Increased platelet activation in sleep apnea subjects with intermittent hypoxemia.

PURPOSE: Obstructive sleep apnea (OSA) is independently associated with increased risk for stroke and other cardiovascular diseases. Since activated platelets play an important role in cardiovascular disease, the objective of this study was to determine whether platelet reactivity was altered in OSA subjects with intermittent nocturnal hypoxemia. METHODS: Thirty-one subjects, without hypertension or cardiovascular disease and not taking medication, participated in the study. Subjects were stratified based on OSA-related oxygen desaturation index (ODI) recorded during overnight polysomnography. Platelet reactivity to a broad panel of agonists (collagen, thrombin, protease-activated receptor1 hexapeptide, epinephrine, ADP) was measured by monitoring platelet aggregation and ATP secretion. Expression of platelet activation markers CD154 (CD40L) and CD62P (P-selectin) and platelet-monocyte aggregates (PMA) was quantified by flow cytometry. RESULTS: Epinephrine-induced platelet aggregation was substantially decreased in OSA subjects with significant intermittent hypoxemia (ODI ≥ 15) compared with subjects with milder hypoxemia levels (ODI < 15) (area under curve, p = 0.01). In addition, OSA subjects with ODI ≥ 15 exhibited decreased thrombin-induced platelet aggregation (p = 0.02) and CD40L platelet surface expression (p = 0.05). Platelet responses to the other agonists, CD62P platelet surface expression, and PMA levels were not significantly different between groups. Reduction in platelet responses to epinephrine and thrombin, and decreased CD40L surface marker expression in significant hypoxemic OSA individuals, is consistent with their platelets being in an activated state. CONCLUSIONS: Increased platelet activation was present in otherwise healthy subjects with intermittent nocturnal hypoxemia due to underlying OSA. This prothrombotic milieu in the vasculature is likely a key contributing factor toward development of thrombosis and cardiovascular disease. TRIAL REGISTRATION: NCT00859950.

Elucidation of the thromboregulatory role of CD39/ectoapyrase in the ischemic brain.

Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4-6 (apyrase-conserved regions 2-4) with a PGKneo cassette. Although CD39 mRNA 3' of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39(-/-) mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke.

CD39/NTPDase-1 activity and expression in normal leukocytes.

INTRODUCTION: CD39/NTPDase-1 is a cell surface enzyme expressed on leukocytes and endothelial cells that metabolizes ATP to ADP and AMP. CD39 is expressed on numerous different types of normal leukocytes, but details of its expression have not been determined previously. METHODS: We examined CD39 expression and activity in leukocytes isolated from healthy volunteers. Expression of CD39 on leukocytes was measured by FACS and activity of CD39 in lymphocytes and neutrophils was determined by an enzymatic radio-TLC assay. RESULTS: We established that CD39 is expressed on neutrophils, lymphocytes, and monocytes. The enzyme is found on >90% of monocytes, neutrophils, and B-lymphocytes, and 6% of T-lymphocytes and natural killer cells. Per cell density of expression varied, with the highest expression on monocytes and B-lymphocytes. ATPase and ADPase activities were highest on B-lymphocytes, lower on neutrophils, lowest on T-lymphocytes. The ratio of ADPase:ATPase activity was 1.8 for neutrophils and B-lymphocytes and 1.4 for T-lymphocytes. Hypertensive volunteers had lower levels of CD39 on their T-lymphocytes and NK cells. No correlation between age, gender, ethnic background, or cholesterol level and CD39 expression was observed. CONCLUSIONS: We conclude that CD39 activity and expression are present to varying degrees on all leukocytes types examined. Differences between leukocyte types should be considered when examining CD39 in disease states.

The ratio of ADP- to ATP-ectonucleotidase activity is reduced in patients with coronary artery disease.

INTRODUCTION: CD39 (NTPDase1), an endothelial cell membrane glycoprotein, is the predominant ATP diphosphohydrolase (ATPDase) in vascular endothelium. It hydrolyses both triphosphonucleosides and diphosphonucleosides at comparable rates, thus terminating platelet aggregation and recruitment responses to ADP and other platelet agonists. This occurs even when nitric oxide (NO) formation and prostacyclin production are inhibited. Thus, CD39 represents the main control system for platelet reactivity. Reduced or deficient local ecto-nucleotidase activity may predispose to development of vascular disease. Based on data in animal models and in vitro, CD39 constitutes a new therapeutic modality for vascular disease with a novel and unique mode of action. MATERIALS AND METHODS: Lymphocytes were isolated from 46 patients with angiographically proven coronary artery disease (CAD) as well as from matched healthy control subjects. Ectonucleotidase ADPase and ATPase activities (prototypical for the ATPDase activity of endothelial cells) were measured using established radio-TLC procedures. RESULTS AND DISCUSSION: In the patients, a decreased ratio of ADPase to ATPase activities (from 1.26 to 1.04) was observed despite increases in both ADPase and ATPase activities. Coronary artery disease was the only independent predictor of a difference in the ADPase/ATPase activity ratio by multivariate linear regression analysis (P=0.0035). This altered ADPase/ATPase activity ratio in patients may represent a reduction in endogenous defense systems against platelet-driven thrombotic events. These data may identify a population of patients with excessive platelet reactivity in their circulation. Increased generation of prothrombotic ADP in these patients implies a potential benefit from therapeutic intervention with soluble forms of CD39.

Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

CD39 expression on T lymphocytes correlates with severity of disease in patients with chronic lymphocytic leukemia.

INTRODUCTION: Chronic lymphocytic leukemia (CLL) is a B-cell disorder, but it is also associated with abnormalities in T-lymphocyte function. In this study we examine changes in T-lymphocyte CD39 and CD73 expression in patients with CLL. METHODS: Blood samples were drawn from 34 patients with CLL and 31 controls. The cells were stained for CD3, CD4, CD8, CD19, CD39, and CD73 and analyzed by flow cytometry. RESULTS: Overall, patients with CLL had a higher percentage of CD39(+) T lymphocytes than did controls. The percentage of cells expressing CD39 was higher in both CD4(+) cells and CD8(+) cells. Higher CD3/CD39 expression was associated with a later disease stage. No correlations between T-lymphocyte CD39 levels and CD38 or Zap-70 expression were observed. In contrast, the percentage of T lymphocytes and B lymphocytes that expressed CD73 was decreased in patients with CLL. Average B-lymphocyte CD73 expression was decreased in CLL because the majority of CLL clones were CD73. However a minority of CLL clones were CD73(+), and patients with CD73(+) clones tended to have earlier stage disease. CONCLUSION: T-lymphocyte CD39 and CD73 expression may be useful prognostic markers in patients with CLL. Expression of CD73 on the malignant cell population in CLL may be a marker of better prognosis.

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

Role of CD39 (NTPDase-1) in thromboregulation, cerebroprotection, and cardioprotection.

Blood platelets maintain vascular integrity and promote primary and secondary hemostasis following interruption of vessel continuity. Biochemical or physical damage to coronary, carotid, or peripheral arteries promotes excessive platelet activation and recruitment culminating in vascular occlusion and tissue ischemia. Currently, inadequate therapeutic approaches to stroke and coronary artery disease (CAD) are a public health issue. Following our demonstration of neutrophil leukotriene production from arachidonate released from activated aspirin-treated platelets, we studied interactions among platelets and other blood cells. This led to concepts of transcellular metabolism and thromboregulation. Thrombosis has a proinflammatory component whereby biologically active substances are synthesized by different cell types that could not individually synthesize the metabolite(s). Endothelium controls platelet reactivity via at least three biochemical systems: autacoids leading to production of prostacyclin and nitric oxide (NO) and endothelial ecto-adenosine phosphatase (ADPase)/CD39/nucleoside triphosphate diphosphohydrolase (NTPDase-1). The autacoids are fluid phase reactants, not produced by tissues in the basal state, but are only synthesized intracellularly and released upon interactions of cells with an agonist. When released, they exert fleeting actions in the immediate milieu and are rapidly inactivated. CD39 is an integral component of the endothelial cell (EC) surface and is substrate activated. It maintains vascular fluidity in the complete absence of prostacyclin and NO, indicating that the latter are ancillary components of hemostasis. Therapeutic implications for the autacoids have not been compelling because of their transient and local action and limited potency. Conversely, CD39, acting solely on the platelet releasate, is efficacious in animal models. It metabolically neutralizes a prothrombotic releasate via deletion of ADP-the major recruiting agent responsible for formation of an occlusive thrombus. In addition, solCD39 reduced adenosine triphosphate (ATP)- and ischemia-induced norepinephrine release in the heart. This action can prevent fatal arrhythmia. Moreover, solCD39 ameliorated the sequelae of stroke in cd39 null mice. Thus, CD39 represents the next generation of cardioprotective and cerebroprotective molecules. This article focuses on our interpretations of recent data and their implications for therapeutics.

Roles of Asp54 and Asp213 in Ca2+ utilization by soluble human CD39/ecto-nucleotidase.

Soluble human CD39 (solCD39) rapidly metabolizes nucleotides, especially ADP released from activated platelets, thereby inhibiting further platelet activation and recruitment. Using alanine substitution mutagenesis, we established a functional role for aspartates D54 and D213 in solCD39. Kinetic analyses of D54A and D213A indicated decreased K(m)s of the mutants, compared to wild type, for the cofactor calcium and for the substrates ADP and ATP. These decreases in calcium and nucleotide affinity of the mutants were accompanied by increases in their rate of catalysis. The decreased affinity of the mutants for calcium was responsible for their diminished ability to reverse platelet aggregation in plasma anticoagulated with citrate, a known calcium chelator. Their ADPase activity in the presence of citrated plasma was also decreased, although this could be overcome with excess calcium. Thus, aspartates 54 and 213 are involved in calcium utilization and potentially involved in cation coordination with substrate in the catalytic pocket of solCD39.

EctoNucleotidase in cardiac sympathetic nerve endings modulates ATP-mediated feedback of norepinephrine release.

ATP, coreleased with norepinephrine, affects adrenergic transmission by acting on purinoceptors at sympathetic nerve endings. Ectonucleotidases terminate the actions of ATP. Previously, we had preliminary evidence for ectonucleotidase activity in cardiac sympathetic nerve terminals. Therefore, we investigated whether this ectonucleotidase might influence norepinephrine release in the heart. Sympathetic nerve endings isolated from guinea pig heart (cardiac synaptosomes) were rich in Ca(2+)-dependent ectonucleotidase activity, as measured by metabolism of exogenously added radiolabeled ATP or ADP. By its inhibitor profile, ectonucleotidase resembled ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1). Exogenous ATP elicited concentration-dependent norepinephrine release from cardiac synaptosomes (EC(50) 0.96 microM). This release was antagonized by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) and potentiated by the P2Y receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) (30 nM). Norepinephrine release promoted by ATP was also potentiated by the nucleotidase inhibitor 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) (30 microM) and blocked by a recombinant, soluble form of human E-NTPDase1 (solCD39). In contrast, ARL67156 had no effect on norepinephrine release induced by the nonhydrolyzable analog, alpha, beta-methyleneadenosine-5'-triphosphate (alpha,beta-MeATP). Depolarization of cardiac synaptosomes with K(+) elicited release of endogenous norepinephrine. This was attenuated by PPADS and solCD39 and potentiated by MRS 2179 and ARL67156. Importantly, our results demonstrate that facilitation of ATP-induced norepinephrine release from cardiac sympathetic nerves is a composite of two autocrine components: positive, mediated by P2X receptors, and negative, mediated by P2Y receptors. Modulation of norepinephrine release by coreleased ATP is terminated by endogenous as well as exogenous ectonucleotidase. We propose that ectonucleotidase control of norepinephrine release should provide cardiac protection in hyperadrenergic states such as myocardial ischemia.

Role of a novel soluble nucleotide phospho-hydrolase from sheep plasma in inhibition of platelet reactivity: hemostasis, thrombosis, and vascular biology.

Ecto- and exoenzymes that metabolize extracellular adenosine diphosphate (ADP), the major promoter of platelet activation and recruitment, are of potential clinical importance because they can metabolically prevent excessive thrombus growth. An ecto-ADPase (CD39, NTPDase1) has been identified on endothelial cells. We demonstrate that ADP and adenosine triphosphate (ATP) are rapidly metabolized to adenosine monophosphate (AMP) in sheep plasma at pH 7.4. This hydrolysis is sensitive to P(1), P(5)-di-(adenosine-5') pentaphosphate (Ap(5)A), and ethylene glycol bis (beta-aminoethyl ether) - N, N, N(-), N(-) tetra-acetate (EGTA) but insensitive to tetramisole (an alkaline phosphatase inhibitor). A specific phosphodiesterase substrate, p -nitrophenol-5'-thymidine monophosphate (TMP) (p -Nph-5'-TMP), was readily hydrolyzed in sheep plasma at a rate of approximately 0.25 nmol/min/mg protein, and this hydrolysis was inhibited by ADP, ATP, and Ap(5)A. Furthermore, 200-fold purified p -Nph-5'-TMP-hydrolyzing activity also hydrolyzed ATP and ADP directly to AMP. When ADP was preincubated in plasma, its ability to induce platelet aggregation was inhibited in a time-dependent manner. This effect was abolished by Ap(5)A. The inhibitory effects on platelet aggregation correlated with hydrolysis of the ADP in plasma. These data suggest that the endogenous soluble plasma phosphohydrolase metabolizes ATP and ADP by means of cleavage of the alpha-beta-phosphodiester bond of nucleoside 5'-phosphate derivatives. This novel biochemical activity inhibits platelet reactivity through hydrolysis of extracellular nucleotides released by activated platelets during (patho)physiological processes, serving a homeostatic and antithrombotic function in vivo.

Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases.

Platelets are responsible for maintaining vascular integrity. In thrombocytopenic states, vascular permeability and fragility increase, presumably due to the absence of this platelet function. Chemical or physical injury to a blood vessel induces platelet activation and platelet recruitment. This is beneficial for the arrest of bleeding (hemostasis), but when an atherosclerotic plaque is ulcerated or fissured, it becomes an agonist for vascular occlusion (thrombosis). Experiments in the late 1980s cumulatively indicated that endothelial cell CD39-an ecto-ADPase-reduced platelet reactivity to most agonists, even in the absence of prostacyclin or nitric oxide. As discussed herein, CD39 rapidly and preferentially metabolizes ATP and ADP released from activated platelets to AMP, thereby drastically reducing or even abolishing platelet aggregation and recruitment. Since ADP is the final common agonist for platelet recruitment and thrombus formation, this finding highlights the significance of CD39. A recombinant, soluble form of human CD39, solCD39, has enzymatic and biological properties identical to the full-length form of the molecule and strongly inhibits human platelet aggregation induced by ADP, collagen, arachidonate, or TRAP (thrombin receptor agonist peptide). In sympathetic nerve endings isolated from guinea pig hearts, where neuronal ATP enhances norepinephrine exocytosis, solCD39 markedly attenuated norepinephrine release. This suggests that NTPDase (nucleoside triphosphate diphosphohydrolase) could exert a cardioprotective action by reducing ATP-mediated norepinephrine release, thereby offering a novel therapeutic approach to myocardial ischemia and its consequences. In a murine model of stroke, driven by excessive platelet recruitment, solCD39 reduced the sequelae of stroke, without an increase in intracerebral hemorrhage. CD39 null mice, generated by deletion of apyrase-conserved regions 2 to 4, exhibited a decrease in postischemic perfusion and an increase in cerebral infarct volume when compared with controls. "Reconstitution" of CD39 null mice with solCD39 reversed these changes. We hypothesize that solCD39 has potential as a novel therapeutic agent for thrombotic diatheses.

Role of extracellular ATP metabolism in regulation of platelet reactivity.

Extracellular adenosine triphosphate (ATP) regulates platelet reactivity by way of direct action on platelet purinergic receptors or by hydrolysis to adenosine diphosphate (ADP). Subsequent metabolism of ATP and ADP to adenosine monophosphate (AMP) and adenosine inhibits platelet aggregation. Endothelial cell membrane-bound ecto-ATP/ADPase (CD39, E-NTPDase1) is thought to be the main regulator of platelet responsiveness. However, the findings in studies of CD39-knockout mice imply that nucleotidase(s) in plasma regulates circulating adenine nucleotides levels. Understanding extracellular ATP metabolism by CD39 and plasma nucleotidases is therefore important. In this study, alpha-phosphorus 32- and gamma-phosphorus 32-labeled ATP were rapidly metabolized directly to AMP and pyrophosphate in human plasma at pH 7.4, suggesting the presence of pyrophosphatase/phosphodiesterase-like activity. A specific phosphodiesterase substrate, p-nitrophenol-5'-TMP (p-Nph-5'-TMP), was readily hydrolyzed in human plasma. The antiaggregatory action of beta,gamma-methylene-ATP (AMPPCP) (5 micromol/L) was blocked by DMPX, an adenosine-receptor antagonist, suggesting that in plasma, AMPPCP was metabolized to AMP and adenosine. Recombinant soluble CD39 (solCD39) was used to assess the role of CD39 in ATP metabolism. As little as 0.25 microg/mL of solCD39 inhibited ADP-induced platelet aggregation. However, in the presence of ADP-free ATP (10 micromol/L), solCD39 induced platelet aggregation in a dose-dependent manner. Because AMPPCP could not substitute for ATP in solCD39-stimulated platelet aggregation, it is likely that ADP formation from ATP was required. Endogenous CD39 may thus have a hemostatic function by promoting ADP formation from released ATP, in addition to its antiaggregatory properties. A plasma nucleotidase hydrolyzes ATP directly to AMP. This prevents ADP accumulation and generates adenosine, a potent, locally acting inhibitor of platelet reactivity. The presence of both endothelial CD39 and plasma nucleotidase appears to be important in the maintenance of normal hemostasis and prevention of excessive platelet responsiveness.

Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We report here that induction of ectoATPase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is cell-type specific and not a generalized response to aryl hydrocarbon (Ah) receptor activation. TCDD increased [14C]-ATP and -ADP metabolism in two mouse hepatoma lines, Hepa1c1c7 and Hepa1-6 cells, but not in human hepatoma HepG2 or HuH-7 cells, human umbilical vein endothelial cells (HUVEC), chick hepatoma (LMH) cells, or chick primary hepatocytes or cardiac myocytes, even though all of those cell types were Ah receptor-responsive, as evidenced by cytochrome P4501A induction. To determine whether the differences in ectonucleotidase responsiveness to TCDD might be related to differences in cell-type ectonucleotidase expression, ATP and ADP metabolite patterns, the products of several classes of ectonucleotidases including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectophosphodiesterase/pyrophosphatases (E-NPP enzymes) and ectoalkaline phosphatase activities were examined. Those patterns, together with results of enzyme assays, Western blotting, or semiquantitative RT-PCR show that NTPDase2 is the main ectonucleotidase for murine and human hepatoma cells, NTPDase3 for chick hepatocytes and LMH cells, and an E-NPP enzyme for chick cardiac myocytes. Evidence for NTPDase2 expression was lacking in all cells except the mouse and human hepatoma cells. TCDD increased expression of the NTPDase2 gene but only in the mouse and not in the human hepatoma cells. TCDD did not increase NTPDase3, NTPDase1, E-NPP, or alkaline phosphatase in any of the cell types examined. The failure of TCDD to increase ATP metabolism in HUVEC, chick LMH cells, hepatocytes, and cardiac myocytes can be attributed to their lack of NTPDase2 expression, while the increase in ATP metabolism by TCDD in the mouse but not the human hepatoma cells can be explained by differences in TCDD effects on mouse and human hepatoma NTPDase2 gene expression. In addition to characterizing effects of TCDD on ectonucleotidases, these studies reveal major differences in the complements of ectonucleotidases present in different cell types. It is likely that such differences are important for cell-specific susceptibility to extracellular nucleotide toxicity and responses to purinergic signaling.