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1.
Platelets ; 27(2): 159-67, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26247099

RESUMO

High on-clopidogrel platelet reactivity (HPR) is a predictor of ischemic events after percutaneous coronary intervention. We conducted a prospective cohort study to identify variables related to HPR in acute coronary syndrome patients who are at high thrombotic risk. We enrolled 463 patients undergoing urgent coronary angiography. Platelet reactivity was measured 12-36 hours after 600 mg clopidogrel loading with multiple electrode aggregometry (Multiplate® analyzer, Roche, Basel, Switzerland, 6.4 µM ADP). HPR was defined by the consensus cut-off area under the curve >46 U. The rate of HPR was 16.0%. We analyzed simple clinical and laboratory parameters with backward multivariate logistic regression and identified the following predictors of HPR: platelet count (per G/L, OR: 1.0073, 95% CI: 1.0035-1.0112, p = 0.0002), CRP level (per mg/L, OR: 1.0077, 95% CI: 1.0016-1.01372, p = 0.01), and active smoking (OR: 0.51, 95% CI: 0.29-0.89, p = 0.02). We developed and internally validated a risk prediction model demonstrating moderate discriminative capacity (area-under-the-receiver operating characteristic curve = 0.67). In conclusion, we found a relatively low rate of high on-clopidogrel platelet reactivity (16.0%) even in an acute patient cohort. HPR measured by Multiplate was associated with high platelet count and CRP level on admission and was inversely related to active smoking. The model with rapidly available simple parameters might help to identify individuals at risk for HPR in the acute setting.


Assuntos
Síndrome Coronariana Aguda/diagnóstico , Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Trombose/diagnóstico , Ticlopidina/análogos & derivados , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/complicações , Síndrome Coronariana Aguda/diagnóstico por imagem , Área Sob a Curva , Plaquetas/metabolismo , Plaquetas/patologia , Proteína C-Reativa/metabolismo , Clopidogrel , Angiografia Coronária , Modelos Logísticos , Intervenção Coronária Percutânea , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Estudos Prospectivos , Fatores de Risco , Fumar/fisiopatologia , Trombose/sangue , Trombose/complicações , Trombose/diagnóstico por imagem , Ticlopidina/uso terapêutico , Resultado do Tratamento
2.
Front Cardiovasc Med ; 11: 1338066, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38450368

RESUMO

Introduction: Kinetics of stress-related biological parameters were determined in acute coronary syndrome (ACS) patients undergoing complex cardiovascular rehabilitation. Methods: We determined platelet functionality in the absence/presence of a selective alpha-2 adrenergic receptor inhibitor, atipemazole parallel with salivary cortisol levels at enrolment, and at 3- and 12-months follow-up in 75 ACS patients with percutaneous coronary intervention. Results: Pharmacological/non-pharmacological secondary prevention methods have been efficiently applied. Baseline aggregometry indicated platelet hyperactivity, decreasing gradually and being significantly reduced late, at 12 months (p < 0.05). Cortisol levels followed similar kinetics (p < 0.05). Baseline epinephrine-induced aggregations (EIA) significantly correlated with most of the other platelet agonists, even at subsequent time-points. Patients with upper-quartile EIA at enrolment (EIA-UQ) had significantly higher ADP- and collagen-induced aggregations at enrolment, at 3- and 12-months follow-up as well, indicating that high adrenergic response in the acute phase is accompanied by general platelet hyperactivity and predicts sustained platelet activation. In the EIA-UQ group higher cardiac biomarker release, elevated C-reactive protein and cortisol levels, and lower baseline left ventricular ejection fraction were detected.Atipemazole significantly reduced platelet aggregation induced by several platelet agonists, being most potent and comparable to full in vitro P2Y12 inhibition on collagen-induced aggregations (p < 0.05), indicating that catecholamines might serve as promt/long-term modulators of platelet function. Discussion: Despite effective CCR programme and dual antiplatelet therapy, prolonged activation of sympathetic neuroendocrine system and general platelet hyperactivity can be detected up to one year in ACS patients with high adrenergic platelet activity. Moreover, initial high adrenergic activity is accompanied by clinical parameters associated to increased cardiovascular risk, therefore early identification of these patients might support complex optimal long-term therapy.

3.
Thromb Haemost ; 100(5): 829-38, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18989527

RESUMO

Combined antiplatelet therapy reduces recurrent atherothrombotic events in stable coronary disease patients; however, high residual platelet reactivity measured ex vivo still raises concerns as a condition related to treatment failure. Alpha-2 adrenoceptor enhances platelet reactivity and might contribute to this phenomenon. For the present study, 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid) were recruited. Born aggregometry was performed with adenosine diphosphate (ADP), collagen and epinephrine. To verify platelet adrenergic activity, potentiation by low-dose epinephrine and inhibition by selective alpha-2 receptor blocker atipamezole were determined. To assess the P2Y(12)-specific residual activity, cangrelor was used. Plasma norepinephrine, soluble CD40-ligand, high-sensitivity-C-reactive protein (hsCRP) - and in 24 subjects platelet P-selectin positivity were measured. Epinephrine - at very low concentration (10(-9)g/ml) - significantly potentiates (1.25 microM ADP: 26.5% vs. 43%; 5 microM ADP: 53% vs. 64.5%; collagen: 17% vs 42%, p < 0.001) while atipamezole inhibits ADP- and collagen-induced platelet aggregations (1.25 microM ADP: 26.5% vs. 23%; 5 microM ADP: 53% vs. 47%; collagen: 17% vs. 11%, p < 0.001). Patients with high adrenergic activity have significantly increased baseline ADP- and collagen-induced platelet aggregation. Based on cangrelor's efficacy, these patients have significantly more residual P2Y(12) activity as well. HsCRP and soluble CD40-ligand levels were similar. In conclusion, stable coronary heart disease patients with prominent adrenoceptor activity in vitro have significantly increased platelet aggregability and more functional P2Y(12) receptor, indicating poor inhibitory response to thienopyridines. Therefore, platelet adrenergic receptor represents a considerable, dynamic factor of high residual platelet reactivity and might contribute to cardiovascular events indicating failure of antiplatelet therapy.


Assuntos
Plaquetas/efeitos dos fármacos , Doença da Artéria Coronariana/tratamento farmacológico , Resistência a Medicamentos , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Ticlopidina/análogos & derivados , Difosfato de Adenosina , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Idoso , Aspirina , Plaquetas/metabolismo , Clopidogrel , Colágeno , Doença da Artéria Coronariana/sangue , Quimioterapia Combinada , Feminino , Humanos , Imidazóis/farmacologia , Masculino , Pessoa de Meia-Idade , Norepinefrina/sangue , Testes de Função Plaquetária , Receptores Adrenérgicos alfa 2/sangue , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2Y12 , Ticlopidina/uso terapêutico , Falha de Tratamento
4.
Eur J Prev Cardiol ; 24(11): 1148-1156, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28438028

RESUMO

Background Acute coronary syndrome is associated with platelet hyperactivity, which in its persistent form, promotes recurrent thrombotic events. Complex cardiac rehabilitation after acute coronary syndrome improves clinical outcome; however, its effect on platelet hyperactivity is unknown. Design and methods We enrolled 84 acute coronary syndrome patients on dual antiplatelet therapy, who underwent a new complex cardiac rehabilitation programme (NovaCord physiotherapy, lifestyle counselling, strict diet, stress management and regular coaching) and 51 control acute coronary syndrome patients with traditional cardiac rehabilitation. Platelet functionality was determined at enrolment and at three months follow-up by aggregometry, serum platelet-derived growth factor levels, total- and platelet-derived microvesicle counts (PMV; CD41a+/CD61+, CD62P+). Results Platelet aggregation parameters and platelet-derived growth factor levels were significantly decreased in the complex cardiac rehabilitation group at three months (1 µg/ml collagen, median (interquartile range): 22 (10-45) vs 14 (7.5-25.5)%, p = 0.0015; 2 µg/ml collagen: 36 (22-60) vs 26.5 (16-37)%, p = 0.0019; 1.25 µM adenosine-diphosphate: 4.5 (1-10) vs 1 (0-3)%, p = 0.0006; 5 µM adenosine-diphosphate: 27 (16-38) vs 22 (12-31)%, p = 0.0078; epinephrine: 33 (15-57) vs 27 (12-43)%, p = 0.01; platelet-derived growth factor: 434.6 (256.0-622.7) vs 224.8 (148.5-374.1) pg/ml, p = 0.0001). In contrast, these changes were absent or did not reach statistical significance in the traditional cardiac rehabilitation group. Platelet-derived microvesicle counts were significantly decreased in both groups, while total microvesicle count was significantly reduced only in the complex cardiac rehabilitation group (median (interquartile range): 3945.5 (2138-5661) vs 1739 (780-2303) count/µl; p = 0.0001). Conclusions Platelet hyperactivity three months after acute coronary syndrome significantly decreased in patients undergoing complex cardiac rehabilitation. Besides dual antiplatelet therapy, effective management and comprehensive control of cardiovascular risk factors might represent a new, non-pharmacological approach to influence platelet functionality.


Assuntos
Síndrome Coronariana Aguda/reabilitação , Plaquetas/fisiologia , Reabilitação Cardíaca/métodos , Dietoterapia/métodos , Estilo de Vida Saudável , Modalidades de Fisioterapia , Agregação Plaquetária/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Sobreviventes/estatística & dados numéricos , Síndrome Coronariana Aguda/sangue , Feminino , Seguimentos , Humanos , Integrina alfa2/sangue , Integrina beta3/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Testes de Função Plaquetária , Estudos Prospectivos
5.
Atherosclerosis ; 188(1): 142-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16297392

RESUMO

Systemic factors and blood flow velocity related to atherosclerosis have been examined mainly separately or by in vitro studies. The aim of our study was to investigate the association between local coronary blood flow (corrected TIMI frame count, CTFC) and systemic atherosclerosis-related inflammatory parameters such as soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (Il-6), high sensitivity C-reactive protein (hsCRP) and von Willebrand factor (vWF) in humans. We enrolled the following groups of ischemic heart disease (IHD) patients: patients with coronary stenosis and stable (CAD, n = 96) or unstable angina (ACS, n = 27), patients with documented myocardial ischemia and normal coronary angiogram (NEG, n = 68). Patient groups showed only marginal differences in CTFC or sICAM-1 levels. In contrast, when IHD patients were studied individually, general positive correlation was found between CTFC and sICAM-1 level (r = 0.33; in NEG r = 0.25; in CAD r = 0.37; in ACS r = 0.61), being the strongest in ACS. The relation was independent from age, gender, BMI, smoking, hypertension, diabetes, previous myocardial infarction, family history of IHD, medication, hsCRP, IL-6 and vWF levels. (odds ratio, OR = 6.4; CI 95%: 2.43-16.84; p < 0.05). Nevertheless, correlation between CTFC and IL-6, hsCRP, vWF levels was not found. These results indicate inverse correlation between coronary blood flow and adhesion molecule production independently from conventional cardiovascular risk factors and inflammatory markers.


Assuntos
Molécula 1 de Adesão Intercelular/sangue , Isquemia Miocárdica/fisiopatologia , Velocidade do Fluxo Sanguíneo/fisiologia , Proteína C-Reativa/análise , Circulação Coronária/fisiologia , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Fator de von Willebrand/análise
6.
Clin Chim Acta ; 463: 138-144, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27794429

RESUMO

The effect of invasive percutaneous coronary procedures on complement activation has not been elucidated. We enrolled stable angina patients with elective percutaneous coronary intervention (SA-PCI, n=24), diagnostic coronary angiography (CA, n=52) and 23 patients with ST segment elevation myocardial infarction and primary PCI (STEMI-PCI). Complement activation products (C1rC1sC1inh, C3bBbP and SC5b-9) were measured on admission, 6 and 24h after coronary procedures. The alternative pathway product, C3bBbP significantly and reversibly increased 6h after elective PCI (baseline: 7.81AU/ml, 6h: 16.09AU/ml, 24h: 4.27AU/ml, p<0.01, n=23) and diagnostic angiography (baseline: 6.13AU/ml, 6h: 12.08AU/ml, 24h: 5.4AU/ml, p<0.01, n=52). Six hour C3bBbP values correlated with post-procedural CK, creatinine level and the applied contrast material volume (r=0.41, r=0.4, r=0.3, p<0.05, respectively). In STEMI-PCI, baseline C3bBbP level was higher, compared to SA-PCI or CA patients (11.33AU/ml vs. 7.81AU/ml or 6.13AU/ml, p<0.001). Similarly, the terminal complex (SC5b-9) level was already elevated at baseline compared to SA-PCI group (3.49AU/ml vs. 1.87AU/ml, p=0.011). Complement pathway products did not increase further after primary PCI. Elective coronary procedures induced transient alternative complement pathway activation, influenced by the applied contrast volume. In STEMI, the alternative complement pathway is promptly activated during the atherothrombotic event and PCI itself had no further detectable effect.


Assuntos
Angina Pectoris/imunologia , Angina Pectoris/cirurgia , Procedimentos Cirúrgicos Cardíacos , Ativação do Complemento , Via Alternativa do Complemento/imunologia , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/cirurgia , Doença Aguda , Angina Pectoris/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue
7.
FEBS Lett ; 524(1-3): 15-9, 2002 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-12135734

RESUMO

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.


Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico , Receptores Purinérgicos P2/fisiologia , Tirosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Homologia de Sequência de Aminoácidos
8.
Thromb Res ; 133(2): 257-64, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24359966

RESUMO

UNLABELLED: The benefit of adjusted antiplatelet therapy in patients with myocardial infarction after primary percutaneous coronary intervention is not well elucidated. We aimed to identify patients with high on treatment platelet reactivity and to gradually adjust antiplatelet therapy. MATERIALS AND METHODS: We enrolled 133 acute myocardial infarction and 67 stable angina patients undergoing intracoronary stenting into our study. Maximal aggregation was determined with light transmission aggregometry. Aggregation >50% induced by 5 µM ADP was indexed with high on-clopidogrel treatment platelet reactivity. In these cases 75 mg clopidogrel was doubled and control test was performed. Patients effectively inhibited with 150 mg clopidogrel were defined as clopidogrel pseudo non-responders. Patients with high platelet reactivity even on 150 mg clopidogrel were considered as clopidogrel real non-responders and were switched to ticlopidine. RESULTS: Aggregations (5ADP; p=0.046) and the ratio of real non-responders (p=0.013) were significantly higher in the myocardial infarction group. Most real non-responders were effectively treated with switch of therapy. The ratio of pseudo non-responders also tended to be higher in myocardial infarction. Platelet reactivity remained constant during follow-up; however, a new appearance of high platelet reactivity was observed at 6 and at 12 months. CONCLUSIONS: Patients with acute myocardial infarction undergoing percutaneous coronary intervention may benefit from prospective platelet function testing, because of higher platelet reactivity and much higher ratio of clopidogrel real non-response. Switch of therapy may effectively overcome clopidogrel non-response. A new appearance of high platelet reactivity with unknown clinical significance is observed in both groups among the patients on clopidogrel.


Assuntos
Plaquetas/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Stents , Ticlopidina/análogos & derivados , Idoso , Clopidogrel , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Stents/efeitos adversos , Ticlopidina/uso terapêutico
9.
Can J Cardiol ; 25(4): 229-32, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19340348

RESUMO

BACKGROUND: Drug-eluting coronary stent implantation emerged as a safe and effective therapeutic approach by preventing coronary restenosis and reducing the need for further revascularization. However, in contrast to bare metal stents, recent data suggest a unique underlying pathology, namely late coronary stent thrombosis and delayed endothelial healing. OBJECTIVE: To report a case of very late coronary stent thrombosis (834 days after implantation) requiring repeat urgent target-vessel revascularization. Importantly, six days before the acute coronary event, combined nonsteroidal anti-inflammatory drug therapy was initiated. RESULTS: Although a dual antiplatelet regimen was continuously maintained, aggregation measurements indicated only partial antiplatelet effect, which returned to the expected range when nonsteroidal anti-inflammatory drugs were omitted. CONCLUSIONS: The observation indicates that, even 834 days after drug-eluting stent implantation, effective combined antiplatelet therapy might be crucial in certain individuals and the possible impact of drug interactions should not be underestimated. Further efforts should focus on the challenging task of identifying patients or medical situations with prolonged, increased risk of stent thrombosis.


Assuntos
Trombose Coronária/etiologia , Stents Farmacológicos/efeitos adversos , Inibidores da Agregação Plaquetária/administração & dosagem , Adulto , Angioplastia Coronária com Balão , Aspirina/administração & dosagem , Clopidogrel , Trombose Coronária/fisiopatologia , Trombose Coronária/prevenção & controle , Inibidores de Ciclo-Oxigenase/administração & dosagem , Inibidores de Ciclo-Oxigenase/efeitos adversos , Angiopatias Diabéticas/terapia , Diclofenaco/administração & dosagem , Diclofenaco/efeitos adversos , Interações Medicamentosas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Humanos , Deslocamento do Disco Intervertebral/tratamento farmacológico , Masculino , Meloxicam , Infarto do Miocárdio/terapia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Tiazinas/administração & dosagem , Tiazinas/efeitos adversos , Tiazóis/administração & dosagem , Tiazóis/efeitos adversos , Ticlopidina/administração & dosagem , Ticlopidina/análogos & derivados , Fatores de Tempo
10.
Mol Med ; 12(9-10): 221-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17225870

RESUMO

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.


Assuntos
Leucócitos/enzimologia , Isquemia Miocárdica/enzimologia , Reperfusão Miocárdica/métodos , Poli(ADP-Ribose) Polimerases/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Idoso , Angina Pectoris/enzimologia , Angina Pectoris/patologia , Fator de Indução de Apoptose/metabolismo , Dano ao DNA , Demografia , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangue , Ativação Enzimática , Feminino , Humanos , Imuno-Histoquímica , Leucócitos/imunologia , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/patologia , Oxirredução , Peróxidos/sangue , Transporte Proteico , Tirosina/análogos & derivados , Tirosina/biossíntese
11.
Blood ; 100(7): 2499-505, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12239162

RESUMO

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Plaquetas/fisiologia , Colágeno/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Agregação Plaquetária/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/sangue , Apirase/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Ácido Egtázico/farmacologia , Humanos , Técnicas In Vitro , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Cardiovasculares , Agregação Plaquetária/efeitos dos fármacos , Proteína Quinase C/sangue , Receptores Purinérgicos P2X
12.
J Biol Chem ; 278(47): 46661-7, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-14500714

RESUMO

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Plaquetas/metabolismo , Degranulação Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/farmacologia , Plaquetas/citologia , Cálcio/metabolismo , Calmodulina/metabolismo , Tamanho Celular , Colágeno/farmacologia , Ativação Enzimática , Humanos , Microscopia Eletrônica , Fosforilação/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2X
13.
Blood ; 101(10): 3969-76, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12521992

RESUMO

We have generated transgenic mice overexpressing the human P2X(1) ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X(1) ionotropic activity as determined by more prominent P2X(1)-mediated Ca(2+) influx and platelet shape change. P2X(1) overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A(2) mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds(-1) resulted in increased P2X(1)-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X(1) ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A(2)-, and shear stress-triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.


Assuntos
Plaquetas/fisiologia , Receptores Purinérgicos P2/genética , Trombose/genética , Difosfato de Adenosina/farmacologia , Animais , Contagem de Células Sanguíneas , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Tamanho Celular , Contagem de Eritrócitos , Humanos , Canais Iônicos/genética , Canais Iônicos/fisiologia , Cinética , Megacariócitos/fisiologia , Camundongos , Camundongos Transgênicos , Fenótipo , Contagem de Plaquetas , Receptores Purinérgicos P2X , Trombina/farmacologia
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