abcArquivos Brasileiros de CardiologiaArq. Bras. Cardiol.0066-782X1678-4170Sociedade Brasileira de Cardiologia - SBCSão Paulo, SP, BraziltkMDGB7ZBccND9CMmYDjKwt00008S0066-782X2006001700008S0066-782X(06)0870040810.1590/S0066-782X2006001700008Artigos OriginaisAtivação plaquetária em formas clínicas distintas da doença arterial coronariana (papel da P-selectina e de outros marcadores nas anginas estável e instável)VenturinelliMargareth L.HovnanAndréSoeiroAlexandre de MatosNicolauJosé CarlosRamiresJosé A. F.D'AmicoÉlbio A.Serrano JúniorCarlos VicenteSão PauloSão PauloUniversidade de São PauloInstituto do Coração do Hospital das Clínicas FMBrazil1020068744464500107200509052005This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
OBJETIVO: Os marcadores da ativação plaquetária em geral se apresentam elevados na doença arterial coronariana. Desse modo, procuramos identificar a presença e as potenciais associações de diferentes marcadores da ativação plaquetária. MÉTODOS: Estudamos pacientes com angina instável (n=28), pacientes com angina estável (n=36) e pacientes sem doença arterial coronariana (n=30); sexo e idade foram estratificados. Os níveis sangüíneos da molécula de adesão P-selectina, do thromboxane B2 e de serotonina foram medidos por imunoensaios enzimáticos. RESULTADOS: Quando comparamos os grupos, os resultados foram: a P-selectina, o thromboxane B2 e os níveis do serotonina apresentaram-se significativamente mais elevados nos pacientes com angina instável do que nos pacientes com angina estável. CONCLUSÃO: Estes marcadores da ativação plaquetária podem, portanto, identificar formas instáveis de doença arterial coronariana.
Ativação plaquetária em formas clínicas distintas da doença arterial coronariana (papel da P-selectina e de outros marcadores nas anginas estável e instável)
Margareth L. Venturinelli; André Hovnan; Alexandre de Matos Soeiro; José Carlos Nicolau; José A. F. Ramires; Élbio A. D'Amico; Carlos Vicente Serrano Júnior
Instituto do Coração do Hospital das Clínicas – FMUSP - São Paulo, SP
Correspondência
RESUMO
OBJETIVO: Os marcadores da ativação plaquetária em geral se apresentam elevados na doença arterial coronariana. Desse modo, procuramos identificar a presença e as potenciais associações de diferentes marcadores da ativação plaquetária.
MÉTODOS: Estudamos pacientes com angina instável (n=28), pacientes com angina estável (n=36) e pacientes sem doença arterial coronariana (n=30); sexo e idade foram estratificados. Os níveis sangüíneos da molécula de adesão P-selectina, do thromboxane B2 e de serotonina foram medidos por imunoensaios enzimáticos.
RESULTADOS: Quando comparamos os grupos, os resultados foram: a P-selectina, o thromboxane B2 e os níveis do serotonina apresentaram-se significativamente mais elevados nos pacientes com angina instável do que nos pacientes com angina estável.
CONCLUSÃO: Estes marcadores da ativação plaquetária podem, portanto, identificar formas instáveis de doença arterial coronariana.
A doença arterial coronariana, representada pela aterosclerose, é a doença mais predominante dos dias de hoje, e suas complicações trombóticas são responsáveis por um número altíssimo de mortes e incapacitações. Ao longo dos últimos anos, estudos experimentais e observações clínicas e patológicas proporcionaram uma maior compreensão da formação do trombo e da sua incidência na doença arterial coronariana1.
A presença de plaquetas foi identificada no início e na propagação da trombose coronariana2-9. As plaquetas formam uma monocamada inicial no local de rompimento da placa aterosclerótica, e diversos ativadores (colágeno, difosfato de adenosina (ADP), epinefrina, serotonina) promovem a ativação plaquetária.
A agregação plaquetária e a formação de trombo decorrentes da ruptura da placa também foram identificadas como importantes mecanismos patogênicos subjacentes à síndrome coronariana de angina instável. A fisiopatologia da angina instável difere da fisiopatologia da angina induzida por esforço, devido ao aumento da demanda de oxigênio do miocárdio. A hipótese de processo patológico na angina instável foi fortemente corroborada pelos resultados de estudos anteriores10-16. O processo patológico da angina instável parece envolver a formação temporária de trombo mediada por uma complexa cascata de interações celulares entre o endotélio vascular e as plaquetas na artéria coronária aterosclerótica15.
As moléculas de adesão celular desempenham um papel primordial nas interações celulares em diversos processos patológicos, inclusive trombose coronariana, aterosclerose, reestenose após angioplastia coronariana e lesão por reperfusão15,16. A P-selectina é um membro da família da selectina e uma glicoproteína de membrana encontrada tanto nos grânulos alfa das plaquetas como nos corpúsculos de Weibel-Palade das células endoteliais. Essas moléculas contêm um domínio N-terminal semelhante à lectina, um domínio semelhante ao fator de crescimento epidérmico, um número variável de repetições de consenso de uma seqüência encontrada nas proteínas reguladoras do sistema complemento, um domínio transmembrana e uma curta cauda citoplasmática16,17. Após a ativação celular por agonistas, como a trombina, a P-selectina é rapidamente redistribuída para a superfície celular, ligando-se uma estrutura de carboidrato sializada expressa nos neutrófilos e monócitos por meio de um mecanismo dependente de cálcio semelhante ao da lectina. Assim, a P-selectina medeia as interações entre plaquetas e células endoteliais com leucócitos17-19.
Neste estudo, examinamos os níveis plasmáticos de P-selectina solúvel, serotonina e tromboxano B2 em pacientes com angina instável e estável para compreender melhor a fisiopatologia dessas síndromes distintas.
Métodos
Pacientes estudados – A população do estudo consistiu de um grupo de pacientes com angina instável (n=28), um grupo de pacientes com angina estável (n = 36) com indicação para revascularização coronariana e um grupo de controle sem DAC (n = 30). Informações clínicas detalhadas, inclusive historia de angina pectoris, fatores de risco para doença arterial coronariana, medicação e intervenções anteriores foram coletadas prospectivamente. Todos os pacientes forneceram um termo de consentimento livre e esclarecido para participar do estudo. Pacientes com infecção, diabete melito, câncer, hepatopatia crônica, insuficiência renal ou doença do tecido conjuntivo, assim como os pacientes que estavam sendo tratados com medicamentos antiinflamatórios ou anticoagulantes (inclusive clopidogrel, aspirina ou inibidores dos receptores da glicoproteína IIb/IIIa), foram excluídos do estudo. Os pacientes receberam tratamento farmacológico convencional para angina estável. Todos os pacientes aguardavam cirurgia de revascularização miocárdica sem tomar aspirina, para evitar interação com a ativação plaquetária. Quando havia indicação de anticoagulação com heparina nos casos de angina instável, eram selecionados apenas os pacientes que estavam recebendo heparina de baixo peso molecular, que não afeta a função plaquetária. A tabela 1 apresenta o perfil clínico desses pacientes.
Grupo de angina instável – Os pacientes admitidos no pronto-socorro com angina típica em repouso e eletrocardiograma com supradesnivelamento do segmento ST tinham diagnóstico de angina instável. Do grupo de 48 pacientes submetidos à triagem, 20 pacientes com níveis elevados de enzima CK-MB e troponina I foram excluídos do estudo. Os 25 homens e 3 mulheres restantes tinham estenose > 75% do diâmetro luminal de pelo menos uma artéria coronária.
Grupo de angina estável – Os 30 homens e 6 mulheres incluídos no grupo de angina estável apresentavam angina de esforço típica, ausência de angina em repouso, teste ergométrico positivo e estenose > 75% do diâmetro luminal de pelo menos uma artéria coronária.
Grupo de controle – Vinte e três homens e 7 mulheres sem doença arterial coronariana.
Coleta de sangue e dosagem dos marcadores plaquetários – Nos pacientes com angina instável, a coleta de sangue venoso foi feita imediatamente após a entrada no pronto-socorro, antes de ser instituída a terapia com anticoagulante. Nos pacientes com angina estável, as amostras de sangue foram coletadas antes da angiografia. Todas as amostras de sangue foram retiradas de uma veia antecubital. As amostras anticoaguladas foram separadas e armazenadas a – 80°C para a dosagem de marcadores plaquetários. As concentrações de sP-selectina, serotonina e tromboxano B2 no plasma armazenado foram mensuradas com kits de kits de teste imonoenzimático (Bender MedSystems Diagnostics GmbH, Immunotech/Coulter e Amersham Pharmacia, respectivamente).
Análises estatísticas – Os níveis plasmáticos de sP-selectina, serotonina e tromboxano B2 foram comparados por ANOVA unidirecional com o programa de computador SYSTAT 9. Um valor de P <0,05 foi considerado estatisticamente significante.
Resultados
Níveis plasmáticos de sP-selectina – Os níveis plasmáticos de P-selectina solúvel foram de 274,2 ± 131,9 ng/mL no grupo de angina instável, 199,7 ± 15,78 ng/mL no grupo de angina estável e 176,20 ± 43,90 no grupo de controle. Esses níveis são apresentados na figura 1. Os níveis plasmáticos de sP-selectina foram significativamente mais elevados no grupo de angina instável do que no grupo de angina estável e no grupo de controle (p <0,002).
Níveis de tromboxano B2 – Os níveis plasmáticos de tromboxano B2 foram de 62,89 ± 14,58 pg/mL no grupo de angina instável, 21,03 ± 6,44 pg/mL no grupo de angina estável e 19,09 ± 3,28 no grupo de controle, e são apresentados na figura 2. Os níveis plasmáticos de tromboxano B2 foram significativamente mais elevados no grupo de angina instável do que no grupo de angina estável e no grupo de controle (p <0,001).
Níveis de serotonina - Os níveis plasmáticos de serotonina foram de 2692,61 ± 358,42 nM no grupo de angina instável, 1631,36 ± 315,2 nM no grupo de angina estável e 1462,20 ± 166,36 no grupo de controle, e são apresentados na figura 3. Os níveis plasmáticos de serotonina foram significativamente mais elevados no grupo de angina instável do que no grupo de angina estável e no grupo de controle (p <0,001).
Discussão
No presente estudo, os níveis plasmáticos de P-selectina, tromboxano B2 e serotonina foram mais elevados em pacientes com angina instável do que nos pacientes com angina estável. Essas observações indicam que a ativação de plaquetas ocorre na circulação durante a angina instável.
P-selectina solúvel – A ruptura da placa coronariana, que resulta na formação de trombo e/ou agregação plaquetária, é apontada como principal mecanismo responsável pelo desenvolvimento de síndromes coronarianas agudas, inclusive angina instável19. Estudos recentes19-21 mostraram que o aumento dos níveis de sP-selectina em pacientes com angina instável pode ser conseqüência da ruptura da placa e da formação de trombo, bem como de uma interação de plaquetas pelos leucócitos ativados antes da ruptura da placa, e que os níveis de sP-selectina podem ser usados como marcadores de desestabilização da placa na angina instável22. No entanto, os níveis de sP-selectina poderiam ser mais elevados nos pacientes com acometimento de múltiplos vasos do que nos pacientes com acometimento de um único vaso23. Concluindo, a sP-selectina pode refletir indiretamente o quadro clínico dos pacientes com doença arterial coronariana, com possíveis implicações diagnósticas e terapêuticas2-26.
Segundo Ault e cols.21 existem indícios de ativação plaquetária contínua após evento coronariano isquêmico agudo. A P-selectina associada às plaquetas constitui um marcador sensível de ativação plaquetária; esse marcador permaneceu elevado por até um mês após a estabilização clínica de pacientes com angina instável ou infarto do miocárdio. A ativação plaquetária persistente pode ser conseqüência de estímulos inflamatórios sustentados 20,21. Os autores também descobriram uma fraca correlação entre parâmetros de ativação plaquetária e níveis séricos elevados de proteína C reativa21.
Tromboxano B2 – Os níveis de TXB2 foram estimados pela mensuração de 11-desidrotromboxano B2, o mais abundante metabólito enzimático de TXA2 no plasma. Este estudo demonstrou aumento dos níveis de tromboxano em pacientes com angina instável comparado com pacientes com angina estável. Esses achados são compatíveis com o conceito de que a angina estável reflete o fluxo de sangue restringido através de uma estenose fixa e com a observação de que os inibidores e antagonistas do tromboxano A2 não alteram a isquemia miocárdica induzida por exercício27. Indicam também que o aumento de tromboxano B2 na angina instável pode ser um evento de importância primária, e não apenas secundário à isquemia miocárdica. Além disso, a incidência de infarto do miocárdico e morte é reduzida em 30% a 50% nos pacientes com angina instável tratados com aspirina21,27.
Serotonina – Estudos clínicos28-30 demonstraram que as plaquetas são ativadas e agregadas nos locais de estenose da artéria coronária e de lesão endotelial. As plaquetas ativadas liberam serotonina em quantidades substanciais, causando vasoconstrição e agregação recidivante de plaquetas com reduções cíclicas do fluxo. A serotonina também atua como um fator de crescimento, estimulando a mitogênese e a migração de células da musculatura lisa arterial31. Estudos recentes indicam a utilidade da serotonina como um novo marcador de doença vascular aterosclerótica32-34.
Este estudo mostra que os níveis de P-selectina, tromboxano B2 e serotonina são mais elevados na angina instável do que na angina estável. Portanto, esses marcadores de ativação plaquetária podem nos ajudar a compreender a fisiopatologia da doença arterial coronariana, por refletir indiretamente o quadro clínico de pacientes com doença arterial coronariana, com possíveis implicações diagnósticas e terapêuticas.
Potencial Conflito de Interesses
Declaro não haver conflitos de interesses pertinentes.
Alexandre de Matos Soeiro
Rua Maranhão, 680/81
09545-001 – São Caetano do Sul, SP
E-mail:
portuga88@hotmail.com
Recebido em 09/05/05
Aceito em 01/07/05
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OBJECTIVE: Markers of platelet activation are elevated in coronary artery disease. We sought to identify the presence and the potential associations of different markers of platelet activation. METHODS: We studied patients with unstable angina (n=28), patients with stable angina (n=36) and patients without coronary artery disease (n=30); sex and age matched. Blood levels of the adhesion molecule P-selectin, Thromboxane B2 and Serotonin were measured by enzyme immunoassays. RESULTS: When we compared the groups the results were: sP-selectin, thromboxane B2 and serotonin levels were significantly higher in patients with unstable angina than in patients with stable angina. CONCLUSION: These markers of platelet activation were able to identify unstable forms of coronary artery disease.
Platelet activationcoronary artery disease
ORIGINAL ARTICLE
Platelet activation in different clinical forms of the coronary artery disease (roll of P-selectin and others platelet markers in the stable and unstable angina)
Margareth L. Venturinelli; André Hovnan; Alexandre de Matos Soeiro; José Carlos Nicolau; José A. F. Ramires; Élbio A. D'Amico; Carlos Vicente Serrano Júnior
Instituto do Coração do Hospital das Clínicas – FMUSP - São Paulo, SP - Brazil
Mailing Address
ABSTRACT
OBJECTIVE: Markers of platelet activation are elevated in coronary artery disease. We sought to identify the presence and the potential associations of different markers of platelet activation.
METHODS: We studied patients with unstable angina (n=28), patients with stable angina (n=36) and patients without coronary artery disease (n=30); sex and age matched. Blood levels of the adhesion molecule P-selectin, Thromboxane B2 and Serotonin were measured by enzyme immunoassays.
RESULTS: When we compared the groups the results were: sP-selectin, thromboxane B2 and serotonin levels were significantly higher in patients with unstable angina than in patients with stable angina.
CONCLUSION: These markers of platelet activation were able to identify unstable forms of coronary artery disease.
The coronary artery disease, represented by the atherosclerosis, is the most prevalent disease of our time and its trombotic complications are responsible for an exceedingly high number of deaths and disabilities. Over the past few years, experimental investigation, clinical and pathologic observations have led to a better understanding of how a thrombus forms and also of its incidence in coronary artery disease1.
Platelets have been recognized as being in the initiation and propagation of coronary thrombosis2-9. At the site of the ruptured plaque, platelets form an initial monolayer and a variety of activators (collagen, adenosine diphosphate [ADP], epinephrine, serotonin) promote platelet activation.
Platelet aggregation and thrombus formation secondary to plaque disruption also have been implicated as major pathogenic mechanisms underlying the acute coronary syndrome of unstable angina. The pathophysiology of unstable angina is different from that of effort-induced angina due to increased myocardial oxigen demand14. The hypothesis of disease process in unstable angina has been strongly supported by results of previous studies10-16. The disease process of unstable angina appears to involve a transient thrombus formation mediated by a complex cascade of cellular interactions between the vascular endothelium and platelets at the atherosclerotic coronary artery15.
Cell adhesion molecules play a key role in cellular interactions in diverse disease processes, including coronary thrombosis, atherosclerosis, restenosis after coronary angioplasty, and reperfusion injury15,16. P-selectin is a member of the selectin family and is an integral membrane glycoprotein found in both -granules of platelets and Weibel-Palade bodies of endothelial cells. These molecules contain na N-terminal lectin-like domain, an epidermal growth factor-like domain, a variable number of consensus repeats of a sequence found in complement regulatory proteins, a transmembrane domain, and a short cytoplasmic tail16,17. After cellular activation by agonists such as thrombin, P-selectin is rapidly redistributed to the cell surface and binds a sialylated carbohydrate structure expressed on neutrophils and monocytes through a calcium dependent lectin-like mechanism. Thus, P-selectin mediates platelet-leukocyte and endothelium cell-leukocyte adhesive interactions17-19.
In the present study, we examined the plasma levels of soluble P-selectin, serotonin and thromboxane B2 in patients with unstable and stable angina to further understand the pathophysiology of these distinct syndromes.
Methods
Study patients - The study population consisted of a group of patients with unstable angina (n=28), a group of patients with stable angina (n=36) with indication for coronary revascularization and a control group without CAD (n=30). Detailed clinical information, including history of angina pectoris, coronary artery disease risk factors, medication, and prior interventions were prospectively colected. All patients had granted their written informed consent to participate. Patients with ongoing infections, diabetes mellitus, malignancy, chronic liver disease, renal insufficiency, connective tissue disease, or being treated with anti-inflammatory or anticoagulant drugs (including clopidogrel, aspirin or glycoprotein IIbIIIa receptor inhibitor), were excluded from the study. Patients received conventional drug therapy for unstable angina or for stable angina. All patients were waiting a surgical myocardial revascularization without aspirin to avoid the interaction with the platelet activation. When anticoagulation was indicated with heparin in patients with unstable angina, were selected only patients in use of the low-molecular-weight heparin, that does not affect the platelet function. Their clinical profile is shown in Table I.
Unstable angina group - Patients admited to the emergency department with typical angina at rest and ST segment depression on the eletrocardiogram had a diagnosis of unstable angina. From a group of 48 patients screened, 20 patients with elevated CK-MB enzyme and troponin I were excluded from the study. The remaining 25 men and 3 women had > 75% luminal diameter stenosis of at least one coronary artery.
Stable angina group - The 30 men and 6 women included in the stable angina group had typical exertional angina, no angina at rest, a positive exercise test, and > 75% luminal diameter stenosis of at least one coronary artery.
Control group - The 23 men and 7 women without coronary artery disease.
Blood sampling and measurent of the platelet markers - In patients with unstable angina, venous blood was collected immediately after their admission to the emergency department, before initiation of anticoagulant therapy. In patients with stable angina blood samples were collected before angiography. All blood samples were obtained from an antecubital vein. The anticoagulated samples was separated and stored at –800C for measurements of platelet markers. Concentrations of sP-selectin, Serotonin and Thromboxane B2 in stored plasma were measured with enzyme-linked immunosorbent assay kits (Bender MedSystems Diagnostics GmbH, Immunotech/Coulter and Amersham Pharmacia, respectively).
Statistical analysis - The plasma levels of sP-selectin, serotonin and thromboxane B2 were compared by one-way ANOVA with the computer program SYSTAT 9. A P-value <0,05 was considered statiscally significant.
Results
Plasma sP-selectin levels - The plasma soluble P-selectin levels in the unstable angina group were 274,2 ± 131,9 ng/mL, in the stable angina group were 199,7 ± 15,78 ng/mL and in the control group were 176,20 ± 43,90 and are presented in figure 1. The plasma sP-selectin levels were significantly higher in the unstable angina group than stable angina or control group (P <0,002).
Thromboxane B2 levels - The plasma thromboxane levels in the unstable angina group were 62,89 ± 14,58 pg/mL, in the stable angina group were 21,03 ± 6,44 pg/mL and in the control group were 19,09 ± 3,28 and are presented in figure 2. The plasma thromboxane levels were significantly higher in the unstable angina group than stable angina or control group (P <0,001).
Serotonin levels - The plasma serotonin levels in the unstable angina group were 2692,61 ± 358,42 nM, in the stable angina group were 1631,36 ± 315,2 nM and in the control group were 1462,20 ± 166,36 and are presented in figure 3. The plasma serotonin levels were significantly higher in the unstable angina group than stable angina or control group (P <0,001).
Discussion
In the present study, plasma levels of soluble P-selectin, thromboxane B2 and serotonin were higher in patients with unstable angina than in patients with stable angina. These observations suggest that activation of platelets takes place in the circulation during unstable angina.
Soluble P-selectin - Coronary plaque disruption resulting in thrombus formation and/or platelet aggregation is considered the most important mechanism responsible for the development of acute coronary syndromes, including unstable angina19. Recent studies19-21 have show that the increase levels of sP-selectin in patients with unstable angina may be due to plaque rupture and thrombus formation, as well as to an interaction of platelets by activated leukocytes before plaque disruption, and sP-selectin level could be used as a marker of plaque destabilization in unstable angina22. Nevertheless, the level of sP-selectin in patients with multivessel disease could be higher than in those with single-vessel disease23. In conclusion, sP-selectin may indirectly reflect clinical condition of patients with coronary artery disease, with potential diagnostic and therapeutic implications22-26.
Ault et al21 reported that there is evidence of continued activation of platelets after an acute ischemic coronary event. Platelet associated P-selectin is a sensitive measure of platelet activation; this marker remained elevated for up to one month after clinical stabilization after unstable angina or myocardial infarction. Persistent platelet activation may be a consequence of sustained inflammatory stimuli20,21. The authors also found a weak correlation between platelet activation parameters and levels of serum C-reactive protein21.
Thromboxane B2 - The levels of TXB2 were estimated by measurement of 11-dehydro-tromboxane B2, the most abundant enzymatic metabolite of TXA2 in plasma. This study demonstrated increase of thromboxane levels in patients with unstable angina when compared with stable angina. These findings are consistent with the concept that stable angina reflects restricted blood flow through a fixed stenosis and with the observation that tromboxane A2 inhibitors and antagonists do not alter exercise induced myocardial ischemia27. They also suggest that the increase in thromboxane B2 in unstable angina is likely to be an event of primary importance and is not merely secondary to myocardial ischemia. In addition, the incidence of myocardial infarction and death is decreased by 30 to 50 percent in patients with unstable angina treated with aspirin21,27.
Serotonin - Clinical studies28-30 have demonstraded that platelets are activated and aggregate at the sites of coronary artery stenosis and endothelial injury. Activated platelets release serotonin in substantial quantities causing vasoconstriction and recurrent aggregation of platelets with cyclic flow reductions. Serotonin also acts as a growth factor stimulating mitogenesis and migration of arterial smooth muscle cells31. Recent studies propose that serotonin is useful as a novel marker for atherosclerotic vascular disease32-34.
The present study shows that sP-selectin, thromboxane B2 and serotonin are increased in the unstable angina when compared with stable angina. Thus, these markers of platelet activation may help us to understand the pathophysiology of unstable coronary artery disease, and may indirectly reflect clinical condition of patients with coronary artery disease, with potential diagnostic and therapeutic implications.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.