Gene mutations of platelet glycoproteins and response to tirofiban in acute coronary syndrome / Mutações gênicas das glicoproteínas plaquetárias e resposta ao tirofiban na síndrome coronariana aguda

CONTEXT AND OBJECTIVES: Glycoprotein inhibitors (abciximab, eptifibatide and tirofiban) are used in patients with unstable angina and non-ST-segment elevation myocardial infarction before percutaneous coronary intervention. Of these, tirofiban is the least effective. We hypothesized that the response to tirofiban might be associated with glycoprotein gene mutations. DESIGN AND SETTING: Prospective study at Emergency Unit, Heart Institute (InCor), University of São Paulo. METHOD: Intrahospital evolution and platelet aggregation in response to tirofiban were analyzed in relation to four glycoprotein mutations in 50 patients indicated for percutaneous coronary intervention: 17 (34%) with unstable angina and 33 (66%) with non-ST-segment elevation myocardial infarction. Platelet aggregation was analyzed using the Born method. Blood samples were obtained before and one hour after tirofiban infusion. Glycoproteins Ia (807C/T ), Ib (Thr/Met) , IIb (Ile/Ser ) and IIIa (PIA ) were the mutations selected. RESULTS: Hypertension, dyslipidemia, diabetes, smoking, previous coronary artery disease and stroke were similar between the groups. Mutant glycoprotein IIIa genotypes had lower platelet aggregation before tirofiban administration than that of the wild genotype (41.0% ± 22.1% versus 55.9% ± 20.8%; P = 0.035). Mutant glycoprotein IIIa genotypes correlated moderately with lower platelet inhibition (r = -0.31; P = 0.030). After tirofiban administration, platelet glycoprotein Ia, Ib, IIb and IIIa mutations did not influence the degree of inhibition of platelet aggregation or intrahospital mortality. CONCLUSIONS: Mutations of glycoproteins Ia, Ib, IIb and IIIa did not influence platelet aggregation in response to tirofiban in patients with unstable angina and non-ST-segment elevation myocardial infarction.

RESUMO CONTEXTO E OBJETIVOS: Inibidores da glicoproteína (abciximab, eptifibatide, tirofiban) são utilizados em pacientes com angina instável e infarto do miocárdio sem elevação do segmento ST (IAMSSST) antes da intervenção coronária percutânea. Dentre eles, o tirofiban é o menos eficaz. Nossa hipótese é que a resposta ao tirofiban possa estar associada a mutações no gene da glicoproteína. DESENHO E LOCAL: Estudo prospectivo na Unidade de Emergência do Instituto do Coração (InCor), Universidade de São Paulo (USP). MÉTODOS: Foram analisadas a evolução intra-hospitalar e agregabilidade plaquetária em resposta ao tirofiban de 4 mutações da glicoproteína em 50 pacientes com indicação para intervenção coronária percutânea, 17 (34%) com angina instável e 33 (66%) com IAMSSST. A agregação plaquetária foi analisada pelo método de Born. Amostras de sangue foram obtidas antes e uma hora após infusão do tirofiban. As glicoproteínas Ia (807C/T ), Ib (Thr/Met ), IIb (Ile/Ser ) e IIIa (PIA ) foram as mutações selecionadas. RESULTADOS: Hipertensão, dislipidemia, diabetes, tabagismo, doença coronariana e acidente vascular cerebral prévios foram semelhantes entre os grupos. Observou-se menor agregabilidade plaquetária dos genótipos mutantes da glicoproteína IIIa antes da administração de tirofiban do genótipo selvagem (41% ± 22% versus 56% ± 21%; P = 0,035). Genótipos mutantes da glicoproteína IIIa correlacionaram-se moderadamente com menor inibição plaquetária (r = -0,31; P = 0,030). Após a administração tirofiban, as mutações das glicoproteínas Ia, Ib, IIb, e IIIa não influenciaram o grau de inibição da agregação plaquetária e mortalidade intra-hospitalar. CONCLUSÕES: Mutações das glicoproteínas Ia, Ib, IIb e IIIa não influenciaram a agregação plaquetária em resposta ao tirofiban nos pacientes com angina instável e IAMSSST.

Seguimiento de gestante con síndrome de plaquetas pegajosas: primer caso comunicado en Cuba / Gestational follow-up of pregnant woman with sticky platelets syndrome: first patient reported in Cuba

El síndrome de plaquetas pegajosas es una entidad en la que se presentan hiperagregabilidad plaquetaria y manifestaciones clínicas trombóticas. Fue observada hace más de 30 años en personas jóvenes con eventos oclusivos, arteriales, venosos, miorcárdicos, cerebrales y, años más tarde, se describió en mujeres con pérdidas recurrentes de embarazos y complicaciones obstétricas. El tratamiento de elección es la aspirina, con la cual se logra revertir la hiperagregabilidad plaquetaria. Se presenta el seguimiento de una gestante con diagnóstico de síndrome de plaquetas pegajosas tipo II, con antecedentes de dos pérdidas de embarazos. Durante la gestación recibió terapia antiagregante plaquetaria y heparina de bajo peso molecular. A pesar de esta terapéutica surgieron complicaciones en el tercer trimestre de la gestación que condujeron a la interrupción temprana del embarazo. Se logró una recién nacida viva, con bajo peso, sin otras complicaciones perinatales. Actualmente la paciente se mantiene con aspirina y no ha referido eventos oclusivos. Este trabajo demuestra que la atención a estas pacientes por un equipo multidisciplinario permite obtener resultados alentadores.

Sticky platelet syndrome is an entity where patients have platelet hiperaggregability and thrombotic clinical manifestations. It was observed more than 30 years ago in young people with occlusive arterial, venous, myocardial infarctions, stroke, and later it was reported in women with recurrent pregnancy loss and obstetric complication events. The treatment of choice is aspirin which reverted platelet hyperaggregability. We present a pregnant woman diagnosed with type II sticky platelets syndrome with a history of two pregnancy loss. During this pregnancy, she received antiplatelet therapy and molecular weight heparin. Despite this therapy, complications arose in the third quarter that led to the early termination of pregnancy, but we obtained an alive underweight newborn without any perinatal complications. Currently, the patient maintains aspirin and she has no occlusive events. These results demonstrate that the care of these patients by a multidisciplinary team has successful outcome.

Clonagem, expressão e caracterização de um inibidor de agregação plaquetária proveniente dos complexos salivares de sanguessugas Haementeria depressa / Cloning, expression and characterization of a platelet aggregation inhibitor from Haementeria depressa leech salivary complexes

Animais hematófagos possuem em sua saliva substâncias que permitem a fluidez do sangue, para o sucesso de sua alimentação. Com isso, têm sido descritos diversos componentes com atividades nos diferentes processos hemostáticos (coagulação, fibrinólise e agregação plaquetária). O complexo salivar da sanguessuga Haementeria depressa vem sendo estudado através de bioquímica clássica e análises transcriptômica e proteômica deste tecido determinaram o perfil dos transcritos e das proteínas produzidas. Dentre os transcritos mais abundantes foram encontrados três clones (H06A09, H06A02 e L02F02) que apresentaram 45%, 87% e 94% de similaridade ao LAPP, um inibidor de agregação plaquetária da sanguessuga Haementeria officinallis, a produção destes componentes pelo tecido foi confirmada pela análise proteômica. O LAPP é um inibidor que age pela via do colágeno e possui cerca de 14 kDa e pI de 4,0 e inibe a ligação da plaqueta ao colágeno tanto pelo epítopo do FvW quanto pelo domínio a2b1. Assim, o objetivo do presente trabalho foi clonar, expressar e caracterizar a proteína recombinante ativa, a partir do clone H06A09 para estudos de atividade desta molécula. Para obter a proteína recombinante de interesse inicialmente a clonagem do transcrito foi realizada com sucesso em vetor pAE, porém, a expressão em sistema procarioto apresentou alguns obstáculos já que a molécula não tinha atividade. Uma nova estratégia foi proposta, sendo realizada clonagem em vetor pPIC9K e expressão em sistema eucariótico (leveduras Pichia pastoris - GS115). Desta forma, o presente trabalho caracteriza o primeiro inibidor recombinante de agregação plaquetária pela via do colágeno proveniente de sanguessugas Haementeria depressa, e comprova que apesar de apresentar 45% de similaridade estrutural ao LAPP é um inibidor com características funcionais diferentes, e com grande potencial a ser estudado.

Hematophagous animals have in their saliva substances that maintain the blood fluidity to the success of their feeding. Therefore, components have been described by their activities in the hemostatic processes (coagulation, fibrinolysis and platelet aggregation).The salivary complex of Haementaria depressa leech has been studied by classical biochemical and transcriptomic and proteomic analysis of this tissue determined the profile of transcripts and proteins produced by it. Among the most abundant transcripts were found three clones (H06A09, H06A02 e L02F02) that showed 45%, 87% e 94% of similarity to LAPP, an inhibitor of platelet aggregation from Haementeria officinallis, the components production was confirmed by proteomic analysis. LAPP is a inhibitor that acts by collagen pathway and has around 14 kDa and pI of 4.0, and inhibits the binding of platelet to collagen by both the epitope domain of vWF as the a2b1. Thereby, the aim of this study was to clone, express and characterize the active recombinant protein from the clone H06A09 for studies of activity of this molecule. To obtain the recombinant protein initially cloning of transcript was successfully performed in pAE vector, however, the protein expressed in prokaryotic system presented some obstacles not presenting activity. A new strategy was proposed, being held in pPIC9K vector and expression in eukaryotic system - yeast Pichia pastoris (GS115). Thus, this study characterized the first recombinant inhibitor of platelet aggregation through collagen pathway from Haementeria depressa leeches, and proves that despite having 45% structural similarity to the LAPP is an inhibitor with different functional characteristics, and great potential to be studied.

Polimorfismo del receptor P2Y12 de ADP plaquetario en pacientes con enfermedad coronaria y variabilidad en agregación plaquetaria: resultados preliminares / Platelet P2Y12 ADP receptor polymorphism in patients with coronary artery disease and varying degrees of platelet

Introducción: Uno de los mecanismos que explicaría la variabilidad en la agregación plaquetaria observada en la respuesta inhibitoria a clopidogrel es el polimorfismo del receptor P2Y12 de ADP plaquetario, específicamente, el haplotipo H2 y H1/H2. No se ha descrito la prevalencia del haplotipo H1/H2 del receptor plaquetario en pacientes con enfermedad coronaria. Objetivo: Evaluar la presencia del haplotipo H1/H2 del receptor P2Y12 en un grupo de pacientes con enfermedad coronaria Métodos: Estudio prospectivo en pacientes sometidos en forma electiva a angioplastía coronaria con stent. Todos recibieron aspirina y una dosis de carga de clopidogrel de 600 mg, seguido de dosis de mantención de 75 mg/día. En todos se midió agregación plaquetaria previo a la dosis de carga de clopidogrel (día 0) y luego entre 6º y 8º día det ratamiento. La agregación plaquetaria se expresó de acuerdo al porcentaje de cambio respecto del valor basal. Se utilizó test t student pareado para evaluar el porcentaje de cambio. Se amplificó el segmento de interés del ADN de los pacientes mediante PCR y se determinó la presencia del haplotipo H1/H2 usando enzimas de restricción. Resultados: Se enrolaron 40 pacientes, 34 (85 por ciento) hombres, edad promedio 61 +/-12 años. El promedio de agregación plaquetaria, previo y durante terapia con clopidogrel fue de 64 +/-10 por ciento y 41 +/-14 por ciento, respectivamente (p<0.0001) frente a ADP 8 µM. La respuesta de agregación a clopidogrel presentó una distribución normal según el test de Kolmogorov-Smirnov (p=0.58). Los pacientes se estratificaron de acuerdo al porcentaje de cambio en cuartiles y el cuartil de menor cambio representó una diferencia menor a 10 por ciento. De estos pacientes, 30 por ciento (3 pacientes) tenían el haplotipo H1/H2. En total, se demostró la presencia del haplotipo H1/H2 en 4 (10 por ciento) pacientes...

Background: One factor influencing the variability in the anti aggregation effect of clopidogrel is the polymorphism of the platelet P2Y12 ADP receptor, specifically the H2 and H1/H2 haplotypes. The prevalence of the H1/H2 haplotype has not been described in patients with coronary artery disease. Aim: To evaluate the presence of H1/H2 haplotype of P2Y12 receptor in patients with coronary artery disease. Methods: A prospective study was conducted in patients undergoing elective PTCA with stenting. All received aspirin followed by a loading dose of clopidogrel 600 mg and a maintenance dose of 75 mg daily. Platelet aggregation was measured prior to the loading clopidogrel dose and at 6 and 8 days post treatment. Platelet aggregation was indicated as the percent change over the basal value. The Student’s t test was used to evaluate the response. The DNA segment involved was amplified by PCR and the H1/H2 haplotype was determined using restriction enzymes. Results: Fourty patients (85 percent males) with mean age 61 years (SD 12) were studied. Mean platelet aggregation changed from a basal value of 64+/-10 to a post clopidogrel value of 41 +/-14 percent (p<0.0001) with an ADP level of 8 µM. The platelet aggregation response was normal according to Kolmogorov-Smirnov. The lowest quartile of platelet aggregation showed a <10 percent change. Three patients in this group (30 percent) had the H1/H2 haplotype. The overall incidence of this haplotype was 10 percent (4 patients). Conclusion: Clopidogrel does not induce a significant decreased platelet aggregation in 25 percent of patients subjected to coronary angioplasty. A third of this patients exhibited the H1/H2 haplotype for the P2Y12 receptor. This group of patients might be at increased risk from subsequent cardiovascular events.