furthermore, any general functional interpretation of the cytosolic enzymes requires both intimate knowledge of other cytosolic enzymes capable of hydrolysing 5'-mononucleotides as well as of the actual free concentrations of the metabolites concerned. Thc surface-located enzyme is involved in a number of functions which presumably do not mutually exclude each other. They rather supplement each other in their contribution to the physiology of cellular metabolism and tissue function. It is important to note that expression of ecto-5'-nucleotidase does not occur on all cells and can be developmentally regulated. Ecto-5'-nucleotidase is involved in the salvage of extracellular nucleotides. In addition, the enzyme has a major role in the control of tissue homeostasis. It effects the final step in the complete hydrolysis of thc extracellular messenger ATP. and, at the same time, produces adenosine as a further extracellular messenger. But this polysialyated GPI-anchored metalloprotein might not only function as an enzyme. The increased expression of the protein at the surface of activated cells and during certain stages of cell maturation, its capability to bind fibronectin as well as the presence of the HNK-I epitope in some tissues implies an involvement in cellular interactions such as cell recognition and cell adhesion or in cell matrix relations. This calls for a detailed analysis also of the carbohydrate chains, including possible developmental changes in the glycosylation pattern. Studies with lymphocytes even imply a function in signal transfer from the cell surface to the interior. Possibly a new mechanism for activation of intracellular messengers via GPI-anchored proteins will evolve. Finally, consensus should be sought on 5'-nucleotidase nomenclature. Since conventional enzyme nomenclature groups all forms described into one category the literature has become confusing. For example, at the bacterial level, periplasmic 5'nuclcotidase is multispecific and its relation to the vertebrate ecto-5'-nucleotidase can only be deduced from the primary structure. The cytolic 5'-nucleotidases have kinetic properties and physiological functions completely different from those of the ectoenzyme and the soluble form derived from it. It is suggested that current enzyme nomenclature is extended by introducing subclasses of EC 3.1.3.5: 1 for c-N-I, 2 for c-N-II, and 3 for e-N and e-N(s). 5'-Nucleotidases from bacteria and plants could then be grouped according to their sequence identities with one of the vertebrate enzymes.
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The distribution, biochemical properties, and function of CD39 were characterized with the use of a new mAb termed 400. CD39 is an acidic (isoelectric point, approximately 4.2) glycoprotein of Mr approximately 78,000, containing approximately 24 kDa of N-linked oligosaccharide but no detectable O-linked sugars. CD39 was not expressed by resting blood T, B, or NK cells, neutrophils, or monocytes, but was expressed on activated NK cells, B cells, subsets of T cells, and T cell clones. Furthermore, the pattern of expression of CD39 was distinct from the "classic" activation Ag CD25 and CD71, inasmuch as it was expressed long after expression of CD25 and CD71 had returned to basal levels. CD39 was easily detectable on EBV-transformed B cell lines but was absent from pre-B and non-EBV-transformed B cell lines, most myeloid cell lines, and leukemic T cell lines. In lymphoid tissues, germinal center cells expressed little or no CD39, whereas some paracortical lymphocytes and most macrophages and dendritic cells were positive. CD39 was strongly expressed by endothelium in all tissues examined, including skin, and was present on some, but not all, endothelial cell lines propagated in vitro. Interestingly, mAb binding to certain epitopes on CD39 induced rapid homotypic adhesion that appeared to involve LFA-1 (CD11a/CD18), but was morphologically and kinetically distinct from that induced by PMA. Anti-CD39 mAb also induced homotypic adhesion in an CD11/CD18-EBV-transformed B cell line derived from a patient with severe leukocyte adhesion deficiency. This adhesion was unaffected by EDTA, suggesting that this pathway of anti-CD39-induced homotypic adhesion was not mediated by any of the known integrins. These studies suggest that CD39 is involved in the cellular signaling that regulates adhesion.
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In previous views the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. However, research of the last decades has revealed that nucleotides also occur in the extracellular space. This survey deals with the sources, metabolism and the role in blood of the extracellular adenine mononucleotides ATP, ADP, AMP and the dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). The latter two are novel compounds, which have recently been discovered in human platelets. The mononucleotides originate from damaged tissues, from red blood cells during haemolysis, from activated platelets, the working muscle and from the nervous system, whereas the dinucleotides are exclusively released from stimulated platelets. Both the adenine mono- and the dinucleotides act as signal molecules on blood cells as well as on cells of the vascular wall, thereby modulating physiological processes such as platelet aggregation, histamine release from mast cells, regulation of vascular tone and white cell functions. In order to limit the signal effects of extracellular nucleotides, blood cells, plasma and the interior of the vessel walls are provided with nucleotide splitting enzymes: ATP, ADP and AMP are mainly degraded by ectoenzymes present on blood cells, endothelial and on smooth muscle cells, whereas dinucleotides are primarily metabolized by plasma enzymes. This review closes with the presentation of the clinical utility of Ap3A and Ap4A as tools for the diagnosis of platelet storage pool defects.