RESUMEN
Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.
Asunto(s)
Factor de Activación Plaquetaria/fisiología , Agregación Plaquetaria , Ácido Araquidónico , Ácidos Araquidónicos/sangre , Plaquetas/metabolismo , Calcio/sangre , Citosol/metabolismo , Fibrinógeno/sangre , Humanos , Cinética , Peso Molecular , Fosfatidilinositoles/sangre , Fosfoproteínas/sangre , Fosforilación , Temperatura , Tromboxano B2/sangreRESUMEN
The effect of 23 antiphospholipid antibody positive SLE sera, 4 antiphospholipid antibody negative SLE sera and 17 control sera on endothelial prostacyclin and platelet thromboxane A2 production was studied. Endothelial cells and platelets were stimulated with different agonists. Depending on the stimulus used, 4-19% of the SLE sera inhibited the prostacyclin release, whereas 4-28% enhanced prostacyclin production. Our data suggest that the pathophysiological mechanisms underlying decreased prostacyclin production are heterogeneous. Follow-up of two patients showed that prostacyclin inhibitory activity was variable in time. Platelet thromboxane production was normal or increased, but never decreased in the presence of the SLE sera. An imbalance in thromboxane A2/prostacyclin ratio was present in some patients, but did not correlate with a history of thrombosis. We conclude that, in general, interference of antiphospholipid antibodies with endothelial or platelet prostanoid synthesis does not explain the occurrence of thromboembolic manifestations in antiphospholipid antibody positive SLE patients.
Asunto(s)
Autoanticuerpos/inmunología , Factores de Coagulación Sanguínea/inmunología , Plaquetas/metabolismo , Ácidos Grasos/biosíntesis , Lupus Eritematoso Sistémico/inmunología , Ácidos Prostanoicos/biosíntesis , Trombosis/etiología , Adulto , Factores de Coagulación Sanguínea/análisis , Endotelio/inmunología , Endotelio/metabolismo , Epoprostenol/biosíntesis , Femenino , Humanos , Inhibidor de Coagulación del Lupus , Lupus Eritematoso Sistémico/sangre , Masculino , Persona de Mediana Edad , Trombosis/inmunología , Tromboxano A2/biosíntesisRESUMEN
The interrelationship of antibodies directed against cardiolipin (CL), double stranded DNA (dsDNA), endothelial cells (EC) and blood platelets was investigated. IgG fractions, reactive with these antigens, were isolated from the plasmas from 8 patients with systemic lupus erythematosus and tested for crossreactivity with anionic phospholipids (CL, phosphatidylserine, phosphatidylinositol), zwitterionic phospholipids (phosphatidylethanolamine, phosphatidylcholine), dsDNA, EC and platelets by enzyme-linked immunosorbent assays and for lupus anticoagulant (LAC) activity with a coagulation assay. Our results demonstrate the frequent occurrence of crossreactivity between antibodies to anionic phospholipids, EC and platelets. Crossreactivities between these antibodies and antibodies to dsDNA or zwitterionic phospholipids are exceptional. LAC activity was found in the anti-CL, anti-EC and anti-platelet fractions of only one patient. These findings support the hypothesis that subpopulations of antibodies directed against negatively charged phospholipids can bind to EC and blood platelets, which may have implications for the pathogenetic potential of antiphospholipid antibodies.
Asunto(s)
Autoanticuerpos/inmunología , Plaquetas/inmunología , Cardiolipinas/inmunología , ADN/inmunología , Endotelio Vascular/inmunología , Especificidad de Anticuerpos/inmunología , Factores de Coagulación Sanguínea/análisis , Factores de Coagulación Sanguínea/inmunología , Células Cultivadas , Reacciones Cruzadas , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/análisis , Inhibidor de Coagulación del LupusRESUMEN
The effect of 15 antiphospholipid antibody (APA) positive SLE sera, 13 APA negative SLE sera, 10 APA negative sera from patients with other connective tissue diseases (CTD) and 15 control sera on the expression of endothelial procoagulant activity (PCA) was studied. Endothelial cells (EC) were stimulated with tumor necrosis factor (TNF) and 20% serum for 4 hr and the surface PCA expression was measured. Without TNF, none of the sera stimulated PCA expression. With suboptimal TNF stimulation, 14/15 APA positive SLE sera, 7/13 APA negative SLE sera, 2/10 CTD sera and 2/15 control sera enhanced PCA expression. This stimulating effect resided in the IgG fraction and was associated with the presence of APA, but not with a history of thrombosis. Purified APA had no PCA stimulating activity. PCA expression was inhibited by cycloheximide and heat treatment (30 min, 56 degrees C) of serum. In conclusion, 21/28 (75%) SLE sera increase the TNF-induced endothelial PCA expression. Although this effect predominantly occurs with APA positive serum, a causative role of APA was not demonstrated.
Asunto(s)
Factores de Coagulación Sanguínea/biosíntesis , Endotelio Vascular/metabolismo , Lupus Eritematoso Sistémico/sangre , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Autoanticuerpos/aislamiento & purificación , Autoanticuerpos/fisiología , Factores de Coagulación Sanguínea/inmunología , Factores de Coagulación Sanguínea/fisiología , Pruebas de Coagulación Sanguínea , Cardiolipinas/inmunología , Células Cultivadas , Compuestos Cromogénicos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Inmunoglobulina G/aislamiento & purificación , Inhibidor de Coagulación del Lupus , Masculino , Persona de Mediana EdadAsunto(s)
Anticuerpos/fisiología , Fosfolípidos/inmunología , Coagulación Sanguínea , Factores de Coagulación Sanguínea/fisiología , Plaquetas/fisiología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Epoprostenol/fisiología , Fibrinólisis , Humanos , Proteína C/fisiología , Receptores de Superficie Celular/fisiología , Receptores de Trombina , Trombina/fisiologíaRESUMEN
Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose-dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N- and C-terminal Ca(2+)-binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell-associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high-affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]-thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum-free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.
Asunto(s)
Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Osteonectina/farmacología , Animales , Aorta , Bovinos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Fragmentos de Péptidos/farmacologíaRESUMEN
Strains of bovine aortic endothelial cells, grown on plastic under conventional culture conditions and in the absence of growth factor supplementation, exhibited a sprouting phenotype and a predisposition toward the formation of cords and tubular structures. We examined endothelial cells at different stages of tube formation. Analysis of metabolically labeled proteins showed that the synthesis of type I collagen was initiated in sprouting cells and during the formation of tubular structures. SPARC (secreted protein, acidic and rich in cysteine) a Ca2(+)-binding protein associated with cellular shape change and morphogenetic processes (Sage H, Vernon RB, Funk SE, Everitt EA, Angello J: J Cell Biol 109:341, 1989), was upregulated during spontaneous tube formation. Levels of messenger RNA for type I collagen and SPARC corroborated the stage-specific increases observed for these proteins. Differential levels of transcription were apparent in multilayered cells directly involved in tube formation, in comparison with cells comprising either the tubes or the confluent monolayers at a distance from the tubes. Analysis of DNA synthesis indicated that multilayered sprouting cells in the proximity of the endothelial tubes were actively proliferating, whereas cells that had been incorporated into tubes showed low levels of DNA synthesis. Immunolabeling studies revealed a dense accumulation of SPARC and type I collagen in the cytoplasm of cells that were situated near the growing tubes. Two other secreted proteins, type III collagen and thrombospondin, were expressed constitutively by subconfluent cultures and were increased in those cells contributing to tube formation. We propose that type I collagen and SPARC are specifically related to the angiogenesis-like phenomenon displayed by bovine aortic endothelial cells in vitro. Type I collagen might facilitate the active migration of endothelial cells, or the stabilization of the resulting tubes, with SPARC directing the re-organization and dynamic assembly of the tubular network.
Asunto(s)
Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Neovascularización Patológica , Osteonectina/genética , Animales , Bovinos , División Celular , Células Cultivadas , Colágeno/análisis , Colágeno/genética , Endotelio Vascular/citología , Matriz Extracelular/ultraestructura , Expresión Génica , Técnicas para Inmunoenzimas , Cinética , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genéticaRESUMEN
A combination of CN- and 2-deoxy-D-glucose decreases the binding of fibrinogen to platelets stimulated with PAF-acether (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine). Decreased binding is found after pretreatment with metabolic inhibitors, thereby lowering the energy content before stimulation as well as at various stages after stimulation of undisturbed cells. Binding and ATP hydrolysis occur in parallel, suggesting tight coupling between both phenomena. Energy appears to be predominantly required for exposure and maintenance of accessible binding sites, whereas the interaction between fibrinogen and the exposed sites does not depend on metabolic energy.
Asunto(s)
Plaquetas/metabolismo , Fibrinógeno/metabolismo , Factor de Activación Plaquetaria/análogos & derivados , Agregación Plaquetaria/efectos de los fármacos , Plaquetas/efectos de los fármacos , Cianuros/farmacología , Desoxiglucosa/farmacología , Metabolismo Energético/efectos de los fármacos , Humanos , Factor de Activación Plaquetaria/farmacología , Unión Proteica/efectos de los fármacos , Estimulación QuímicaRESUMEN
SPARC, a Ca(2+)-binding glycoprotein that is expressed during tissue morphogenesis and functions as an inhibitor of cell spreading in vitro, was found to induce the secretion of an Mr = 45,000 protein in bovine aortic endothelial (BAE) cells. This protein was identified as type 1 plasminogen activator inhibitor (PAI-1) on Western blots with anti-PAI-1 antiserum. SPARC stimulated the secretion of PAI-1 protein into the medium of subconfluent BAE cells, but not confluent BAE cells, in a dose- and time-dependent manner. Secretion of PAI-1 into the culture medium was progressive and exhibited an increase of 3- to 7-fold over control values within 24 h after the addition of SPARC. Levels of PAI-1 mRNA were elevated 2-fold within 4 to 24 h after the addition of SPARC and did not increase with higher concentrations of SPARC. Since the induction of PAI-1 mRNA by SPARC was not blocked by cycloheximide, de novo protein synthesis was apparently not required for this stimulation. Control experiments showed that the induction of PAI-1 was not due to contamination of the SPARC preparations with endotoxin. These data demonstrate that SPARC induces the biosynthesis of PAI-1 in BAE cells and suggest a role for SPARC in the regulation of fibrinolysis and in the control of proteolytic events in remodeling tissues.
Asunto(s)
Endotelio Vascular/fisiología , Osteonectina/farmacología , Inactivadores Plasminogénicos/metabolismo , Animales , Aorta , Northern Blotting , Bovinos , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Cinética , Lipopolisacáridos/farmacología , Hibridación de Ácido Nucleico , Osteonectina/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
In 111 lupus patients we compared the potential of the IgG and IgM anticardiolipin antibody (ACA) enzyme linked immunosorbent assay (ELISA) and four different lupus anticoagulant (LAC) assays (partial thromboplastin time (PTT) of a 1:1 mixture of patient and control plasma with phospholipids from animal (PTT-st) or human brain (PTT-HB); PTT with dilutions of human brain phospholipids (PL dilution); and kaolin clotting time of mixtures of patient and control plasma (KCT] to identify patients with thrombosis (26/111), fetal loss (19/46), and/or thrombocytopenia (11/106). The highest specificity for thrombosis (87%) was found with PTT-HB and PL dilution (sensitivity 65%, detection rate 61%); for fetal loss (93%) with PL dilution (sensitivity 47%; detection rate 82%), and for thrombocytopenia (83%) with KCT (sensitivity 82%; detection rate 36%). Compared with LAC assays, the sensitivity of ACA-ELISA was high (greater than or equal to 77%), but specificity (less than or equal to 51%) and detection rate (less than or equal to 52%) were low. So, a panel of three LAC assays (PTT-HB, PL dilution, and KCT) can identify lupus patients apparently at risk for thrombosis, fetal loss, and/or thrombocytopenia, whereas the ACA-ELISA is insufficiently specific.
Asunto(s)
Autoanticuerpos/análisis , Factores de Coagulación Sanguínea/inmunología , Cardiolipinas/inmunología , Lupus Eritematoso Sistémico/complicaciones , Trombosis/diagnóstico , Adolescente , Adulto , Anciano , Factores de Coagulación Sanguínea/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Muerte Fetal/diagnóstico , Muerte Fetal/etiología , Humanos , Inhibidor de Coagulación del Lupus , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Fosfolípidos , Embarazo , Trombocitopenia/diagnóstico , Trombocitopenia/etiología , Trombosis/etiologíaRESUMEN
In 53 patients with systemic lupus erythematosus sequential blood samples obtained during a four year period (range 6-47 months) were screened for anticardiolipin antibodies (ACAs). Disease activity and treatment with prednisone were also assessed and related to ACA concentrations. During follow up only 21 patients for ACA IgG (40%) and 25 for ACA IgM (47%) remained in the ACA category (negative, low positive, high positive) found at the first sample taken at entrance. Marked increases from negative to high positive concentrations were sometimes seen and were not accompanied by typical events such as thrombosis or thrombocytopenia (the ACA syndrome). Shifts in ACA concentrations could not always be explained by changes in prednisone dose. Also, in patients with low dose prednisone treatment or none at all (n = 22) 10 patients (45%) changed ACA IgG category and 12 patients (55%) fluctuated in ACA IgM categories during follow up. As a consequence of the variability in ACA titres relations of ACAs with the ACA syndrome depended on the blood sample studied. In the second sample, randomly taken half way through follow up, no significant relations with the ACA syndrome could be found. Anticardiolipin antibody IgG was significantly associated with disease activity in 11/47 patients (23%) and in the group as a whole. During remission ACA IgG was significantly associated with the ACA syndrome, whereas during moderate/severe disease activity in the same patients that correlation was not significant. Anticardiolipin antibody IgM was much less influenced by disease activity, and in only 4/47 patients (9%) could a significant relation with disease activity be shown. Associations of ACA IgM with the ACA syndrome were significant during both lupus flares and remission.
Asunto(s)
Autoanticuerpos/metabolismo , Cardiolipinas/inmunología , Lupus Eritematoso Sistémico/inmunología , Adolescente , Adulto , Anciano , Femenino , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Prednisolona/uso terapéutico , Estudios ProspectivosRESUMEN
Lupus anticoagulant, concentrations of anticardiolipin antibodies, antithrombin III, plasminogen, (free) protein S, protein C, prothrombin, platelet counts, and bleeding times were determined in 74 lupus patients (58 with systemic lupus erythematosus; 16 with lupus-like disease) to establish the presence of risk factors for thrombosis in these patients. Of the variables evaluated, lupus anticoagulant had the strongest association with a history of thrombosis. Both positive anticardiolipin antibody concentrations and the presence of (mild) thrombocytopenia were significantly associated with a history of thrombosis and the presence of lupus anticoagulant. Reduced concentrations of antithrombin III, plasminogen, (free) protein S, and protein C were found in some patients but were not associated with either thrombosis or lupus anticoagulant. Mean concentrations of total protein S were significantly lower in patients with thrombosis than in those without and in patients with lupus anticoagulant than in those without. The antigenic concentration of prothrombin was reduced in 3/74 (4%) lupus patients. These three patients had lupus anticoagulant but no history of thrombosis, which suggests that a low prothrombin concentration protects patients with lupus anticoagulant from the development of thrombosis. A prolonged bleeding time was associated with the presence of lupus anticoagulant but not with a history of thrombosis. Analysis by stepwise logistic regression did not disclose additional risk factors for thrombosis in lupus patients with lupus anticoagulant. Increased antithrombin III concentrations and decreased free protein S concentrations are often found in lupus patients, unrelated to lupus anticoagulant or thrombosis.
Asunto(s)
Autoanticuerpos/análisis , Factores de Coagulación Sanguínea/inmunología , Lupus Eritematoso Sistémico/complicaciones , Tromboflebitis/etiología , Adolescente , Adulto , Anciano , Antitrombina III/análisis , Factores de Coagulación Sanguínea/análisis , Cardiolipinas/inmunología , Femenino , Humanos , Inhibidor de Coagulación del Lupus , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Persona de Mediana Edad , Plasminógeno/análisis , Recuento de Plaquetas , Pruebas de Función Plaquetaria , Proteína C/análisis , Protrombina/análisis , Factores de Riesgo , Tromboflebitis/inmunología , Tromboflebitis/metabolismoRESUMEN
We found that levels of antiphospholipid antibodies (aPLA), measured with an ELISA increase if serum or plasma samples are heated. The phenomenon is dependent on duration and degree of heating, optimum levels being reached at 3 h at 56 degrees C. Negative samples become positive after heating. The heating effect is more pronounced for IgG-aPLA than for IgM-aPLA and is not observed for adsDNA, atetanus or lymphocytotoxic antibodies. The presence of serum/plasma components in addition to IgG is essential for the phenomenon to occur. Ultracentrifugation and mixing experiments with isolated IgG did not enable us to explain our observations. Nevertheless, knowledge of this phenomenon is of practical importance.