RESUMEN
1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+ concentration ([Ca2+]in) due to both Ca2+ entry and internal Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]in in Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLC gamma 2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin alpha IIb beta 3 is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin alpha IIb beta 3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLC gamma 2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin alpha IIb beta 3.
Asunto(s)
Venenos de Crotálidos/farmacología , Isoenzimas/metabolismo , Lectinas Tipo C , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Humanos , Técnicas In Vitro , Cinética , Fosfolipasa C gamma , Fosforilación , Conejos , Transducción de Señal , Soluciones , Estaurosporina/farmacologíaRESUMEN
Canatoxin (CNTX), a toxic protein isolated from seeds of Canavalia ensiformis, induces Ca2+ influx across the platelet plasma membrane, mobilization of arachidonic acid mediated by phospholipase A2, ATP secretion, and platelet aggregation. All these events depend on the presence of extracellular Ca2+ and are blocked by methoxyverapamil, a calcium-channel blocker. CNTX does not activate phospholipase C, and the intracellular calcium mobilization mediated by IP3 does not play a role in platelet activation by this toxin. Preincubation of rabbit platelets with 8-Br-cGMP inhibited the CNTX-evoked calcium influx, arachidonate release, ATP secretion, and cell aggregation. Our data suggest that the calcium influx is a prior step on platelet activation by CNTX, being modulated by cGMP.
Asunto(s)
Plaquetas/metabolismo , Calcio/metabolismo , GMP Cíclico/metabolismo , Galopamilo/antagonistas & inhibidores , Lectinas/farmacología , Proteínas de Plantas , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Toxinas Biológicas , Animales , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/metabolismo , Regulación hacia Abajo , Interacciones Farmacológicas , Galopamilo/farmacología , Transporte Iónico/efectos de los fármacos , ConejosRESUMEN
Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Plaquetas/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/sangre , 3',5'-AMP Cíclico Fosfodiesterasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , GMP Cíclico/farmacología , Cartilla de ADN/química , ADN Complementario/genética , Expresión Génica , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/genética , Proteínas Recombinantes , Saccharomyces cerevisiae , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
Camp is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH-rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzyme activity. N-Ethylmaleimide (NEM) and 5.5'-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not only by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Plaquetas/enzimología , AMP Cíclico/metabolismo , GMP Cíclico/farmacología , Inhibidores de Fosfodiesterasa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/química , Adenosina Monofosfato/farmacología , Sitios de Unión , Catálisis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Cisteína/química , Dietil Pirocarbonato/farmacología , Ácido Ditionitrobenzoico/farmacología , Etilmaleimida/farmacología , Histidina/química , Humanos , CinéticaRESUMEN
cGMP-inhibited phosphodiesterase (cGI-PDE) has been found to require a divalent metal cation for cAMP hydrolysis. The cGI-PDE isolated from human platelets exhibited significantly higher enzymatic activity when incubated with Mn2+, Mg2+, and Co2+. The addition of Zn2+, Cd2+, Ca2+, K+, or Na+ to the enzyme did not enhance the activity and, when present in high concentration (> 1.0 microM), Zn2+ and Cd2+ inhibited the enzymatic activity of cGI-PDE. The inhibition by Zn2+ (and Cd2+) was partially prevented by preincubation of the enzyme with Mn2+. The enzyme was also inhibited by metal chelators EDTA and 1,10-phenanthroline and not by their non-metal-chelating analogs. The partial protection against chelation (and inhibition) was afforded by AMP (the product of cAMP hydrolysis).
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Plaquetas/enzimología , Cationes Bivalentes/metabolismo , Metaloendopeptidasas/clasificación , 3',5'-GMP Cíclico Fosfodiesterasas/clasificación , AMP Cíclico/metabolismo , HumanosRESUMEN
Canatoxin (CNTX), the toxic protein from Canavalia ensiformis seeds, injected into the peritoneal cavities of mice (10 micrograms/cavity) induced a significant neutrophil migration (10.5 +/- 0.5 x 10(6) cells/cavity) after 4 h. A later migratory effect (48 h) on mononuclear cells, predominantly macrophages, was also observed (controls: 7 +/- 0.9; CNTX: 17 +/- 2.0 x 10(6) cells/cavity). These CNTX-elicited macrophages, when compared to resident cells (R) or cells elicited by thioglycollate (TG), had an increased content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (R: 4.5 +/- 0.5; TG: 7.2 +/- 1.0; CNTX: 20.2 +/- 3.0 mU/10(6) cells) and a greater (> or = 100%) phagocytic activity. The data suggest that CNTX-stimulated macrophages presented some characteristics of activated cells.
Asunto(s)
Lectinas/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Proteínas de Plantas , Toxinas Biológicas/farmacología , Acetilglucosaminidasa/efectos de los fármacos , Acetilglucosaminidasa/metabolismo , Animales , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Ratones , Fagocitosis/efectos de los fármacos , Tioglicolatos/farmacología , Factores de TiempoRESUMEN
Canatoxin (CNTX), the toxic protein from Canavalia ensiformis seeds, injected into the peritoneal cavities of mice (10 micrograms/cavity) induced a significant neutrophil migration (10.5 +/- 0.5 x 10(6) cells/cavity) after 4 h. A later migratory effect (48 h) on mononuclear cells, predominantly macrophages, was also observed (controls: 7 +/- 0.9; CNTX: 17 +/- 2.0 x 10(6) cells/cavity). These CNTX-elicited macrophages, when compared to resident cells (R) or cells elicited by thioglycollate (TG), had an increased content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (R: 4.5 +/- 0.5; TG: 7.2 +/- 1.0; CNTX: 20.2 +/- 3.0 mU/10(6) cells) and a greater (> or = 100%) phagocytic activity. The data suggest that CNTX-stimulated macrophages presented some characteristics of activated cells