RESUMO
Aldosterone plays a key role in maintaining the homeostasis of the whole organism. Under some circumstances, aldosterone can contribute to the progression of cardiovascular diseases, including coronary artery disease. This study demonstrates that aldosterone associates negatively with some lipidogram parameters and positively with the concentration of homocysteine. These associations are characteristic for coronary artery disease and are not present in control subjects. The findings also indicate that in vitro aldosterone stimulates homocysteine production by rat adrenal glands, which may explain the associations observed with coronary artery disease. Moreover, we have found that aldosterone significantly modulates in vitro platelet reactivity to arachidonate and collagen - aldosterone increases the pro-aggregatory action of collagen, but decreases the pro-aggregatory potential of arachidonate. Therefore, the findings of these in vitro and ex vivo experiments indicate the existence of new pathways by which aldosterone modulates lipid- homocysteine- and platelet-dependent atherogenesis.
Assuntos
Aldosterona/sangue , Colesterol/sangue , Doença da Artéria Coronariana/sangue , Homocisteína/sangue , Trombose/sangue , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Idoso , Aldosterona/farmacologia , Animais , Ácido Araquidônico/metabolismo , Colágeno/metabolismo , Creatinina/sangue , Feminino , Homocisteína/biossíntese , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/sangueRESUMO
Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI2) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR-/- mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF1α, nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR-/- mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR-/- but not in WT mice, strenuous exercise partially inhibited TXB2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR-/- mice; however, only 7-month-old ApoE/LDLR-/- mice showed lower TXB2 production after exercise. In female 4-6-month-old ApoE/LDLR-/- but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO2-) and nitrate (NO3-) were decreased in ApoE/LDLR-/- as compared to that in age-matched WT mice. In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR-/- as compared to WT mice. However, platelet activation in ApoE/LDLR-/- mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI2-dependent anti-platelet mechanisms in ApoE/LDLR-/- mice.
Assuntos
Envelhecimento/sangue , Apolipoproteínas E/deficiência , Aterosclerose/sangue , Plaquetas/metabolismo , Esforço Físico , Ativação Plaquetária , Receptores de LDL/deficiência , 6-Cetoprostaglandina F1 alfa/sangue , Envelhecimento/genética , Envelhecimento/patologia , Animais , Apolipoproteínas E/sangue , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Plaquetas/patologia , Modelos Animais de Doenças , Feminino , Fibrinogênio/genética , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout para ApoE , Nitratos/sangue , Nitritos/sangue , Selectina-P/sangue , Selectina-P/genética , Condicionamento Físico Animal/métodos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores de LDL/sangue , Receptores de LDL/genética , Comportamento Sedentário , Tromboxano B2/sangue , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Modified C-reactive protein (mCRP) has been reported to non-specifically bind to immunoglobulins; notwithstanding, the nature of these interactions is not clear. The aim of this study was to investigate the binding of antibodies directed against HSA and IgG to mCRP, fibrinogen (Fg), IgG, fibronectin (Fn) and C1q and its contaminants. We also studied the binding of mCRP to the antibodies directed towards receptors involved in CRP signalling (anti-CD32, anti-CD16). For the analysis of such interactions, a combination of ELISA and Western immunoblotting has been applied. The tested antibodies powerfully bound to either the contaminations of purified proteins (Fg, IgG, Fn and mCRP) or interacted directly with some of these proteins (C1q, mCRP, Fg). The effectiveness of anti-HSA binding to immobilized proteins was influenced by the antigenic specificity of the antibody, the content of various protein fractions in the contaminants of a given protein (albumin augmented the interactions), overall protein purity and a natural avidity of a given protein towards immunoglobulins. The relative binding of anti-HSA or anti-IgG to immobilized mCRP was considerably lower than that observed for plasma proteins. Furthermore, the strength of the direct interaction between immunoglobulins and mCRP varied from the lack of response (anti-HSA) or a negligible response (anti-IgG) to the relatively high signal (human IgG, anti-CD16, anti-CD32), as compared to the control. Based on these observations, we conclude that the binding of mCRP to immunoglobulins cannot be easily generalized as a kind of some universal phenomenon.
Assuntos
Proteína C-Reativa/imunologia , Imunoglobulina G/imunologia , Albumina Sérica/imunologia , Adulto , Western Blotting , Complemento C1q/imunologia , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinogênio/imunologia , Fibronectinas/imunologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The work is focused on clarifying the impact of diabetes and natural plant polyphenols contained in Pycnogenol® (PYC) on the activity and synthesis of Cu/Zn-SOD and synthesis of nNOS and eNOS in the cerebellum and cerebral cortex in rats with induced diabetes. Rats included in the study (n=38) were divided into three groups: the controls (C), (n=7), untreated diabetics (D) (n=19) and diabetic rats treated with PYC (DP) (n=12). Diabetes significantly decreased synthesis, as well as the activity of Cu/Zn-SOD in both studied parts of the brain. PYC significantly increased the synthesis of Cu/Zn-SOD but had no effect on its activity. Diabetes also reduced the synthesis of nNOS in cerebral cortex and administered PYC elevated its amount to the level of controls. In the cerebellum, diabetes does not affect the synthesis of nNOS and PYC reduces synthesis of NOS. Diabetes as well as PYC had no influence on the synthesis of eNOS in both, the cerebellum and cerebral cortex. PYC modulated level of Cu/Zn-SOD and nNOS in cerebellum and cerebral cortex of diabetic rats, but in a different way.
Assuntos
Cerebelo/enzimologia , Córtex Cerebral/enzimologia , Diabetes Mellitus Experimental/enzimologia , Flavonoides/farmacologia , Óxido Nítrico Sintase/metabolismo , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Superóxido Dismutase/metabolismo , Animais , Masculino , Pinus , Polifenóis , RatosRESUMO
The subject of this work is the characterisation of the metric features of deciduous dentition in a Medieval population of central Poland with the use of the jackknife technique leave one out (LOO)-supporting multivariate methods, which are important for deriving discrimination equations that would result in sex determination of children's skeletal remains. The sex of the individuals was assessed through analysis of sex-specific DNA sequences (AMELY/AMELX, SRY and alpha satellite sequences). Discriminant analysis concerned only teeth of those individuals whose sex was confirmed by the primary structure of three DNA sequences. The deciduous tooth diameters of males were found to be significantly larger than those of females in four respects: MD diameter of the maxillary second molar, MD and BL diameters of the mandibular first molar and BL diameter of the mandibular second molar. A two-group discriminant analysis considered all those measurements as independent variables. A multiple regression procedure produced a linear equation predicting the sex of children's skeletons with a significant probability amounting to approximately 78%. The accuracy of the sex assessment of an individual, using dental measurements, was established at 69% in deciduous male and 88% in deciduous female teeth.
Assuntos
Arqueologia , Dente Molar/anatomia & histologia , Determinação do Sexo pelo Esqueleto/métodos , Dente Decíduo , Criança , Feminino , História Medieval , Humanos , Modelos Lineares , Masculino , Análise Multivariada , PolôniaRESUMO
The present state of knowledge unequivocally indicates that chronic diabetes is associated with impaired function of coronary vessels. Langendorff retrograde perfusion is one of the most frequently employed methods to study dysfunction of coronary vasculature in animal models of diabetes mellitus. However, because of methodological discrepancies in experimental protocols, the reliability of this technique is limited. In the current study, we propose the novel technique of vasoactive drug administration and aim to evaluate its usefulness in detecting coronary dysfunction in diabetes. Using Langendorff model, we compared the results of coronary endothelium-dependent (bradykinin) and -independent (diethylamine/nitric oxide, DEA/NO) vasodilatation obtained from experimental model utilizing automatically corrected-rate infusion with commonly used, constant-rate infusion of vasoactive drug. The infusion of bradykinin at constant rate failed to reveal coronary endothelium-dependent dysfunction typical for diabetes mellitus. Induction of endothelium-independent vasodilatation by constant infusion demonstrated augmented response in diabetic hearts. The administration of bradykinin or DEA/NO at the corrected rate was associated with significantly increased maximal responses in comparison with constant infusion experiments. This phenomenon was observed particularly in the control group. We conclude that only corrected-rate infusion of vasoactive agents to actual value of coronary flow enables the reliable detection of endothelial dysfunction in diabetes mellitus.
Assuntos
Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/fisiopatologia , Animais , Glicemia/análise , Bradicinina/administração & dosagem , Bradicinina/farmacologia , Peptídeo C/análise , Colesterol/sangue , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Endotélio Vascular/efeitos dos fármacos , Jejum/sangue , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hidrazinas/administração & dosagem , Hidrazinas/farmacologia , Técnicas In Vitro , Infusões Intravenosas , Insulina/sangue , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Doadores de Óxido Nítrico/administração & dosagem , Doadores de Óxido Nítrico/farmacologia , Perfusão , Ratos , Ratos Wistar , Triglicerídeos/sangue , Vasodilatação/efeitos dos fármacos , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Low-dose acetylsalicylic acid (ASA) treatment is a standard therapeutic approach in diabetes mellitus for prevention of long-term vascular complications. The aim of the present work was to investigate the effect of long-term ASA administration in experimental diabetes on activities of some liver enzymes: glutathione peroxidase (GSHPx), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST). Blood glucose, glycated hemoglobin, as well as plasma ALT and AST activities increased in rats with streptozotocin-induced experimental diabetes. The long-term hyperglycemia resulted in decreased activities of GSHPx (by 26%), catalase (by 34%), GST (by 38%) and G6PDH (by 27%) in diabetic animals. We did not observe increased accumulation of membrane lipid peroxidation products or altered levels of reduced glutathione in livers. The linear correlation between blood glucose and glycated hemoglobin in diabetic animals was distorted upon ASA treatment, which was likely due to a chemical competition between nonenzymatic protein glycosylation and protein acetylation. The long-term ASA administration partially reversed the decrease in GSHPx activity, but did not influence the activities of catalase and GST in diabetic rats. Otherwise, some decrease in these parameters was noted in ASA-treated nondiabetic animals. Increased ASA-induced G6PDH activity was recorded in both diabetic and nondiabetic rats. While both glycation due to diabetic hyperglycemia and ASA-mediated acetylation had very similar effects on the activities of all studied enzymes but G6PDH, we conclude that non-enzymatic modification by either glucose or ASA may be a common mechanism of the observed convergence.
Assuntos
Antioxidantes/metabolismo , Aspirina/uso terapêutico , Diabetes Mellitus Experimental , Angiopatias Diabéticas/prevenção & controle , Glutationa Transferase/metabolismo , Inibidores da Agregação Plaquetária/uso terapêutico , Animais , Aspirina/administração & dosagem , Aspirina/farmacologia , Glicemia/análise , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Relação Dose-Resposta a Droga , Hemoglobinas/análise , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos WistarRESUMO
The main objective of this retrospective study was to investigate relations between cyclin E and pathoclinical factors in patients with operable breast cancer. Expression of cyclin E was analyzed by immunohistochemistry in specimens of invasive ductal breast cancer tissue obtained from 189 women during radical mastectomy. Overall, 110 tumor samples were regarded to be cyclin E positive. Cyclin E expression was more often seen in tumors with: negative steroid receptor status (p<0.0001), higher proliferative index (p=0.0014), higher tumor grade (p=0.0017), and presence of HER2 (p=0.0171). With a median follow-up of 58 months, expression of cyclin E together with negative steroid receptor status determined poor prognosis with a 5-year cancer-specific survival rate of 58%. It differed significantly from a survival curve of cyclin E negative and steroid receptor positive patients (87%, p=0.0005). No significant difference was observed in comparison with survival of cyclin E positive and steroid receptor positive patients (68%, p=0.221). We demonstrated that cyclin E expression in breast cancer cells was associated with negative steroid receptor status, HER2 presence, higher tumor grade and higher proliferation index. Expression of cyclin E together with lack of steroid receptors determined poor prognosis.
Assuntos
Neoplasias da Mama/metabolismo , Ciclina E/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptor ErbB-2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Receptores de Esteroides/metabolismo , Estudos RetrospectivosRESUMO
BACKGROUND: Laser light irradiation is assumed to have biostimulating effect in various cell types. However, there is still a lack of information concerning response of blood platelets to laser light irradiation. METHODS: In our study we used flow cytometry to monitor the effect of a green Nd-YAG laser (532 nm, 30 mW) irradiation on platelet activation and the expression of activated GPIIbIIIa glycoprotein complex (fibrinogen receptor) of whole blood platelets stained with fluorolabelled monoclonal antibody PAC-1. Also the formation of platelet microparticles and aggregates in a population of whole blood platelets following such irradiation was evaluated. RESULTS: Effects of laser light on platelet activation and reactivity were significant over a wide range of applied energies (p<0.01). While low and medium laser light energies (18 and 54 J) increased platelet activation, the irradiation with a high-energy laser light (108 J) resulted in depressed platelet reactivity and attenuated platelet response to activators. In addition, laser light irradiation had significant influence on the formation of platelet microparticles in either resting (p<0.05) or ADP-activated (p<0.05) platelets, while no significant effect was observed in collagen-activated platelets. On the other hand, laser light irradiation significantly increased the formation of platelet aggregates both in resting (p<0.01) and agonists-activated (p<0.05) platelets. CONCLUSIONS: Our results clearly point that the laser light irradiation of blood platelets can trigger signal transduction, leading to platelet activation, as well as the gradual loss of natural platelet reactivity and platelets' ability to respond to activating agents.
Assuntos
Plaquetas/efeitos da radiação , Lasers , Relação Dose-Resposta à Radiação , Citometria de Fluxo/métodos , HumanosRESUMO
Two polymorphisms of platelet glycoprotein Ib, VNTR and Thr/Met(145), regarded as the possible inherited risks factors for thromboembolic complications, have been suggested to underlie platelet response to activating stimuli. This study examined the functional significance of these polymorphisms in platelet reactivity and sensitivity to aurintricarboxylic acid (the antagonist of von Willebrand factor, vWF). To evaluate platelet function at low and high flow conditions we monitored the ristocetin-induced and vWF-mediated aggregation of isolated platelets and the platelet function analyzer collagen/ADP closure time (PFA-100 CT(CADP)), which reflects platelets' ability to adhere and aggregate in whole blood. Aurintricarboxylic acid significantly reduced ristocetin-induced platelet agglutination in a dose-dependent manner (IC(50)=3.5+/-1.9 microM). At the concentration of 100 microM it also markedly prolonged PFA-100 CT(CADP) (up to 147+/-32 s vs. 94+/-17 s in control). The efficacy of this antagonist in the inhibition of vWF-mediated platelet agglutination was approximately 1.5-fold higher in the VNTR B/Met145(+) carriers than in VNTR B/Met145(-) carriers ( P<0.05). Otherwise, no significant differences occurred between VNTR B/Met(145)-positive and B/Met(145)-negative individuals in the prolongation of closure time by ATA. These findings indicate that under certain experimental conditions VNTR-B and Met(145) alleles may contribute to the increased platelet sensitivity to some antagonists of platelet natural ligands.
Assuntos
Ácido Aurintricarboxílico/farmacologia , Agregação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Polimorfismo Genético , Fator de von Willebrand/metabolismo , Adulto , Alelos , Antibacterianos/farmacologia , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Genótipo , Humanos , Concentração Inibidora 50 , Ligantes , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Agregação Plaquetária/efeitos dos fármacos , Ristocetina/farmacologia , Fator de von Willebrand/antagonistas & inibidoresRESUMO
Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.
Assuntos
Ácido Aurintricarboxílico/farmacologia , Degranulação Celular/efeitos dos fármacos , Piperazinas/farmacologia , Piperidinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Difosfato de Adenosina/química , Adulto , Colágeno Tipo I/química , Feminino , Citometria de Fluxo , Hemostasia/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidoresRESUMO
We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.
Assuntos
Plaquetas/metabolismo , Oligonucleotídeos/farmacologia , Receptores de Trombina/metabolismo , Trombina/antagonistas & inibidores , Adulto , Aptâmeros de Nucleotídeos , Testes de Coagulação Sanguínea , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Fibrina/biossíntese , Fibrina/efeitos dos fármacos , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Receptor PAR-1 , Trombina/metabolismo , Trombina/farmacologiaRESUMO
UNLABELLED: Thrombospondin (TSP), which is secreted from alpha-granules of activated platelets, binds to its surface receptor (CD36) in the presence of Ca2+. OBJECTIVES: We monitored how the modulation of intraplatelet Ca2+ affects TSP binding to CD36 on platelets from healthy donors and patients with type 2 diabetes mellitus. We also aimed to verify whether the impaired Ca2+ mobilisation in diabetes influences TSP binding upon the pharmacological modulation of calcium transport. METHODS: Whole blood cytometry was used to monitor TSP release/binding and CD36 presentation in platelets from 28 type 2 patients and 33 healthy donors. RESULTS: No significant changes in TSP and CD36 levels were revealed between the groups in circulating platelets and TRAP-, collagen- or thrombin-activated platelets. In healthy donors, 1 microM thapsigargin (TG) elevated the TRAP-activated TSP binding (by up to 50%, p<0.001), 5 mM EGTA reversed the effect (by up to 85%, p<0.001), and overcame the effect of TG when used together. Less profoundly expressed effects occurred in the NIDDM group. In both groups TG increased the presentation of CD36 in TRAP-stimulated platelets (p<0.05), whereas EGTA lowered the TRAP-stimulated increase in CD36 (p<0.001). The inhibition of CD36 by EGTA was stronger in healthy volunteers (41% vs. 32%, respectively, p<0.05), whereas the activation by TG was higher in the NIDDM group (11% vs. 27%, p<0.05). When acting together the suppressive effects of EGTA on TG-dependent Ca2+ mobilisation were much attenuated in diabetic subjects (p<0.05). CONCLUSION: Both the release of TSP and CD36 presentation are under the influence of agents modulating intracellular Ca2+. Diabetic platelets seem more vulnerable to the releasers of cytosolic [Ca2+] and more resistant to the blockers of cytosolic [Ca2+] mobilisation.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/sangue , Trombospondinas/metabolismo , Adulto , Idoso , Plaquetas/efeitos dos fármacos , Antígenos CD36/efeitos dos fármacos , Antígenos CD36/metabolismo , Feminino , Humanos , Masculino , Análise por Pareamento , Pessoa de Meia-Idade , Selectina-P/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Ligação Proteica , Trombospondinas/efeitos dos fármacosRESUMO
Coronary artery bypass grafting (CABG) surgery impairs platelet function and reactivity to a considerable extent. However, variability in the individual patients' responses makes any generalised statement uncertain. The observed variability is nowadays thought to relate to platelet glycoprotein polymorphisms. Our objective was to investigate the association between platelet reactivity and the restoration of platelet functional response to agonists during the period following cardiosurgical operation and some genetic polymorphisms of selected platelet membrane glycoproteins. Platelet reactivity was monitored in 32 IHD patients (56 +/- 8 years) subjected to CABG surgery by means of whole blood impedance aggregometry and concurrently using the platelet function analyser (PFA-100 at four time intervals: prior to operation (A), 2 h after administration of protamine sulfate (B), 3 days after (C) and 7 days after CABG surgery (D). Three important findings were made. First, in all patients platelet reactivity became decreased 2 h postoperatively (aggregation with 20 microM ADP reduced by up to 49%, P < 0.02) and vastly increased 7 days after CABG surgery (CT(CADP) reduced down to 87% of initial value, P < 0.05, ADP-induced aggregation enhanced up to 167%, P < 0.001, and that with collagen up to 131% of the initial value, P < 0.01). Second, the frequencies of the 'prothrombotic' phenotype variants of platelet membrane glycoproteins were higher in patients referred to as the carriers of more reactive platelets compared to those with less reactive platelets (GPIa (807)T-positive, 50 vs. 28%; GPIIIa Pl(A2)-positive, 27 vs. 21%; GPIb Met(145)-positive and GPIb VNTR B-positive, 13 vs. 0%. Lastly, the restoration in platelet hyperreactivity in CABG surgery patients was recorded more often in patients who underwent postoperative myocardial ischaemic episode(s), and was associated with significantly higher frequency of the 'prothrombotic' allele (807)T of the collagen receptor glycoprotein Ia (GPIa) in these subjects (83 vs. 61%). In conclusion, in patients with ischaemic episodes after CABG, we demonstrated a fast postoperative restoration of haemostatic capacity and evidence of platelet hyperreactivity at 7 days after CABG surgery. The platelet hyperfunction seems to relate to the occurrence of platelet glycoprotein polymorphisms GPIa(807)C/T and GPIIIa PlA(1/A2) and may be important in predicting postoperative vascular complications in CABG patients.
Assuntos
Ponte de Artéria Coronária , Glicoproteínas da Membrana de Plaquetas/genética , Alelos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/genética , Polimorfismo GenéticoRESUMO
Abnormal platelet function has been hypothesised to play a role in the haemostatic abnormalities in cyanotic congenital heart disease (CCHD) patients. Using whole blood flow cytometry we found that platelets from cyanotic patients were hyperreactive and we related such hyperreactivity directly to young age, unoperated state, high haematocrit, reduced saturation with oxygen and low platelet count. Circulating platelets from CCHD children showed significantly enhanced P-selectin expression (P<0.004) and remained more reactive to 0.2 IU/ml thrombin, 1-8 microM TRAP and 2-4 microM ADP (P<0.04), especially in younger (0-3-year-olds) patients. Such a platelet 'priming' largely concerned CCHD children who were not subjected to modified Blalock-Taussig shunts in the past (non-MBTS). Only non-MBTS cyanotic children, but not MBTS-operated patients, showed significantly higher platelet reactivity compared to controls in response to ADP or 1 microM TRAP with respect to P-selectin expression (p<0.05) and in response to all examined agonists with respect to GPIb expression (P<0.045). The enhanced P-selection expression in MBTS-operated CCHD children and reduced GPIb expression in non-MBTS patients, especially in younger patients, were positively associated with the occurrence of the polymorphic variant Pl(A2) of platelet membrane glycoprotein IIIa gene. Altered blood morphology parameters (elevated RBC, Hb, Hct and MCHC, for all P<0.0005) in CCHD children correlated with the enhanced degranulation of circulating blood platelets and their hyperreactivity in response to some agonists (P<0.05). Overall, our data encourage the reasoning that circulating platelets are remarkably hyperreactive in non-MBTS cyanotic children, which are at higher risk to often encounter platelets activation in circulation. It seems unlikely that the apparently unchanged platelet reactivity in MBTS-operated children is due to the advantageous effects of the shunt, since these patients showed neither altered haematological parameters nor improved oxygen carrying capacity. Otherwise, it may rather result from more frequent episodes of platelet degranulation and preactivation in the past, and/or post-operative enhanced platelet consumption.
Assuntos
Prótese Vascular , Cardiopatias Congênitas/sangue , Ativação Plaquetária/fisiologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/fisiopatologia , Humanos , Lactente , Masculino , Fragmentos de Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Polimorfismo Genético/genéticaRESUMO
Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) and ADP in 42 healthy 1-2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectin-positive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F(1+2) in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.
Assuntos
Plaquetas/efeitos dos fármacos , Recém-Nascido/sangue , Ativação Plaquetária/efeitos dos fármacos , Receptores de Trombina/agonistas , Difosfato de Adenosina/farmacologia , Adulto , Fatores Etários , Antitrombina III/análise , Plaquetas/química , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/análise , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases/análise , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Proteínas/farmacologia , Protrombina/análise , Receptor PAR-1 , Trombina/farmacologiaRESUMO
The contribution of platelets to the pathogenesis and progression of vascular complications in diabetes is supported by several studies. In general, platelets obtained from diabetic subjects show increased adhesiveness and an exaggerated aggregation, both spontaneous and in response to stimulating agents. The causes for this activation are multifold: altered exposure and/or abundance of glycoprotein receptors for agonists and adhesive proteins on the platelet surface, increased binding of fibrinogen, decreased membrane fluidity, altered platelet metabolism and changes in intraplatelet signalling pathways. The altered biophysical state of platelet membrane components in diabetes mellitus may be one of the major determinants of platelet hypersensitivity and hyperfunction and may contribute to impairments in various metabolic pathways, like intensified calcium mobilisation and accentuated thromboxane synthesis and release. Activated platelets interact with other cells, such as endothelial cells and leukocytes as well with the coagulation system in the process of atherosclerosis. Some studies indicated that platelet dysfunction was especially apparent in diabetic subjects with macro- or microangiopathy, while others showed that it may be related to the presence of diabetes mellitus per se. Several pharmaceutical compounds have been developed for the inhibition of platelet activation. However, aspirin treatment is cheap and effective, and aspirin remains to be the drug of choice for diabetic patients. It should be prescribed widely for patients who are at high risk of cardiovascular events.
Assuntos
Plaquetas/fisiologia , Angiopatias Diabéticas/sangue , Arteriosclerose/sangue , Arteriosclerose/fisiopatologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/fisiopatologia , Progressão da Doença , Produtos Finais de Glicação Avançada/sangue , Humanos , Inibidores da Agregação Plaquetária/uso terapêuticoRESUMO
Intima-Media Thickness (IMT) of Common Carotid Artery (CCA) could be seen as the atherosclerotic risk factors' final morphological effect. We investigated the hypothesis that IMT of CCA is significantly different in sex- and age-matched groups of persons with stroke and healthy subjects. 47 patients with first-ever atherothrombotic stroke proven by CT were investigated. Patients with atrial fibrillation, valvular heart disease and left ventricular hyperthrophy were excluded. The IMT of CCA were estimated by High-Resolution B-Mode Ultrasonography. All the patients had bilateral IMT measurement within 20 mm proximal to the carotid bulb on the far wall in the anterioposterior and laterolateral plane. The results were compared with those obtained in 50 healthy sex- and age-matched subjects. We found a strong association between IMT and stroke (p < 0.0001). Mean IMT was 0.96 mm (SD 0.18) in patients and 0.70 mm (SD 0.09) in controls. The presence of atherosclerotic plaques was 0.34 and 0.08 for patients and controls respectively (p = 0.0025). IMT of CCA is strongly positively associated with the risk for stroke. The frequency of atherosclerotic plaques in CCAs is statistically significantly higher in stroke patients than in control group.
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Artérias Carótidas/anatomia & histologia , Acidente Vascular Cerebral/diagnóstico , Idoso , Arteriosclerose/complicações , Artérias Carótidas/diagnóstico por imagem , Feminino , Humanos , Masculino , Valores de Referência , Fatores de Risco , Acidente Vascular Cerebral/etiologia , UltrassonografiaRESUMO
Introduction of the antiplatelet agents of new generations and the occurrence of the phenomenon of "aspirin-resistance" triggered the search for better, simpler and more reliable routine diagnostic methods to monitor platelet reactivity. Our objective was to evaluate the usefulness and reliability of two simple methods: platelet function analyzer (PFA-100) and whole blood platelet aggregometry for monitoring of platelet function in 18 healthy blood donors and 35 patients with ischaemic heart disease (IHD) subjected to small doses/75 mg and 150 mg a day) of acetylsalicylic acid (aspirin). In 50% of healthy blood donors the intake of 75 mg ASA a day resulted in the prolongation of PFA-100 collagen/epinephrine closure time (CEPI = (relevant to reduced platelet reactivity) of over 150 s, whereas 75% donors responded to 150 mg ASA-. Otherwise, the daily dose of 150 mg ASA resulted in a prolonged CEPI merely in 23% of in IHD patients. At both doses ASA completely inhibited the arachidonic acid-induced whole blood platelet aggregation in all healthy donors and in all but 3 IHD patients. Collagen-induced platelet aggregation was only negligibly affected by either dose of ASA. Our results point that the simultaneous monitoring of the PFA-100 collagen/epinephrine closure time and whole blood platelet aggregometry (Chrono-Log) enables to reliably evaluate the inhibition of platelet function by ASA and discriminate the partial or complete platelet insensitivity to aspirin. The phenomenon of more frequent platelet aspirin-resistance in IHD patients requires to be verified in randomised clinical prospective studies.
Assuntos
Aspirina/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Agregação Plaquetária/efeitos dos fármacos , Adulto , Resistência a Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica/métodos , Isquemia Miocárdica/sangue , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Excessive blood loss, as a result of augmented postoperative drainage, is considered one of the most serious cardiosurgical complications. The compounding constitutive anemia seems particularly harmful for patients with coronary artery disease. Aprotinin (Trasylol), a non-specific serine protease inhibitor, is successfully used to reduce excessive postoperative bleeding in such patients. The aim of our study was to verify the hypothesis whether aprotinin used during cardiopulmonary bypass procedure affects hemostatic parameters, which might be crucial for the elevated risk of thromboembolic complications. MATERIAL AND METHODS: The group of 54 patients subjected to coronary artery surgical treatment included 30 patients, who were given intraoperatively 3 million KIU aprotinin each, and 24 subjects non-treated with aprotinin. Aliquots of blood were withdrawn at several time intervals, until the 5th day after the operation. Whole blood platelet activation and reactivity (the expressions of P-selectin and glycoprotein Ib) were monitored by means of flow cytometry. In addition, several plasma parameters, like PAI-1, t-PA, D-dimers, prothrombin fragment F1 + 2, fibrinogen, ATIII activity, troponin I and CK-MB, as well as platelet count were determined at each time point. RESULTS: In this study we confirmed the essential advantage of the use of aprotinin: both the postoperative blood drainage and the blood units to be transfused postoperatively to cardiosurgical patients were vastly reduced in the aprotinin-treated subjects. The enhanced overall frequency of perioperative myocardial infarction events was not attributed to this group of patients, nor the non Q-wave infarctions were observed more often in patients treated with aprotinin. In these patients, fibrinolysis parameters tended to be depressed (with increased PAI-1 dominating over elevated t-PA) on the first day after the operation, and no significant differences with regard to fibrinogen, prothrombin fragment F1 + 2, troponin I and platelet count. There was a continuous rise in D-dimers in all the postoperative patients, which lasted until the third day and tended to reach plateau at the 5th day after the operation. We failed to reveal the preventive effects of aprotinin on platelet function: both platelet activation and reactivity remained apparently unchanged. Overall, our results rather support the reasoning on the advantageous effects of low doses of aprotinin. The use of this inhibitor reduces the risk of postoperative undesirable bleeding and results in a decreased postoperative drainage and reduced transfused blood units. On the other hand, however, a higher incidence of perioperative Q-wave infarction in the aprotinin-treated patients, although purely apparent and not statistically significant, might question the unlimited safety of the use of aprotinin in cardiovascular operations.