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1.
Platelets ; 21(4): 260-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20218907

RESUMO

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8) platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01-300 microg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 microg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca(2+) platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca(2+), independently of cGMP generation and activation of GPIIb/IIIa complex.


Assuntos
Plaquetas , Lipopolissacarídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Cálcio/metabolismo , Catalase/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Cicloeximida/metabolismo , Fosfatase 2 de Especificidade Dupla/metabolismo , Fibrinogênio/metabolismo , Humanos , Camundongos , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
2.
Eur J Pharmacol ; 627(1-3): 229-34, 2010 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-19895807

RESUMO

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.


Assuntos
Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Molsidomina/análogos & derivados , Superóxidos/farmacologia , Trombina/farmacologia , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Adesão Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Sequestradores de Radicais Livres/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Humanos , Molsidomina/farmacologia , Nitratos/metabolismo , Ácido Peroxinitroso/farmacologia , Solubilidade , Tirosina/análogos & derivados , Tirosina/metabolismo
3.
Proc Natl Acad Sci U S A ; 103(9): 3434-9, 2006 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-16492779

RESUMO

The nitric oxide-mediated actions are mostly due to cyclic GMP (cGMP) formation, but cGMP-independent mechanisms, such as tyrosine nitration, have been suggested as potential signaling pathways modulating the NO-induced responses. However, the mechanisms that lead to tyrosine nitration in platelets are poorly studied, and the protein targets of nitration have not been identified in these cells. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the cGMP-independent mechanisms of the NO-donor sodium nitroprusside (SNP) that leads to inhibition of platelet adhesion. SNP concentration-dependently inhibited platelet adhesion, as observed at 15-min and 60-min adhesion. Additionally, SNP markedly increased the cGMP levels, and the soluble guanylate inhibitor ODQ nearly abolished the SNP-mediated cGMP elevations in all experimental conditions used. Nevertheless, ODQ failed to affect the adhesion inhibition obtained with 1.0 mM SNP at 15 min. On the other hand, superoxide dismutase or peroxynitrite (ONOO(-)) scavenger epigallocatechin gallate significantly reversed the inhibition of platelet adhesion by SNP (1 mM, 15 min). Western blot analysis in SNP (1 mM, 15 min)-treated platelets showed a single tyrosine-nitrated protein with an apparent mass of approximately 105 kDa. Nanospray LC-MS/MS identified the human alpha-actinin 1 cytoskeletal isoform (P12814) as the protein contained in the nitrated SDS gel band. Thus, tyrosine nitration of alpha-actinin, through ONOO(-) formation, may be a key modulatory mechanism to control platelet adhesion.


Assuntos
Actinina/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , GMP Cíclico/metabolismo , Nitratos/metabolismo , Doadores de Óxido Nítrico/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Actinina/química , Actinina/isolamento & purificação , Plaquetas/química , Plaquetas/efeitos dos fármacos , Catequina/análogos & derivados , Catequina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibrinogênio/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Humanos , Doadores de Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Solubilidade , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo , Trombina/metabolismo , Tirosina/química , Tirosina/metabolismo
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