CD39 activity correlates with stage and inhibits platelet reactivity in chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is characterized by accumulation of mature appearing lymphocytes and is rarely complicated by thrombosis. One possible explanation for the paucity of thrombotic events in these patients may be the presence of the ecto-nucleotidase CD39/NTDPase-1 on the surface of the malignant cells in CLL. CD39 is the major promoter of platelet inhibition in vivo via its metabolism of ADP to AMP. We hypothesize that if CD39 is observed on CLL cells, then patients with CLL may be relatively protected against platelet aggregation and recruitment and that CD39 may have other effects on CLL, including modulation of the disease, via its metabolism of ATP. METHODS: Normal and malignant lymphocytes were isolated from whole blood from patients with CLL and healthy volunteers. Enzyme activity was measured via radio-TLC assay and expression via FACS. Semi-quantitative RT-PCR for CD39 splice variants and platelet function tests were performed on several samples. RESULTS: Functional assays demonstrated that ADPase and ATPase activities were much higher in CLL cells than in total lymphocytes from the normal population on a per cell basis (p-value < 0.00001). CD39 activity was elevated in stage 0-2 CLL compared to stage 3-4 (p < 0.01). FACS of lymphocytes demonstrated CD39 expression on > 90% of normal and malignant B-lymphocytes and approximately 8% of normal T-lymphocytes. RT-PCR showed increased full length CD39 and splice variant 1.5, but decreased variant 1.3 in CLL cells. Platelet function tests showed inhibition of platelet activation and recruitment to ADP by CLL cells. CONCLUSION: CD39 is expressed and active on CLL cells. Enzyme activity is higher in earlier stages of CLL and decreased enzyme activity may be associated with worsening disease. These results suggest that CD39 may play a role in the pathogenesis of malignancy and protect CLL patients from thrombotic events.

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.

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.

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.

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.

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.