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Ectonucleotide Triphosphate Diphosphohydrolase-1 (CD39) Mediates Resistance to Occlusive Arterial Thrombus Formation after Vascular Injury in Mice

  • Zachary M. Huttinger
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • Michael W. Milks
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • Michael S. Nickoli
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • William L. Aurand
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • Lawrence C. Long
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • Debra G. Wheeler
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
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  • Karen M. Dwyer
    Affiliations
    Immunology Research Centre, St. Vincent's Hospital Melbourne, Australia

    Department of Medicine, University of Melbourne, Australia
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  • Anthony J.F. d'Apice
    Affiliations
    Immunology Research Centre, St. Vincent's Hospital Melbourne, Australia

    Department of Medicine, University of Melbourne, Australia
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  • Simon C. Robson
    Affiliations
    Transplant Institute, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts
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  • Peter J. Cowan
    Affiliations
    Immunology Research Centre, St. Vincent's Hospital Melbourne, Australia

    Department of Medicine, University of Melbourne, Australia
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  • Richard J. Gumina
    Correspondence
    Address reprint requests to Richard J. Gumina, M.D., Ph.D., Interventional Cardiovascular Research, Division of Cardiovascular Medicine, The Ohio State University, Davis Heart and Lung Research Institute, Suite 200, 473 W. 12th Ave., Columbus, OH 43210-1252
    Affiliations
    Division of Cardiovascular Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio

    Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio
    Search for articles by this author
Open AccessPublished:May 21, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.03.024
      Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl3-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5′-0-(2-thiodiphosphate) trilithium salt, Ado-5′-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5′-0-(2-thiodiphosphate) trilithium salt, Ado-5′-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein αIIb3, in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A2A or A2B adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.
      Arterial thrombosis secondary to rupture of an atherosclerotic plaque is the underlying event in the majority of acute myocardial infarctions and the leading cause of death in the westernized world.
      • Libby P.
      Current concepts of the pathogenesis of the acute coronary syndromes.
      Atherosclerotic plaque rupture exposes the subendothelial matrix, which leads to platelet activation secondary to the convergence of numerous signaling cascades that release platelet-dense granules, increasing local concentrations of the purinergic mediators, ATP and ADP.
      • Massberg S.
      • Schulz C.
      • Gawaz M.
      Role of platelets in the pathophysiology of acute coronary syndrome.
      • Freedman J.E.
      Molecular regulation of platelet-dependent thrombosis.
      Engagement of specific receptors on platelets results in further activation and recruitment of platelets into the growing thrombus.
      • Massberg S.
      • Schulz C.
      • Gawaz M.
      Role of platelets in the pathophysiology of acute coronary syndrome.
      • Freedman J.E.
      Molecular regulation of platelet-dependent thrombosis.
      After thrombotic coronary arterial occlusion, myocardial ischemia and subsequent necrosis ensues, resulting in myocardial damage and dysfunction if blood flow is not restored promptly.
      An extracellular purinergic regulatory pathway is positioned uniquely to modulate thrombosis, inflammation, and myocardial ischemia-reperfusion injury.
      • Eltzschig H.K.
      • Ibla J.C.
      • Furuta G.T.
      • Leonard M.O.
      • Jacobson K.A.
      • Enjyoji K.
      • Robson S.C.
      • Colgan S.P.
      Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium.
      A key component of this pathway is ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD-1; CD39), a 70- to 100-kDa transmembrane protein expressed on platelets, endothelium, and leukocytes that hydrolyzes the proinflammatory prothrombotic molecules ATP and ADP to AMP.
      • Kaczmarek E.
      • Koziak K.
      • Sevigny J.
      • Siegel J.B.
      • Anrather J.
      • Beaudoin A.R.
      • Bach F.H.
      • Robson S.C.
      Identification and characterization of CD39/vascular ATP diphosphohydrolase.
      • Koziak K.
      • Sevigny J.
      • Robson S.C.
      • Siegel J.B.
      • Kaczmarek E.
      Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.
      AMP subsequently is converted by ecto-5′-nucleotidase (CD73) to the anti-inflammatory, anti-thrombotic, and cardiac protective compound, adenosine,
      • Colgan S.P.
      • Eltzschig H.K.
      • Eckle T.
      • Thompson L.F.
      Physiological roles for ecto-5′-nucleotidase (CD73).
      with multiple receptors existing for ATP, ADP, and adenosine.
      • Erlinge D.
      • Burnstock G.
      P2 receptors in cardiovascular regulation and disease.
      • Headrick J.P.
      • Peart J.N.
      • Reichelt M.E.
      • Haseler L.J.
      Adenosine and its receptors in the heart: regulation, retaliation and adaptation.
      Our laboratory recently showed that ENTPD-1 expression reduces myocardial infarct size after ischemia-reperfusion injury in both mouse and pig.
      • Cai M.
      • Huttinger Z.M.
      • He H.
      • Zhang W.
      • Li F.
      • Goodman L.A.
      • Wheeler D.G.
      • Druhan L.J.
      • Zweier J.L.
      • Dwyer K.M.
      • He G.
      • d'Apice A.J.
      • Robson S.C.
      • Cowan P.J.
      • Gumina R.J.
      Transgenic over expression of ectonucleotide triphosphate diphosphohydrolase-1 protects against murine myocardial ischemic injury.
      • Wheeler D.G.
      • Joseph M.E.
      • Mahamud S.D.
      • Aurand W.L.
      • Mohler P.J.
      • Pompili V.J.
      • Dwyer K.M.
      • Nottle M.B.
      • Harrison S.J.
      • d'Apice A.J.
      • Robson S.C.
      • Cowan P.J.
      • Gumina R.J.
      Transgenic swine: expression of human CD39 protects against myocardial injury.
      Furthermore, decreased ENTPD-1 activity has been shown in atherectomy specimens from patients with acute coronary syndromes, suggesting a pivotal role for ENTPD-1 in modulating the balance between a prothrombotic and antithrombotic milieu.
      • Hatakeyama K.
      • Hao H.
      • Imamura T.
      • Ishikawa T.
      • Shibata Y.
      • Fujimura Y.
      • Eto T.
      • Asada Y.
      Relation of CD39 to plaque instability and thrombus formation in directional atherectomy specimens from patients with stable and unstable angina pectoris.
      Given the complex regulation of the purinergic system in thrombosis, we hypothesized that overexpression of ENTPDase-1 may modulate in vivo large-conduit vessel arterial thrombosis by affecting both platelet reactivity and tissue factor expression levels. Here, we show that overexpression of human ENTPDase-1 in mice modulates purinergic signaling, which attenuates activation of the platelet fibrinogen receptor, glycoprotein αIIb3 (GP IIb/IIIa; CD41/CD61), and conveys resistance to in vivo thrombosis not only via hydrolysis of ADP but also through specific adenosine-receptor engagement.

      Materials and Methods

      Transgenic Mice

      The generation of the human ENTPDase-1–expressing mice has been described previously.
      • Dwyer K.M.
      • Robson S.C.
      • Nandurkar H.H.
      • Campbell D.J.
      • Gock H.
      • Murray-Segal L.J.
      • Fisicaro N.
      • Mysore T.B.
      • Kaczmarek E.
      • Cowan P.J.
      • d'Apice A.J.
      Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.
      The human ENTPD-1 (CD39) transgene is expressed from the mouse H-2Kb promoter, resulting in global expression of ENTPD-1 in these mice. Transgenic mice expressing human (ENTPD-1-Tg) were back-crossed for more than 10 generations onto the BALB/c background and compared with littermate controls. The investigations described conform to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by The Ohio State University Institutional Animal Care and Use Committee.

      Chemicals

      The following inhibitor of CD73 activity was used in these studies: α-β-methylene-ADP (APCP; Sigma-Aldrich, St. Louis, MO).
      • Eckle T.
      • Krahn T.
      • Grenz A.
      • Kohler D.
      • Mittelbronn M.
      • Ledent C.
      • Jacobson M.A.
      • Osswald H.
      • Thompson L.F.
      • Unertl K.
      • Eltzschig H.K.
      Cardioprotection by ecto-5′-nucleotidase (CD73) and A2B adenosine receptors.
      The following chemical antagonists of adenosine receptors were used in this study
      • Ralevic V.
      • Burnstock G.
      Receptors for purines and pyrimidines.
      • Auchampach J.A.
      • Kreckler L.M.
      • Wan T.C.
      • Maas J.E.
      • van der Hoeven D.
      • Gizewski E.
      • Narayanan J.
      • Maas G.E.
      Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.
      : 8-(p-sulfophenyl) theophylline (8-SPT), a nonselective antagonist for adenosine receptors A1, A2A, A2B, and A3 (Sigma-Aldrich); 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), a highly selective A2A adenosine antagonist (Tocris, Ellisville, MI); N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (MRS 1754), a selective A2B-receptor antagonist (Sigma-Aldrich). The following ADP mimetic, adenosine-5′-0-(2-thiodiphosphate) trilithium salt, Ado-5′-PP[S] (ADP-β-S; Sigma-Aldrich) was used.
      • Ralevic V.
      • Burnstock G.
      Receptors for purines and pyrimidines.

      In Vivo Carotid Thrombosis

      Ferric chloride (FeCl3)-induced carotid artery thrombosis was used.
      • Wang X.
      • Xu L.
      An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research.
      Wild-type (WT) or ENTPD-1-Tg mice were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg). Atropine (0.05 mg s.c.) was administered to reduce airway secretions. Animals were intubated and ventilated with room air (tidal volume, 250 μL; 150 breaths/minute) with a mouse respirator (Harvard Apparatus, Holliston, MA). Rectal temperatures were maintained at 37°C by a thermo-regulated heating pad. The left common carotid artery was dissected gently, a flow probe was placed on the artery (MA0.5PSB; Transonic Systems; Ithaca, NY), and blood flow was measured with a pulsed Doppler flow system. After obtaining baseline flow recordings, carotid artery injury was induced by application of filter paper saturated with 10% FeCl3 solution on the adventitial surface proximal to the flow probe for 3 minutes. The flow as a percentage of baseline and the time to thrombotic occlusion (blood flow, 0 mL/minute) was measured from the placement of the FeCl3-saturated filter paper. The surgeon was blinded to the animal genotype and any drug treatment during all experiments. Animals were reanesthetized as needed and at each hour under anesthesia the animals were administered normal saline intraperitoneally. For those animals treated with pharmacologic antagonists, the antagonists were dissolved in a final concentration of 0.5% dimethyl sulfoxide and administered at the designated dose as a single intraperitoneal injection 15 to 30 minutes before the application of FeCl3.
      For visualization of thrombus formation, mice were injected intravenously with 5 μL of a 100-μmol/L solution/gram of body weight of the cationic lipophilic dye 3,3′-dihexyloxacarbocyanine iodide 10 minutes before application of FeCl3 to induce vessel injury.
      • Hechler B.
      • Freund M.
      • Alame G.
      • Leguay C.
      • Gaertner S.
      • Cazenave J.P.
      • Petitou M.
      • Gachet C.
      The antithrombotic activity of EP224283, a neutralizable dual factor Xa inhibitor/glycoprotein IIbIIIa antagonist exceeds that of the coadministered parent compounds.
      The carotid artery was video recorded continuously using a Leica M165 FC fluorescent stereomicroscope (Leica, Wetzlar, Germany) equipped with a Hamamatsu ORCA-R2 Digital CCD camera (Hamamatsu, Hamamatsu City, Japan) for 30 minutes.

      Carotid Histology

      At 15 minutes after the application of FeCl3 animals were perfusion-fixed with 4% buffered paraformaldehyde and the injured and noninjured carotid arteries from WT or ENTPD-1-Tg mice were isolated. The samples were embedded in paraffin, sectioned at 3 μm, and stained with H&E.

      Whole-Blood Aggregation

      Blood was collected from the inferior vena cava of anesthetized WT or ENTPD-1-Tg mice into 70 U/mL of sodium heparin and then diluted 1:2 into physiological saline and whole-blood aggregometry was performed on these diluted samples.
      • Henry M.
      • Davidson L.
      • Cohen Z.
      • McDonagh P.F.
      • Nolan P.E.
      • Ritter L.S.
      Whole blood aggregation, coagulation, and markers of platelet activation in diet-induced diabetic C57BL/6J mice.
      One milliliter of diluted blood was placed into 2-mL cuvettes and incubated for 6 minutes at 37°C in the Chrono-Log Whole Blood Lumi-Ionized Calcium Aggregometer (Chrono-Log Corp., Havertown, PA). The cuvettes then were placed into the test chamber and incubated with the impedance electrode at 37°C while stirring at 900 rpm. After 2 minutes of stabilization, ADP (20 μmol/L final concentration; Chrono-Log Corp., Havertown, PA) or ADP-β-S (20 μmol/L final concentration; Sigma-Alrich) was added. Aggregation, measured as ohms of electrical impedance, was recorded for up to 6 minutes. All whole-blood aggregometry tests were performed within 2 hours of blood acquisition.

      Flow Cytometric Analysis

      Whole blood was obtained from the inferior vena cava of anesthetized WT or ENTPD-1-Tg–expressing mice. All antibodies were obtained from EMFRET Analytics GmbH & Co. KG (Eibelstadt, Germany). Whole-blood fluorescence-activated cell sorting analysis for the levels of the total fibrinogen receptor, glycoprotein αIIb3 complex expression (clone Leo.H4; Rat IgG2b), was examined at baseline to ensure that there was no difference in the level of these critical platelet membrane proteins involved in platelet activation and thrombosis. Platelet activation was analyzed by activation of diluted whole blood with 20 μmol/L ADP and activated GP αIIb3 complex (clone JON/A; Rat IgG2b)
      • Bergmeier W.
      • Schulte V.
      • Brockhoff G.
      • Bier U.
      • Zirngibl H.
      • Nieswandt B.
      Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody.
      expression was examined by flow cytometric analysis at specified times to examine the level of platelet activation (BD LSR II Flow Cytometer; Becton Dickinson, Franklin Lakes, NJ). Nonspecific staining was determined with isotype control antibodies.

      ENTPDase Activity Assay

      Total platelet lysates were assayed for ENTPDase activity as previously described.
      • Dwyer K.M.
      • Robson S.C.
      • Nandurkar H.H.
      • Campbell D.J.
      • Gock H.
      • Murray-Segal L.J.
      • Fisicaro N.
      • Mysore T.B.
      • Kaczmarek E.
      • Cowan P.J.
      • d'Apice A.J.
      Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.
      One unit of ATP diphosphohydrolase activity corresponds to the release of 1 μmol inorganic phosphate/minute at 37°C.
      • Imai M.
      • Kaczmarek E.
      • Koziak K.
      • Sevigny J.
      • Goepfert C.
      • Guckelberger O.
      • Csizmadia E.
      • Schulte Am
      • Esch 2nd, J.
      • Robson S.C.
      Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells.

      Quantification of Vascular Tissue Factor Expression and Platelet P2Y1- and P2Y12-Receptor Levels

      Vascular tissue factor (TF) was measured semiquantitatively using Western blot analysis as published previously.
      • White T.A.
      • Pan S.
      • Witt T.A.
      • Simari R.D.
      Murine strain differences in hemostasis and thrombosis and tissue factor pathway inhibitor.
      Aortas were removed, cleaned of adventitia, placed in ice-cold phosphate-buffered saline, and then homogenized using an Omni tissue homogenizer (model TH-794; Omni International, Marietta, GA) in lysis buffer consisting of 30 mmol/L 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, 10 μmol/L E64, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L EDTA in PBS (pH 7.4). For platelet homogenates, whole blood was obtained from the inferior vena cava of anesthetized WT or ENTPD-1-Tg–expressing mice, diluted 1:1 in physiological saline, and layered onto platelet Fico/Lite (Atlanta Biologicals, Lawrenceville, GA). Platelet-rich plasma was generated by centrifugation at 350 × g for 15 minutes. Platelets then were washed with physiological saline and pelleted. Samples from 3 mice were pooled and homogenized in lysis buffer consisting of 50 mmol/L Tris (pH 7.4), 150 μmol/L NaCl, 0.5% Nonidet P-40, 1 mmol/L sodium pyrophosphate, 5 mmol/L sodium vanadate, 1 mmol/L benzamidine, and 1 mmol/L sodium fluoride with protease inhibitor cocktail (Sigma-Aldrich).
      The crude homogenates were centrifuged at 10,000 × g, and supernatants were transferred and stored at −80°C until analyzed. Protein content of supernatants was measured using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (12 μg for TF, 15 μg for P2Y1 and P2Y12) were diluted 1:2 in Laemmli sample buffer (Bio-Rad) and placed in a boiling water bath for 5 minutes. Samples then were run on 12.5% Criterion ready gels (Bio-Rad) using SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes, and blocked overnight in Tris-buffered saline (pH 7.4) with 0.05% Tween 20 (Sigma-Aldrich) and 5% powdered milk. Membranes were incubated with primary antibodies to mouse TF (1:500, 12 hours; American Diagnostica, Stamford, CT) or mouse P2Y12 receptor (1:1000, 12 hours; AnaSpec, Freemont, CA), mouse P2Y1 (1:400, 12 hours; Alomone, Jerusalem, Israel), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:15,000, 12 h; Abcam, Inc, Cambridge, MA), and after washing were incubated for 45 minutes with horseradish-peroxidase–conjugated secondary antibody. Immunoblots were developed using Supersignal (Pierce, New Haven, CT) and quantified by densitometry (ChemiDoc; Bio-Rad). TF, P2Y1, and P2Y12 were normalized to GAPDH and data are presented as the relative quantification of TF P2Y1 and P2Y12 (relative to each individual sample GAPDH).

      Statistical Analysis

      The results of experiments were analyzed by several statistical methods using standard software (eg, GraphPad Prism, version 4.0, San Diego, CA). Results were expressed as mean ± standard error of the mean. For comparison between 2 groups, significance was determined by an unpaired Student's t-test. For comparison of multiple groups, multifactorial analysis of variance with post hoc comparison of the means with Bonferroni correction was used to determine statistical significance. For all evaluations, probability values less than 0.05 were considered significant.

      Results

      Overexpression of ENTPDase-1 Markedly Delays Formation of Occlusive Thrombus Formation in Vivo

      Analysis of platelets from human ENTPDase-1–expressing mice revealed a twofold increase in ENTPDase activity and increased surface level expression of ENTPD-1 compared with WT control platelets consistent with a prior study showing increased expression on the platelets, endothelium, and leukocytes of the transgenic mice on a different genetic background (C57BL/6) (data not shown).
      • Dwyer K.M.
      • Robson S.C.
      • Nandurkar H.H.
      • Campbell D.J.
      • Gock H.
      • Murray-Segal L.J.
      • Fisicaro N.
      • Mysore T.B.
      • Kaczmarek E.
      • Cowan P.J.
      • d'Apice A.J.
      Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.
      No differences in the complete blood count were observed between WT and ENTPD-1-Tg mice (Table 1). To determine whether ENTPDase-1 overexpression translated to a resistance to in vivo conduit artery thrombosis, WT or ENTPD-1-Tg mice were evaluated in a model of FeCl3-induced carotid artery thrombosis.
      • Wang X.
      • Xu L.
      An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research.
      In WT animals there was an initial increase in carotid flow followed by a precipitous decrease in flow to zero that correlated in vivo with the formation of an acute occlusive thrombus. However, in ENTPD-1–expressing animals, although there was no increase in flow observed after ferric chloride application and there was a gradual decrease in carotid flow to approximately 70% of the baseline flow, no occlusive thrombus was formed within the injured vessel (Figure 1, A and B). These findings were confirmed by histologic assessment of the carotid arteries after FeCl3 treatment that revealed occlusive thrombus present in the injured WT animals by 15 minutes, but not in the injured carotid arteries of ENTPD-1-Tg animals (data not shown).
      Table 1Complete Blood Count from WT and ENTPD-1-Tg Mice
      ParameterWT (n = 6)ENTPD-1-Tg (n = 6)
      WBC (K/μL)5.7 ± 0.87.5 ± 1
       NE (%)26.9 ± 1.625.6 ± 1.6
       LY (%)60.7 ± 2.161.7 ± 1.7
       MO (%)9.2 ± 1.29.6 ± 1.2
       EO (%)2.4 ± 0.32.4 ± 0.6
       BA (%)0.8 ± 0.20.6 ± 0.1
      HCT (%)51.9 ± 5.354.9 ± 5.4
      RBC (K/μL)10.1 ± 1.010.8 ± 1.2
      Hb (g/dL)17.1 ± 0.816.2 ± 0.8
      MCV (fL)51.4 ± 0.451.2 ± 0.8
      MCHC (g/dL)34.2 ± 2.630.9 ± 3.0
      RDW (%)15.4 ± 0.215.6 ± 0.3
      Retics (%)1.3 ± 0.21.1 ± 0.4
      PLT (K/μL)1295.7 ± 52.61128.7 ± 98.5
      MPV (fL)5.3 ± 0.15.1 ± 0.2
      None of the data were found to be statistically significantly different.
      BA, basophills; EO, eosinophils; Hb, hemoglobin; HCT, hematocrit; LY, lymphocytes; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MO, monocytes; MPV, mean platelet volume; NE, neutrophils; PLT, platelets; RBC, red blood cells; RDW, red cell differential width; Retics, reticulocyte; WBC, white blood cells.
      Figure thumbnail gr1
      Figure 1ENTPD-1 conveys resistance to in vivo thrombosis. A: Carotid flow after FeCl3 application is expressed as a percentage of baseline flow in WT and ENTPD-1-Tg animals. B: In vivo real-time fluorescence imaging of thrombus formation in WT and ENTPD-1-Tg animals after FeCl3 application. C: Time to thrombosis after FeCl3 application in WT and ENTPD-1-Tg mice (WT: 13.7 ± 0.9 minutes; ENTPD-1-Tg: 281.5 ± 57.3 minutes; N = 8 per group) and the effect of ADP-β-S treatment on the time to thrombosis after FeCl3 application in WT and ENTPD-1-Tg mice (ADP- β-S: WT: 11.3 ± 1.53 minutes versus ENTPD-1-Tg: 17.1 ± 2.59 minutes; N = 3 per group). D: Tissue factor expression on aortae from WT and ENTPD-1 mice. Representative immunoblot and normalized densitometry (relative TF/GAPDH densitometric ratios: WT: 0.20 ± 0.044; ENTPD-1-Tg: 0.21 ± 0.033; P = 0.85; N = 3 per group). Values are mean ± standard error of the mean. *P < 0.001; P < 0.05; TG, ectonucleoside triphosphate diphosphohydrolase-1 expressing animals.
      To determine the absolute extension of the time to thrombosis conveyed by ENTPD-1 expression, a separate group of animals was monitored until occlusion was observed. ENTPD-1 expression profoundly delayed the time to occlusion (WT: 13.7 ± 0.88 minutes, N = 14 versus ENTPD-1-Tg: 281.5 ± 57.31 minutes, N = 8; P < 0.001; Figure 1C). Given the role of TF in ferric chloride–induced carotid thrombosis,
      • Wang L.
      • Miller C.
      • Swarthout R.F.
      • Rao M.
      • Mackman N.
      • Taubman M.B.
      Vascular smooth muscle-derived tissue factor is critical for arterial thrombosis after ferric chloride-induced injury.
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      • Mackman N.
      Tissue factor and thrombosis: the clot starts here.
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      Endothelial-derived tissue factor pathway inhibitor regulates arterial thrombosis but is not required for development or hemostasis.
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      • Ruggeri Z.M.
      • Ruf W.
      P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice.
      as well as prior studies showing an increase in TF levels in ENTPD-1 knockout mice
      • Enjyoji K.
      • Sevigny J.
      • Lin Y.
      • Frenette P.S.
      • Christie P.D.
      • Esch 2nd, J.S.
      • Imai M.
      • Edelberg J.M.
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      • Wagner D.D.
      • Robson S.C.
      • Rosenberg R.D.
      Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.
      and decreased expression of TF on endothelial cell cultures treated with adenosine,
      • Deguchi H.
      • Takeya H.
      • Urano H.
      • Gabazza E.C.
      • Zhou H.
      • Suzuki K.
      Adenosine regulates tissue factor expression on endothelial cells.
      the level of TF in the vasculature was measured and found not to differ between WT and ENTPD-1-Tg aortae at baseline (relative TF/GAPDH densitometric ratios were as follows: WT, 0.20 ± 0.044; ENTPD-1-Tg, 0.21 ± 0.033; P > 0.05; N = 3 per group; Figure 1D). Furthermore, strain variations with regard to coagulation and thrombosis parameters did not account for the resistance to occlusive thrombus formation because C57BL/6 background mice overexpressing human ENTPD-1 showed a comparable prolongation in the time to thrombosis (data not shown). Thus, overexpression of ENTPD-1 conveys a marked protection against the generation of an occlusive arterial thrombus in vivo.

      ADP-Receptor Engagement in ENTPDase-1–Mediated Resistance to Thrombosis

      As stated earlier, ENTPDase-1 sequentially converts extracellular ATP and ADP to AMP. ADP is a potent platelet agonist required for the continued expression of activated glycoprotein αIIb3-complex. To determine whether the ENTPDase-1–mediated in vivo resistance to thrombosis involved ADP hydrolysis, animals were treated with the nonhydrolyzable ADP analog (ADP-β-S). Treatment with 6.25 mg/kg ADP-β-S, administered intravenously, 10 minutes before the application of ferric chloride, resulted in abrogation of the antithrombotic efficacy observed in ENTPD-1-Tg mice (ADP-β-S–treated mice: WT: 11.3 ± 1.53 minutes versus ENTPD-1-Tg: 17.1 ± 2.59 minutes; P > 0.05; N = 3 per group; Figure 1C), consistent with the interpretation that hydrolysis of ADP is the critical step in the protection from in vivo thrombosis in ENTPD-1-Tg mice.

      ENTPDase-1 Modulates ex Vivo Platelet Aggregation and Activation

      To examine the effect of ENTPD-1 overexpression in an ex vivo model, whole-blood aggregation was performed. In response to ADP stimulation (20 μmol/L), both WT and ENTPD-1-Tg whole blood displayed a rapid initiation of aggregation that persisted in WT blood. In contrast, although an initial aggregatory response was observed after ADP stimulation, there was a rapid and near-complete disaggregation observed in ENTPD-1-Tg whole blood (Figure 2A). This resulted in a decrease in the total area under the curve in response to 20 μmol/L ADP stimulation in ENTPD-1-Tg versus WT (area under the curve was as follows: WT: 20,900 ± 746.2 Ω · seconds; ENTPD-1-Tg: 5586 ± 1544 Ω · seconds; N = 4 per group; P = 0.0001) and a decrease in the total aggregation at 6 minutes (Figure 2B; aggregation at 6 minutes: WT: 69.8 ± 6.35 Ω; N = 4 per group: ENTPD-1-Tg: 4.5 ± 1.55 Ω; N = 4 per group; P < 0.001). These results differ slightly from what was reported previously for the transgenic mice on a different genetic background using whole blood anticoagulated with citrate.
      • Dwyer K.M.
      • Robson S.C.
      • Nandurkar H.H.
      • Campbell D.J.
      • Gock H.
      • Murray-Segal L.J.
      • Fisicaro N.
      • Mysore T.B.
      • Kaczmarek E.
      • Cowan P.J.
      • d'Apice A.J.
      Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.
      Heparin, an in vivo anticoagulant frequently used in the treatment of acute coronary syndromes, may increase platelet responsiveness to agonists.
      • Xiao Z.
      • Theroux P.
      Platelet activation with unfractionated heparin at therapeutic concentrations and comparisons with a low-molecular-weight heparin and with a direct thrombin inhibitor.
      However, regardless of the anticoagulant used, the data are consistent with the interpretation that ENTPD-1 expression inhibits platelet reactivity in whole blood. Next, to determine whether the differences in whole-blood aggregation are caused by ADP hydrolysis, whole blood from ENTPD-1-Tg mice was stimulated with ADP-β-S (20 μmol/L), which resulted in a persistent aggregatory response in ENTPD-1-Tg whole blood compared with ADP stimulation with a corresponding increase in the total area under the curve (Figure 2C; ADP: 5358 ± 443.7 Ω · second; ADP-β-S: 15,380 ± 680.2 Ω · second; N = 3 per group; P = 0.0002) and an increase in the total aggregation at 6 minutes (Figure 2D; ADP: 1.5 ± 0.75 Ω; N = 3 per group; ADP-β-S: 55.8 ± 3.38 Ω; N = 3 per group; P < 0.0001), consistent with ENTPDase-1–mediated hydrolysis of ADP inhibiting the whole-blood aggregation.
      Figure thumbnail gr2
      Figure 2ENTPDase-1 attenuates whole-blood aggregation and ADP-β-S treatment restores whole-blood aggregation in ENTPDase-1 mice. A: Whole-blood aggregation curve in response to 20 μmol/L ADP in WT and ENTPD-1-Tg blood. Area under the curve for whole-blood aggregation in WT and ENTPD-1-Tg blood in response to 20 μmol/L ADP (WT: 20,900 ± 746.2 Ω · second; ENTPD-1-Tg: 5586 ± 1544 Ω · second; N = 4 per group; P = 0.0001). B: Aggregation at 6 minutes in WT and ENTPD-1-Tg blood in response to 20 μmol/L ADP (WT: 69.9 ± 6.35 Ω; ENTPD-1-Tg: 4.5 ± 1.55 Ω; N = 4 per group). ADP-β-S restores whole-blood aggregation in ENTPDase-1 mice. C: Whole-blood aggregation in ENTPD-1-Tg blood in response to 20 μmol/L ADP or 20 μmol/L ADP-β-S, a nonhydrolyzable ADP analog. Area under the curve for whole-blood aggregation in ENTPD-1-Tg blood in response to 20 μmol/L ADP or 20 μmol/L ADP-β-S (ADP: 5358 ± 443.7 Ω · second; ADP-β-S: 15,380 ± 680.2 Ω · second; N = 3 per group; P = 0.0002). D: Aggregation at 6 minutes in ENTPD-1-Tg blood in response to 20 μmol/L ADP or 20 μmol/L ADP-β-S (ADP: 1.5 ± 0.75 Ω; ADP-β-S: 55.8 ± 3.38 Ω; N = 3 per group). Values are mean ± standard error of the mean. *P < 0.001; TG, ectonucleoside triphosphate diphosphohydrolase-1 expressing animals.
      To investigate the effects of overexpression of ENTPD-1 further, platelet activation in whole blood from either WT or ENTPD-1-Tg mice treated with ADP was compared. Prior studies have shown a time-dependent increase in activated platelet fibrinogen receptor, the integrin glycoprotein (GP) αIIb3 (CD41/CD61) with ADP stimulation.
      • Bergmeier W.
      • Schulte V.
      • Brockhoff G.
      • Bier U.
      • Zirngibl H.
      • Nieswandt B.
      Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody.
      Basal expression levels of GPIX, GPVI (not shown), and the integrin glycoprotein αIIb3 did not differ between WT and ENTPD-1-Tg platelets (Figure 3, A and B). Although ADP stimulation resulted in an increase in surface expression of activated glycoprotein αIIb3 (Figure 3C) in a time-dependent manner in WT platelets, the level of activated GP αIIb3 on platelets from ENTPD-1-Tg mice was reduced significantly at all time points. However, stimulation of WT or ENTPD-1 blood with ADP-β-S showed comparable expression of activated GP αIIb3 on platelets at 6 minutes after stimulation (mean fluorescence intensity was as follows: WT: 207.3 ± 22.41 versus ENTPD-1: 229.0 ± 22.94; P > 0.05), consistent with intact expression and signaling mechanism via P2Y1 and P2Y12 receptors in ENTPD-1 platelets. Furthermore, to determine whether differences in the level of P2Y1- and P2Y12-receptor expression on platelets from ENTPD-1-Tg mice contributed to the observed effects, immunoblot analysis was conducted. No differences in either P2Y1 (relative P2Y1/GAPDH densitometric ratios were as follows: WT: 0.80 ± 0.158; ENTPD-1-Tg: 0.92 ± 0.116; P > 0.05; N = 3 per group; Figure 3D) or P2Y12-receptor levels (relative P2Y12/GAPDH densitometric ratios were as follows: WT: 1.83 ± 0.122; ENTPD-1-Tg: 2.03 ± 0.069; P > 0.05; N = 3 per group; Figure 3E) were observed. Together with the in vivo data of treatment of mice with ADP-β-S, these data suggest that ENTPD-1 activity mediates antiaggregatory efficacy by hydrolyzing ADP, thus limiting the activation state of platelet glycoprotein αIIb3, preventing the acute generation of an occlusive thrombus in vivo.
      Figure thumbnail gr3
      Figure 3ENTPDase-1 expression attenuates fibrinogen receptor activation. A: Representative fluorescence-activated cell sorter analysis of expression of total GP αIIb3 on WT (A) and ENTPD-1-Tg (B) resting platelets (isotype control: WT: 24.2 ± 1.40; ENTPD-1-Tg: 23.3 ± 0.59; GP αIIb3: WT: 3,215 ± 273; ENTPD-1-Tg: 3,488 ± 203; N = 4 per group). C: Fluorescence-activated cell sorter analysis of activated GP αIIb3 on WT and ENTPD-1-Tg platelets after stimulation with 20 μmol/L ADP. Geometric mean ± standard error of the mean from 10,000 platelet events are shown: baseline (WT: 17.5 ± 3.40; ENTPD-1-Tg: 18.2 ± 2.62; N = 3 per group; P = 0.8732); 1 minute (WT: 226.0 ± 9.74; ENTPD-1-Tg: 114.3 ± 0.75; N = 3 per group); 3 minutes (WT: 171.4 ± 12.28; ENTPD-1-Tg: 90.4 ± 20.36; N = 3 per group), and 6 minutes (WT: 136.3 ± 12.95; ENTPD-1-Tg: 67.05 ± 6.074; N = 3 per group). D: P2Y1-receptor levels on platelets from WT and ENTPD-1 mice. Representative immunoblot and normalized densitometry (relative P2Y1/GAPDH densitometric ratios: WT: 0.80 ± 0.158; ENTPD-1-Tg: 0.92 ± 0.116; P = 0.57; N = 3 per group) E: P2Y12-receptor levels expression on platelets from WT and ENTPD-1 mice. Representative immunoblot and normalized densitometry levels (relative P2Y12/GAPDH densitometric ratios: WT: 1.83 ± 0.122; ENTPD-1-Tg: 2.03 ± 0.069; P = 0.23; N = 3 per group). *P < 0.001; P < 0.05; TG, ectonucleoside triphosphate diphosphohydrolase-1 expressing animals.

      Adenosine in ENTPDase-1–Mediated Resistance to Occlusive Thrombosis

      As outlined earlier, ENTPDase-1 converts extracellular ATP and ADP to AMP and ecto-5′-nucleotidase (CD73) converts extracellular AMP to adenosine, which can regulate platelet reactivity and in vivo thrombosis.
      • Bullough D.A.
      • Zhang C.
      • Montag A.
      • Mullane K.M.
      • Young M.A.
      Adenosine-mediated inhibition of platelet aggregation by acadesine.
      • Kitakaze M.
      • Hori M.
      • Sato H.
      • Takashima S.
      • Inoue M.
      • Kitabatake A.
      • Kamada T.
      Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs.
      To determine whether the in vivo resistance to carotid thrombosis observed in ENTPD-1-Tg animals also involves the generation of adenosine by ecto-5′-nucleotidase, mice were treated with APCP, a specific ecto-5′-nucleotidase antagonist. Treatment with APCP also abrogated the resistance to occlusive thrombus formation mediated by ENTPD-1 overexpression (APCP-treated WT: 11.9 ± 1.01 minutes versus APCP-treated ENTPD-1-Tg: 15.5 ± 1.08 minutes; N = 4 per group; P < 0.05; Figure 4, A and B), suggesting the possibilities that APCP inhibits ENTPDase-1 activity directly, or that inhibition of ecto-5′-nucleotidase activity inhibits ENTPDase-1 activity via accumulation of AMP, or that ecto-5′-nucleotidase–mediated generation of adenosine is involved in the in vivo resistance to thrombosis.
      Figure thumbnail gr4
      Figure 4Adenosine in ENTPDase-1–mediated resistance to occlusive thrombosis. A: Administration of the ecto-5′-nucleotidase inhibitor APCP or the nonselective adenosine receptor antagonist 8-SPT abrogates ENTPDase-1–mediated resistance to occlusive thrombus formation in vivo (APCP-treated WT: 11.9 ± 1.01 minutes; APCP-treated ENTPD-1-Tg: 15.5 ± 1.08 minutes; N = 4 per group, P = 0.045 versus APCP-treated WT; P < 0.001 versus untreated ENTPD-1-Tg; 8-SPT–treated WT: 10.5 ± 1.42 minutes; 8-SPT–treated ENTPD-1-Tg: 14.3 ± 1.57 minutes; N = 8 per group, P > 0.05 versus 8-SPT–treated WT; P < 0.001 versus untreated ENTPD-1-Tg). B: Carotid flow after FeCl3 application expressed as a percentage of baseline flow in WT and ENTPD-1-Tg animals treated with APCP. C: Carotid flow after FeCl3 application expressed as a percentage of baseline flow in WT and ENTPD-1-Tg animals treated with 8-SPT. D: Administration of the adenosine A2A-receptor antagonist ZM 241385 or the adenosine A2B-receptor antagonist MRS-1754 abrogates ENTPDase-1–mediated resistance to occlusive thrombus formation in vivo (ZM-treated WT: 11.1 ± 0.58 minutes; ZM 241385-treated ENTPD-1-Tg: 14.4 ± 1.31 minutes; N = 5 per group, P > 0.05 versus ZM 241385-treated WT; P < 0.001 versus untreated ENTPD-1-Tg; MRS-1754–treated WT: 12.3 ± 1.88 minutes; MRS-1754–treated ENTPD-1-Tg: 20.5 ± 11.05 minutes; N = 5 per group, P > 0.05 versus MRS-1754–treated WT; P < 0.001 versus untreated ENTPD-1-Tg). E: Carotid flow after FeCl3 application expressed as a percentage of baseline flow in WT and ENTPD-1-Tg animals treated with ZM 241385. F: Carotid flow after FeCl3 application expressed as a percentage of baseline flow in WT and ENTPD-1-Tg animals treated with MRS-1754. Values are mean ± standard error of the mean. P < 0.05; TG, ectonucleoside triphosphate diphosphohydrolase-1 expressing animals.
      To address if adenosine-receptor–mediated signaling contributes to the resistance to in vivo thrombosis observed in ENTPDase-1–expressing mice, mice were treated with the nonselective adenosine-receptor antagonist 8-SPT. Treatment with 8-SPT abrogated the resistance to occlusive thrombus formation observed in mice overexpressing ENTPDase-1, consistent with the interpretation that adenosine receptor engagement is required for the resistance to occlusive thrombus formation mediated by ENTPDase-1 (8-SPT–treated WT: 10.5 ± 1.42 minutes versus 8-SPT–treated ENTPD-1-Tg: 14.3 ± 1.57 minutes; N = 8 per group; P > 0.05; Figure 4, A and C). Thus, APCP and 8-SPT, both with distinct molecular structures, either inhibit ENTPDase-1 activity directly or these pharmacologic data are consistent with an interpretation that, in the mouse, generation of adenosine and engagement of adenosine receptors contributes to the in vivo antithrombotic efficacy conveyed with ENTPD-1 expression. Prior studies have shown that adenosine alters platelet reactivity via interaction with the A2A receptor. To delineate if occupation of the adenosine A2A receptor is involved in the ENTPDase-1–mediated resistance to thrombosis, the acute effect of ZM 241385, a selective antagonist of the A2A-adenosine receptor, was examined. Acute pretreatment of mice with ZM 241385 completely abrogated the in vivo resistance to thrombosis conveyed by ENTPDase-1 overexpression, consistent with an interpretation that the in vivo ENTPDase-1–mediated effects require A2A-receptor occupancy (ZM 241385 1 mg/kg: WT: 11.1 ± 0.58 minutes versus ENTPD-1: 14.4 ± 1.31 minutes; N = 5 per group; P > 0.05; Figure 4, D and E). Given recent data suggesting an inhibitory role of adenosine A2B-receptor activation on platelet reactivity ex vivo,
      • Yang D.
      • Chen H.
      • Koupenova M.
      • Carroll S.H.
      • Eliades A.
      • Freedman J.E.
      • Toselli P.
      • Ravid K.
      A new role for the A2b adenosine receptor in regulating platelet function.
      the effect of MRS 1754, a selective A2B-receptor antagonist, also was examined. At all doses examined, pretreatment with MRS 1754 (0.1 mg/kg) abrogated the resistance to thrombosis achieved with ENTPD-1 overexpression (0.1 mg/kg MRS 1754–treated: WT: 12.3 ± 1.88 minutes versus ENTPD-1-Tg: 20.5 ± 11.05 minutes; P > 0.05; N = 5 per group; Figure 4, D and F), consistent with the interpretation that adenosine A2B-receptor occupation also is required for the in vivo ENTPDase-1–mediated resistance to thrombosis. Thus, 8-SPT, ZM 241385, or MRS 1754, all structurally distinct molecules, attenuate ENTPDase-1 activity directly, or these pharmacologic data are consistent with the interpretation that, in the mouse, engagement of both the A2A and A2B adenosine receptors contribute to the antithrombotic efficacy conveyed with ENTPD-1 expression.

      Discussion

      Acute arteriosclerotic plaque rupture with subsequent adhesion and aggregation of platelets results in thrombus formation and coronary vessel occlusion, the pathophysiologic basis of acute coronary syndromes.
      • Libby P.
      Current concepts of the pathogenesis of the acute coronary syndromes.
      The current studies show that increased ENTPD-1 (CD39) activity can modulate purinergic signaling, thereby attenuating activation of the platelet fibrinogen receptor, the integrin GP αIIb3 (CD41/CD61), resulting in a profound resistance to in vivo occlusive thrombus formation. Increased expression of ENTPDase-1 pacifies the response to vascular injury. Thus, overexpression of ENTPD-1 conveys a marked protection against in vivo arterial thrombosis and treatments aimed at increasing ENTPD-1 levels might convey vascular protection.
      The accumulation of platelets at sites of vascular disruption via engagement of specific platelet receptors to the exposed subendothelial matrix leads to platelet activation. On activation, numerous signaling cascades converge to release of ATP and ADP from platelet-dense granules, which further activate and recruit platelets into the growing thrombus. ATP and ADP also affect vascular reactivity, inducing vasodilation when administered abluminally to intact carotid arteries.
      • Kaul S.
      • Waack B.J.
      • Heistad D.D.
      Asymmetry of vascular responses of perfused rabbit carotid artery to intraluminal and abluminal vasoactive stimuli.
      ENTPDase-1 appears to control nucleotide-dependent vasoconstriction because knockout of the ENTPDase-1 gene leads to increased aortic ring constriction in response to UDP or UTP.
      • Kauffenstein G.
      • Drouin A.
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      • Thorin E.
      • Sevigny J.
      NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse.
      • Kauffenstein G.
      • Furstenau C.R.
      • D'Orleans-Juste P.
      • Sevigny J.
      The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation.
      Thus, ATP and ADP mediate prothrombotic and inflammatory signaling through interactions with specific purinergic receptors on endothelium, vascular smooth muscle, platelets, and inflammatory cells.
      • Kauffenstein G.
      • Drouin A.
      • Thorin-Trescases N.
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      • Thorin E.
      • Sevigny J.
      NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse.
      • Kauffenstein G.
      • Furstenau C.R.
      • D'Orleans-Juste P.
      • Sevigny J.
      The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation.
      • Kahner B.N.
      • Shankar H.
      • Murugappan S.
      • Prasad G.L.
      • Kunapuli S.P.
      Nucleotide receptor signaling in platelets.
      Modulation of platelet function is achieved in part through the action of several purinergic receptors on platelets, P2Y1, P2Y12, and P2X1.
      • Kahner B.N.
      • Shankar H.
      • Murugappan S.
      • Prasad G.L.
      • Kunapuli S.P.
      Nucleotide receptor signaling in platelets.
      The P2Y1 is a Gαq G-protein–coupled receptor critical to ADP-induced shape change, aggregation, thromboxane A2 generation, and thrombus formation under shear conditions.
      • Offermanns S.
      • Toombs C.F.
      • Hu Y.H.
      • Simon M.I.
      Defective platelet activation in G alpha(q)-deficient mice.
      • Jin J.
      • Dasari V.R.
      • Sistare F.D.
      • Kunapuli S.P.
      Distribution of P2Y receptor subtypes on haematopoietic cells.
      • Jin J.
      • Quinton T.M.
      • Zhang J.
      • Rittenhouse S.E.
      • Kunapuli S.P.
      Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors.
      Knockout of the P2Y1 receptor results in absence of platelet shape change and reduced aggregation in response to ADP as well as reduced thrombus size and stability.
      • Fabre J.E.
      • Nguyen M.
      • Latour A.
      • Keifer J.A.
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      • Coffman T.M.
      • Koller B.H.
      Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.
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      • LeMeur M.
      • Cazenave J.P.
      • Gachet C.
      Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.
      Overexpression of the P2Y1 receptor results in increased platelet reactivity both ex vivo and in vivo.
      • Hechler B.
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      • Evans R.
      • Gachet C.
      A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo.
      The P2Y12 receptor is a Gαi-coupled G-protein–coupled receptor that suppresses cAMP formation,
      • Ohlmann P.
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      • Spicher K.
      • Schultz G.
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      The human platelet ADP receptor activates Gi2 proteins.
      thereby potentiating platelet activation by a number of stimuli.
      • Kahner B.N.
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      • Prasad G.L.
      • Kunapuli S.P.
      Nucleotide receptor signaling in platelets.
      Knockout of the P2Y12 receptor results in reduced ADP-induced platelet aggregation, an inability to inhibit adenylyl cyclase activity, and prolonged bleeding time, however, platelet shape change and intracellular aggregation are not affected.
      • Foster C.J.
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      • Chintala M.S.
      Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.
      • Andre P.
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      • Phillips D.R.
      • Conley P.B.
      P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.
      Interestingly, the P2Y12 receptor, the target of the clinically approved antiplatelet agents clopidogrel and prasugrel, has a lower affinity for ADP than the P2Y1 receptor. Furthermore, the prodrug form of clopidogrel appears to facilitate platelet aggregation by inhibition of ENTPDase-1.
      • Lecka J.
      • Rana M.S.
      • Sevigny J.
      Inhibition of vascular ectonucleotidase activities by the pro-drugs ticlopidine and clopidogrel favours platelet aggregation.
      In contrast to the P2Y1 and P2Y12 receptors, the P2X1 receptor is stimulated by ATP, resulting in an influx of calcium that does not lead to aggregation but is necessary for full activation of platelets. Knockout of the P2X1 receptor results in resistance to thrombosis of small arteries,
      • Hechler B.
      • Lenain N.
      • Marchese P.
      • Vial C.
      • Heim V.
      • Freund M.
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      • Cattaneo M.
      • Ruggeri Z.M.
      • Evans R.
      • Gachet C.
      A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo.
      whereas overexpression of P2X1 receptors results in increased thrombus formation in response to various agonists.
      • Oury C.
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      • Vermylen J.
      • Heemskerk J.W.
      • Hoylaerts M.F.
      Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothrombotic phenotype.
      Thus, genetic studies have shown that activation of each of the purinergic receptors is necessary for full platelet activation.
      In the current study, increased ENTPDase-1 activity reduced ADP-mediated whole-blood aggregation. Similarly, in response to ADP activation, ENTPDase-1 expression decreased activation of the platelet fibrinogen receptor, GP αIIb3 complex. These data are consistent with prior work showing that persistent ADP-mediated P2Y1- and P2Y12-receptor stimulation is required to maintain calcium-mediated activation of the platelet fibrinogen receptor, glycoprotein αIIb3.
      • Goto S.
      • Tamura N.
      • Ishida H.
      • Ruggeri Z.M.
      Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5′-diphosphate receptor stimulation and cyclic calcium signaling.
      Thus, ENTPDase-1 not only maintains basal vascular function, but in pathologic states, ENTPDase-1–mediated hydrolysis of ATP and ADP released from damaged tissue regulates the purinergic pathways involved in platelet activation and thrombosis. However, the antithrombotic effects conveyed by ENTPD-1 expression were reversed by stimulation with the nonhydrolyzable ADP analog, ADP-β-S, suggesting that continued ADP stimulation is required to maintain maximal aggregation and platelet activation, a well-known phenomenon,
      • Bourgain R.H.
      • Vermarien H.
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      • Vereecke F.
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      • Rennies J.
      • Blockeel E.
      • Six F.
      A standardized ‘in vivo’ model for the study of experimental arterial thrombosis: description of a method.
      • Born G.V.
      Adenosine diphosphate as a mediator of platelet aggregation in vivo.
      • Cattaneo M.
      • Canciani M.T.
      • Lecchi A.
      • Kinlough-Rathbone R.L.
      • Packham M.A.
      • Mannucci P.M.
      • Mustard J.F.
      Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates.
      • Trumel C.
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      • Plantavid M.
      • Hechler B.
      • Viala C.
      • Presek P.
      • Martinson E.A.
      • Cazenave J.P.
      • Chap H.
      • Gachet C.
      A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase.
      and that P2Y1 and P2Y12 expression and signaling are intact and capable of mediating platelet activation in ENTPD-1 platelets.
      ATP and ADP are metabolized to AMP, which is converted to adenosine by the actions of ecto-5′-nucleotidase (CD73). Recent work has shown that soluble 5′-nucleotidase derived from Crotalus atrox venom inhibits ex vivo platelet aggregation, suggesting a role for adenosine-mediated signaling in the inhibition of platelet function.
      • Hart M.L.
      • Kohler D.
      • Eckle T.
      • Kloor D.
      • Stahl G.L.
      • Eltzschig H.K.
      Direct treatment of mouse or human blood with soluble 5′-nucleotidase inhibits platelet aggregation.
      However, in our current model, we observed no effect with antagonism of ecto-5′-nucleotidase or of adenosine-receptor blockade in whole-blood aggregation (data not shown). Indeed, prior studies have shown no effect of adenosine on whole-blood aggregation except under conditions in which dipyridamole is added to prevent red blood cell uptake and metabolism of adenosine.
      • Klabunde R.E.
      Dipyridamole inhibition of adenosine metabolism in human blood.
      • Dawicki D.D.
      • Agarwal K.C.
      • Parks Jr, R.E.
      Role of adenosine uptake and metabolism by blood cells in the antiplatelet actions of dipyridamole, dilazep and nitrobenzylthioinosine.
      • Iyu D.
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      Adenosine derived from ADP can contribute to inhibition of platelet aggregation in the presence of a P2Y12 antagonist.
      The current data suggest that in our model inhibition of whole-blood aggregation is dependent on ADP degradation rather than adenosine generation. However, a more complex situation appears to be involved in the cascade leading to in vivo thrombosis in which both ADP degradation and adenosine generation via ecto-5′-nucleotidase (CD73) activity appear capable of modulating occlusive thrombus formation after arterial injury. Indeed, prior studies
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      Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs.
      have demonstrated that endogenous adenosine production appears to inhibit in vivo platelet aggregation. It has been proposed that localized effects might explain the observation reported here and in other studies regarding adenosine modulation of platelet activation and thrombus growth in vivo; adenosine-receptor effects may be more pronounced in pathologic processes, such as a growing thrombus.
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      Adenosine derived from ADP can contribute to inhibition of platelet aggregation in the presence of a P2Y12 antagonist.
      In our in vivo model, ENTPD-1–mediated resistance to formation of an occlusive thrombus appears dependent on ecto-5′-nucleotidase activity because treatment of ENTPD-1-Tg mice with the CD73 antagonist APCP normalized the time to thrombosis. These data are consistent with prior studies that have shown that knockout of ecto-5′-nucleotidase results in a shortened time to thrombosis in a murine model of FeCl3-induced carotid arterial thrombosis.
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      Targeted disruption of cd73/ecto-5′-nucleotidase alters thromboregulation and augments vascular inflammatory response.
      Future studies examining the effect of genetic ablation of ecto-5′-nucleotidase in mice that overexpress ENTPDase-1 will provide further insight into this complex signaling cascade both ex vivo and in vivo. Our data suggest that not only ADP hydrolysis but also adenosine generation are necessary for ENTPD-1–mediated resistance to in vivo occlusive thrombus formation after vascular injury. Thus, therapies that lead to increased ENTPD-1 activity may have profound vascular protective efficacy.
      Platelets possess both A2A adenosine receptors and A2B adenosine receptors,
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      • Amisten S.
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      • Erlinge D.
      Gene expression profiling for the identification of G-protein coupled receptors in human platelets.
      and both couple to adenylate cyclase, causing an increase in cAMP levels, which inhibits platelet activation.
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      Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses.
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      • Rubin P.C.
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      Adenosine receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine release in human platelets.
      Genetic ablation of either the A2A adenosine receptor
      • Ledent C.
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      • Schiffmann S.N.
      • Pedrazzini T.
      • El Yacoubi M.
      • Vanderhaeghen J.J.
      • Costentin J.
      • Heath J.K.
      • Vassart G.
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      Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor.
      or the A2B adenosine receptor
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      • Chen H.
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      • Carroll S.H.
      • Eliades A.
      • Freedman J.E.
      • Toselli P.
      • Ravid K.
      A new role for the A2b adenosine receptor in regulating platelet function.
      results in higher platelet aggregation in response to ADP. The A2A and A2B adenosine receptors also are expressed on circulating neutrophils and monocytes. Indeed, not only platelet-platelet interactions but also platelet-leukocyte adhesion occur with acute arterial injury; circulating platelet-leukocyte aggregates are increased in acute coronary syndromes.
      • Sarma J.
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      • Fox K.A.
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      Increased platelet binding to circulating monocytes in acute coronary syndromes.
      Thus, one might hypothesize that ENTPDase-1 expression on leukocytes may modulate platelet/leukocyte interactions and affect thrombosis. Prior studies have shown that in patients with leukocytosis, ENTPDase-1 activity is increased and platelet aggregation is reduced in response to various stimuli.
      • Glenn J.R.
      • White A.E.
      • Johnson A.J.
      • Fox S.C.
      • Myers B.
      • Heptinstall S.
      Raised levels of CD39 in leucocytosis result in marked inhibition of ADP-induced platelet aggregation via rapid ADP hydrolysis.
      Recent work has shown that ENTPDase-1 polymorphisms regulate the level of expression of ENTPDase-1 on leukocytes.
      • Friedman D.J.
      • Kunzli B.M.
      • A-Rahim Y.I.
      • Sevigny J.
      • Berberat P.O.
      • Enjyoji K.
      • Csizmadia E.
      • Friess H.
      • Robson S.C.
      CD39 deletion exacerbates experimental murine colitis and human polymorphisms increase susceptibility to inflammatory bowel disease.
      Furthermore, ENTPDase-1 regulates leukocyte chemotaxis by hydrolyzing released ATP to adenosine, which coordinate to modulate chemotaxis via activation of purinergic nucleotide and adenosine receptors,
      • Chen Y.
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      • Inoue Y.
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      • Hashiguchi N.
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      • Nizet V.
      • Insel P.A.
      • Junger W.G.
      ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.
      • Linden J.
      New insights into the regulation of inflammation by adenosine.
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      • Linden J.
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      Adenosine receptors: therapeutic aspects for inflammatory and immune diseases.
      • Corriden R.
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      • Junger W.G.
      Ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) regulates neutrophil chemotaxis by hydrolyzing released ATP to adenosine.
      • Chen Y.
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      • Li A.
      • To U.K.
      • Elkhal A.
      • Inoue Y.
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      Purinergic signaling: a fundamental mechanism in neutrophil activation.
      • Kronlage M.
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      • Robaye B.
      • Conley P.B.
      • Kim H.-C.
      • Sargin S.
      • Schon P.
      • Schwab A.
      • Hanley P.J.
      Autocrine purinergic receptor signaling is essential for macrophage chemotaxis.
      and modulates P2X purinoreceptor 7–dependent function in murine macrophages.
      • Lévesque S.A.
      • Kukulski F.
      • Enjyoji K.
      • Robson S.C.
      • Sévigny J.
      NTPDase1 governs P2X7-dependent functions in murine macrophages.
      The regulation of TF procoagulant activity is complex, with a number of pathways capable of switching TF from a cryptic, nonactive state, to a decrypted, active state.
      • Bach R.R.
      Tissue factor encryption.
      Recent work has shown a critical role for activation of the macrophage P2X purinoreceptor 7 in a protein disulfide isomerase-regulated thiol pathway that controls the release of procoagulant TF–positive microparticles and also TF-dependent thrombosis.
      • Furlan-Freguia C.
      • Marchese P.
      • Gruber A.S.
      • Ruggeri Z.M.
      • Ruf W.
      P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice.
      Although the level of TF does not differ between WT and ENTPD-1-Tg aortae, further work will be required to determine whether ENTPDase-1 influences TF decryption. Indeed, ongoing work is investigating the influence of the cellular expression of ENTPDase-1 on in vivo thrombosis.
      In conclusion, these results show a pivotal role of ectonucleoside triphosphate diphosphohydrolase-1 in the modulation of purinergic-mediated platelet activation. ENTPDase-1 expression results in an attenuation of activation of the platelet fibrinogen receptor, glycoprotein αIIb3, which translates to a resistance to in vivo occlusive thrombus formation. Our data suggest that not only ADP removal, but also adenosine-receptor engagement, delays in vivo thrombosis because antagonism of CD73 or nonselective adenosine-receptor antagonism abrogates the resistance to occlusive thrombus formation conveyed by ENTPDase-1 expression. Supporting our work is recent data showing that in mice subjected to hypothermia, a condition used frequently to treat patients with out-of-hospital arrest, ENTPDase-1 activity was decreased, expression of the platelet activation marker P-selectin was increased, and platelet thrombus formation in FeCl3-injured murine mesenteric arteries was increased.
      • Straub A.
      • Krajewski S.
      • Hohmann J.D.
      • Westein E.
      • Jia F.
      • Bassler N.
      • Selan C.
      • Kurz J.
      • Wendel H.P.
      • Dezfouli S.
      • Yuan Y.
      • Nandurkar H.
      • Jackson S.
      • Hickey M.J.
      • Peter K.
      Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.
      These effects were reversed by administration of recombinant soluble ENTPD-1.
      • Straub A.
      • Krajewski S.
      • Hohmann J.D.
      • Westein E.
      • Jia F.
      • Bassler N.
      • Selan C.
      • Kurz J.
      • Wendel H.P.
      • Dezfouli S.
      • Yuan Y.
      • Nandurkar H.
      • Jackson S.
      • Hickey M.J.
      • Peter K.
      Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.
      We acknowledge that a limitation of the current studies was the specificity and selectivity of the pharmacologic agents used and that the generalizability of the current findings to other models of vascular injury cannot be inferred. However, the ferric chloride model has been used to show the efficacy of agents used clinically to treat acute arterial thrombosis, including tirofiban, eptifibatide, and clopidogrel.
      • Wang X.
      • Xu L.
      An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research.
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa.
      Continuing investigations using in vivo thrombus imaging, alternate methods of arterial vascular injury, and adenosine-receptor knockout animals will help define the interplay between ENTPDase-1 activity and adenosine receptor–mediated signaling on platelet activation and in vivo thrombus formation and stability further. The current data show that ENTPDase-1–mediated modulation of purinergic signaling is a key determinant of the formation of an occlusive arterial thrombus after vascular injury and supports the hypothesis that therapy focused on increasing ENTPD-1 expression and activity could have profound in vivo antithrombotic efficacy.

      Acknowledgments

      We thank Dr. Peter J. Newman (Blood Research Institute, Blood Center of Wisconsin) for his critical reading of the manuscript relative to the regulation of platelet activation and thrombosis.

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