RESUMEN
Essentials Mitochondrial hyperpolarization enhances the conversion of platelets to a procoagulant phenotype. Mitochondrial calcium uniporter (MCU) function is essential in procoagulant platelet formation. Mitochondrial calcium uniporter deletion does not impact other aspects of platelet activation. Ablation of MCU results in the emergence of a permeability transition pore-independent pathway. SUMMARY: Background Procoagulant platelets comprise a phenotypically distinct subpopulation of activated platelets with high-level phosphatidylserine externalization. When initiated by co-stimulation with thrombin and a glycoprotein VI (GPVI) agonist, the transition to the procoagulant phenotype is mediated by extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation. Objectives The intracellular mechanisms coordinating these distinct cytoplasmic and mitochondrial processes remain unclear. The mitochondrial calcium uniporter (MCU) protein is a central component of the transmembrane ion channel that allows the passage of Ca2+ from the cytosol into the mitochondrial matrix. Here we investigate the role of the MCU in the regulation of procoagulant platelet formation. Results Procoagulant platelet formation was directly correlated with pre-stimulatory mitochondrial transmembrane potential, a key determinant of calcium flux from the cytoplasm to the mitochondria. The role of MCU in the regulation of procoagulant platelet formation was investigated using MCU null platelets. Procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation. In contrast, neither granule release nor initial integrin activation was altered in response to stimulation. In the genomic absence of MCU, developmental induction of an alternative intracellular pathway partially rescued procoagulant platelet formation. Conclusion These results identify a key role for the mitochondrial calcium uptake channel in the regulation of mPTP-mediated procoagulant platelet formation and suggest a novel pharmacologic target for procoagulant-platelet-related pathologies.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Activación Plaquetaria/efectos de los fármacos , Animales , Antimicina A/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Péptidos/metabolismo , Fenotipo , Fosfatidilserinas/metabolismo , Glicoproteínas de Membrana Plaquetaria/agonistas , Rotenona/farmacología , Transducción de Señal/efectos de los fármacos , Trombina/agonistasRESUMEN
Activated protein C (APC) is a critical regulator of thrombin formation and thereby protects against thrombosis. On the other hand, overwhelming formation of APC increases the risk of bleeding such as in trauma-induced coagulopathy. Thus, pharmacological inhibition of APC activity may improve blood clottability in certain clinical situations. In this study, we demonstrate that the DNA aptamer HS02-52G binds with fast onset (1.118 ± 0.013 × 105 M-1 s-1) to APC and possesses a long residence time of 13.5 min within the aptamer-APC complex. Functional analysis revealed HS02-52G as a highly potent and specific inhibitor of APC in plasma and whole blood with IC50 values ≤30 nM, whose activity can be readily neutralized by the short complementary DNA molecule AD22. These features qualify the novel aptamer-antidote pair as a candidate treatment option for acute APC-related bleedings.
Asunto(s)
Anticoagulantes/química , Aptámeros de Nucleótidos/química , Oligonucleótidos Antisentido/química , Proteína C/antagonistas & inhibidores , Trombina/química , Anticoagulantes/síntesis química , Aptámeros de Nucleótidos/síntesis química , Emparejamiento Base , Humanos , Cinética , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/síntesis química , Tiempo de Tromboplastina Parcial , Unión Proteica , Proteína C/química , Proteínas Recombinantes/química , Termodinámica , Trombina/agonistas , Trombina/antagonistas & inhibidores , Tiempo de Coagulación de la Sangre TotalRESUMEN
MMP9-responsive bivalirudin-HPMA copolymers were synthesized for direct, local administration in rat spinal cord contusion injury models. Polymer-conjugated bivalirudin peptides maintained activity while demonstrating enzyme-mediated release upon MMP9 exposure and prolonged release from hyaluronic acid/methylcellulose (HAMC) hydrogels compared to free bivalirudin peptide. Localized administration of bivalirudin copolymers in vivo at the site of rat spinal cord injury decreased cellular proliferation and astrogliosis, suggesting the bivalirudin copolymer and HAMC hydrogel system are a promising therapeutic intervention for reducing immediate inflammatory responses and long term scarring.
Asunto(s)
Hirudinas/síntesis química , Ácido Hialurónico/química , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapéutico , Metaloproteinasa 9 de la Matriz/química , Metilcelulosa/química , Metilcelulosa/uso terapéutico , Fragmentos de Péptidos/síntesis química , Traumatismos de la Médula Espinal/tratamiento farmacológico , Trombina/agonistas , Animales , Hirudinas/química , Ácido Hialurónico/uso terapéutico , Hidrogeles/química , Hidrogeles/uso terapéutico , Metaloproteinasa 9 de la Matriz/metabolismo , Fragmentos de Péptidos/química , Ratas , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Trombina/químicaRESUMEN
The effect of antiaggregants on the system of regulation of the aggregative state of the whole blood in healthy volunteers has been studied using the global test of low-frequency piezothrombelastography. It is established that specific effects of antiaggregants are manifested in all stages of fibrinogenesis (initiation/amplification and propagation). According to the results of the global test at initial stages of hemocoagulation (initiation/amplification), a pronounced specific antiaggregative effect of COX-1 blockers and ADP receptors is accompanied by the intensification of thrombin activity and the formation of chronometric hypercoagulation. The blocker of phosphodiesterase, while losing a specific antiaggregative effect, has a lower influence on the change of thrombin activity, thus ensuring hronometric and structural hypocoagulation at the subsequent stages of fibrinogenesis. The proposed method can be used for real-time monitoring of the results of pharmacotherapy and drug effects at various stages of fibrinogenesis in the program of personalized antiaggregant therapy.
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Aspirina/farmacología , Coagulación Sanguínea/efectos de los fármacos , Pentoxifilina/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Tromboelastografía/métodos , Ticlopidina/análogos & derivados , Adulto , Aspirina/análogos & derivados , Clopidogrel , Ciclooxigenasa 1/metabolismo , Fibrinógeno/metabolismo , Voluntarios Sanos , Humanos , Cinética , Masculino , Medicina de Precisión , Receptores Purinérgicos P2/metabolismo , Trombina/agonistas , Trombina/metabolismo , Ticlopidina/farmacologíaRESUMEN
Glanzmann's thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl(2) and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor XIII/farmacología , Fibrinógeno/farmacología , Trombastenia/sangre , Trombina/metabolismo , Área Bajo la Curva , Cloruro de Calcio/farmacología , Estudios de Casos y Controles , Eptifibatida , Humanos , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Plasma Rico en Plaquetas/química , Tromboelastografía , Trombina/agonistas , Tiempo de Trombina , Tromboplastina/farmacología , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
PURPOSE: The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer bloodretinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the bloodretinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1. METHODS: Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels. RESULTS: Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion. CONCLUSIONS: Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.
Asunto(s)
Endotelina-1/biosíntesis , Endotelina-1/metabolismo , Células Epiteliales/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismo , Trombina/metabolismo , Barrera Hematorretinal/metabolismo , Calcio/metabolismo , Calcio/farmacología , Endotelina-1/análisis , Endotelina-1/farmacología , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/farmacología , Receptor PAR-1/metabolismo , Epitelio Pigmentado de la Retina/fisiopatología , Pigmentos Retinianos/farmacología , Transducción de Señal/efectos de los fármacos , Trombina/agonistas , Trombina/farmacología , Quinasas Asociadas a rho/análisis , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/farmacologíaRESUMEN
We assessed the effect of the intercellular mediator of inflammation, platelet activating factor (PAF), on platelet function. The interaction between PAF and the platelet agonists ADP, thrombin and convulxin was analyzed in vitro in whole blood with the use of flow cytometry and was further characterized with the use of receptor antagonists to PAF (ABT-491), P2Y1 (MRS-2179), and P2Y12 (cangrelor) as well as a monoclonal anti-PSGL-1 antibody (anti-CD162). Low concentrations of PAF (0.1 nM) synergistically augmented platelet activation induced by other agonists (P < 0.01). Augmentation by PAF was receptor mediated and did not require platelet-leukocyte interaction. With >99% inhibition of P2Y receptor-mediated platelet activation, greater than additive activation was still observed with the combination of ADP plus PAF. Accordingly, PAF synergistically augments platelet activation in response to ADP and thrombin, and the extent of inhibition exerted by P2Y receptor antagonists is decreased in the presence of PAF.
Asunto(s)
Adenosina Difosfato/farmacología , Plaquetas/metabolismo , Factor de Activación Plaquetaria/farmacología , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Trombina/farmacología , Adenosina Difosfato/agonistas , Adulto , Anciano , Anciano de 80 o más Años , Venenos de Crotálidos/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Lectinas Tipo C , Masculino , Persona de Mediana Edad , Factor de Activación Plaquetaria/agonistas , Inhibidores de Agregación Plaquetaria/agonistas , Pruebas de Función Plaquetaria , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Trombina/agonistasRESUMEN
Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Aspirina/farmacología , Plaquetas/metabolismo , Epinefrina/farmacología , Hemostáticos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas de Receptores Adrenérgicos alfa 2 , Aspirina/agonistas , Aspirina/antagonistas & inhibidores , Plaquetas/citología , Calcio/farmacología , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Antagonismo de Drogas , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Epinefrina/agonistas , Epinefrina/antagonistas & inhibidores , Humanos , Morfolinas/farmacología , Oligopéptidos/agonistas , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/farmacología , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Agregación Plaquetaria/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Agonistas Purinérgicos , Agonistas del Receptor Purinérgico P2 , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P2X , Receptores de Trombina/agonistas , Vesículas Secretoras/metabolismo , Trombina/agonistas , Trombina/antagonistas & inhibidoresAsunto(s)
Fluoxetina/farmacología , Activación Plaquetaria/efectos de los fármacos , Receptor PAR-1/agonistas , Receptores de Trombina/agonistas , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Antígenos CD/sangre , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Sinergismo Farmacológico , Humanos , Metisergida/farmacología , Oligopéptidos/farmacología , Selectina-P/sangre , Fragmentos de Péptidos/farmacología , Glicoproteínas de Membrana Plaquetaria , Receptor de Serotonina 5-HT2A/efectos de los fármacos , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Serotonina/sangre , Tetraspanina 30 , Trombina/agonistas , Trombina/farmacología , Valeratos/farmacologíaRESUMEN
Wild type of bovine thrombin has been crystallized in a ligand-free form by the hanging drop vapor diffusion method with polyethylene glycol 4000 and 2-propanol. The crystals belong to space group P4 3 2 12 with unit cell parameters of a = b = 87.7 A, c = 195.9 A. X-ray diffraction data were collected to 2.8 A resolution.
Asunto(s)
Trombina/química , Animales , Bovinos , Cristalización , Cristalografía por Rayos X , Ligandos , Mutagénesis , Sodio , Trombina/agonistas , Trombina/antagonistas & inhibidores , Trombina/genéticaRESUMEN
Platelets have been implicated in accelerated bone regeneration in grafting applications. The beneficial effects of platelets may involve their ability to stimulate the proliferation of osteoblasts. We therefore determined the mitogenic response of human trabecular bone-derived cells to human platelets and supernatants of thrombin-activated platelets. We can show a approximately 50-fold increase in DNA-synthesis of bone cells (BC) cultured in the presence of platelets as determined by [3H]-thymidine incorporation. Preventing cell-to-cell contact by a membrane filter did not abrogate the stimulatory effect, indicating the release of soluble factor(s) that are mitogenic for BC. The lipid fraction of the platelets had no effect on [3H]-thymidine uptake into the DNA of BC. Platelet-released supernatant (PRS) increased the rate of [3H]-thymidine incorporation to approximately 20-fold and retained 56% of their activity after incubation at 56 degrees C, and 27% at 100 degrees C, respectively. Neutralizing antibodies raised against platelet-derived growth factor (PDGF) partially suppressed the mitogenic potential of PRS. Gel exclusion chromatography analysis showed that molecules ranging from 25 kDa to more than 70 kDa within the PRS can stimulate BC proliferation. The highest amount of PDGF was detected in fractions corresponding to a molecular weight of 28-37 kDa as determined by immunoassay. The mitogenic activity was not restricted to soluble growth factors because microparticles in the PRS and platelet membranes also increased BC proliferation. Our data indicate that native platelets, the respective PRS, microparticles, and platelet membranes can stimulate the mitogenic activity of BC, thereby contributing to the regeneration of mineralized tissue.
Asunto(s)
Plaquetas/fisiología , Osteoblastos/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Anciano , Anciano de 80 o más Años , Anticuerpos , Plaquetas/metabolismo , Plaquetas/ultraestructura , Regeneración Ósea/fisiología , Comunicación Celular/fisiología , División Celular/fisiología , Membrana Celular/fisiología , Cromatografía en Gel , Citosol/química , ADN/biosíntesis , Humanos , Lípidos/fisiología , Persona de Mediana Edad , Mitógenos , Peso Molecular , Activación Plaquetaria/fisiología , Factor de Crecimiento Derivado de Plaquetas/análisis , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Radiofármacos , Estadística como Asunto , Temperatura , Trombina/agonistas , Timidina/metabolismo , TritioRESUMEN
A thrombin-like enzyme with a new amino-terminal sequence was isolated from the venom of Trimeresurus flavoviridis using Q-Sepharose, CM-Cellulose, and HW55 column chromatographies. Homogeneity was confirmed by the formation of a single band in polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 29,000 Da. This thrombin-like enzyme was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), and dithiothreitol (DTT) suggesting that serines and disulfide bonds are involved in the expression of the enzyme's clotting activity. This thrombin-like enzyme hydrolyzes the Aalpha-chain and Bbeta-chain of bovine fibrinogen. The enzyme was stable to heat treatment.
Asunto(s)
Venenos de Crotálidos/enzimología , Animales , Bovinos , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/metabolismo , Hidrólisis , Trombina/agonistas , TrimeresurusRESUMEN
BACKGROUND: Thrombin is a serine protease produced following gingival tissue injury or inflammation. It regulates the functional behavior of injury-neighboring cells via the activation of specific protease-activated receptors (PAR). Thrombin's role in gingival tissue healing and inflammatory response processes is not yet well understood. METHODS: We investigated the effects of thrombin on gingival fibroblast (GF) growth [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay], collagen lattice contraction, and associated morphological changes. RESULTS: Thrombin (>1 U/ml), but not thrombin receptor (PAR-1) agonist peptide (SFLLRN, single letter amino acid code, abbreviated as TRAP, 1 to 50 microg/ml), stimulated the growth and clustering of cultured human GF in vitro. Growth-stimulatory effects of thrombin were inhibited by D-Phe-Pro-ArgCH2Cl (PPACK), a serine protease inhibitor. By contrast, trypsin (>10 microg/ml), a PAR-2 activator, suppressed the growth of GF. Thrombin (>0.2 U/ml) and TRAP (10 to 25 microg/ml), but not trypsin, prostaglandin E2 (0.01 to 0.5 microg/ml), or bovine serum albumin (BSA) (1 to 80 microg/ml), induced the GF-populated collagen lattice contraction within 30 to 60 minutes of exposure. The thrombin-induced collagen lattice contraction was inhibited by PPACK (20 microg/ml) and an actin filament polymerization inhibitor, cytochalasin B (1 microg/ml). The collagen lattice contraction induced by TRAP was also inhibited by cytochalasin B, but not by PPACK. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of PAR-1, and to a lesser extent PAR-3, was observed for human GF, although little PAR-2 and PAR-4 expression was noted. CONCLUSIONS: These results indicate that thrombin is important in periodontal wound healing and inflammatory processes by promoting the growth and contraction of GF. The stimulatory effects of thrombin are associated with its protease activation of thrombin receptors.
Asunto(s)
Colágeno/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Receptores de Trombina/efectos de los fármacos , Trombina/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Antitrombinas/farmacología , Agregación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Cristalización , Citocalasina B/farmacología , Dinoprostona/farmacología , Activación Enzimática , Fibroblastos/enzimología , Expresión Génica , Encía/citología , Encía/enzimología , Humanos , Fragmentos de Péptidos/farmacología , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores de Serina Proteinasa/farmacología , Albúmina Sérica Bovina/farmacología , Estadística como Asunto , Sales de Tetrazolio , Tiazoles , Trombina/agonistas , Factores de Tiempo , Tripsina/farmacologíaRESUMEN
Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.
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Endopeptidasas/metabolismo , Receptores de Trombina/antagonistas & inhibidores , Receptores de Trombina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Plaquetas/metabolismo , Señalización del Calcio , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Escherichia coli/genética , Citometría de Flujo , Humanos , Hidrólisis , Espectrometría de Masas , Datos de Secuencia Molecular , Elastasa Pancreática/fisiología , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína/genética , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/química , Receptores de Trombina/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , Trombina/agonistas , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
Thrombopoietin (TPO) regulates stem cell proliferation and maturation of megakaryocytes by activating the c-Mp1-receptor, a member of the hematopoietic cytokine family. As human platelets possess c-Mp1-receptors and supraphysiological concentrations of TPO trigger platelet aggregation and secretion, we searched for the signalling pathways through which the c-Mp1-receptor might activate platelets. A physiological concentration of TPO (20 ng/mL) did not trigger platelet functions, but increased their sensitivity to alpha-thrombin resulting in a 4-fold faster dense granule secretion. The effect of TPO was abolished by indomethacin and caused by synergism with signal generation by alpha-thrombin at the level of the cytosolic phospholipase A2 (cPLA2) pathway resulting in more arachidonate release, cPLA2 phosphorylation and thromboxane A2 formation. A similar synergism was seen at the level of extracellular signal-regulated kinase 2 (ERK2 or p42-MAPK). These data suggest, that TPO increases the sensitivity of platelets to alpha-thrombin by enhancing cPLA2 activation via the ERK2-cPLA2 pathway.
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Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Fosfolipasas A/fisiología , Agregación Plaquetaria/efectos de los fármacos , Trombina/agonistas , Trombopoyetina/farmacología , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Carbazoles/farmacología , Bovinos , Gránulos Citoplasmáticos/metabolismo , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Fosfolipasas A2 Grupo IV , Humanos , Imidazoles/farmacología , Indoles/farmacología , Indometacina/farmacología , Maleimidas/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosfolipasas A2 , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/farmacología , Serotonina/metabolismo , Albúmina Sérica Bovina/farmacología , Tromboxano A2/biosíntesis , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: Sodium arachidonate was used in this study to determine its capacity to generate thrombin through platelet activation. Whether aspirin prevent this effect was also investigated. METHODS AND RESULTS: Seventeen healthy volunteers without and after 160 mg/day aspirin intake for 3-5 days were studied. Lag-time and TG at basal condition and after platelet stimulation by sodium arachidonate (AA) were measured in normal non-aspirinated as well as "in vivo" aspirinated platelet rich plasma. (PRP). The lag-time was statistically significant shorter in non-aspirinated PRP activated with AA compared with non-activated PRP. This effect was inhibited by aspirin. In non-aspirinated PRP, there was an increase of TG at 4 and 6 min. incubation when platelets were activated with AA but the difference disappeared after 8 min. incubation, (84 +/- 71; 148 +/- 58 and 142 +/- 92 nmol/L respectively) compared with non-aspirinated. non-activated platelets (16 +/- 23; 55 +/- 56 and 111 +/- 76 nmol/L at 4,6 and 8 min, p < 0.0001, p < 0.0001 and p = 0.292, respectively). The AUCo-->22 min were 520.6 +/- 545.5 in non-aspirinated, non-stimulated PRP and 808.9 +/- 617, in non-aspirinated PRP activated with sodium arachidonate (p = 0.014). Aspirin administered in vivo produced a decrease of TG in PRP activated with AA. CONCLUSION: Platelet activated by AA trigged TG. This effect was inhibited by aspirin and could be an additional beneficial effect of aspirin in the prevention of thrombosis.
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Ácido Araquidónico/farmacología , Activación Plaquetaria/efectos de los fármacos , Trombina/agonistas , Adulto , Anciano , Área Bajo la Curva , Aspirina/administración & dosificación , Aspirina/farmacología , Femenino , Humanos , Cinética , Masculino , Persona de Mediana Edad , Trombina/antagonistas & inhibidores , Trombina/metabolismoRESUMEN
Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.
Asunto(s)
Plaquetas/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/sangre , Proteínas de Choque Térmico , Proteínas Quinasas Activadas por Mitógenos , Receptores de Trombina/agonistas , Trombopoyetina/farmacología , Plaquetas/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Proteínas de Choque Térmico HSP27 , Humanos , Imidazoles/farmacología , Chaperonas Moleculares , Proteínas de Neoplasias/sangre , Fosfolipasas A/sangre , Fosforilación/efectos de los fármacos , Fosfotirosina/sangre , Piridinas/farmacología , Receptores de Trombina/fisiología , Trombina/agonistas , Trombina/antagonistas & inhibidores , Trombina/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.