Asunto(s)
Enfermedades del Nervio Abducens/etiología , Arteria Carótida Interna/anomalías , Malformaciones Vasculares del Sistema Nervioso Central/complicaciones , Enfermedades del Nervio Abducens/patología , Enfermedad Aguda , Enfermedades de las Arterias Carótidas/complicaciones , Enfermedades de las Arterias Carótidas/patología , Malformaciones Vasculares del Sistema Nervioso Central/patología , Diplopía/diagnóstico , Diplopía/etiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
INTRODUCTION: Recently, rapid immunoassays have been developed to allow the detection of antibodies anti-PF4/heparin. In this prospective study, we evaluated the performances of a automatized immunoassay (HemosIL HIT-Ab) in comparison with an ELISA (Zymutest HIA IgG) used for the diagnosis of heparin-induced thrombocytopenia (HIT) in association with the 4T's score. METHODS: According to the 4T's score, samples with score ≤3 had no further analysis. Two immunological assays Zymutest HIA IgG and HemosIL HIT-Ab were performed in samples with score ≥4. In patients with at least one positive immunological assay or two negative immunological assays but with high-pretest probability (4T's score ≥6), HIT was screened by one functional assay using washed platelets. RESULTS: The sensitivities of both assays were excellent and comparable (100%). The specificity was 92.3% for ELISA and 91.2% for HemosIL HIT-Ab. The analysis of the operating characteristics showed that both assays have almost identical ROCs (AUROC, 0.9951 and 0.9853, respectively, for ELISA and HemosIL HIT-Ab) and the calculating of the κ coefficient revealed a good agreement (0.67). CONCLUSION: Performance characteristics of the HemosIL HIT-Ab are comparable to the Zymutest HIA IgG. The HemosIL HIT-Ab can be used in association with the 4T's score to rule out HIT.
Asunto(s)
Heparina/efectos adversos , Inmunoensayo/métodos , Trombocitopenia/diagnóstico , Trombocitopenia/etiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/sangre , Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Heparina/inmunología , Humanos , Inmunoensayo/instrumentación , Inmunoensayo/normas , Masculino , Persona de Mediana Edad , Factor Plaquetario 4/inmunología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
It has been reported that thrombin generation test (TGT) may be a useful tool to monitor recombinant factor VIIa (rFVIIa). However, TGT does not reflect the stability of fibrin clot and its resistance to fibrinolysis which are crucial. Using whole-blood thromboelastography (TEG) and tissue plasminogen activator (tPA), we developed an in-vitro model to assess fibrin clot stability. Fibrin fibres were thicker in haemophiliacs compared with controls (P < 0.0001). After addition of rFVIIa 90 µg kg(-1), the diameter of fibrin fibres was dramatically decreased (P = 0.006). TEG-tPA assay showed a dose-dependent improvement of clot stability in the presence of rFVIIa. These data demonstrate a significant correlation between fibrin clot structure and its stability (P = 0.001). We also showed a correlation between thrombin generating capacity and clot resistance to fibrinolysis. Despite this overall correlation, a relatively large spreading around a general trend was observed, suggesting that the two assays bring complementary information on the haemostatic effect of rFVIIa.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor VIIa/uso terapéutico , Fibrina/efectos de los fármacos , Hemofilia A/tratamiento farmacológico , Hemostáticos/uso terapéutico , Trombina/biosíntesis , Análisis de Varianza , Hemofilia A/metabolismo , Hemostasis/efectos de los fármacos , Humanos , Microscopía Electrónica de Rastreo , Modelos Biológicos , Proteínas Recombinantes/uso terapéutico , Tromboelastografía/métodos , Activador de Tejido Plasminógeno/análisisRESUMEN
BACKGROUND: Human prothrombin complex concentrates (PCCs) are used for prevention and treatment of bleeding episodes in patients under warfarin therapy. PCCs contain human factor (F) II, FVII, FIX, FX, protein C and protein S. The concentrations of these coagulation factors contained in PCCs are variable and do not reflect entirely the capacity of these drugs to correct hemostasis. Furthermore, commercially available PCCs do not have exactly the same composition, though they are all labelled and prescribed in units per kg of FIX (10-40 IU of FIX/kg). As the final product generated by PCCs is thrombin, a thrombin generation (TG) test could theoretically be used for monitoring the hemostatic correction. METHODS: TG was measured in platelet free plasma in the presence of tissue factor 5 pm and phospholipids 4 microM with a final concentration of PCC of 0-0.1-0.2-0.3-0.4-0.5-0.75-1 IU ml(-1). The activity of vitamin K-dependent coagulation factors (i.e. FII, FVII, FIX, FX, protein C and protein S) were determined for each concentration of two different PCCs available on the French market. RESULTS AND DISCUSSION: Our results showed that the addition of two different PCCs dose-dependently increased the TG capacity in patients with INR of 2-2.5-3-4 and >7 (n = 15 subjects) that reached the normal values. We also found a significant correlation between endogenous thrombin potential (ETP) and INR (Pearson test, P < 0.0001). The two PCCs improved the TG parameters differently with increasing concentrations. The difference in the correction of TG capacity observed between the two drugs could be explained by a variable increase in FX, FVII and protein C with similar doses. These results strongly suggest that TG assay could be used for monitoring the clinical efficacy of PCC and for optimizing the therapeutic regimen towards a more individualized therapy involving the type of the bleeding complications, the level of inhibition of the coagulation system and the molecule content of the PCC.
Asunto(s)
Anticoagulantes/farmacología , Factores de Coagulación Sanguínea/química , Factores de Coagulación Sanguínea/farmacología , Trombina/química , Administración Oral , Adulto , Anticoagulantes/efectos adversos , Anticoagulantes/química , Estudios de Casos y Controles , Precursores Enzimáticos/química , Femenino , Hemorragia/tratamiento farmacológico , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Proteína C/química , Trombina/biosíntesis , Trombofilia/tratamiento farmacológico , Vitamina K/metabolismoRESUMEN
Bypassing agents consist of activated prothrombin complex concentrates (aPCC) and recombinant factor VIIa (rFVIIa). Their main utilization is for prevention and treatment of bleeding complications, which may occur in inhibitor-developing haemophiliacs, although new indications for rFVIIa (e.g. trauma-related and cerebral bleeds) are now under evaluation in clinical trials. The mechanisms of action for these agents are still not fully understood. The relative complexity of the composition of aPCC suggests the possibility of multiple modes of action for achieving haemostasis. Among those possibilities, the contributions of activated factor X and prothrombin have been demonstrated in recent years both in vitro and in animal models for the only aPCC which remains on the market. rFVIIa also exhibits a complex mode of action, improving coagulation through both tissue factor-dependent and -independent pathways. The various mechanisms that occur at the cellular surfaces, particularly on the outer leaflet of the platelet membrane, primarily contribute to Xase complex formation and thrombin generation. The ways in which these agents affect the complex kinetics of fibrin formation at the site of vascular damage need further clarification, although significant progress has been achieved in the last 10 years. In addition, the ex vivo monitoring that would reflect achievement of haemostasis in vivo is still not standardized, although several attempts using thromboelastography, thrombin generation and the kinetics of fibrin formation have been initiated.
Asunto(s)
Factores de Coagulación Sanguínea/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Factor VIIa/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemostáticos/uso terapéutico , Trombina/uso terapéutico , Coagulación Sanguínea/fisiología , Fibrina/efectos de los fármacos , Hemofilia A/fisiopatología , HumanosRESUMEN
BACKGROUND: Hemophilia A patients with inhibitors are generally treated with preparations containing activated coagulation factors to achieve hemostasis by bypassing factor (F)VIII. OBJECTIVES: We developed an assay for monitoring the kinetic of thrombin generation in human FVIII inhibitor plasma reconstituted in vitro with activated prothrombin complex concentrate, FEIBA, and in plasma samples from hemophilia A patients taken after FEIBA treatment. PATIENTS AND METHODS: For pharmacokinetic studies three patients with severe hemophilia A and with a high-titer inhibitor received a single dose of FEIBA. Repeated FEIBA treatment was monitored in one patient with acquired hemophilia A. Coagulation was triggered in citrated plasma by adding a low concentration of tissue factor/phospholipid complex and CaCl2 in the presence of a fluorogenic thrombin substrate. The intensity of the fluorescence signal (FU) was continuously monitored, and the rate of increase in the fluorescence signal for every time point, which reflects the actual thrombin concentrations, was calculated. RESULTS: The maximum rate of substrate conversion, which indicates the highest thrombin concentration, was approximately 1900 FU min(-1) in a normal plasma pool. Practically no thrombin generation was observed in the FVIII inhibitor plasma, but when it was spiked with FEIBA, the rate and the peak of thrombin generation increased dose-dependently to close to normal. Plasma samples from FVIII inhibitor patients treated with a single dose of FEIBA had an improved thrombin maximum within an hour after treatment, which gradually returned to baseline values with a half-life of 4-7 h. Changes in the characteristic parameters of thrombin generation coincided with the repeated administration of FEIBA in a patient with acquired hemophilia A. CONCLUSIONS: This assay enables the pharmacodynamic and pharmacokinetic properties of bypassing therapies to be monitored, thus helping to optimize treatment.
Asunto(s)
Factores de Coagulación Sanguínea/farmacocinética , Monitoreo de Drogas/métodos , Trombina/biosíntesis , Adulto , Anciano , Disponibilidad Biológica , Pruebas de Coagulación Sanguínea/métodos , Evaluación de Medicamentos , Factor VIII/inmunología , Femenino , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Humanos , Isoanticuerpos/sangre , Masculino , FarmacocinéticaRESUMEN
Factor VIII (FVIII)-bypassing agents have complex modes of action but all control bleeding in inhibitor patients by triggering the generation of thrombin. No routine test is available for monitoring this therapy in patients with inhibitors against FVIII. We present an assay that records FEIBA- or FVIIa-mediated changes in thrombin generation (TG) in FVIII inhibitor plasma samples. In plasma samples spiked with FEIBA TG was normalized above 0.4 U/ml, while for recombinant FVIIa (rFVIIa) more than 12.5 microg/ml were required to induce TG in the absence of tissue factor (TF). Addition of TF increased the TG potential of rFVIIa in vitro. This assay seems suitable for monitoring the pharmacokinetics of inhibitor bypassing agents during treatment and possibly for predicting responses to treatment.
Asunto(s)
Autoanticuerpos/inmunología , Factores de Coagulación Sanguínea/farmacología , Factor VIII/antagonistas & inhibidores , Factor VII/farmacología , Hemofilia A/sangre , Isoanticuerpos/inmunología , Proteínas Recombinantes/farmacología , Trombina/biosíntesis , Factores de Coagulación Sanguínea/análisis , Sistemas de Computación , Relación Dosis-Respuesta a Droga , Factor VIII/inmunología , Factor VIII/uso terapéutico , Factor VIIa , Hemofilia A/tratamiento farmacológico , Humanos , Modelos Biológicos , Proteínas Recombinantes/sangre , Trombina/análisis , Tromboplastina/farmacologíaRESUMEN
We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.
Asunto(s)
Factor IX/biosíntesis , Células Madre Hematopoyéticas/metabolismo , Factor IX/genética , Estudios de Factibilidad , Terapia Genética , Células Madre Hematopoyéticas/citología , Hemofilia B/terapia , Humanos , Megacariocitos , Glicoproteína IIb de Membrana Plaquetaria/genética , Regiones Promotoras Genéticas , Transfección , Células Tumorales CultivadasRESUMEN
In this study we have used denaturing gradient gel electrophoresis (DGGE) for identifying sequence alterations in glycoprotein (GP) IIb and IIIa genes from 20 patients affected by Glanzmann's thrombasthenia. These patients were from 16 different families. Using computer modelling, we divided the promoters, coding sequences and flanking splicing regions, in 31 segments for the GPIIb gene and 19 domains for the GPIIIa gene. We were able to find a mutation potentially affecting GPIIb-IIIa expression or function in 16 patients out of 20. In six patients from three families, the gypsy mutation modifying the splice donor site of intron 15 of the GPIIb gene was detected. In the other patients, 10 novel mutations were characterised, which were located either in the GPIIb gene (nine cases) or in the GPIIIa gene (one case). The type of mutation was nonsense mutation (one case), missense mutation (five cases), small insertion of 1 bp (one case) and splicing modifications (three cases). Among these genetic events, three were directly responsible for Glanzmann's thrombasthenia, four were localised in regions known to be involved in GPIIb-IIIa complex expression and three mutations were potentially responsible for Glanzmann's thrombasthenia.
Asunto(s)
Mutación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Trombastenia/genética , Electroforesis en Gel de Poliacrilamida/métodos , Exones , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The effects of the marine fatty acid 20:4n-3, an isomer of arachidonic acid (20:4n-6), have been compared to that of 20:5n-3 on 20:4n-6 oxygenation in human platelets and endothelial cells. In platelets, 20:4n-3 added along with 20:4n-6 was as potent as 20:5n-3 in inhibiting prostaglandin H synthase (PGH synthase) activity. From 2.5- to 10 microM of 20:4n-6, the synthesis of thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid, reflecting the PGH/thromboxane synthase activity, was lowered by 5 and 10 microM of both fatty acids. In contrast, 20:4n-3, but not 20:5n-3, strongly stimulated the lipoxygenase activity at each concentration of 20:4n-6 used whatever the amount of 20:4n-3 added. The effects of both n-3 polyunsaturated fatty acids on endothelial cell PGH/prostacyclin synthases were compared after 2- and 24-hr incubation with the cells, leading to moderate (2 hr) and high (24 hr) concentrations of these fatty acids in membrane phospholipids. The incorporation of 20:4n-3 and 20:5n-3 occurred mostly in phosphatidylcholine and phosphatidylethanolamine and did not alter the 20:4n-6 level of phospholipid classes after 2-hr supplementation, whereas it was drastically decreased after 24 hr. The synthesis of prostacyclin obtained after cell stimulation by 0.1 U/mL thrombin was unaffected by the fatty acid modifications induced after 2-hr supplementation, whereas it was strongly depressed after 24 hr. It was concluded that 20:4n-3 is not an agonist for platelet activation, despite its close structural analogy with 20:4n-6, and is as potent as 20:5n-3 in inhibiting PGH synthase activities, showing that the double bond at C5 is not necessary for inhibition. In contrast, the oxygenation of 20:4n-6 by 12-lipoxygenase was stimulated by 20:4n-3 but not by 20:5n-3, which might be related to the efficient oxygenation of 20:4n-3 by this enzyme compared with 20:5n-3.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Ácidos Araquidónicos/farmacología , Plaquetas/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Plaquetas/enzimología , Células Cultivadas , Endotelio Vascular/enzimología , Activación Enzimática , Epoprostenol/biosíntesis , Ácidos Grasos Insaturados/análisis , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , HumanosRESUMEN
The objective of this study was to evaluate the effects of ticlopidine on the generation of eicosanoids and nitric oxide in heart-transplant recipients. In a randomized double-blind study, we studied the urinary excretion of the stable metabolites of thromboxane, prostacyclin, and nitric oxide before and after ticlopidine (250 mg/day). Platelet aggregation was significantly reduced in ticlopidine-treated patients [from 40.2 +/- 24.2% of maximal aggregation to 14.7 +/- 8.2% in response to adenosine diphosphate (ADP); p < 0.001] but not in the placebo group, confirming the efficacy of the drug with that dosage in these specific patients. The 24-h urinary excretion of prostacyclin metabolites was not modified by ticlopidine (1,865 +/- 833 ng/24 h at day 14 and 1,664 +/- 425 ng/24 h at day 0), whereas the excretion of thromboxane B2 tended to increase in the ticlopidine group (from 3,854 +/- 1,163 ng/24 h at day 0 to 5,014 +/- 2,914 ng/24 h at day 14), although not significantly. The excretion of nitric oxide metabolites (although not different from that of healthy nonimmunosuppressed subjects) was significantly (p < 0.005) increased in the ticlopidine group (from 3,082 +/- 1,683 micromol/24 h at day 0 to 4,133 +/- 2,262 micromol/24 h at day 14), but not in controls. Thus ticlopidine does not reduce prostacyclin but increases the systemic generation of nitric oxide, both substances having major antiplatelet and vasodilator properties. Further studies are warranted to examine whether ticlopidine could reduce the incidence of thromboembolic complications in these patients and whether this possible novel property of ticlopidine is restricted to immunosuppressed heart-transplant recipients.
Asunto(s)
Óxido Nítrico/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Ticlopidina/farmacología , Análisis de Varianza , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Método Doble Ciego , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Epoprostenol/orina , Trasplante de Corazón , Humanos , Terapia de Inmunosupresión , Masculino , Óxido Nítrico/orina , Tromboxanos/orinaAsunto(s)
Grasas Insaturadas en la Dieta/uso terapéutico , Alimentos Fortificados , Aceites de Plantas/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Adolescente , Adulto , Grasas Insaturadas en la Dieta/efectos adversos , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Evaluación de Medicamentos , Humanos , Masculino , Persona de Mediana Edad , Aceites de Plantas/efectos adversos , Valores de Referencia , Ácido gammalinolénicoRESUMEN
This study was conducted to identify the nature of a glycogen-associated compound that had been shown to inhibit glucose-6 phosphatase in vitro. Glycogen was purified from the liver of fed rats by potassium hydroxyde digestion and ethanol precipitation. It inhibited glucose-6 phosphatase in microsomes isolated from rats deprived of food for 48 h. Two glycogen-associated fractions were purified by anion-exchange chromatography on DOWEX 1 (200-400 mesh). These fractions inhibited microsomal glucose-6-phosphatase activity in vitro (80 +/- 2 and 76 +/- 3% of control, respectively). After chromatography, glycogen was no longer inhibitory (101 +/- 3% of control). Because glycogen is associated with endoplasmic reticulum membranes in the liver, we tested the hypothesis that lipids could be involved in the inhibitory process. Lipids were extracted from glycogen by Folch's method and analyzed by thin-layer chromatography and gas chromatography. The glycogen-associated fractions did not contain complex lipids but contained unsaturated fatty acids, which had been shown previously to inhibit glucose-6-phosphatase in vitro. Because the concentration of unsaturated fatty acids in both fractions quantitatively accounted for the inhibition of glucose-6 phosphatase observed, and because noninhibitory chromatographed glycogen reconstituted with equivalent amounts of pure unsaturated fatty acids inhibited the enzyme as glycogen did, we conclude that unsaturated fatty acids likely constitute the glycogen-associated compound that inhibits glucose-6 phosphatase activity.
Asunto(s)
Ácidos Grasos Insaturados/farmacología , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Microsomas Hepáticos/efectos de los fármacos , Animales , Cromatografía por Intercambio Iónico , Ácidos Grasos Insaturados/clasificación , Ácidos Grasos Insaturados/metabolismo , Privación de Alimentos , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/metabolismo , Masculino , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
In this study, we investigate the application of the fuzzy clustering to the anatomical localization and quantitation of brain lesions in Positron Emission Tomography (PET) images. The method is based on the Fuzzy C-Means (FCM) algorithm. The algorithm segments the PET image data points into a given number of clusters. Each cluster is an homogeneous region of the brain (e.g. tumor). A feature vector is assigned to a cluster which has the highest membership degree. Having the label affected by the FCM algorithm to a cluster, one may easily compute the corresponding spatial localization, area and perimeter. Studies concerning the evolution of a tumor after different treatments in two patients are presented.
Asunto(s)
Neoplasias Encefálicas/diagnóstico por imagen , Lógica Difusa , Aumento de la Imagen/métodos , Tomografía Computarizada de Emisión , Algoritmos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Análisis por Conglomerados , Terapia Combinada , Desoxiglucosa/metabolismo , Radioisótopos de Flúor , HumanosRESUMEN
This study was conducted to determine whether inhibition of hepatic glucose-6 phosphatase is involved in the mechanism of suppression of hepatic glucose production during the postprandial period. We studied the time course of changes in the enzyme activity by refeeding food-deprived rats with nonpurified diet. The Vmax of the enzyme, assayed in homogenates from livers freeze-clamped in situ in anesthetized 48-h unfed rats (12.3 +/- 0.15 U/g wet liver, mean +/- SEM, n = 6) was progressively decreased upon refeeding: 11.1 +/- 0.5, 8.5 +/- 0.4 and 7.9 +/- 0.5 U/g, in rats refed for 90, 180 (P < 0.01) and 360 min (P < 0.01), respectively. The Km of the enzyme was not affected by refeeding. No inhibition of the enzyme was observed in microsomes purified from these homogenates, suggesting a metabolite-induced inhibition mechanism. To assess the role of insulin in the inhibition, we assayed the glucose-6 phosphatase activity in similarly processed liver homogenates from food-deprived rats perfused with insulin at physiological and supraphysiological concentrations, whereas plasma glucose was maintained at the basal level by adapted glucose perfusion (euglycemic clamps). No inhibition of glucose-6 phosphatase was found under these conditions, suggesting that insulin cannot by itself account for the inhibition observed in the refeeding experiments. These data constitute the first demonstration of the inhibition of glucose-6 phosphatase activity during the postprandial period.
Asunto(s)
Ingestión de Alimentos/fisiología , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Hígado/enzimología , Animales , Glucemia/análisis , Glucosa/biosíntesis , Técnica de Clampeo de la Glucosa , Insulina/sangre , Insulina/farmacología , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
It is now well established that monocytes adhere to endothelial cells activated by oxidized low-density lipoproteins (LDL). However, the adhesive receptors on endothelial cells involved in binding monocytes, following an insult by oxidized LDL, remains to be elucidated. In this study we have looked at the effect of native or oxidized LDL on the expression of P-selectin. Native LDL (N-LDL) was oxidized by incubation with either endothelial cells (EC-LDL) or copper (Cu-LDL), or in culture medium as a control (C-LDL). Expression of P-selectin was assayed with an anti-P-selectin (CD62) monoclonal antibody (LYP20). Results show that EC-LDL and Cu-LDL, but not N-LDL or C-LDL, induce the expression of P-selectin by human umbilical-vein endothelial cells (HUVECs). Induction of P-selectin by low concentrations (20 micrograms/ml) of LDL is directly related to the state of oxidation of the LDL particles. In addition, high concentrations (100 micrograms/ml) of N-LDL also activate HUVECs by inducing P-selectin expression. This expression was sustained for a period of over 1 h on LDL-activated endothelial cells, in contrast with thrombin- or histamine-activated endothelial cells, whose P-selectin levels fall within 15-20 min after induction. E-selectin, in contrast with P-selectin, could not be induced by endothelial cells treated with low or high concentrations of oxidized LDL. Results in this study show that P-selectin expressed by oxidized-LDL-treated endothelial cells are involved in mediating the adhesion of a monocytic cell line (U937) or monocytes in peripheral-blood mononuclear cells. An anti-P-selectin monoclonal antibody (LYP20) inhibited the binding of U937 cells and monocytes. These results strongly suggest that P-selectin is involved in the early stages of atherogenesis.
Asunto(s)
Endotelio Vascular/metabolismo , Lipoproteínas LDL/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Células Cultivadas , Selectina E , Endotelio Vascular/efectos de los fármacos , Humanos , Leucocitos Mononucleares/fisiología , Lipoproteínas LDL/administración & dosificación , Monocitos/fisiología , Oxidación-Reducción , Selectina-P , Glicoproteínas de Membrana Plaquetaria/fisiología , Venas UmbilicalesRESUMEN
In this study we have investigated the presence on endothelial cells of potential glycoprotein receptors, other than P-selectin, which are involved in the adhesion of monocytes at the early stages of activation. We report that the majority of cells binding to thrombin-activated endothelial cells from a peripheral blood mononuclear cell (PBMC) preparation are monocytes. The adhesion of PBMC to thrombin-activated, but not resting, endothelial cells was inhibited (66%) by a monoclonal antibody (mAb) directed against alpha v beta 3. Elutriated monocytes or a monocytic cell line (U937) were also inhibited by this antibody, its F(ab)'2 fragments and three other anti-(alpha v beta 3) mAbs. alpha v beta 3 isolated from endothelial-cell lysates significantly inhibited the adhesion of monocytes and U937 cells to endothelial cells. A peptide motif (RGDF) known to interact with alpha v beta 3 inhibited U937 cell adhesion to activated endothelial cells by 53%. Finally, an anti-(P-selectin) mAb (LYP20) or a platelet-activating factor (PAF)-receptor antagonist (WEB 2086) inhibited monocyte adhesion to activated endothelial cells. This study shows for the first time that alpha v beta 3 is implicated, in addition to P-selectin and PAF, in the adhesion of monocytes to activated endothelial cells.
Asunto(s)
Adhesión Celular , Endotelio Vascular/citología , Integrinas/fisiología , Monocitos/fisiología , Factor de Activación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores de Citoadhesina/fisiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Azepinas/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Integrinas/inmunología , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Selectina-P , Factor de Activación Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/inmunología , Receptores de Citoadhesina/inmunología , Receptores de Vitronectina , Trombina/farmacología , Triazoles/farmacología , Venas UmbilicalesRESUMEN
Glycoprotein CD36, also known as GPIIIb or GPIV, is a major platelet glycoprotein that bears the newly identified Naka alloantigen. The aim of this study was to clone platelet CD36 and investigate other forms of CD36-cDNA present in monocytes, endothelial and HEL cells. RNA from above mentioned cells were reverse transcribed (RT), using specific primers for CD36, and amplified by the polymerase chain reaction (PCR) technique. Sequencing the different amplified platelet derived cDNA fragments, spanning the whole coding and flanking regions, showed the near identity between platelet and CD36-placenta cDNA. Platelet CD36-cDNA cross-hybridized, in Southern blots, with RT-PCR amplified cDNA originating from monocytes, endothelial and HEL cells. However, monocytes showed a RT-PCR amplified cDNA fragment (561 bp) that was present in platelets and placenta but not on endothelial on HEL-cells. Northern blot analysis of platelet RNA hybridized with placenta CD36 indicated the presence of a major (1.95 kb) and a minor (0.95 kb) transcript. The 1.95 kb transcript was the only one observed on Northern blots of monocytes, endothelial and HEL cells. These results indicate that the structure of CD36 expressed in platelets is similar, with the exception of the 3' flanking region, to that of placenta. Differences in apparent molecular weight between CD36 and CD36-like glycoproteins may be due to post-translational modifications.
Asunto(s)
Antígenos CD/sangre , ADN Complementario/genética , Endotelio Vascular/inmunología , Código Genético , Monocitos/inmunología , Glicoproteínas de Membrana Plaquetaria/inmunología , Secuencia de Bases , Antígenos CD36 , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Eicosapentaenoic acid (EPA), a major polyunsaturated fatty acid of fish has been widely proposed as a potential nutrient for decreasing platelet-endothelial cell interactions and the subsequent atherogenesis and thrombogenesis. This is mainly based upon the decrease of arachidonic acid (AA) oxygenation into bioactive molecules like thromboxane A2. In addition, EPA may be oxygenated into its own active derivatives via cell dioxygenases. We report evidence for the requirement of specific peroxides, adequately provided by AA, to allow EPA to be oxygenated into its bioactive products like prostaglandin I3, a prostacyclin mimetic. On the other hand, we present some data that argue for a decreased basal AA dioxygenation (specific peroxidation) by small concentrations of EPA. The interactions between AA and EPA are then dual, EPA being able to counteract AA oxygenation whereas EPA requires AA to be efficiently oxygenated.