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1.  Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk 
Science translational medicine  2014;6(248):248ra105.
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.
doi:10.1126/scitranslmed.3009246
PMCID: PMC4291186  PMID: 25100739
2.  CD39 Expression on T Lymphocytes Correlates With Severity of Disease in Patients With Chronic Lymphocytic Leukemia 
Introduction
Chronic lymphocytic leukemia (CLL) is a B-cell disorder, but it is also associated with abnormalities in T-lymphocyte function. In this study we examine changes in T-lymphocyte CD39 and CD73 expression in patients with CLL.
Methods
Blood samples were drawn from 34 patients with CLL and 31 controls. The cells were stained for CD3, CD4, CD8, CD19, CD39, and CD73 and analyzed by flow cytometry.
Results
Overall, patients with CLL had a higher percentage of CD39+ T lymphocytes than did controls. The percentage of cells expressing CD39 was higher in both CD4+ cells and CD8+ cells. Higher CD3/CD39 expression was associated with a later disease stage. No correlations between T-lymphocyte CD39 levels and CD38 or Zap-70 expression were observed. In contrast, the percentage of T lymphocytes and B lymphocytes that expressed CD73 was decreased in patients with CLL. Average B-lymphocyte CD73 expression was decreased in CLL because the majority of CLL clones were CD73. However a minority of CLL clones were CD73+, and patients with CD73+ clones tended to have earlier stage disease.
Conclusion
T-lymphocyte CD39 and CD73 expression may be useful prognostic markers in patients with CLL. Expression of CD73 on the malignant cell population in CLL may be a marker of better prognosis.
doi:10.1016/j.clml.2011.06.005
PMCID: PMC3590911  PMID: 21816376
CD39; Chronic lymphocytic leukemia; Prognosis; T lymphocyte
3.  P2X1 receptor inhibition and soluble CD39 administration as novel approaches to widen the cardiovascular therapeutic window 
Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclo-oxygenase inhibitor, and a thienopyridine such as clopidogrel (Plavix), which inactivates the ADP-stimulated P2Y12 receptor. Whilst these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial “resistance”. Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X1 receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at pro-thrombotic locations. Since both proteins affect platelet activation at an early stage in the events leading to thrombosis, but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety.
doi:10.1016/j.tcm.2009.01.005
PMCID: PMC2866184  PMID: 19467446
CD39; NTPDase1; apyrase; P2X1; platelet; thrombosis; drug therapy
4.  Homocysteine inhibits neoangiogenesis in mice through blockade of annexin A2–dependent fibrinolysis 
The Journal of Clinical Investigation  2009;119(11):3384-3394.
When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 μM, individuals are at increased risk of developing large vessel atherothrombosis and small vessel dysfunction. The annexin A2 complex (termed “A2”) is the cell surface coreceptor for plasminogen and TPA and accelerates the catalytic activation of plasmin, the major fibrinolytic agent in mammals. We previously showed that HC prevents A2-mediated, TPA-dependent activation of plasminogen in vitro by disulfide derivatization of the “tail” domain of A2. We also demonstrated that fibrinolysis and angiogenesis are severely impaired in A2-deficient mice. We now report here that, although hyperhomocysteinemic mice had a normal coagulation profile and normal platelet function, fibrin accumulated in their tissues due to reduced perivascular fibrinolytic activity and angiogenesis was impaired. A2 isolated from hyperhomocysteinemic mice failed to fully support TPA-dependent plasmin activation. However, infusion of hyperhomocysteinemic mice with fresh recombinant A2, which localized to neoangiogenic endothelial cells, resulted in normalization of angiogenesis and disappearance of peri- and intravascular fibrin. We therefore conclude that hyperhomocysteinemia impairs postnatal angiogenesis by derivatizing A2, preventing perivascular fibrinolysis, and inhibiting directed endothelial cell migration. These findings provide a mechanistic explanation for microvascular dysfunction and macrovascular occlusion in individuals with hyperhomocysteinemia.
doi:10.1172/JCI39591
PMCID: PMC2769184  PMID: 19841537
5.  Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase CD39 
The Journal of Clinical Investigation  2009;119(5):1136-1149.
Leukocyte and platelet accumulation at sites of cerebral ischemia exacerbate cerebral damage. The ectoenzyme CD39 on the plasmalemma of endothelial cells metabolizes ADP to suppress platelet accumulation in the ischemic brain. However, the role of leukocyte surface CD39 in regulating monocyte and neutrophil trafficking in this setting is not known. Here we have demonstrated in mice what we believe to be a novel mechanism by which CD39 on monocytes and neutrophils regulates their own sequestration into ischemic cerebral tissue, by catabolizing nucleotides released by injured cells, thereby inhibiting their chemotaxis, adhesion, and transmigration. Bone marrow reconstitution and provision of an apyrase, an enzyme that hydrolyzes nucleoside tri- and diphosphates, each normalized ischemic leukosequestration and cerebral infarction in CD39-deficient mice. Leukocytes purified from Cd39–/– mice had a markedly diminished capacity to phosphohydrolyze adenine nucleotides and regulate platelet reactivity, suggesting that leukocyte ectoapyrases modulate the ambient vascular nucleotide milieu. Dissipation of ATP by CD39 reduced P2X7 receptor stimulation and thereby suppressed baseline leukocyte αMβ2-integrin expression. As αMβ2-integrin blockade reversed the postischemic, inflammatory phenotype of Cd39–/– mice, these data suggest that phosphohydrolytic activity on the leukocyte surface suppresses cell-cell interactions that would otherwise promote thrombosis or inflammation. These studies indicate that CD39 on both endothelial cells and leukocytes reduces inflammatory cell trafficking and platelet reactivity, with a consequent reduction in tissue injury following cerebral ischemic challenge.
doi:10.1172/JCI36433
PMCID: PMC2673847  PMID: 19381014
6.  CD39/NTPDase-1 Activity and Expression in Normal Leukocytes 
Thrombosis research  2007;121(3):309-317.
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 previously determined.
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.
doi:10.1016/j.thromres.2007.04.008
PMCID: PMC2255570  PMID: 17555802
CD39; thrombosis; lymphocytes
7.  Aspirin Therapy for Inhibition of Platelet Reactivity in the Presence of Erythrocytes in Patients with Vascular Disease 
Inhibition of erythrocyte (RBC) promotion of platelet reactivity could improve the antiplatelet effect of aspirin (ASA). We tested different ASA regimens for optimal inhibition of platelets and the effects of RBC in patients with a history of vascular diseases. Collagen-induced platelet activation (14C-5HT, TXA2 release) and platelet recruitment (proaggregatory activity of cell-free releasates from activated platelets) were measured in PRP, platelet-RBC (Hct 40%) and whole blood (WB) in 206 patients initially on 200–300 mg ASA/day. Their regimen was modified to bi-weekly 500 mg (loading dose, L) plus daily or twice-daily low-dose ASA (50 or 100 mg). TXA2 was inhibited with all regimens. Percentage of patients with suboptimal inhibition of platelet recruitment in WB was: 200–300 ASA/day (41%), L-50/day (87%), L-100/day (58%), L-50/twice-daily (39%) and L-100/twice-daily (20%; p<0.05 vs. other regimens). 14C-5HT release was inhibited to the greatest extent with L-100/twice-daily in PRP+RBC or WB (p<0.05 vs. other regimens) due to greater inhibition of the RBC prothrombotic effect. Compared to other ASA regimens, L-100 twice-daily (equivalent to 221 mg ASA/day in the 14 day cycle), reduced by >50% the proportion of patients with suboptimal inhibition of platelet recruitment in WB and inhibited 14C-5HT release to the greatest extent.
doi:10.1016/j.lab.2005.12.005
PMCID: PMC1600016  PMID: 16697769
aspirin; blood cells; platelets; erythrocytes; cardiovascular diseases; cerebrovascular disorders; thrombosis; ADP: adenosine 5′-diphosphate; ASA: acetylsalicylic acid; BID: twice-daily; 14C-5HT: 14C-serotonin; COX-1: cyclooxygenase-1; L: loading dose; OD: daily; PRP: platelet-rich plasma; RBC: erythrocyte; TXA2: thromboxane A2; WB: whole blood
8.  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-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.
doi:10.1186/1479-5876-5-23
PMCID: PMC1885243  PMID: 17480228
9.  Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization 
Journal of Clinical Investigation  2006;116(12):3277-3291.
Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell–derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.
doi:10.1172/JCI29314
PMCID: PMC1679710  PMID: 17143334
10.  Elucidation of the thromboregulatory role of CD39/ectoapyrase in the ischemic brain 
The Journal of Clinical Investigation  2002;109(8):1031-1040.
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.
doi:10.1172/JCI10649
PMCID: PMC150939  PMID: 11956240
11.  Synthesis of Prostacyclin from Platelet-derived Endoperoxides by Cultured Human Endothelial Cells 
Journal of Clinical Investigation  1980;66(5):979-986.
We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI2) from the purified prostaglandin endoperoxide PGH2 (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI2 from endoperoxides released by stimulated platelets, [3H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [3H]PGI2 as [3H]6-keto-PGF1α and [3H]thromboxane A2 (TXA2) as [3H]TXB2, as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all three agents was inhibited. [3H]6-keto-PGF1α was recovered from the supernates of the combined cell suspensions after stimulation by all three agents. The order of PGI2 production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [3H]TXB2 recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF1α and TXB2 were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF1α measured by radioimmunoassay represented amounts of PGI2 sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/μl were combined with 3,000-6,000 aspirin-treated endothelial cells/μl. At higher platelet levels the proportion of 6-keto-PGF1α to TXB2 decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI2 from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. Therefore the endothelial cell suspensions could only have used endoperoxides from stimulated platelets.
Thus, under our experimental conditions, production by endothelial cells of PGI2 from endoperoxides derived from activated platelets could be demonstrated by two independent methods. These experimental conditions included: (a) enhanced platelet-endothelial cell proximity, as attainable in stirred cell suspensions; (b) use of increased endothelial cell/platelet ratios; and (c) utilization of arachidonate of high specific activity in radiolabeling experiments. Furthermore, when a mixture of platelets and endothelial cells that were not treated with aspirin was stimulated with thrombin, more than twice as much 6-keto-PGF1α was formed than when endothelial cells were stimulated alone. These results indicate that endothelial cells can utilize platelet endoperoxides for PGI2 formation to a significant extent.
PMCID: PMC371534  PMID: 6776148
13.  Quantification of human platelet inositides and the influence of ionic environment on their incorporation of orthophosphate-32P 
Journal of Clinical Investigation  1971;50(4):762-772.
Platelets are a rich source for the study of inositol lipids in man. The substitution of an EDTA-KCl solution for the water component of the Bligh and Dyer procedure permitted quantitative extraction of polyphosphoinositides. The latter, with monophosphoinositide, were found to comprise, on a molar basis, 6.7% of total platelet phospholipids. Study of the incorporation of orthophosphate-32P into platelet phospholipids was further simplified by separating eight 32P-labeled lipids, including the inositides, with a single chromatographic development on formaldehyde-treated paper. Particular attention was paid to the influence of ionic environment on the pattern and degree of labeling.
In 300 mOsm media major phospholipids other than the inositides were not labeled. Small amounts of label appeared in certain trace phospholipids, notably phosphatidic acid. In 150 mOsm media, labeling of inositides was moderately increased, that of trace phospholipids enormously so. The increased labeling was not solely due to thrombocytolysis since (a) platelet disruption by sonication or freeze-thawing abolished 32P incorporation into phospholipids and (b) in timed studies, restoration of osmolarity to 300 mOsm by addition of hypertonic sorbitol blunted the enhancement effect of previous 150 mOsm exposure. Lowering K and compensatorily increasing Na concentration of 300 mOsm media also stimulated 32P labeling of inositides and, to a lesser extent, the trace phospholipids. However, the pattern and degree of stimulation were not as strikingly altered as in the osmolarity studies.
These data show that drastic alterations of ionic environment can sharply influence the platelet's ability to incorporate orthophosphate-32P into its phospholipids.
Images
PMCID: PMC291990  PMID: 4323126
14.  Phospholipid Metabolism in Stimulated Human Platelets 
Journal of Clinical Investigation  1980;66(2):275-283.
Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A2 activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation.
These studies of endogenous phospholipid metabolism provide information supporting the existence of two previously postulated pathways for liberation of arachidonic acid from platelet phospholipids: (a) the combined action of PI-specific phospholipase C plus diglyceride lipase yielding arachidonate derived from PI; and (b) a phospholipase A2 acting primarily on diacyl ethanolamine phosphoglyceride.
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PMCID: PMC371708  PMID: 7400315

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