Low triiodothyronine syndrome is associated with platelet function in patients with nephrotic syndrome

SUMMARY OBJECTIVE The objective of this study was to investigate the effects of low triiodothyronine syndrome (LT3S) on platelet function and clotting factors in patients with nephrotic syndrome(NS). METHODS Patients with primary nephrotic syndrome were divided into two groups, normal thyroid function (group A) and LT3S (group B), based on whether they had LT3S or not. Healthy subjects were selected as the control group (group C). Blood coagulation function was detected in each group. The platelet activation function (CD62P, CD63) was determined by flow cytometry. The platelet aggregation rate was detected by an optical method using adenosine diphosphate and arachidonic acid as inducers. RESULTS The proportion of primary nephrotic syndrome with LT3S was 23.2% (69/298). Compared with group C, group A had higher CD62P and PAgTADP, and group B had higher CD62P, CD63, PAgTAA, and PAgTADP; the difference was statistically significant (all P < 0.05). There was no significant difference in renal pathology between group A and group B (X2 = 4.957, P = 0.421). Compared with group A, the 24-hour urine protein, CD63, PAgTAA, and PAgTADP were higher in group B, and APTT and Alb were lower. The difference was statistically significant (P < 0.05). Logistic regression analysis showed that LT3S was associated with CD36 (OR: 3.516; 95% CI: 1.742~8.186; P = 0.004) and PAgTAA (OR: 0.442; 95% CI: 1.001~1.251; P = 0.037). CONCLUSION NS patients are prone to LT3S. Patients with LT3S may have abnormal platelet activation and increase of platelet aggregation.

RESUMO OBJETIVO O objetivo deste estudo foi investigar os efeitos da síndrome do baixo triiodotironina (LT3S) na função plaquetária e nos fatores de coagulação em pacientes com síndrome nefrótica (SN). MÉTODOS Pacientes com síndrome nefrótica primária foram divididos em dois grupos, função tireoidiana normal (grupo A) e LT3S (grupo B), com base na presença ou não de LT3S. Indivíduos saudáveis foram selecionados como grupo de controle (grupo C). A função de coagulação do sangue foi analisada em cada grupo. A função de ativação plaquetária (CD62P, CD63) foi determinada por citometria de fluxo. A taxa de agregação plaquetária foi detectada por um método óptico usando adenosina difosfato e ácido araquidônico como indutores. RESULTADOS A proporção de síndrome nefrótica primária com LT3S foi de 23,2% (69/298). Em comparação com o grupo C, o grupo A apresentou níveis mais altos de CD62P e PAgTADP, e o grupo B apresentou maiores CD62P, CD63, PAgTAA e PAgTADP; a diferença teve significância estatística (P < 0,05). Não houve diferença significativa na patologia renal entre o grupo A e o grupo B (X2 = 4,957, P = 0,421). Em comparação com o grupo A, a proteína em urina de 24 horas, CD63, PAgTAA e PAgTADP foram maiores no grupo B, já APTT e Alb foram mais baixos. A diferença apresentou significância estatística (P < 0,05). A análise de regressão logística mostrou uma associação entre LT3S e CD36 (OR: 3,516; 95% IC: 1,742~8,186; P = 0,004) e PAgTAA (OR: 0,442; 95% IC: 1,001~1,251; P = 0,037). CONCLUSÃO Pacientes com síndrome nefrótica estão propensos à síndrome do baixo triiodotironina (LT3S). Pacientes com LT3S podem ter ativação plaquetária anormal e aumento da agregação plaquetária.

Detection of Clopidogrel Hyporesponsiveness Using a Point-of-Care Assay and the Impact of Additional Cilostazol Administration after Coronary Stent Implantation in Diabetic Patients

BACKGROUND/AIMS: Impaired responsiveness to clopidogrel is common in patients with type 2 diabetes mellitus (DM). The aim of this study was to evaluate the clinical application of a point-of-care assay to detect impaired responsiveness to clopidogrel after coronary stent implantation in patients with type 2 DM. METHODS: We measured P2Y12 reaction units (PRU) with the VerifyNow point-of-care assay in 544 consecutive patients undergoing dual or triple (i.e., dual plus cilostazol) anti-platelet therapy after coronary stent implantation. High platelet reactivity (HPR) was defined as a PRU value > or = 240. RESULTS: The mean PRU values were 233.5 +/- 83.2 and 190.3 +/- 85.5 in patients undergoing dual or triple anti-platelet therapy, respectively (p < 0.001). Patients with DM manifested higher post treatment PRU values (238.3 +/- 82.4 vs. 210.8 +/- 86.8, p = 0.001) and a higher frequency of HPR (44.8% vs. 31.0%, p = 0.003) as compared to patients without DM. We also found that higher PRU values and a higher frequency of HPR were present in patients with DM who were undergoing both triple and dual anti-platelet therapy. However, the higher post-treatment PRU values observed in patients with DM decreased with triple anti-platelet therapy (219.4 +/- 82.5 vs. 247.9 +/- 81.1, p = 0.044). CONCLUSIONS: A point-of-care assay can detect elevated platelet reactivity and impaired responsiveness to clopidogrel in patients with type 2 DM. The addition of cilostazol to dual anti-platelet therapy may decrease post-treatment PRU values in patients with type 2 DM.

Ausência de interação clopidogrel-estatina em pacientes submetidos a implante de "stent" coronário / Lack of clopidogrel-statin interaction in patients undergoing coronary stent implantation / Ausencia de interacción clopidogrel-estatina en pacientes sometidos a implante de "Stent" coronario

FUNDAMENTO: Alguns estudos têm sugerido redução da atividade do clopidogrel sobre a ativação e adesão plaquetárias em pacientes em uso de estatinas. OBJETIVO: Avaliar se a ativação e agregação plaquetárias diminuem com clopidogrel, e se ocorre redução da ação do clopidogrel quando associado à atorvastatina ou à sinvastatina. MÉTODOS: Estudo prospectivo que incluiu 68 pacientes com angina estável em uso prévio de sinvastatina, atorvastatina, ou nenhuma estatina (grupo controle), com indicação prévia eletiva de realização de intervenção coronária percutânea. Foi analisada a ativação plaquetária através do número de plaquetas, níveis de P-selectina e glucoproteína IIb/IIIa (com e sem estímulo de ADP) através de citometria de fluxo. Os resultados foram analisados antes e após a intervenção coronária percutânea e da administração de clopidogrel. RESULTADOS: Observamos redução da atividade plaquetária com uso de clopidogrel. Além disso, não houve diferenças entre as variáveis analisadas que comprovassem redução da atividade do clopidogrel quando associado à estatinas. Observou-se níveis de p-selectina (pré-angioplastia: 14,23±7,52 x 11,45±8,83 x 7,65±7,09; pós angioplastia: 21,49±23,82 x 4,37±2,71 x 4,82±4,47, ρ<0,01) e glicoproteína IIb/IIIa (pré-angioplastia: 98,97±0,43 x 98,79±1,25 x 99,21±0,40; pós angioplastia: 99,37±0,29 x 98,50±1,47 x 98,92±0,88, ρ=0,52), respectivamente nos grupos controle, atorvastatina e sinvastatina. CONCLUSÃO: Concluímos que a ativação plaquetária diminui com a administração de clopidogrel, e que o clopidogrel não tem seu efeito antiplaquetário reduzido na presença de sinvastatina ou atorvastatina.

BACKGROUND: Some studies have suggested reduced activity of clopidogrel on platelet activation and adherence in patients using statins. OBJECTIVE: To assess whether platelet activation and aggregation decrease with clopidogrel, and whether there is a reduction of the action of clopidogrel when associated with atorvastatin or simvastatin. METHODS: This prospective study included 68 patients with stable angina with previous use of simvastatin, atorvastatin, or no statin (control group), with previous elective indication of percutaneous coronary intervention (PCI). Platelet activation was analyzed by means of platelet count, levels of P-selectin and glycoprotein IIb/IIIa (with and without ADP stimulation) by flow cytometry. The findings were analyzed before and after percutaneous coronary intervention and the administration of clopidogrel. RESULTS: We observed reduction in platelet activity with use of clopidogrel. Furthermore, no differences were found between the variables analyzed to prove reduced activity of clopidogrel when combined with statins. We observed levels of p-selectin (pre-angioplasty: 14.23 ± 7.52 x 8.83 x 11.45 ± 7.65 ± 7.09; after angioplasty: 21.49 ± 23.82 x 4 37 ± 2.71 x 4.82 ± 4.47, ρ < 0.01) and glycoprotein IIb/IIIa (pre-angioplasty: 98.97 ± 0.43 ± 1.25 x 98.79 x 99.21 ± 0.40 after angioplasty: 99.37 ± 0.29 ± 1.47 x 98.50 x 98.92 ± 0.88, ρ = 0.52), respectively, in the control, atorvastatin and simvastatin groups. CONCLUSION: We concluded that platelet activation decreases with administration of clopidogrel, and clopidogrel has no antiplatelet effect reduced in the presence of simvastatin or atorvastatin.

FUNDAMENTO: Algunos estudios han sugerido reducción de la actividad del clopidogrel sobre la activación y adhesión plaquetarias en pacientes en uso de estatinas. OBJETIVO: Evaluar si la activación y agregación plaquetarias disminuyen con clopidogrel, y si ocurre reducción de la acción del clopidogrel cuando está asociado a la atorvastatina o a la sinvastatina. MÉTODOS: Estudio prospectivo que incluyó 68 pacientes con angina estable en uso previo de sinvastatina, atorvastatina, o ninguna estatina (grupo control), con indicación previa electiva de realización de intervención coronaria percutánea. Fue analizada la activación plaquetaria a través del número de plaquetas, niveles de P-selectina y glucoproteína IIb/IIIa (con y sin estímulo de ADP) a través de citometría de flujo. Los resultados fueron analizados antes y después de la intervención coronaria percutánea y de la administración de clopidogrel. RESULTADOS: Observamos reducción de la actividad plaquetaria con uso de clopidogrel. Además de eso, no hubo diferencias entre las variables analizadas que comprobasen reducción de la actividad del clopidogrel cuando está asociado a las estatinas. Se observaron niveles de p-selectina (pre-angioplastia: 14,23±7,52 x 11,45±8,83 x 7,65±7,09; post angioplastia: 21,49±23,82 x 4,37±2,71 x 4,82±4,47, ρ<0,01) y glicoproteína IIb/IIIa (pre-angioplastia: 98,97±0,43 x 98,79±1,25 x 99,21±0,40; post angioplastia: 99,37±0,29 x 98,50±1,47 x 98,92±0,88, ρ=0,52), respectivamente en los grupos control, atorvastatina y sinvastatina. CONCLUSIÓN: Concluimos que la activación plaquetaria disminuye con la administración de clopidogrel, y que el clopidogrel no tiene su efecto antiplaquetario reducido en la presencia de sinvastatina o atorvastatina.

Expression of p-selectin [CD62p] on platelets as activated marker in preeclampsia

Pre-eclampsia is a medical condition in which hypertension arises in pregnancy [pregnancy-induced hypertension] in association with significant amounts of protein in the urine. Pre-eclampsia may develop from 20 weeks gestation [it is considered early onset before 32 weeks, which is associated with increased morbidity]. Platelets play an important role in the pathophysiologic mechanisms of preeclampsia. The aim of this work was to study the platelet activation state by flow cytometer analysis of platelet expression of CD62p in patients with preeclampsia. This study was conducted on ten cases of mild preeclampsia [group I], their ages range was 22- 36 years and ten cases of severe preeclampsia [group II] their ages range was 20-35 years .Also ten normotensive pregnant women were included as a control group [group III] . The percentage of platelets expression of the CD61, CD62p and MFI were analyzed by the flow- cytometr . The mean percentage of CD62p expression on platelets and MFI were 67. 3% and 6.5 respectively in mild preeclampsia compared with 3.7% and 1.5 in normotensive pregnant as control [p < 0.01 and p < 0.015 respectively]. Also the mean percentage of CD62p expression on platelets and MFI were 73.3% and 2.1 respectively in severe preeclampsia, they showed significant increase when compared with normotensive pregnant as control [p < 0.01 and p < 0.015 respectively]. There were a positive significant correlation between% of expression of CD 62p on platelets and SBP, DBP, protein in urine, and% CD61. While a negative significant correlation between% of expression of CD 62p on platelets and age, platelet count and CD62P MFI was found. High levels of platelet glycoprotein CD62p expressions in patients with mild and severe preeclampsia, could be a compensatory mechanism for the preeclampsia induced thrombocytopenia

Flowcytometric evidence of platelet activation in patients on aspirin following myocardial infarction.

BACKGROUND: Following a myocardial infarction, patients are usually started on long term antiplatelet therapy with aspirin in a dose of 80-150 mg/day. However, there are no quick and easy methods to assess the efficacy of the antiplatelet activity of aspirin. METHODS: We studied 60 consecutive patients (men, < 40 years of age) 8-10 weeks after they had had acute myocardial infarction. These patients were receiving 100 mg aspirin daily orally with or without b-blockers. We measured P-selectin expression and fibrinogen binding by flowcytometry at least 3 times over a period of 2 years in all the patients. We also studied 100 age- and sex-matched controls. RESULTS: Of the 60 patients, 30 (50%) showed both increased P-selectin and fibrinogen binding by platelets, suggesting platelet activation. Fourteen other patients had increased fibrinogen binding but normal P-selectin expression. Sixteen patients and all the controls had normal results of both tests. CONCLUSION: Our data show evidence of platelet activation in at least 50% of patients receiving 100 mg of aspirin daily. Flowcytometry for P-selectin expression and fibrinogen binding to platelets can be used to monitor antiplatelet therapy with aspirin following acute myocardial infarction.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Pharmacology of platelet activation-inhibitory drugs.

The role of blood platelets in the pathogenesis of atherosclerosis, thrombosis, thromboembolism and stroke (hemorrhagic/thrombotic) is well established. In view of this recognized role played by platelets in the complications associated with coronary artery disease and cerebrovascular disease, there is considerable interest in the pharmacology of platelet activation inhibitory drugs. These drugs exert their effect by blocking several different activation signalling mechanisms. Some of the known compounds that modulate platelet function include: inhibitors of arachidonic acid metabolism (nonsteroidal anti-inflammatory drugs and thromboxane synthetase inhibitors), drugs that alter membrane phospholipid composition (omega 3 fatty acids), stimulators of adenylyl cyclase and guanylyl cyclase (PGE1, PGI2, PGD2/ERRF [nitric oxide], nitroglycerin, nitroprusside), phosphodiesterase inhibitors (dipyridamole and methylxanthines) and calcium antagonists (verapamil, nifedipine, diltiazem). Current research on the pharmacology of platelet activation inhibitory drugs is focused on the development of specific receptor antagonists (antibodies, peptides, receptor antagonists). Since platelets have multiple mechanisms for achieving activation, and the process of thrombosis involves multicellular modulation of platelet activity, it will be rather difficult to develop a compound that is capable of causing complete inhibition of activation mechanisms. Therefore, future research will be devoted to development of designer drugs that will be used for preventing discrete platelet responses. This approach may be useful as total inhibition of platelet activation, although it may prevent thrombotic events, may possibly precipitate hemorrhagic conditions. A better understanding of cell signalling pathways and the mechanisms involved in the pathogenesis of cardiovascular cerebrovascular disease will facilitate the development of efficient antiplatelet drugs.

Physiology of blood platelet activation.

Blood platelets interact with a variety of soluble agonists such as epinephrine and adenosine diphosphate (ADP); many insoluble cell matrix components, including collagen and laminin, and biomaterials used for construction of invasive medical devices. These interactions stimulate specific receptors and glycoprotein-rich domains (integrins and nonintegrin) on the plasma membrane and lead to the activation of intracellular effector enzymes. The majority of regulatory events appear to require free calcium. Ionized calcium is the primary bioregulator, and a variety of biochemical mechanisms modulate the level and availability of free cytosolic calcium. Major enzymes that regulate the free calcium levels via second messengers include phospholipase C, phospholipase A2, and phospholipase D, together with adenylyl and guanylyl cyclases. Activation of phospholipase C results in the hydrolysis of phosphatidyl inositol 4,5-bisphosphate and formation of second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (IP3). Diglyceride induces activation of protein kinase C, whereas IP3 mobilizes calcium from internal membrane stores. Elevation of cytosolic calcium stimulates phospholipase A2 and liberates arachidonic acid. Free arachidonic acid is transformed to a novel metabolite, thromboxane A2, by fatty acid synthetases. Thromboxane A2 is the major metabolite of this pathway and plays a critical role in platelet recruitment, granule mobilization and secretion. Up-regulation in signalling pathways will increase the risk for clinical complications associated with thromboembolic episodes. Down-regulation of signal transduction mechanisms may precipitate bleeding diathesis or stroke.