Joan H.F. Drosopoulos

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.

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.

Thromboregulation by Endothelial Cells: Significance for Occlusive Vascular Diseases

-During their 7- to 9-day lifespan in the circulation, platelets perform an ill-defined baseline function that maintains the integrity of the vasculature. In thrombocytopenic states, there is an increase in vascular permeability and fragility, which is presumably due to absence of this platelet function. In sharp contrast, biochemical or physical injury in the coronary, carotid, or peripheral arteries induces platelet activation and platelet recruitment, which can progress to thrombotic vascular occlusion. Because there is 1 death every 33 seconds from vascular occlusion in the United States, this problem has critical public health implications. In this review, we describe the characterization of a novel potential antithrombotic agent with a unique mode of action-biochemical deletion of ADP from an activated platelet releasate, which thereby inhibits platelet recruitment and further activation.

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

INTRODUCTIONnChronic 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.nnnMETHODSnBlood 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.nnnRESULTSnOverall, 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.nnnCONCLUSIONnT-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.

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.

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.

Human SolCD39 Inhibits Injury-induced Development of Neointimal Hyperplasia

Blood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolise nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADPase activity in plasma peaked 1 hour (h) post-injection, with an elimination half-life of 43 h. Accordingly, mice were administered solCD39 or saline 1 h prior to vessel injury, then either sacrificed 24 h post-injury or treated with solCD39 or saline (three times weekly) for an additional 18 days. Twenty-four hours post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and smooth muscle cell (SMC) proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56 +/- 0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimising platelet deposition and leukocyte recruitment, and suppressing neointimal hyperplasia.

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

APT102, an optimized human apyrase designed for treatment of myocardial infarction, improves recanalization of occluded arteries and decreases heart muscle damage, without an increased bleeding risk. A Better Antithrombotic, Without Bleeding The drugs that prevent blood clots—the cause of heart attacks and strokes—almost always also increase the risk of dangerous bleeding. Now, Moeckel et al. report that the enzyme apyrase can be engineered to degrade the prothrombotic molecule adenosine diphosphate (ADP) faster and longer than the native version, thereby preventing clots without increasing bleeding. Delivered to dogs or mice with heart attacks, the optimized apyrase kept the concentration of ADP low, averting the blood cell aggregation that initiates a clot. At the highest dose tested, the animals showed less heart damage but did not bleed. Clopidogrel, a common drug used currently in this situation, was less effective in minimizing heart damage and caused bleeding. 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.

Control of Platelet Reactivity by an Ecto-ADPase on Human Endothelial Cells

Arterial thrombosis, which is a misdirected form of hemostasis, is the major complication of atherosclerosis. Thrombosis accounts for 50% of mortal illnesses in the United States, Europe, and Japan1. Thrombi occur at sites of pathologic vascular damage. These sites take the form of necrotic or fissured atherosclerotic plaques in the coronary or cerebral circulation. The damaged surfaces act as agonists for at least three interacting biologic systems which are responsible for complete occlusion of the vessel: 1) structural components of the blood vessels, 2) blood platelets and, 3) plasma proteins of the coagulation system. Thus far, therapeutic measures to induce blockade of these interacting prothrombotic systems have only been partially successful1,2.