Ischemia/reperfusion (I/R) elicits renin release from cardiac mast cells (MC), thus activating a local renin-angiotensin system (RAS), culminating in ventricular fibrillation. We hypothesized that in I/R, neurogenic ATP could degranulate juxtaposed MC and that ecto-nucleoside triphosphate diphosphohydrolase 1/CD39 (CD39) on MC membrane could modulate ATP-induced renin release. We report that pharmacological inhibition of CD39 in a cultured human mastocytoma cell line (HMC-1) and murine bone marrow-derived MC with ARL67156 (100 mu M) increased ATP-induced renin release (>= 2-fold), whereas purinergic P2X(7) receptors (P2X(7)R) blockade with A740003 (3 mM) prevented it. Likewise, CD39 RNA silencing in HMC-1 increased ATP-induced renin release (>= 2-fold), whereas CD39 overexpression prevented it. Acetaldehyde, an I/R product (300 mM), elicited an 80% increase in ATP release from HMC-1, in turn, causing an autocrine 20% increase in renin release. This effect was inhibited or potentiated when CD39 was overexpressed or silenced, respectively. Moreover, P2X(7)R silencing prevented ATP- and acetaldehyde-induced renin release. I/R-induced RAS activation in ex vivo murine hearts, characterized by renin and norepinephrine overflow and ventricular fibrillation, was potentiated (similar to 2-fold) by CD39 inhibition, an effect prevented by P2X(7)R blockade. Our data indicate that by regulating ATP availability at the MC surface, CD39 modulates local renin release and thus, RAS activation, ultimately exerting a cardioprotective effect.

Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-differentiated PC12 cells, a cellular model of sympathetic neuron, in which dopamine is the predominant catecholamine. We report that E-NTPDase1/CD39 deletion markedly increases depolarization-induced exocytosis of ATP and dopamine and increases ATP-induced dopamine release. Moreover, overexpression of E-NTPDase1/CD39 resulted in enhanced removal of exogenous ATP, a marked decrease in exocytosis of ATP and dopamine, and a large decrease in ATP-induced dopamine release. Administration of a recombinant form of E-NTPDase1/CD39 reproduced the effects of E-NTPDase1/CD39 overexpression. Exposure of PC12 cells to simulated ischemia elicited a release of ATP and dopamine that was markedly increased in E-NTPDase1/CD39-silenced cells and decreased in E-NTPDase1/CD39-overexpressing cells. Therefore, transmitter ATP acts in an autocrine manner to promote its own release and that of dopamine, an action that is controlled by the level of E-NTPDase1/CD39 expression. Because ATP availability greatly increases in myocardial ischemia, recombinant E-NTPDase1/CD39 therapeutically used may offer a novel approach to reduce cardiac dysfunctions caused by excessive catecholamine release.

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

Abstract Abstract 2110 HUVECs, neutrophils, monocyte/macrophages and T cell subsets all express ectonucleoside triphosphate diphosphohydrolase 1 (CD39) on the cell surface. In addition, they all express both P2X and P2Y receptors and dose dependently respond to ATP. ATP enhances superoxide production in activated neutrophils and supports chemotaxis of macrophages responding to a chemoattractant. ATP also induces apoptosis of anti-inflammatory T regulatory cells and supports the differentiation of pro-inflammatory Th17 cells. Lastly, ADP drives thrombus formation by activation of platelet P2Y12 receptors. An increase in the activity of expressed CD39 would result in increased metabolism of these pro-thrombotic and pro-inflammatory nucleotides. We examined the reported relationship between CD39 cleavage and cell surface enzymatic activity. We cloned N-terminal and C-terminal V5 and VP16 tagged CD39 into eukaryotic expression vectors as well as lentiviral genomic vectors for analysis of CD39 expression in 293 cells and HUVECs. Western blots of membrane fractions prepared from HUVECs and transiently and stably transfected 293 cells identified the previously described CD39 cleavage fragments. Streptavidin precipitation of biotinylated membrane proteins demonstrated that both fragments are present on the cell surface of both transduced HUVECs and transfected 293 cells. We next separated membranes on a discontinuous sucrose gradient to yield ER, Golgi/plasma membrane, early endosome and late endosome enriched membrane fractions. Surprisingly, full length CD39 and the N- and C-terminal fragments appeared in both the Golgi/plasma membrane and early endosome fractions. In addition, the early endosome CD39 exhibited enzyme activity equal to that of the Golgi/plasma membrane CD39. We then prepared membranes from cells treated with either chloroquine or bafilomycin, reagents known to interfere with endosomal acidification and/or maturation. In each case, we observed a decrease in the fractional cleavage of full length CD39 and a proportional decrease in associated enzymatic activity. When sonicated membranes were resolved on a continuous sucrose gradient, the N- and C-terminal fragments and a fraction of full length CD39 as well as maximal enzymatic activity were found in the low density, “raft” fractions. These results suggest that the formation of an enzymatically active CD39 complex requires N- and C-terminal CD39 fragments as well as membrane cholesterol. When 293 cells that stably expressed CD39 were transiently transfected with dominant negative dynamin 2, we observed a decrease in fractional cleavage as well as a proportional decrease in enzymatic activity. This result suggested that the cleavage event occurs following endocytosis of plasma membrane expressed CD39. Finally, we treated both stably transfected 293 cells and HUVECs with the cell permeable cysteine protease inhibitor zLLY.fmk. Prepared membranes analyzed by Western blot showed a decrease in fractional cleavage of full length CD39. Apyrase assays showed a corresponding decrease in ATPase and ADPase activity. We then examined the cholesterol dependence of CD39 activity by depleting membrane cholesterol with MβCD. As expected, ATPase activity decreased in a dose dependent manner. The predominant “active” species appeared as a 1.4 megadalton complex on a 3–12% BN gel of Digitonin solubilized membranes prepared from cultured cells treated with the cleavable cross-linker DTSSP. As a result of membrane cholesterol depletion, there was a proportional decrease in the amount of full length CD39 and N- and C-terminal fragments present in the DTSSP cross-linked HMW complex. Interestingly, there was a marked increase in the abundance of lower molecular weight complexes in cholesterol depleted cells. In conclusion, we provide evidence that CD39 enzymatic activity resides in a megadalton complex formed by protein-protein interactions between full length CD39 and C- and N-terminal fragments generated by cleavage of the full length molecule. Assembly of the oligomeric protein complex requires membrane cholesterol and likely occurs on intracellular membranes. Moreover, approximately 50% of the active enzyme complex remains sequestered on intracellular membranes. These results suggest that up-regulation of CD39 metabolism of pro-thrombotic and pro-inflammatory nucleotides involves pathways independent of gene transcription. Disclosures: No relevant conflicts of interest to declare.

We have previously shown that CD39 undergoes limited cleavage and that inhibition of proteolysis results in a decrease in ATPase activity. The reduction in enzymatic activity correlated with a decrease in the fraction of full-length CD39 present in active membrane raft-localized oligomeric complexes. We exploited N-and C-terminal VP16-and V5-tagged CD39, both transiently and stably expressed in 293 cells, to further elucidate the role of cleavage in the regulation of CD39 processing and activity. To characterize the complexes generated by cross-linking, N-terminal VP16-tagged and C-terminal V5-tagged CD39 were co-expressed in 293 cells. Following crosslinking of membranes with DTSSP and immunoprecipitation with anti-V5, DTT-cleaved species were visualized by Western Blot using VP16 antibody. Interestingly, both VP16-tagged full-length and N-terminal fragments (30 kDa) were immunoprecipitated by anti-V5. This indicates that both full-length CD39 and the N-terminal cleavage fragment are present in raft-localized complexes. The composition of raft-localized CD39 complexes was studied by separating membrane fractions on a discontinuous sucrose gradient using a non-detergent method. When overexpressed, CD39 and its C-terminal fragment distribute across the gradient as visualized by Western with anti-VP16. Importantly, specific activity (expressed as ATPase activity divided by total CD39 content) was 8 times greater in low-density raft-enriched fractions than in high density raft-free fractions. In addition, relative ADPase activity was higher in fractions containing a higher proportion of C-terminal CD39 relative to full-length CD39. Thus, CD39 forms oligomeric complexes and possesses optimal enzyme activity in lipid rafts. The relationship between CD39 cleavage, ATPase activity and raft localization was further studied in 293 cells transfected with C-or N-terminal VP16-tagged CD39. Subcellular fractionation on a discontinuous sucrose gradient yielded membrane fractions enriched in endoplasmic reticulum (ER), early endosomes (EE) and plasma membrane/Golgi (PM-Golgi). Importantly, the EE fraction contained both full-length and C-terminal (or N-terminal) CD39 at the same level as seen in the PM-Golgi fraction, suggesting that near 50% of CD39 resides in the EE compartment. Furthermore, EE-expressed CD39 exhibited an ATPase and ADPase activity equivalent to that seen in Golgi-PM fractions. This led us to examine effects of NH4Cl and bafilomycin (which block acidification of EE), and chloroquine (blocks EE maturation) on CD39 cleavage, activity and raft localization. Each treatment inhibited CD39 cleavage and correspondingly decreased ATPase activity. A shift of ~50% of full-length CD39 from raft fractions to high density membrane fractions was observed upon sucrose gradient fractionation following chloroquine treatment of cells transfected with N-terminal VP16 tagged CD39. This redistribution of CD39 in the membrane correlated with a 40% decrease in ATPase activity and a striking inhibition of CD39 cleavage. Here, at a lower level of expression than cited above, ATPase activity in low-density raft fractions was ~100-fold greater than in high density fractions. Thus, cleavage of a portion of CD39 molecules is required for both raft localization of full-length CD39 and optimal enzyme activity. Regulated proteolytic cleavage of CD39 would allow for rapid upregulation of CD39 activity in response to alterations in cell environment. This would occur via cycling of CD39 between plasma membrane and endosomal compartments, the proposed site of CD39 cleavage and assembly of fully active oligomeric complexes.

In the US, 780,000 people are diagnosed with stroke annually, of which 180,000 are recurrent. Nearly 90% of strokes are ischemic and 10% hemorrhagic. Roughly 30% of ischemic strokes are of unknown etiology or “cryptogenic”; this figure is even higher in younger adults. We sought to determine whether platelet activation and recruitment are increased in younger patients with cryptogenic stroke. In addition, we postulated a prothrombotic change in their endogenous CD39/NTPDase1 expression and nucleotidase activities (metabolism of ATP and ADP to AMP). Our sample consists of patients with cryptogenic stroke (n=40) and healthy controls (n=35), ages 18 to 64, participating in the THrombophilia In Cryptogenic stroKe (THICK) study. Extensive preliminary testing for humoral prothrombotic disorders did not account for almost half of the cerebral infarcts. This suggests that cryptogenic stroke may in part represent a disorder of enhanced platelet reactivity. We did indeed find that markers of platelet activation were higher in cases than controls as determined by platelet aggregation in platelet-rich plasma (lumiaggregometry) and FACS analyses. ATP secretion, a measure of platelet recruitment, was higher in stroke vs. control (no ASA) with 5 and 0.5 μg/ml collagen, and with 5 and 0.5 μM epinephrine. FACS analyses revealed that CD154, CD63, and monocyte-platelet aggregates were increased (P=0.18, 0.048 and 0.034). By contrast, neither platelet aggregation (expressed as area under the curve) in response to 0.5 μg/ml collagen nor circulating tissue factor activity were significantly higher in cases as compared to controls. Together these findings suggest a potential role for enhanced platelet activation and recruitment in younger cryptogenic stroke patients. In addition, we established that CD39/NTPDase1 is expressed on neutrophils (PMN), lymphocytes, and monocytes with ATPase and ADPase activities highest on B-lymphocytes, lower on PMN, lowest on T-lymphocytes. In stroke subjects, trends to higher total ADPase activities were observed in lymphocytes (P=0.09) and PMN (P=0.09). In contrast, ATPase activities were similar (P=0.81 and 0.68). Thus, the ratio of ADPase to ATPase activity was greater in stroke patients than controls (P=0.003 for lymphocytes; 0.13 for PMN) in apparent compensation for prothrombotic propensities. In general, these same trends were observed for direct comparisons of stroke patients to controls, whether on ASA or not. This is in apparent contrast to the data obtained previously with CAD patients in the acute phase (El-Omar et al, Thrombosis Res. 116:199–206, 2005), and supports the need for additional studies of cryptogenic and atherothrombotic stroke patients in both acute and convalescent phases. Total CD39 expression (FACS, assessed with mAb BU61) was only marginally higher in lymphocytes of patients as compared to controls. The finding of increased activities, but similar expression supports our hypothesis that the enzymatic nucleotidase activities of CD39 are regulated via expression of our newly discovered CD39 splice variants. Ultimately, therapy with solCD39 to block platelet activation and recruitment may hold promise for patients with cryptogenic stroke.

Introduction: CD39/NTPDase-I 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. Published by Elsevier Ltd.

Brief trypsin exposure increases apyrase activity in hCD39 expressing cells, as previously reported (Schulte am Esch et al, Biochemistry 38:2248, 1999). Since regulated proteolytic cleavage of CD39 would allow for a rapid response to extracellular stimuli, we studied the relationship between observed CD39 cleavage and enzymatic activity. We generated N- and C-terminal VP16-tagged hCD39 to study CD39 expression, processing, and activity in transiently transfected HEK 293 cells. We found that optimal enzymatic activity of CD39 indeed depends on incorporation into cholesterol-rich plasma membrane domains (lipid "rafts"). Membrane fractions from hCD39 -transfected 293 cells readily hydrolyze ATP. Pretreatment of 293 cells with the cholesterol-depleting agent methyl β cyclodextrin (MBCD) results in a dose-dependent decrease in ATPase activity. In addition, treatment of isolated membranes with MBCD also decreases enzymatic activity. We next performed Western blot analyses of membranes prepared from hCD39-transfected 293 cells treated with membrane-impermeant crosslinking agents. These experiments demonstrated a dose-dependent, MBCD-reversible decrease in monomeric CD39. Taken together, these data demonstrate that CD39 enzyme activity resides in raft-localized CD39. Western blots of membrane fractions from cells transfected with N- or C-terminal VP16-tagged hCD39 show partial cleavage of full-length CD39 to yield a 20kDa N-terminal and 50 kDa C-terminal fragments. Biotinylation studies established that both fragments are expressed on the cell surface. As with full-length CD39, crosslinking results in dose-dependent decreases of both monomeric species. Moreover, prior cholesterol depletion with MBCD abolishes crosslinking. Since the cleavage products of full-length CD39 are expressed on the cell surface and localize to lipid rafts, we examined the relation between CD39 cleavage, ATPase activity and lipid raft localization using a panel of cell permeable protease inhibitors. 293 cells transfected with N-terminal VP16-tagged CD39 were treated with AEBSF (serine protease inhibitor), zYVAD.fmk (caspase inhibitor), zLLY.fmk (calpain inhibitor) or the furin inhibitor Furin I. All inhibitors resulted in dose-dependent decreases in formation of the VP16-tagged N-terminal fragment. Concomitantly, ATPase assays of the membrane fractions demonstrated a corresponding dose-dependent decrease in enzymatic activity. Finally, we established that CD39 cleavage promotes raft localization, since protease inhibition decreased the fraction of CD39 susceptible to crosslinking with all inhibitors tested. In summary, we have established that generation of optimally active, raft-localized CD39 requires prior limited proteolysis of the full-length molecule. Activation of caspase-1 by exposure of cells to ATP leads to processing and release of interleukin family members. We propose that purinergic signaling might also enhance CD39 cleavage in vascular cells by an as yet unidentified protease. Our data suggest that subsequent increased cell surface apyrase activity leads to dampening of purinergic signaling and a resulting increase in antithrombotic activity. Of note, we identified an alternately spliced isoform of CD39 which inhibits cleavage of the full-length molecule.

This new training method is based on developing a sound understanding of the sequence in which electrical excitation spreads through both the normal and the infarcted myocardium. The student is made aware of the cardiac electrical performance through a series of 3-dimensional pictures during the excitation process.

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-quantititative 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 ~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 is an ecto-ATP/ADPase expressed on leukocytes and endothelial cells. CD39 is the main control system for blood fluidity. CD39 on lymphocytes was first reported in 1991 by Kansas et al. However, studies of CD39 expression and activity on leukocytes have not been done. We characterized levels of CD39 expression and enzymatic activity on neutrophils (PMN), lymphocytes and lymphocyte subsets. Since inflammatory responses occur in arterial vascular disease, we also examined expression of CD39 on naive versus activated and memory lymphocytes. Lymphocytes were isolated by a histopaque procedure, and PMN by dextran gradient. B-lymphocytes were isolated using the RosetteSep B-cell kit. All cell types were confirmed to have purities of >90%. CD39 activity was assayed via our radio-thin-layer chromatographic system. CD39 expression was measured on leukocytes via FACS. PMN, monocytes, and lymphocytes were identified by their forward and side-scatter characteristics. Subsets of lymphocytes were examined via double staining for CD39 and antibodies against specific sub-types. CD39 localized to the surface of greater than 95% of neutrophils, monocytes, and B-lymphocytes. It was also present on a minority (~8%) of T-lymphocytes with no difference in frequency of expression between CD4+ and CD8+ cells. Geometric mean (GM) expression of CD39 per cell was greatest in B-lymphocytes and monocytes, lower in CD4+ cells, and lowest in CD8+ cells and PMN. Interestingly, incubation of T- lymphocytes with PHA up-regulated CD39 in CD8+ cells both in terms of number of cells expressing and GM, with expression rising to 65%. The GM increased 4-fold after 6d of stimulation with PHA. A similar but less dramatic increase was seen with LPS. This is the first time we have accomplished up-regulation of CD39 expression and enzymatic activity. Radio-TLC measurement of nucleotidase activity showed B-lymphocytes>PMN>T-lymphocytes. B-lymphocyte ADPase and ATPase activities (in pmol/min/50K cells) were 75 and 43, respectively. PMN displayed 39 (ADPase) and 22 (ATPase), while T-lymphocytes had enzymatic activity of 16 and 11.5, respectively. ADPase:ATPase ratios were similar for B-lymphocytes and PMN, but lower for T-lymphocytes (1.8 for B-lymphocytes and PMN, vs 1.45 for T-lymphocytes, p=0.03). Lymphocytes stimulated with PHA demonstrated an increase in enzyme activity of 10–20X baseline that peaked at 7–10d. ADPase:ATPase ratio was unchanged. FACS measurement showed that CD39+ lymphocytes were more often activated than CD39− lymphocytes in both CD3+ (p=0.06) and CD4+ (p=0.02) subgroups. Preliminary experiments indicated that >85% of CD39+ T-lymphocytes are CD45RO+. Importantly, this suggests that CD39 is expressed primarily on activated or memory cells in the T-lymphocyte population. Thus, CD39 is expressed on a broad variety of leukocytes. T-lymphocyte expression can be induced by stimulation with mitogens. Moreover, CD39 is present primarily on CD45RO+ T-lymphocytes. We conclude that CD39 expression can be induced by activation of the immune system. The up-regulation of CD39 on activated and memory T-lymphocytes may be a compensatory mechanism for protection from thrombosis as a consequence of inflammation. It may serve as a mechanism for metabolizing extracellular ATP and therefore decreasing the inflammatory stimulus. Abnormalities in CD39 may result in decreased nucleotidase activity and increased vulnerability to thrombosis as a consequence of inflammation.

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 thrombo-regulation. 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 pro-thrombotic 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.

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of large numbers of mature lymphocytes. However, thrombotic events in CLL are relatively rare. CD39, the main control system for blood fluidity, was identified on the surface of CLL cells in about 80% of patients in two recent studies. This suggests that CD39 could play a role in prevention of thrombotic events in CLL. Lymphocytes were prepared from peripheral blood of an initial 13 patients with CLL by histopaque isolation and purified to at least 95%. The ADPase and ATPase activities in these lymphocytes was measured by our radio-thin-layer chromatographic assay. Additionally, cells were stained with antibodies to CD3, CD19, and CD39, and analyzed by FACS. Remaining lymphocytes were pelleted and frozen for semi-quantitative RT-PCR analysis of the CD39 transcipt. Lymphocytes from patients with CLL showed increased ATPase and ADPase activities compared with lymphocytes from normal donors. Average ADPase activity per 50,000 cells from CLL patients was 57pmol/min, compared with 14pmol/min in lymphocytes from normal donors. Average ATPase activity was 43.6pmol/min in CLL patients, and 10.6 pmol/min in normal lymphocytes. Semi-quantitative RT-PCR of CLL lymphocytes showed upregulation of CD39 RNA compared with normal lymphocytes. FACS data showed that the percentage of cells expressing CD39 was greater in CLL cells (62.5%) than in normal lymphocytes (11%). Additionally, some CLL cells appear to express CD39 at higher densities than normal lymphocytes. Interestingly, one patient in whom lymphocytes were isolated before and during an accelerated phase of the disease showed a decrease in CD39 expression on his lymphocytes as the disease progressed. Two other patients with advanced disease, requiring chemotherapy, showed relatively low CD39 levels as well. We conclude that CD39 is present and functions as an ecto-nucleotidase on the surface of most malignant lymphocytes in CLL. The quantities are much higher than observed in normal lymphocytes. CD39 may be protective from thrombotic events in patients with CLL. In addition, lymphocyte CLL may be a marker of earlier or less aggressive disease and a decrease of CD39 on the cell surface may signify a worsening of the disease.