RESUMEN
In this study we present a comprehensive evaluation of the molecular interactions between human T cells and porcine aortic endothelial cells (PAEC) that contribute to human T cell activation. Binding assays demonstrated that porcine erythrocytes (E) and PAEC express ligand(s) for the human T cell glycoprotein CD2. Prior incubation of human T cells with a blocking monoclonal antibody directed against CD2 (alpha CD2-BL) completely inhibited T cell/E and T cell/PAEC interaction. Xenogeneic mixed lymphocyte reactions (XMLR) revealed that human PBMC, or highly purified T cells were activated by PAEC in the absence of human antigen-presenting cells (APC). Addition of alpha CD2-BL or alpha LFA-1 to these assays inhibited PAEC-mediated human T cell activation. Furthermore, we demonstrated that highly purified human CD4+ and CD8+ T cells proliferated in response to PAEC and that this response was blocked by monoclonal antibodies directed against LFA-1 and CD2. Addition of alpha SLA class I blocked the proliferation of CD8+ but not CD4+ T cells, indicating direct presentation of SLA class I antigens to human T cells. We have recently shown that expression of the human complement inhibitor (CD59) on PAEC (PAEC-LXSNCD59) rendered these cells resistant to human complement-mediated activation and lysis, suggesting that human CD59 expression on PAEC could be an effective therapy for hyperacute rejection (HAR). However, recent studies have shown that in addition to its role as a complement inhibitor, CD59 binds human T cell CD2 and contributes to T cell activation. We therefore examined whether human CD59 expression on PAEC augmented the human antiporcine T cell response. We demonstrated that human T cells do not display increased binding to or activation by PAEC-LXSNCD59 relative to PAEC controls. Taken together, our data establish that PAEC directly stimulate human T cells in vitro and that interactions between the human accessory molecules CD2, LFA-1 and their PAEC surface ligands contribute to human T cell activation. In addition, the expression of human CD59 on porcine donor organs may confer resistance to human complement-mediated HAR without exacerbating the human antiporcine cellular response.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Aorta , Antígenos CD2 , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Células Cultivadas , Endotelio Vascular/inmunología , Eritrocitos/inmunología , Citometría de Flujo , Humanos , Ligandos , Mitomicina/farmacología , Formación de Roseta , Ovinos , Porcinos , Linfocitos T/efectos de los fármacosRESUMEN
Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.
Asunto(s)
Antígenos CD/inmunología , Endotelio Vascular/inmunología , Glicoproteínas de Membrana/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antígenos CD59 , Activación de Complemento , Citotoxicidad Inmunológica , Activación Enzimática , Glicosilfosfatidilinositoles , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes , Especificidad de la Especie , Porcinos , Tromboplastina/metabolismoRESUMEN
BMY 42393, (2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid), is a new prostacyclin partial agonist that inhibited ADP, collagen and thrombin-induced platelet aggregation (IC50 range 0.3 - 2.0 microM). BMY 42393 stimulated platelet adenylate cyclase activity (EC50 = 25 nM), however, the maximal activation was 75-80% of that observed with maximal iloprost or PGE1. Platelets treated with BMY 42393 showed an elevation of cAMP levels and activation of cAMP-dependent protein kinase. BMY 42393 also inhibited thrombin-induced elevation of intracellular free calcium. BMY 42393 competed for radiolabeled iloprost and PGE1 binding to platelet membranes (IC50; 170 nM and 130 nM, respectively); however, it had little effect on radiolabeled PGE2, PGD2, or SQ 29548 binding. These studies indicate that BMY 42393 is a novel platelet aggregation inhibitor which acts by stimulation of platelet prostacyclin receptors to elevate platelet cAMP levels.
Asunto(s)
Epoprostenol , Oxazoles/farmacología , Fenoxiacetatos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Prostaglandina/efectos de los fármacos , Adenilil Ciclasas/sangre , Plaquetas/enzimología , Calcio/sangre , AMP Cíclico/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Humanos , Estructura Molecular , Oxazoles/química , Fenoxiacetatos/química , Inhibidores de Agregación Plaquetaria/química , Ensayo de Unión Radioligante , Receptores de Epoprostenol , Relación Estructura-ActividadRESUMEN
The oral activity and antithrombotic efficacy of BMY 42393 was examined in ex vivo platelet aggregation studies and arterial thrombosis animal models. In a heterologous ex vivo platelet aggregation assay, ADP-induced human platelet aggregation was inhibited when washed human platelets were combined with rat platelet-poor plasma, taken from rats previously orally-dosed with BMY 42393. The IC50 for platelet aggregation inhibition was approximately 10 mg/kg. In a laser-induced thrombosis model, thrombus formation in a revascularized rabbit ear chamber was prevented in a dose-dependent fashion with an ED50 of about 2 mg/kg. A relatively long duration of anti-thrombotic activity was observed in the rabbit ear laser-induced thrombus study and the ex vivo platelet studies. Inhibition of thrombus formation was also demonstrated in a canine model of electrically-induced coronary artery thrombosis. BMY 42393 also prevented cyclic flow reductions in a monkey stenotic renal artery model. These studies indicate that BMY 42393 is orally active and capable of preventing laser and electric current-induced thrombus formation in animal models of arterial thrombosis.
Asunto(s)
Trombosis Coronaria/tratamiento farmacológico , Oxazoles/uso terapéutico , Fenoxiacetatos/uso terapéutico , Administración Oral , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Perros , Humanos , Macaca fascicularis , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Obstrucción de la Arteria Renal/tratamiento farmacológicoRESUMEN
The 4,5-diphenyloxazole derivatives 2-4 were previously identified as nonprostanoid prostacyclin (PGI2) mimetics. A series of derivatives of 2-4 bearing substitutents at the carbon atom alpha to the oxazole ring were synthesized and evaluated as inhibitors of ADP-induced aggregation of human platelets in vitro. In the unsaturated series, the alpha-carbethoxy derivative 10a, evaluated as an equal mixture of geometrical isomers, inhibited platelet aggregation with an IC50 of 0.36 microM. Evaluation of the individual methyl ester derivatives (E)-9a and (Z)-9a revealed that (E)-9a was 10-fold more potent than (Z)-9a. In the saturated series, the alpha-carbomethoxy-substituted compound 12a inhibited platelet aggregation with an IC50 of 0.08 microM, 15-fold more potent than the unsubstituted prototype 2. The potency of 12a was found to be sensitive to variation of the methoxy moiety. The ethyl (12b) and isopropyl (12d) esters were less effective as were the acid 12e and a series of amides (12f-h). Other substituents introduced at this site of the pharmacophore included P(O)(OEt)2 (25), SCH3 (31a), S(O)CH3 (31b), SO2CH3 (31c), isopropyl (31d), phenyl (31f), and CH2OH (31i). However, none were significantly more potent inhibitors of platelet function than the parent compound 2. The results indicate the presence of a pocket in the PGI2 receptor protein that preferentially recognizes small, polar but uncharged substituents. The structure-activity correlates are suggestive of a hydrogen-bond interaction between a donor moiety on the PGI2 receptor and the methoxycarbonyl functionality of 12a that is sensitive to both the size of the substituent and its stereochemical presentation in this structural class of PGI2 mimetic. The ethyl ester 12b dose-dependently displaced [3H]iloprost from human platelet membranes and stimulated adenylate cyclase. However, the maximal stimulation was less than that recorded for iloprost, indicating that 12b functions as a partial agonist at the PGI2 receptor.
Asunto(s)
Epoprostenol/farmacología , Oxazoles/síntesis química , Inhibidores de Agregación Plaquetaria/síntesis química , Receptores de Prostaglandina/efectos de los fármacos , Adenosina Difosfato/farmacología , Humanos , Enlace de Hidrógeno , Estructura Molecular , Oxazoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Epoprostenol , Receptores de Prostaglandina/fisiología , Relación Estructura-ActividadRESUMEN
1-(Cyclohexylmethyl)-4-[4-[(2,3-dihydro-2-oxo-1H-imidazo[4,5-b] quinolin-7-yl)oxy]-1-oxobutyl]piperazine (2) was previously identified as a potent, water-soluble inhibitor of human blood platelet cAMP phosphodiesterase and of induced aggregation in vitro that demonstrated effective antithrombotic activity in animal models of thrombosis. Although 2 exhibited 25% oral bioavailability in rats, pharmacokinetic studies conducted in monkeys revealed that the parent compound was less than 5% bioavailable, the result of extensive first-pass biotransformation in the liver. In an effort to identify potent platelet aggregation inhibitors with enhanced metabolic stability, the side-chain amide moiety of 2 was replaced with chemically more stable urea (6a-s), sulfonamide (13a-m), sulfone (19a-r), and tetrazole (23a-s) moieties. Many representatives from each of these structural types effectively combined potent inhibition of ADP-induced human platelet aggregation in vitro with excellent aqueous solubility, and several are superior to 2. Within each series, the N-(cyclohexylmethyl)-, N-(2-ethylbutyl)-, N-benzyl-, and N-(4-fluorobenzyl)-substituted derivatives were evaluated for in vitro metabolic stability by incubating with the S-9 fraction of monkey liver for 2 h, and the extent of biotransformation was compared with that of the prototype 2. The sulfone 19e and the tetrazoles 23e, 23g, 23j, and 23q were significantly more stable than 2 under these conditions, and 19e and 23e were selected for evaluation in vivo. Tetrazole 23e exhibited 72% bioavailability following ip administration to rats compared with 35% bioavailability for 2 and 19e under the same conditions. However, the oral bioavailability of 19e and 23e in the rat was estimated to be only 3%, suggesting that 19e and 23e are less readily absorbed from the gastrointestinal tract than 2.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Plaquetas/enzimología , Imidazoles/síntesis química , Imidazoles/farmacología , Inhibidores de Agregación Plaquetaria/síntesis química , Quinolonas/síntesis química , Quinolonas/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/sangre , Adenosina Difosfato/farmacología , Animales , Plaquetas/efectos de los fármacos , Chlorocebus aethiops , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Sulfonas/síntesis química , Sulfonas/farmacología , Tetrazoles/síntesis química , Tetrazoles/farmacología , Urea/análogos & derivados , Urea/síntesis química , Urea/farmacología , AguaRESUMEN
Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester- but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8 micrograms/ml for 5-15 min at 37 degrees C) which allow the introduction of small extracellular molecules without the loss of large intracellular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Endotelio Vascular/metabolismo , Transducción de Señal/fisiología , Trombina/farmacología , Factor de von Willebrand/metabolismo , Secuencia de Aminoácidos , Azepinas/farmacología , Calmodulina/antagonistas & inhibidores , Permeabilidad de la Membrana Celular , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Naftalenos/farmacología , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/metabolismo , Sulfonamidas/farmacología , Venas Umbilicales/citologíaRESUMEN
4,5-Diphenyl-2-oxazolenonanoic acid (18b) was synthesized and found to inhibit ADP-induced aggregation of human platelets with an IC50 of 2.5 microM. Acid 18b displaced [3H]iloprost from human platelet membranes in a concentration-dependent fashion, consistent with 18b inhibiting platelet function by acting as a prostacyclin mimetic. By inserting a phenoxy ring into the side-chain moiety of 18b and systematically varying the pattern of substitution and length of the tethers, more potent inhibitors of platelet aggregation were identified. A phenoxy ring inserted centrally in the side chain proved to be the optimal arrangement but significant activity was observed when the aromatic ring was bound directly to the 2 position of the heterocycle. The meta-substituted cis-(ethenylphenoxy)acetic acid 37 is the most potent platelet aggregation inhibitor synthesized as part of this study with an IC50 of 0.18 microM. Acid 37 displaces [3H]iloprost from human platelet membranes with an IC50 of 6 nM. The trans-olefinic isomer of 37 (25p) is 72-fold weaker as an inhibitor of ADP-induced platelet aggregation, but the saturated derivative 25w (BMY 42393) is intermediate in potency. Structure-activity studies using 25w as a template focused on modification of the tethers intervening between the side-chain phenyl ring and the oxazole and carboxylate termini and substitution of the phenyl ring. These studies revealed that biological activity was sensitive to both the identity of the concatenating atoms and the pattern of ring substitution. The structure-activity relationships provide insight into the topographical relationship between the diphenylated oxazole ring and the carboxylic acid terminus that comprise the nonprostanoid prostacyclin mimetic pharmacophore.
Asunto(s)
Oxazoles/síntesis química , Inhibidores de Agregación Plaquetaria/síntesis química , Adenosina Difosfato/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Humanos , Oxazoles/química , Oxazoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Relación Estructura-ActividadRESUMEN
Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the "tethered-ligand" thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.
Asunto(s)
Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Secuencia de Aminoácidos , Tolerancia a Medicamentos , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Transducción de Señal , Trombina/antagonistas & inhibidoresRESUMEN
We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.
Asunto(s)
Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Fosfatidilinositoles/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Plaquetas/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Diglicéridos/análisis , Activación Enzimática/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Nigericina/farmacología , Ésteres del Forbol/farmacología , Ácidos Fosfatidicos/análisis , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles/análisis , Fosforilación , Agregación Plaquetaria , Proteína Quinasa C/metabolismo , Intercambiadores de Sodio-Hidrógeno , Trombina/farmacologíaRESUMEN
Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.
Asunto(s)
Endotelio Vascular/citología , Epoprostenol/metabolismo , Ionomicina/farmacología , Ésteres del Forbol/farmacología , Factor de Activación Plaquetaria/metabolismo , Trombina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Calcimicina/farmacología , Células Cultivadas , Dinoprost/metabolismo , Dinoprostona/metabolismo , Endotelio Vascular/metabolismo , Humanos , Isoquinolinas/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Piperazinas/farmacología , Prostaglandina D2/metabolismo , Prostaglandinas H/metabolismo , Prostaglandinas H/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.
Asunto(s)
Ácidos Araquidónicos/sangre , Plaquetas/metabolismo , Ionóforos/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Proteína Quinasa C/metabolismo , Adenosina Trifosfato/metabolismo , Alcaloides/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Ácidos Araquidónicos/metabolismo , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Calcimicina/farmacología , Proteínas Portadoras/metabolismo , Activación Enzimática , Humanos , Indoles/farmacología , Ionomicina/farmacología , Cinética , Lactamas/farmacología , Fosfolipasas A2 , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.
Asunto(s)
Endotelio Vascular/fisiología , Epoprostenol/biosíntesis , Interleucina-1/farmacología , Factor de Activación Plaquetaria/biosíntesis , Trombina , Factor de von Willebrand/fisiología , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Células Cultivadas , Cromatografía en Capa Delgada , Cicloheximida/farmacología , Sinergismo Farmacológico , Endotelio Vascular/efectos de los fármacos , Epoprostenol/antagonistas & inhibidores , Epoprostenol/metabolismo , Histamina , Humanos , Endoperóxidos de Prostaglandinas Sintéticos/metabolismo , Prostaglandina H2 , Prostaglandinas H/metabolismoRESUMEN
Phosphatidic acid (PA) is synthesized as the result of the receptor-mediated response of platelets to physiologic agonists. The role of PA in platelet signal transduction, however, is largely unknown. We have examined the responses of platelets to 1-stearoyl-2-arachidonoyl phosphatidic acid (SAPA), the predominant molecular species of human platelet PA. SAPA alone causes platelet aggregation, and pretreatment of platelets with SAPA markedly enhances thrombin-induced aggregation and secretion. Addition of SAPA to intact human platelets causes rapid breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the generation of diacylglycerol and endogenous PA. These reactions are associated with mobilization of intracellular calcium and activation of protein kinase C. SAPA also stimulates the release of endogenous arachidonic acid and its conversion to thromboxane A2. Furthermore, platelet activation by SAPA is blocked by indomethacin, indicating that the actions of SAPA are mediated by cyclooxygenase products. These findings suggest that SAPA may play an important role as an endogenous positive feedback signal to amplify receptor-mediated activation of PIP2-specific phospholipase C in human platelets.
Asunto(s)
Plaquetas/fisiología , Ácidos Fosfatidicos/sangre , Agregación Plaquetaria , Sistemas de Mensajero Secundario , Adenosina Trifosfato/sangre , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Calcio/sangre , Humanos , Lípidos/sangre , Ácidos Fosfatidicos/farmacología , Ácidos Fosfatidicos/fisiología , Fosforilación , Tromboxano A2/sangreRESUMEN
In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.
Asunto(s)
Plaquetas/metabolismo , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas/metabolismo , Fosfatos de Azúcar/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Inositol 1,4,5-Trifosfato , Péptidos/metabolismo , FosforilaciónRESUMEN
Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.
Asunto(s)
Plaquetas/metabolismo , Calcio/sangre , Proteínas Portadoras/sangre , Trombina/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Benzofuranos , Plaquetas/efectos de los fármacos , Fluoresceínas , Colorantes Fluorescentes , Fura-2 , Humanos , Concentración de Iones de Hidrógeno , Nigericina/farmacología , Intercambiadores de Sodio-Hidrógeno , Espectrometría de FluorescenciaRESUMEN
Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.
Asunto(s)
Plaquetas/enzimología , AMP Cíclico/farmacología , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/sangre , Adenosina Difosfato/antagonistas & inhibidores , Diglicéridos/farmacología , Epoprostenol/farmacología , Humanos , Técnicas In Vitro , Fosfoproteínas/sangre , Prostaglandina-Endoperóxido Sintasas/sangre , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function. PGI2 and plasmin cause synergistic inhibition of thrombin- and ADP-induced aggregation of washed platelets. Inhibition by PGI2 is similarly potentiated by TPA added to platelet-rich plasma to generate plasmin. Thrombin-stimulated rise in platelet cytosolic Ca2+, measured by fura2 fluorescence, and thromboxane A2 formation, measured by radioimmunoassay (RIA), are likewise synergistically inhibited by PGI2 and plasmin. Plasmin neither increases nor potentiates PGI2-stimulated increases in platelet cyclic AMP. Thus, PGI2 and plasmin cause synergistic inhibition of platelet activation by both cyclic AMP-dependent and independent mechanisms. This interaction between two different endothelium-derived products may play an important role in localizing the hemostatic plug to a site of vascular injury by preventing further thrombin-mediated accrual of platelets.
Asunto(s)
Epoprostenol/farmacología , Fibrinolisina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Calcio/metabolismo , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Humanos , Trombina/farmacología , Tromboxano B2/metabolismoRESUMEN
The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP-ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.