RESUMO
BACKGROUND: Deceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia-reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model. METHODS: The effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4 °C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage. RESULTS: At 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial-mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment. CONCLUSION: Peritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevance The increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome. Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions. This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.
Assuntos
Anticoagulantes/farmacologia , Transplante de Rim/métodos , Polissacarídeos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Constrição , Citocinas/metabolismo , Fondaparinux , Glutationa/farmacologia , Insulina/farmacologia , Rim/efeitos dos fármacos , Rim/fisiologia , Leucócitos/efeitos dos fármacos , Nefrite/fisiopatologia , Soluções para Preservação de Órgãos/farmacologia , Rafinose/farmacologia , Suínos , Transplante Autólogo , Isquemia Quente/métodosRESUMO
Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.
Assuntos
Anticoagulantes/uso terapêutico , Azetidinas/uso terapêutico , Benzilaminas/uso terapêutico , Transplante de Rim/métodos , Adenosina , Alopurinol , Animais , Sequência de Bases , Doença Crônica , Primers do DNA/genética , Fibrose , Glutationa , Humanos , Insulina , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Transplante de Rim/efeitos adversos , Transplante de Rim/patologia , Masculino , Modelos Animais , Preservação de Órgãos , Soluções para Preservação de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rafinose , Transdução de Sinais/efeitos dos fármacos , Suínos , Temperatura , Doadores de Tecidos , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transplante AutólogoRESUMO
Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
Assuntos
Proteínas Aviárias , Compartimento Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Transporte Biológico , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Moléculas de Adesão Celular/metabolismo , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrinas/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Trombina/farmacologia , Tirosina/metabolismoRESUMO
Platelets form heterotypic complexes with circulating monocytes, inducing the expression of the procoagulant Tissue Factor (TF) that leads to thrombin generation. We investigated the potential preventive effect of melagatran, a direct anti-thrombin drug, on TF expression by platelet/monocyte heterotypic complexes (PMHC) from healthy human donors. Flow cytometry and western blot analysis were performed to characterize surface and total TF protein expression in PMHC in venous blood samples drawn in the presence of heparin or heparin and melagatran (4 microM). Addition of melagatran significantly lowered the percentage of TF positive PMHC (2.6+/-0.3 vs. 5.9+/-0.7 %, p<0.01). This was not due to a melagatran-induced decrease in activation of the platelets associated with monocytes in PMHC. Indeed, melagatran effect on TF expression was accompanied by an increase in cell surface P-selectin expression in PMHC (95.6+/-1.9 vs. 48+/-18 %, p<0.001), suggesting that platelet were actually more activated in PMHC from the melagatran-treated samples. Western blot analysis of PBMC extracts suggested that melagatran specifically targeted a (54kD) form of TF in monocytes. Although further investigation is warranted, these data suggest that melagatran decreases TF expression in PMHC.
Assuntos
Anticoagulantes/farmacologia , Azetidinas/farmacologia , Benzilaminas/farmacologia , Plaquetas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Tromboplastina/metabolismo , Adulto , Plaquetas/citologia , Humanos , Pessoa de Meia-Idade , Monócitos/citologia , Selectina-P/metabolismo , Isoformas de Proteínas/metabolismo , Adulto JovemRESUMO
AIMS: Insulin resistance is a key feature of type 2 diabetes. It is also involved in the development and progression of microvascular complications. We analysed the relationship between parental history of diabetes, insulin resistance and diabetic nephropathy (DN) and assessed the specific maternal and paternal influences of history of type 2 diabetes on DN in type 1 diabetic offspring. METHODS: We recorded information regarding family history of type 2 diabetes and of cardiovascular disease in 160 consecutive, unrelated type 1 diabetic patients. Insulin resistance was assessed using a validated estimation of the glucose disposal rate (eGDR). RESULTS: Type 1 diabetic patients with a maternal history of type 2 diabetes were more likely to be insulin-resistant (P=0.043) and to have renal complications (P=0.0041) than those from the reference group (without parental history of diabetes), while patients with a paternal history were not different from those from the reference group, regarding eGDR and DN. Time to development of abnormal albuminuria was significantly affected by maternal history of type 2 diabetes (log-rank=12.66; P=0.0004) and by familial history of premature cardiovascular disease (log-rank=5.48; P=0.0234). In multivariate analysis, a maternal history of type 2 diabetes was independently associated with nephropathy after adjustment for sex, diabetes duration and familial history of premature cardiovascular disease. CONCLUSION: Maternal history of type 2 diabetes is independently associated with DN in type 1 diabetic patients. This might suggest the transmission of a maternal trait related to microvascular complications, raising the hypothesis of imprinted genes predisposing to diabetic renal disease.
Assuntos
Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/epidemiologia , Nefropatias Diabéticas/genética , Mães , Pressão Sanguínea , Índice de Massa Corporal , Tamanho Corporal , Colesterol/sangue , Feminino , França , Hemoglobinas Glicadas/análise , Humanos , Resistência à Insulina/genética , Lipídeos/sangue , Masculino , AnamneseRESUMO
The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.
Assuntos
Ácidos Araquidônicos/sangue , Plaquetas/metabolismo , Lipoxigenase/sangue , Prostaglandina-Endoperóxido Sintases/sangue , Trombina/fisiologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina , Anti-Inflamatórios/farmacologia , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Plaquetas/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase , Humanos , Cinética , Inibidores de Lipoxigenase , Galato de Propila/farmacologia , Pirazóis/farmacologia , TrítioRESUMO
The subcellular distribution of diacylglycerol- and monoacylglycerol-lipases has been studied in human platelets. Using a fractionation procedure on Percoll gradient (Perret, B., Chap, H. and Douste-Blazy, L. (1979) Biochim. Biophys. Acta 556, 434-446), the enzyme activity displayed the same profile as that of [3H]concanavalin A, a plasma membrane marker. This result was confirmed with highly purified platelet plasma membranes prepared by adsorption onto polyethylenimine-bonded polyacrylamide beads (Kinoshita, T., Nachman, R.L. and Minick, R. (1979) J. Cell Biol. 82, 688-696). Studies with isolated membranes or crude homogenate revealed that the enzyme requires calcium or magnesium and displays an optimal pH of 6.2, showing that it is able to hydrolyse diacylglycerol under conditions where phosphatidylinositol-specific phospholipase C is fully active. Using diacylglycerol labelled in the 1- or 2-position, it was found that the two fatty acids are released at the same rate, which is supported by the lack of monoacylglycerol accumulation and by the observation that monoacylglycerol is hydrolysed at a 20-fold faster rate than diacylglycerol. Increasing concentrations of Mg-ATP promote the conversion of diacylglycerol into phosphatidic acid by diacylglycerol kinase, but only high concentrations become inhibitory for diacylglycerol lipase. These results are discussed in the light of our former hypothesis that arachidonic acid release from platelet phospholipids might occur through the sequential action of a phosphatidylinositol-specific phospholipase C coupled to a diacylglycerol lipase (Mauco, G., Chap, H., Simon, M.F. and Douste-Blazy, L. (1978) Biochimie 60, 553-561). The possible role of this enzyme in the regulation of the activity of protein kinase C is also emphasized.
Assuntos
Plaquetas/enzimologia , Hidrolases de Éster Carboxílico/sangue , Diglicerídeos/sangue , Glicerídeos/sangue , Lipase Lipoproteica/sangue , Monoacilglicerol Lipases/sangue , Cátions Bivalentes , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Centrifugação com Gradiente de Concentração , Humanos , Cinética , Lipase Lipoproteica/isolamento & purificação , Monoacilglicerol Lipases/isolamento & purificação , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
A simple method of determination of phosphatidylinositol-specific phospholipase C activity in soluble platelet extracts has been devised. It is based on the use of a total lipid extract from rat liver microsomes incubated with [3H]inositol in the presence of MnCl2. Phosphatidylinositol hydrolysis can thus be detected by determining hydrosoluble radioactivity formed upon incubation with enzyme fractions. Owing to the presence of other phospholipids in the assay system, phospholipase C was inhibited. However, activity was restored by sodium deoxycholate (0.1%, w/v). Optimal conditions also included calcium (1-10 mM) and a pH between 5 and 7, allowing the detection of phospholipase C without the need for purifying the substrate. Using this simplified procedure, platelet phospholipase C was submitted to preparative electrofocusing and to gel filtration chromatography on Sephacryl S-200. Phospholipase C focused in one single peak at pH 6.1. An Mr of 86 000 was found upon gel chromatography of a crude extract, against 68 000 when phospholipase C had been previously purified by electrofocusing. These data indicate that phospholipase C might be associated with lipids or with an Mr 20 000 protein, the significance of which is discussed.
Assuntos
Plaquetas/enzimologia , Diglicerídeos/sangue , Glicerídeos/sangue , Fosfolipases/sangue , Fosfolipases Tipo C/sangue , Humanos , Focalização Isoelétrica , Cinética , Microssomos Hepáticos/análise , Peso Molecular , Fosfatidilinositóis/isolamento & purificação , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
The strong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol, 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the alpha 11bb3 integrin, downstream protein kinase C activation.
Assuntos
Plaquetas/metabolismo , Inibidores Enzimáticos/farmacologia , Mitógenos/farmacologia , Ácido Okadáico/farmacologia , Fosfoproteínas , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Trombina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Plaquetas/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Fibrinogênio/metabolismo , Humanos , Fosfatidilinositol 3-Quinases , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Testes de Precipitina , Tirosina/metabolismoRESUMO
BACKGROUND: Recent studies suggest that HbA1c is an important predictor of the glycometabolic state of patients admitted for acute myocardial infarction (AMI). OBJECTIVE: We aimed at comparing the results of HbA1c concentrations obtained by 2 different methods in patients with AMI. RESEARCH DESIGN AND METHODS: In a first study, HbA1c was measured in all patients consecutively hospitalized for AMI, during a 6 month period using the HPLC method and the DCA 2000 device in the biochemistry laboratory. In a second study, HbA1c measured by the DCA 2000 device in the intensive care unit was compared with HbA1c determined by HPLC in the biochemistry laboratory in a similar sample of patients. In patients without personal history of diabetes, those patients with HbA1c > 6.5% (HPLC method), were classified as possible diabetes. RESULTS: A total of 146 patients were included (119 males, 27 females; mean age: 63 +/- 15 years). Twenty-seven of the patients had a personal history of diabetes. HbA1c determined by 2 techniques were highly correlated (r = 0.939, P < 0.0001). The mean of the differences (Bland and Altman analysis) was 0.4 +/- 0.3%. Compared with the HPLC method, the sensitivity of DCA 2000 device for the detection of possible diabetes was 81.8 +/- 11.6 and the specificity was 99.1 +/- 0.9%. The diagnostic accuracy of DCA method was 97.5 +/- 1.4%. In the second study, the HbA1c concentrations of 21 additional subjects, determined in an intensive care unit, were not different from the first 21 patients of the first study. CONCLUSIONS: HbA1c can be effectively determined using the DCA 2000 device. This method is reliable and easy to be implemented in an intensive care unit.
Assuntos
Hemoglobinas Glicadas/análise , Infarto do Miocárdio/sangue , Autoanálise , Glicemia/metabolismo , Coleta de Amostras Sanguíneas , Cromatografia Líquida de Alta Pressão , Humanos , Pacientes Internados , Análise de RegressãoRESUMO
Value of systematic dosage of biological markers of inflammation for the prognosis at 12 months of patients undergoing programmed coronary angioplasty Systematic dosage of proteins of inflammation has been suggested for assessing the prognosis of athero-thrombotic diseases. The authors undertook a study of plasma C-reactive protein (CRP) and interleukin 6 (IL-6) for evaluating the prognosis of patients undergoing programmed coronary angioplasty. A prospective monocentric study of 117 patients (65 +/- 8 years) was divided into a control group of 28 patients undergoing coronary angiography (Group 1) and 89 patients undergoing programmed coronary angioplasty (Group 2). Serum IL-6 and CRP levels were measured before arterial puncture and at H12 and H24 after coronary catheterisation. The follow-up period was 12 months. The angioplasty did not significantly increase CRP and IL-6 concentrations compared with coronary angiography. Twenty patients (Group 2) (22%) suffered a cardiovascular event in the 12 months' follow-up. These patients had significantly higher CRP levels at H0, H12 and H24 after coronary angioplasty than those who had uncomplicated outcomes. This was not observed for IL-6 concentrations because of the wide dispersion of the results obtained. Increased CRP concentrations between H0 and H24 was also a good predictive factor independently of high basal CRP levels potentially due to other causes than atheroma. Coronary angioplasty is associated with increased CRP at H0, H12 and H24. These values are correlated with the risk of future events at 6 and 12 months. This information is easily obtained and should help management of these patients.
Assuntos
Angioplastia Coronária com Balão , Proteína C-Reativa/análise , Inflamação/sangue , Interleucina-6/sangue , Idoso , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos ProspectivosRESUMO
The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.
Assuntos
Plaquetas/ultraestrutura , Citoesqueleto/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/sangue , 1-Fosfatidilinositol 4-Quinase , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Octoxinol , Polietilenoglicóis/farmacologia , SolubilidadeRESUMO
32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.
Assuntos
Plaquetas/metabolismo , Fosfatidilinositóis/sangue , Fator de Ativação de Plaquetas/análogos & derivados , Animais , Plaquetas/efeitos dos fármacos , Cinética , Fosfatidilinositol 4,5-Difosfato , Fator de Ativação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , CoelhosRESUMO
Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.
Assuntos
Aldeídos/metabolismo , Córtex Cerebral/metabolismo , Ácidos Graxos/metabolismo , Estresse Oxidativo/fisiologia , Plasmalogênios/metabolismo , Animais , Cromatografia Gasosa-Espectrometria de Massas/métodos , Radical Hidroxila/metabolismo , Técnicas In Vitro , Cinética , Masculino , Oxirredução , Ratos , Ratos Wistar , Oxigênio Singlete/metabolismoRESUMO
Beside 4- and 5-phosphatases playing a role in the interconversion between the D-3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonesterasic activity on the D-3 position of inositol lipids is a specific 3-phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3-phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO4(-3) and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3-kinase and 3-phosphatase activities in the pool of phosphotyrosine-containing proteins isolated from EGF-stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células 3T3 , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Hidrólise , Camundongos , Fosfatidilinositol 3-Quinases , Especificidade por SubstratoRESUMO
In thrombin-stimulated platelets alpha IIb beta 3 integrin engagement triggers both phosphatidylinositol 3',4'-bisphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dose-dependent inhibition of phosphatidylinositol 3,4-bisphosphate synthesis by calpeptin from 50 micrograms/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated alpha IIb beta 3 integrin, did not induce additive effects on phosphatidylinositol 3,4-bisphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptin-treated platelets. These data indicate that calpains are involved in the regulation of alpha IIb beta 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.
Assuntos
Plaquetas/metabolismo , Calpaína/fisiologia , Integrinas/fisiologia , Fosfatos de Fosfatidilinositol/biossíntese , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Trombina/farmacologia , Transporte Biológico/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Dipeptídeos/farmacologia , Humanos , Integrinas/metabolismo , Fosfatidilinositol 3-Quinases , Fosfatos de Fosfatidilinositol/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.
Assuntos
Plaquetas/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/síntese química , Agregação Plaquetária , Trombina/farmacologia , Plaquetas/fisiologia , Citocalasina D/farmacologia , Citoesqueleto/fisiologia , Humanos , Técnicas In VitroRESUMO
When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.
Assuntos
Plaquetas/enzimologia , Fosfatos de Fosfatidilinositol/biossíntese , Fosfatidilinositóis/biossíntese , Trombina/farmacologia , Fosfolipases Tipo C/metabolismo , Aminoquinolinas/farmacologia , Células Cultivadas , Ativação Enzimática , Humanos , Hidrólise , Técnicas In Vitro , Cinética , Fosfatidilinositol 4,5-Difosfato , Fosfolipídeos/metabolismoRESUMO
From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.
Assuntos
Lisofosfolipídeos/biossíntese , Fosfolipídeos/metabolismo , Animais , Plaquetas/metabolismo , Membrana Celular/metabolismo , Fosfolipases A2 do Grupo VI , Humanos , Lisofosfolipídeos/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipases A/metabolismoRESUMO
Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.