RESUMEN
Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14â»/â» mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14â»/â» mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14â»/â» mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.
Asunto(s)
Plaquetas/metabolismo , Calgranulina B/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/patología , Antígenos CD36/genética , Antígenos CD36/metabolismo , Calgranulina B/genética , Calgranulina B/farmacología , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones Noqueados , Trombosis/tratamiento farmacológico , Trombosis/genética , Trombosis/patologíaRESUMEN
Bradykinin B2 receptor-deleted mice (Bdkrb2(-/-)) have delayed carotid artery thrombosis times and prolonged tail bleeding time resulting from elevated angiotensin II (AngII) and angiotensin receptor 2 (AT2R) producing increased plasma nitric oxide (NO) and prostacyclin. Bdkrb2(-/-) also have elevated plasma angiotensin-(1-7) and messenger RNA and protein for its receptor Mas. Blockade of Mas with its antagonist A-779 in Bdkrb2(-/-) shortens thrombosis times (58 ± 4 minutes to 38 ± 4 minutes) and bleeding times (170 ± 13 seconds to 88 ± 8 seconds) and lowers plasma nitrate (22 ± 4 µM to 15 ± 5 µM), and 6-keto-PGF1α (259 ± 103 pg/mL to 132 ± 58 pg/mL). Bdkrb2(-/-) platelets express increased NO, guanosine 3',5'-cyclic monophosphate, and cyclic adenosine monophosphate with reduced spreading on collagen, collagen peptide GFOGER, or fibrinogen. In vivo A-779 or combined L-NAME and nimesulide treatment corrects it. Bdkrb2(-/-) platelets have reduced collagen-related peptide-induced integrin α2bß3 activation and P-selectin expression that are partially corrected by in vivo A-779, nimesulide, or L-NAME. Bone marrow transplantations show that the platelet phenotype and thrombosis time depends on the host rather than donor bone marrow progenitors. Transplantation of wild-type bone marrow into Bdkrb2(-/-) hosts produces platelets with a spreading defect and delayed thrombosis times. In Bdkrb2(-/-), combined AT2R and Mas overexpression produce elevated plasma prostacyclin and NO leading to acquired platelet function defects and thrombosis delay.
Asunto(s)
Angiotensina I/sangre , Plaquetas/metabolismo , Epoprostenol/sangre , Óxido Nítrico/sangre , Fragmentos de Péptidos/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas/sangre , Receptores Acoplados a Proteínas G/sangre , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Animales , Tiempo de Sangría , Plaquetas/efectos de los fármacos , Trasplante de Médula Ósea , AMP Cíclico/sangre , GMP Cíclico/sangre , Immunoblotting , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Fragmentos de Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptor de Angiotensina Tipo 2/sangre , Receptor de Bradiquinina B2/deficiencia , Receptor de Bradiquinina B2/genética , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Trombosis/sangre , Factores de TiempoAsunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Mediadores de Inflamación/sangre , Inflamación/complicaciones , Inhibidores de Agregación Plaquetaria/uso terapéutico , Ticlopidina/análogos & derivados , Aspirina/uso terapéutico , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Clopidogrel , Quimioterapia Combinada , Humanos , Inflamación/sangre , Infarto del Miocardio/inmunología , Infarto del Miocardio/prevención & control , Valor Predictivo de las Pruebas , Proteína Plasmática A Asociada al Embarazo/metabolismo , Medición de Riesgo , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/prevención & control , Ticlopidina/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Thrombin potently regulates human platelets by the G protein-coupled receptors protease-activated receptor (PAR) 1 and PAR4. Platelet activation by thrombin and other agonists is broadly inhibited by prostacyclin and nitric oxide acting through adenylyl and guanylyl cyclases to elevate cAMP and cGMP levels, respectively. Using forskolin and YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] to selectively activate the adenylyl and guanylyl cyclases, respectively, and the membrane-permeable analogs N(6),2'-O-dibutyryladenosine-3'-5'-cAMP (dibutyryl-cAMP) and 8-(4-parachlorophenylthoi)-cGMP (8-pCPT-cGMP), we sought to identify key antiplatelet steps for cyclic nucleotide actions in blocking platelet activation by PAR1 versus PAR4. Platelet aggregation by PAR1 or PAR4 was inhibited with similar EC(50) of 1.2 to 2.1 microM forskolin, 31 to 33 microM YC-1, 57 to 150 microM dibutyryl-cAMP, and 220 to 410 microM 8-pCPT-cGMP. There was a marked left shift in the inhibitory potencies of forskolin and YC-1 for alpha-granule release and glycoprotein IIbIIIa/integrin alphaIIbbeta3 activation (i.e., EC(50) of 1-60 and 40-1300 nM, respectively) that was not observed for dibutyryl-cAMP and 8-pCPT-cGMP (i.e., EC(50) of 200-600 and 40-140 microM, respectively). This inhibition was essentially instantaneous, and measurements of cyclic nucleotide levels and kinase activities support a model of compartmentation involving the cyclic nucleotide effectors and regulators and the key molecular targets for this platelet inhibition. The different sensitivities of PAR1 and PAR4 to inhibition of calcium mobilization and dense granule release identify key antiplatelet steps for cyclic nucleotide actions and are consistent with the signaling models for these receptors. Specifically, PAR4 inhibition depends on the regulation of both calcium mobilization and dense granule release, and PAR1 inhibition depends predominantly on the regulation of dense granule release.
Asunto(s)
Plaquetas/fisiología , Nucleótidos Cíclicos/metabolismo , Receptor PAR-1/fisiología , Receptores de Trombina/fisiología , Transducción de Señal/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Bucladesina/farmacología , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/farmacología , Cinética , Proteínas de Microfilamentos/metabolismo , Nucleótidos Cíclicos/agonistas , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptor PAR-1/agonistas , Receptores de Trombina/agonistas , Transducción de Señal/efectos de los fármacos , Tionucleótidos/farmacología , Trombina/farmacologíaRESUMEN
Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through protease-activated receptor (PAR) 1 and PAR4, plays a central role in regulation of human platelet function in that it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intracellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study, we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion after PAR-induced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Furthermore, these inhibitors significantly attenuated activation of Rap1 after stimulation by PAR1-AP but not thrombin or PAR4-AP. Because PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.
Asunto(s)
Ácidos Fosfatidicos/fisiología , Activación Plaquetaria , Receptor PAR-1/fisiología , Receptores de Trombina/fisiología , Gránulos Citoplasmáticos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Nadolol/farmacología , Fosfatidato Fosfatasa/fisiología , Fosfolipasa D/fisiología , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Propranolol/farmacología , Proteína Quinasa C/fisiología , Proteínas de Unión al GTP rap1/fisiologíaRESUMEN
Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.
Asunto(s)
Plaquetas/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Agregación Plaquetaria/fisiología , Receptor PAR-1/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores de Trombina/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Quelantes/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Fibrinógeno/metabolismo , Humanos , Activación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Antagonistas del Receptor Purinérgico P2 , Receptor PAR-1/genética , Receptores Purinérgicos P2Y12 , Receptores de Trombina/genética , Trombina/metabolismoRESUMEN
In this issue of Molecular Pharmacology, Andreeva et al. (p. 975) report a novel functional link between the heterotrimeric G protein G alpha12 and endothelial nitric-oxide synthase (eNOS). Based on studies characterizing the interaction of G alpha12 and the molecular chaperone Hsp90 and the interaction of eNOS and Hsp90, the group proposed an interaction between G alpha12 and eNOS and sought to determine the regulatory mechanisms, including the inferred dependence on Hsp90. Their experiments using an overexpression model lead to the observation that the cotransfection of G alpha12 and eNOS expression vectors increased overall eNOS expression. Additional studies in the overexpression model and in human umbilical vein endothelial cells (HUVEC) provide evidence for a mechanism that involves G alpha12-dependent stabilization of eNOS protein and possibly mRNA. These data present yet another paradigm by which heterotrimeric G proteins, through stabilization of target proteins, can regulate the activity of downstream signaling pathways.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Endotelio Vascular/metabolismo , Estabilidad de Enzimas , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Humanos , Óxido Nítrico Sintasa de Tipo III/genética , Estabilidad del ARN , Transducción de Señal , TransfecciónRESUMEN
In neural crest (NC) cultures cAMP signaling is an instructive signal in catecholaminergic, sympathoadrenal cell development. However, the extracellular signals activating the cAMP pathway during NC cell development have not been identified. We demonstrate that in avian NC cultures, evidenced by tyrosine hydroxylase expression and catecholamine biosynthesis, adenosine and not adrenergic signaling, together with BMP2, promotes sympathoadrenal cell development. In NC cultures, addition of the adenosine receptor agonist NECA in the presence of BMP2 promotes sympathoadrenal cell development, whereas the antagonist CGS 15943 or the adenosine degrading enzyme adenosine deaminase (ADA) suppresses TH expression. Importantly, NC cells express A2A and A2B receptors which couple with Gsalpha increasing intracellular cAMP. Employing the CNS-derived catecholaminergic CAD cell line, we also demonstrate that neuronal differentiation mediated by serum withdrawal is further enhanced by treatment with IBMX, a cAMP-elevating agent, or the adenosine receptor agonist NECA, acting via cAMP. By contrast, the adenosine receptor antagonist CGS 15943 or the adenosine degrading enzyme ADA inhibits CAD cell neuronal differentiation mediated by serum withdrawal. These results support that adenosine is a physiological signal in neuronal differentiation of the CNS-derived catecholaminergic CAD cell line and suggest that adenosine signaling is involved in NC cell development in vivo.
Asunto(s)
Adenosina/metabolismo , Catecolaminas/biosíntesis , Diferenciación Celular/fisiología , Células Cromafines/metabolismo , Cresta Neural/embriología , Neuronas/metabolismo , Sistema Nervioso Simpático/embriología , 1-Metil-3-Isobutilxantina/farmacología , Agonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A2 , Adenosina Desaminasa/metabolismo , Inhibidores de la Adenosina Desaminasa , Adenosina-5'-(N-etilcarboxamida)/farmacología , Médula Suprarrenal/citología , Médula Suprarrenal/embriología , Médula Suprarrenal/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Línea Celular , Células Cultivadas , Células Cromafines/citología , Coturnix , Medio de Cultivo Libre de Suero/farmacología , AMP Cíclico/metabolismo , Cresta Neural/citología , Cresta Neural/metabolismo , Neuronas/citología , Quinazolinas/farmacología , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Transducción de Señal/fisiología , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Triazoles/farmacología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2L dopamine receptor markedly potentiated both forskolin- and A2-adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D2L dopamine receptor-mediated inhibition of Galphas-stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.