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1.
Sleep Breath ; 24(4): 1537-1547, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32036486

RESUMEN

PURPOSE: Obstructive sleep apnea (OSA) is independently associated with increased risk for stroke and other cardiovascular diseases. Since activated platelets play an important role in cardiovascular disease, the objective of this study was to determine whether platelet reactivity was altered in OSA subjects with intermittent nocturnal hypoxemia. METHODS: Thirty-one subjects, without hypertension or cardiovascular disease and not taking medication, participated in the study. Subjects were stratified based on OSA-related oxygen desaturation index (ODI) recorded during overnight polysomnography. Platelet reactivity to a broad panel of agonists (collagen, thrombin, protease-activated receptor1 hexapeptide, epinephrine, ADP) was measured by monitoring platelet aggregation and ATP secretion. Expression of platelet activation markers CD154 (CD40L) and CD62P (P-selectin) and platelet-monocyte aggregates (PMA) was quantified by flow cytometry. RESULTS: Epinephrine-induced platelet aggregation was substantially decreased in OSA subjects with significant intermittent hypoxemia (ODI ≥ 15) compared with subjects with milder hypoxemia levels (ODI < 15) (area under curve, p = 0.01). In addition, OSA subjects with ODI ≥ 15 exhibited decreased thrombin-induced platelet aggregation (p = 0.02) and CD40L platelet surface expression (p = 0.05). Platelet responses to the other agonists, CD62P platelet surface expression, and PMA levels were not significantly different between groups. Reduction in platelet responses to epinephrine and thrombin, and decreased CD40L surface marker expression in significant hypoxemic OSA individuals, is consistent with their platelets being in an activated state. CONCLUSIONS: Increased platelet activation was present in otherwise healthy subjects with intermittent nocturnal hypoxemia due to underlying OSA. This prothrombotic milieu in the vasculature is likely a key contributing factor toward development of thrombosis and cardiovascular disease. TRIAL REGISTRATION: NCT00859950.


Asunto(s)
Hipoxia/complicaciones , Activación Plaquetaria , Apnea Obstructiva del Sueño/sangre , Apnea Obstructiva del Sueño/fisiopatología , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agregación Plaquetaria , Apnea Obstructiva del Sueño/complicaciones
2.
FASEB J ; 29(1): 61-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25318477

RESUMEN

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 µM) increased ATP-induced renin release (≥2-fold), whereas purinergic P2X7 receptors (P2X7R) blockade with A740003 (3 µM) 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 µM), 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, P2X7R 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 (∼2-fold) by CD39 inhibition, an effect prevented by P2X7R 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.


Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Mastocitos/metabolismo , Reperfusión Miocárdica , Renina/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Antígenos CD/genética , Apirasa/antagonistas & inhibidores , Apirasa/genética , Cardiotónicos/metabolismo , Degranulación de la Célula , Línea Celular , Humanos , Masculino , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Miocardio/citología , ARN Interferente Pequeño/genética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología
3.
Sci Transl Med ; 6(248): 248ra105, 2014 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-25100739

RESUMEN

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.


Asunto(s)
Apirasa/uso terapéutico , Hemorragia/etiología , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Trombosis/complicaciones , Trombosis/tratamiento farmacológico , Adenosina Difosfato/farmacología , Animales , Apirasa/efectos adversos , Apirasa/farmacología , Clopidogrel , Circulación Coronaria/efectos de los fármacos , Perros , Fibrinólisis/efectos de los fármacos , Hemorragia/fisiopatología , Humanos , Ratones Endogámicos C57BL , Infarto del Miocardio/complicaciones , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Clorhidrato de Prasugrel , Factores de Riesgo , Tiofenos/farmacología , Trombosis/fisiopatología , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Factores de Tiempo , Activador de Tejido Plasminógeno , Resultado del Tratamiento , Grado de Desobstrucción Vascular/efectos de los fármacos
4.
Blood ; 119(15): 3378-9, 2012 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-22500052

RESUMEN

In a highly interesting, intricate, and novel paper in this issue of Blood, Fung and colleagues have extended their previous pioneering studies and now reveal that molecules such as ATP can promote platelet activation through the P2X1 receptor.


Asunto(s)
Plaquetas/metabolismo , Calcio/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Toll-Like/metabolismo , Animales , Humanos
5.
Clin Lymphoma Myeloma Leuk ; 11(4): 367-72, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21816376

RESUMEN

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.


Asunto(s)
5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Linfocitos B/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Linfocitos T/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Citometría de Flujo , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Recuento de Linfocitos , Pronóstico , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/metabolismo , Linfocitos T/patología
6.
J Pharmacol Exp Ther ; 337(2): 524-32, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21325440

RESUMEN

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.


Asunto(s)
Antígenos CD/biosíntesis , Apirasa/biosíntesis , Exocitosis/fisiología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Sistema Nervioso Simpático/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Apirasa/genética , Western Blotting , Cartilla de ADN , Dopamina/metabolismo , Exocitosis/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Silenciador del Gen , Isquemia/metabolismo , Factores de Crecimiento Nervioso/farmacología , Norepinefrina/metabolismo , Células PC12 , Potasio/farmacología , ARN Interferente Pequeño/metabolismo , Ratas , Receptores Purinérgicos P2X/efectos de los fármacos , Receptores Purinérgicos P2X/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Nervioso Simpático/citología
7.
J Biol Chem ; 286(17): 15428-39, 2011 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-21115493

RESUMEN

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.


Asunto(s)
Anexina A2/metabolismo , Células Endoteliales/metabolismo , Retroalimentación Fisiológica , Fibrinolisina/biosíntesis , Proteína Quinasa C/metabolismo , Animales , Ratones , Ratones Noqueados , Fosforilación , Multimerización de Proteína , Transporte de Proteínas , Serina/metabolismo
8.
J Clin Invest ; 119(11): 3384-94, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19841537

RESUMEN

When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 muM, 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.


Asunto(s)
Anexina A2/metabolismo , Fibrinólisis/fisiología , Homocisteína/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Anexina A2/farmacología , Movimiento Celular/efectos de los fármacos , Neovascularización de la Córnea/fisiopatología , Dieta , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Fibrina/metabolismo , Fibrinólisis/efectos de los fármacos , Homocisteína/farmacología , Hiperhomocisteinemia/fisiopatología , Masculino , Metionina/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/farmacología
9.
Trends Cardiovasc Med ; 19(1): 1-5, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19467446

RESUMEN

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 cyclooxygenase inhibitor, and a thienopyridine such as clopidogrel, which inactivates the adenosine diphosphate-stimulated P2Y(12) receptor. Although 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 P2X(1) receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at prothrombotic locations. Because 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.


Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Enfermedades Cardiovasculares/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptores Purinérgicos P2/efectos de los fármacos , Adenosina Trifosfato/uso terapéutico , Antígenos CD/efectos de los fármacos , Apirasa/efectos de los fármacos , Aspirina/uso terapéutico , Investigación Biomédica , Enfermedades Cardiovasculares/metabolismo , Hemorragia Cerebral/prevención & control , Ensayos Clínicos como Asunto , Clopidogrel , Inhibidores de la Ciclooxigenasa/uso terapéutico , Quimioterapia Combinada , Endotelio Vascular/metabolismo , Humanos , Isquemia Miocárdica/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/efectos adversos , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y12 , Ticlopidina/análogos & derivados , Ticlopidina/uso terapéutico , Resultado del Tratamiento
10.
J Clin Invest ; 119(5): 1136-49, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19381014

RESUMEN

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 alphaMbeta2-integrin expression. As alphaMbeta2-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.


Asunto(s)
Antígenos CD/fisiología , Apirasa/fisiología , Movimiento Celular/fisiología , Leucocitos/citología , 5'-Nucleotidasa/antagonistas & inhibidores , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apirasa/farmacología , Trasplante de Médula Ósea , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Activación Plaquetaria/fisiología , Antagonistas del Receptor Purinérgico P2 , Interferencia de ARN , Receptores Purinérgicos P2/genética , Quimera por Trasplante/fisiología
11.
J Pharmacol Exp Ther ; 322(3): 1269-77, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17565006

RESUMEN

We previously reported that ATP coreleased with norepinephrine from cardiac sympathetic nerves activates presynaptic P2X purinoceptors (P2XR), thereby enhancing norepinephrine exocytosis. Blockade of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) potentiates norepinephrine exocytosis, whereas recombinant soluble CD39 (solCD39) in-hibits it. This suggested that CD39 gene (Entpd1) deletion would enhance purinergic and adrenergic signaling by preserving ATP and its norepinephrine-releasing activity. However, we found that the neurogenic contractile response of vasa deferentia from Entpd1-null (CD39(-/-)) mice was attenuated and accompanied by reduced activity of pre- and postsynaptic P2XR, whereas contractile responses to K(+) or norepinephrine remained intact. In addition, the magnitude of ATP and norepinephrine exocytosis from cardiac synaptosomes was decreased in CD39(-/-) mice. Inhibition of E-NTPDase1/CD39, or solCD39 administration, did not affect the attenuated contractile response of vasa deferentia from CD39(-/-) mice. Notably, Entpd1 deletion and pharmacological P2XR desensitization in control mice similarly attenuated vasa deferentia responses. Thus, excessive and prolonged ATP exposure resulting from CD39 deletion desensitizes pre- and postjunctional P2XR at the sympathetic neuromuscular junction. This diminishes purinergic activity directly and adrenergic activity indirectly. It remains to be determined whether this desensitization results from receptor internalization, changes in receptor conformation or phosphorylation. Shutdown of ATP signaling in CD39(-/-) mice may represent a defense mechanism for the prevention of purinergic overstimulation. Our findings emphasize the cardioprotective role of neuronal CD39: by reducing presynaptic facilitatory effects of neurotransmitter ATP, CD39 attenuates norepinephrine release and its dysfunctional consequences. Moreover, by virtue of its antithrombotic action CD39 can potentially prevent the transition from myocardial ischemia to infarction.


Asunto(s)
Antígenos CD/fisiología , Apirasa/fisiología , Receptores Purinérgicos P2/metabolismo , Sinapsis/química , Adenosina Trifosfato/metabolismo , Animales , Apirasa/deficiencia , Exocitosis , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Norepinefrina/metabolismo
12.
Thromb Res ; 121(3): 309-17, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17555802

RESUMEN

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.


Asunto(s)
Antígenos CD/sangre , Apirasa/sangre , Leucocitos/enzimología , Leucocitos/inmunología , Adulto , Anciano , Linfocitos B/enzimología , Linfocitos B/inmunología , Análisis Químico de la Sangre , Separación Celular , Cromatografía en Capa Delgada , Femenino , Citometría de Flujo , Humanos , Leucocitos/efectos de los fármacos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Mitógenos/farmacología , Monocitos/enzimología , Monocitos/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología
13.
J Transl Med ; 5: 23, 2007 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-17480228

RESUMEN

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.


Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Leucemia Linfocítica Crónica de Células B/fisiopatología , Activación Plaquetaria/fisiología , Adenosina Difosfato/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/genética , Apirasa/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Cromatografía en Capa Delgada , Femenino , Citometría de Flujo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Subgrupos Linfocitarios/metabolismo , Masculino , Persona de Mediana Edad , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Linfocitos T/patología
14.
J Clin Invest ; 116(12): 3277-91, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17143334

RESUMEN

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.


Asunto(s)
Médula Ósea/irrigación sanguínea , Células Madre Hematopoyéticas/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Trombospondinas/metabolismo , Animales , Plaquetas/citología , Médula Ósea/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Células Madre Hematopoyéticas/citología , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Miembro Posterior/patología , Isquemia/metabolismo , Isquemia/fisiopatología , Megacariocitos/citología , Megacariocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estilbenos/metabolismo , Trombopoyesis/fisiología , Trombopoyetina/genética , Trombopoyetina/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo , Trombospondinas/genética
15.
J Lab Clin Med ; 147(5): 220-7, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16697769

RESUMEN

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 biweekly 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 with 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.


Asunto(s)
Aspirina/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Eritrocitos/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Colágeno/farmacología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Eritrocitos/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Serotonina/metabolismo , Tromboxano A2/metabolismo
16.
J Thromb Thrombolysis ; 19(2): 115-22, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16052302

RESUMEN

INTRODUCTION: This study evaluated CD39 in a porcine model of balloon angioplasty and in plasma of patients undergoing percutaneous intervention. CD39 (E-NTPDase1), is the endothelial ecto-ADPase inhibiting platelet function via hydrolysis of released platelet ADP. METHODS AND RESULTS: A recombinant soluble form of CD39 (solCD39) given intravenously to pigs had an elimination half life of 5--7 days, increased the bleeding time to an extent similar to aspirin, and inhibits platelet aggregation by>90%. Platelet counts and clot retraction remained normal following solCD39 administration. In a pig model of acute coronary balloon injury, solCD39 resulted in non-statistically significant decreases in platelet (7.7+/-1.4 versus 11.7+/- 3.4) and fibrin (3.5+/- 0.4 versus 4.2+/- 0.7) deposition ratios. Adding ex vivo to human platelet rich plasma (PRP) solCD39 produced nearly 100% inhibition of ADP-induced platelet aggregation. A dose-response effect of solCD39 on platelet aggregation induced by collagen or a thrombin receptor activating peptide (TRAP(SFLLRN)) was noted in PRP obtained from volunteers and patients receiving aspirin, clopidogrel or ticlopidine. SolCD39 also provided additional and complete inhibition of TRAP-induced platelet aggregation in PRP from patients who had received abciximab, aspirin and clopidogrel. CONCLUSIONS: SolCD39, a novel inhibitor of platelet activation and recruitment with a relatively long half-life appears to be well tolerated and is a potent inhibitor of ADP-, collagen-, or TRAP-induced platelet activation. Its potential use in percutaneous coronary intervention requires further study. ABBREVIATED ABSTRACT: E-NTPDase1/CD39 is the endothelial ecto-ADPase responsible for inhibition of platelet function. A recombinant soluble form (solCD39) had an elimination half life of 5-7 days in pigs, elevated bleeding times similar to aspirin, did not affect clot retraction, and inhibited platelet aggregation by > 90%. When combined with standard heparin therapy in a pig model of acute coronary balloon injury, solCD39 resulted in a trend toward a decrease in platelet and fibrin deposition. SolCD39 added ex vivo to human platelet rich plasma yielded nearly 100% inhibition of ADP-induced platelet aggregation and provided further inhibition when combined with standard therapy.


Asunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Antígenos CD/farmacología , Apirasa/farmacología , Adhesividad Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Animales , Antígenos CD/uso terapéutico , Apirasa/farmacocinética , Apirasa/uso terapéutico , Pruebas de Coagulación Sanguínea , Colágeno/farmacología , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Modelos Animales , Receptores de Trombina , Solubilidad , Porcinos
17.
Thromb Res ; 116(3): 199-206, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15935828

RESUMEN

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.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Enfermedad de la Arteria Coronaria/enzimología , Adenosina Trifosfatasas/análisis , Anciano , Antígenos CD/análisis , Apirasa/análisis , Radioisótopos de Carbono , Estudios de Casos y Controles , Cromatografía en Capa Delgada , Humanos , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Factores de Riesgo
18.
Semin Thromb Hemost ; 31(2): 234-46, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15852226

RESUMEN

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.


Asunto(s)
Antígenos CD/fisiología , Apirasa/fisiología , Cardiotónicos/uso terapéutico , Fibrinolíticos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Agregación Plaquetaria/fisiología , Trombosis/enzimología , Adenosina Difosfato/farmacología , Adenosina Difosfato/fisiología , Animales , Aspirina/farmacología , Aspirina/uso terapéutico , Vasos Sanguíneos/enzimología , Vasos Sanguíneos/fisiopatología , Cardiotónicos/farmacología , Células Cultivadas/efectos de los fármacos , Infarto Cerebral/tratamiento farmacológico , Enfermedad Coronaria/enzimología , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Fibrinolíticos/farmacología , Humanos , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Fármacos Neuroprotectores/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Sinaptosomas/enzimología , Trombosis/tratamiento farmacológico , Trombosis/prevención & control
19.
J Pharmacol Exp Ther ; 313(2): 570-7, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15647328

RESUMEN

Using a guinea pig heart synaptosomal preparation, we previously observed that norepinephrine (NE) exocytosis was attenuated by a blockade of P2X purinoceptors, potentiated by inhibition of ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1)/CD39, and reduced by soluble CD39, a recombinant form of human E-NTPDase1/CD39. This suggests that norepinephrine and ATP are coreleased upon depolarization of cardiac sympathetic nerve endings and that ATP enhances norepinephrine exocytosis by an action modulated by E-NTPDase1/CD39 activity. Whether E-NTPDase1/CD39 is localized to cardiac neurons and modulates norepinephrine exocytosis in intact heart tissue remained untested. We report that E-NTPDase1/CD39 is selectively localized in human and porcine cardiac neurons and that depolarization of porcine heart tissue elicits omega-conotoxin-inhibitable release of both norepinephrine and ATP. Inhibition of E-NTPDase1/CD39 with ARL67156 markedly potentiated ATP release, demonstrating that E-NTPDase1/CD39 is a major determinant of ATP availability at sympathetic nerve terminals. Notably, inhibition of E-NTPDase1/CD39 enhanced both ATP and NE exocytosis, whereas administration of soluble CD39 reduced both ATP and NE exocytosis. The strong correlation between ATP and norepinephrine release was abolished in the presence of the purinergic P2X receptor (P2XR) antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). We conclude that released ATP governs norepinephrine exocytosis by activating presynaptic P2XR and that this action is controlled by neuronal E-NTPDase1/CD39. Clinically, excessive norepinephrine release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, in addition to its effects as an antithrombotic agent, soluble CD39 may constitute a novel therapeutic approach to ischemic complications in the myocardium.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Exocitosis/fisiología , Miocardio/enzimología , Neuronas/enzimología , Norepinefrina/metabolismo , Fosfato de Piridoxal/análogos & derivados , Animales , Exocitosis/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfato de Piridoxal/farmacología , Porcinos
20.
J Pharmacol Exp Ther ; 306(1): 238-44, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12676879

RESUMEN

We recently reported that ATP, coreleased with norepinephrine (NE) from cardiac sympathetic nerves, increases NE exocytosis via a positive feedback mechanism. A neuronal ectonucleotidase (E-NTPDase) metabolizes the released ATP, decreasing NE exocytosis. Excessive NE release in myocardial ischemia exacerbates cardiac dysfunction. Thus, we studied whether the ATP-mediated autocrine amplification of NE release is operative in ischemia and, if so, whether it can be modulated by E-NTPDase and its recombinant equivalent, solCD39. Isolated, guinea pig hearts underwent 10- or 20-min ischemic episodes, wherein NE was released by exocytosis and reversal of the NE transporter, respectively. Furthermore, to restrict the role of E-NTPDase to transmitter ATP, sympathetic nerve endings were isolated (cardiac synaptosomes) and subjected to increasing periods of ischemia. Availability of released ATP at the nerve terminals was either increased via E-NTPDase inhibition or diminished by enhancing ATP hydrolysis with solCD39. P2X receptor blockade with PPADS was used to attenuate the effects of released ATP. We found that, in short-term ischemia (but, as anticipated, not in protracted ischemia, where NE release is carrier-mediated), ATP exocytosis was linearly correlated with that of NE. This indicates that by limiting the availability of ATP at sympathetic terminals, E-NTPDase effectively attenuates NE exocytosis in myocardial ischemia. Our findings suggest a key role for neuronal E-NTPDase in the control of adrenergic function in the ischemic heart. Because excessive NE release is an established cause of dysfunction in ischemic heart disease, solCD39 may offer a novel therapeutic approach to myocardial ischemia and its consequences.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Exocitosis/fisiología , Isquemia Miocárdica/metabolismo , Terminaciones Nerviosas/enzimología , Norepinefrina/metabolismo , Fosfato de Piridoxal/análogos & derivados , Sistema Nervioso Simpático/enzimología , Adenosina Trifosfatasas/efectos de los fármacos , Animales , Cobayas , Masculino , Fosfato de Piridoxal/farmacología , Reperfusión , Sinaptosomas/metabolismo
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