RESUMEN
Increased tendency of cancer patients to develop venous thromboembolism (VTE) is associated with high rates of mortality. Elevation of procoagulant proteins and down regulation of naturally occurring coagulation inhibitors appears to form the basis of high risk of VTE in malignancy. A reduced level of anticoagulant protein like antithrombin (AT) will influence both coagulation and angiogenesis, as its cleaved and latent conformations show potent antiangiogenic activity. We show a concentration dependent perturbation in the secondary and tertiary structures of AT conformers exposed to hypochlorous acid (HOCl). Modulated under a very narrow concentration range of HOCl, native AT undergoes oligomerization, aggregation and fragmentation based on spectroscopic, SDS and native-PAGE studies. Factor Xa inhibition assay demonstrated a progressive decrease in inhibition activity of AT on modification by HOCl. Bis-ANS result showed that hydrophobic patches were more exposed in the case of HOCl-modified AT when assessed fluorometrically. Dosage of HOCl-modified AT in experimental animals induced high titer antibodies showing more specificity towards modified forms in comparison to unmodified forms. Auto-antibodies isolated from cancer patients also showed enhanced binding with HOCl-modified AT in comparison to native counterpart. Compared to normal AT, structurally and functionally altered conformation of HOCl-modified AT showed increased immunogenic sensitivity. HOCl modified AT can contribute to prothrombotic and angiogenic environment during cancer progression/development.
Asunto(s)
Antitrombinas/inmunología , Epítopos/inmunología , Ácido Hipocloroso/química , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/aislamiento & purificación , Antitrombinas/química , Autoanticuerpos/inmunología , Autoanticuerpos/aislamiento & purificación , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Persona de Mediana Edad , Conejos , Adulto JovenRESUMEN
Severe infectious diseases are often characterized by an overwhelming and unbalanced systemic immune response to microbial infections. Human antithrombin (hAT) is a crucial coagulation inhibitor with anti-inflammatory activities. Here we identify three hAT-binding proteins (CD13, CD300f and LRP-1) on human monocytes that are involved in blocking the activity of nuclear factor-κB. We found that the modulating effect is primarily restricted to the less abundant ß-isoform (hßAT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of hßAT and analysis of their blood, which was stimulated ex vivo with lipopolysaccharide, showed a decreased inflammatory response. Similar findings were recorded when heterozygotic mice expressing hAT or hßAT were challenged with lipopolysaccharide or infected with Escherichia coli bacteria. Our results finally demonstrate that in a lethal E. coli infection model, survival rates increased when mice were treated with hßAT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.
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Antitrombinas/química , Antitrombinas/inmunología , Infecciones Bacterianas/inmunología , Animales , Antitrombinas/sangre , Quimiocinas , Citocinas , Modelos Animales de Enfermedad , Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Humanos , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Transgénicos , Monocitos , Mutación , FN-kappa B , Isoformas de Proteínas , Células RAW 264.7RESUMEN
Essentials MicroRNAs (miRNAs) regulate the molecular networks controlling biological functions such as hemostasis. We utilized novel methods to analyze miRNA-mediated regulation of the hemostatic system. 52 specific miRNA interactions with 11 key hemostatic associated genes were identified. Functionality and drugability of miRNA-19b-3p against antithrombin were demonstrated in vivo. SUMMARY: Background microRNAs (miRNAs) confer robustness to complex molecular networks regulating biological functions. However, despite the involvement of miRNAs in almost all biological processes, and the importance of the hemostatic system for a multitude of actions in and beyond blood coagulation, the role of miRNAs in hemostasis is poorly defined. Objectives Here we comprehensively illuminate miRNA-mediated regulation of the hemostatic system in an unbiased manner. Methods In contrast to widely applied association studies, we used an integrative screening approach that combines functional aspects of miRNA silencing with biophysical miRNA interaction based on RNA pull-downs (miTRAP) coupled to next-generation sequencing. Results Examination of a panel of 27 hemostasis-associated gene 3'UTRs revealed the majority to possess substantial Dicer-dependent silencing capability, suggesting functional miRNA targeting. miTRAP revealed 150 specific miRNA interactions with 14 3'UTRs, of which 52, involving 40 miRNAs, were functionally confirmed. This includes cooperative miRNA regulation of key hemostatic genes comprising procoagulant (F7, F8, F11, FGA, FGG and KLKB1) and anticoagulant (SERPINA10, PROZ, SERPIND1 and SERPINC1) as well as fibrinolytic (PLG) components. Bioinformatic analysis of miRNA functionality reveals established and potential novel links between the hemostatic system and other pathologies, such as cancer, bone metabolism and renal function. Conclusions Our findings provide, along with an in-vivo proof of concept, deep insights into the network of miRNAs regulating the hemostatic system and present a foundation for biomarker discovery and novel targeted therapeutics for correction of de-regulated hemostasis and associated processes in the future. A repository of the miRNA targetome covering 14 hemostatic components is provided.
Asunto(s)
Hemostasis , MicroARNs/análisis , Regiones no Traducidas 3' , Animales , Antitrombinas/inmunología , Biomarcadores/metabolismo , Línea Celular Tumoral , Biología Computacional , Silenciador del Gen , Hemostáticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos C57BL , Plásmidos/metabolismo , Trombosis/genéticaRESUMEN
UNLABELLED: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. BACKGROUND: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. OBJECTIVES: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. METHODS: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. RESULTS: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. CONCLUSIONS: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.
Asunto(s)
Antitrombinas/inmunología , Hemorragia/inmunología , Inmunoglobulina A/inmunología , Trombina/química , Anticoagulantes/química , Antitrombinas/química , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Epítopos/química , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/química , Hematoma Subdural/inmunología , Hemostasis/inmunología , Humanos , Inmunoglobulina A/química , Fragmentos Fab de Inmunoglobulinas/química , Persona de Mediana Edad , Fenotipo , Trombina/inmunologíaRESUMEN
BACKGROUND: The anticoagulant effect of dabigatran can be approximated by its prolongation of routine coagulation assays. Consequently, dabigatran also interferes with thrombophilia screening or with diagnosing hemostasis disorders that have developed after the initiation of anticoagulant treatment, such as vitamin K deficiency or acquired hemophilia A. OBJECTIVES: This study was carried out to determine whether idarucizumab, a humanized antibody fragment that binds dabigatran, could fully neutralize dabigatran in routine diagnostic coagulation assays conducted in vitro, thereby preventing false-positive or false-negative diagnostic readouts. METHODS: Preliminary experiments identified coagulation assays that were sensitive to dabigatran, and identified a concentration of idarucizumab that neutralized the effects of dabigatran. These assays were then carried out with patient and control plasma samples spiked with dabigatran, with or without a molar excess of idarucizumab. RESULTS: Dabigatran altered the prothrombin time, activated partial thromboplastin time and thrombin time, and the measurement of intrinsic and extrinsic factor levels. Screening and confirmation tests used for lupus anticoagulant detection were prolonged by dabigatran, falsely suggesting the presence of lupus anticoagulant. Conversely, the addition of dabigatran falsely corrected an abnormal activated protein C resistance ratio. Addition of idarucizumab completely normalized these measurements, and allowed the correct identification of normal and abnormal samples with these assays. CONCLUSIONS: In vitro addition of idarucizumab to plasma samples containing dabigatran fully neutralizes the drug, and facilitates the use of routine coagulation assays to allow the diagnosis of hemostasis disorders that may be concurrently present in patients taking dabigatran.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Neutralizantes/farmacología , Antitrombinas/sangre , Trastornos de la Coagulación Sanguínea/sangre , Pruebas de Coagulación Sanguínea , Dabigatrán/sangre , Resistencia a la Proteína C Activada/sangre , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Antitrombinas/inmunología , Antitrombinas/farmacología , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Dabigatrán/antagonistas & inhibidores , Dabigatrán/inmunología , Dabigatrán/farmacología , Relación Dosis-Respuesta Inmunológica , Reacciones Falso Negativas , Reacciones Falso Positivas , Hemofilia A/sangre , Humanos , Inhibidor de Coagulación del Lupus/sangreRESUMEN
INTRODUCTION: ß-antithrombin, the minor antithrombin glycoform in plasma, is probably the major thrombin inhibitor in vivo because of its high heparin affinity. The levels and variability of this glycoform in general population and its relevance in thromboembolic diseases is unknown since there is no specific method to measure this glycoform in clinical samples. METHODS: Plasma and recombinant α- and ß-antithrombins were purified by heparin affinity chromatography. An anti-FXa chromogenic method in presence of pentassacharide was used with two NaCl concentrations (15mM and 1.1M). This method was applied to plasma samples from 97 healthy subjects and 117 consecutive patients with ischemic cerebrovascular disease during the acute event and one year later. SERPINC1 sequencing was done in cases with antithrombin deficiency. RESULTS: High salt concentrations specifically restricted the pentassacharide-induced activation of antithrombin to the ß glycoform. ß-antithrombin displayed a normal distribution in the general population (89.5%-103.5%), with no significant variations according to age or sex. In patients, whole antithrombin values remained within the normal range. Only five cases had antithrombin deficiency during the thrombotic event, one carrying the L99F mutation in SERPINC1. Interestingly, both ß-antithrombin and the ß/whole antithrombin ratio were significantly higher in patients during the acute event but normalized after one year. CONCLUSIONS: We have developed a rapid, simple, sensitive and specific method to quantify ß-antithrombin activity using 1µL of plasma. ß-antithrombin significantly increases in patients with ischemic cerebrovascular disease during the acute event, probably by its release from the vasculature.
Asunto(s)
Antitrombinas/sangre , Inmunoensayo/métodos , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antitrombinas/clasificación , Antitrombinas/inmunología , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Accidente Cerebrovascular/inmunología , Regulación hacia ArribaRESUMEN
INTRODUCTION: Given its central role in mediating heparin-induced anti-coagulation, antithrombin (AT) gene mutations may result in heparin resistance. This study investigates the relationship between familial AT gene mutations and tolerance to heparin. METHODS: The medical history of a male patient with heparin resistance who received heart surgery and six of his family members was reviewed. Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (Fib), D-dimer (D=D), and platelet count were determined to assess coagulation function. AT activity and the AT gene were also analyzed. For the newly identified gene mutations, polymorphisms were excluded in 120 healthy Kazak controls. RESULTS: Two mutations were identified in exon 7 of the AT gene, SERPINC1: g.1267G>A (p.A391T) found in five participants, including the index patient, and g.1334G>A, a silent mutation, in two family members. The g.1267G>A mutation may alter focal AT protein conformation. Neither of these mutations was observed in the healthy Kazak controls. Although all coagulation parameters and AT activity were within the normal ranges for the index patient and his family members, the platelet levels were significantly lower than that observed for the healthy Kazak controls (p=0.001). There was no significant difference in AT antigen levels between the groups; however, participants with the g.1267G>A mutation had a 44.25% reduction in heparin binding compared to the control group (p<0.001). CONCLUSION: We identified a novel hereditary mutation, g.1267G>A (p.A391T), in the AT gene, which reduces its heparin binding capacity and might be associated with resistance to heparin.
Asunto(s)
Antitrombina III/genética , Antitrombina III/inmunología , Antitrombinas/inmunología , Predisposición Genética a la Enfermedad/genética , Heparina/inmunología , Adulto , Anciano , Anticoagulantes/inmunología , Secuencia de Bases , Femenino , Humanos , Kazajstán , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Unión Proteica , Tiempo de Protrombina , Adulto JovenRESUMEN
The lectin pathway of complement is an important effector arm of innate immunity. It forms a first line of defense against invading pathogens and dangerously altered self structures. Pattern recognition molecules (mannose-binding lectin (MBL), ficolins) bind to the dangerous particles, which is followed by activation of MBL-associated serine proteases, MASP-1 and MASP-2, resulting in the initiation of the complement cascade. The activation of the lectin pathway is strictly controlled by natural inhibitors, since uncontrolled activation can lead to serious self-tissue damage. Recently we have shown that inhibition of either MASP-1 or MASP-2 by in vitro evolved specific inhibitors completely blocks the lectin pathway in human serum. In this study, we examined the inhibitory action of C1-inhibitor (C1-inh), antithrombin (AT) and α(2)-macroglobulin (α(2)M) on MASP-1 and MASP-2, and studied the inhibition of the lectin pathway in normal human serum in the presence and absence of heparin using C3 and C4 deposition assays. We measured the association rate constants for the serpin/protease reactions. We found that in the presence of heparin both C1-inh and AT are equally efficient inhibitors of the lectin pathway. Although α(2)M formed complex with MASP-1 in fluid phase, it could not abolish lectin pathway activation on activator surfaces.
Asunto(s)
Antitrombinas/inmunología , Activación de Complemento/inmunología , Proteína Inhibidora del Complemento C1/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Lectinas/inmunología , alfa-Macroglobulinas/inmunología , Complemento C3/metabolismo , Complemento C4/inmunología , Heparina/inmunología , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunologíaRESUMEN
Recombinant hirudins (desirudin, lepirudin) are direct thrombin inhibitors administered as anticoagulants for heparin-induced thrombocytopenia (HIT) and venous thromboembolism (VTE) prophylaxis. Although these small polypeptides are widely used, concern exists over reports of antigenicity. In the largest study of r-hirudin immunogenicity to-date, we evaluated the prevalence, quantity and specificity of IgG immune responses to desirudin (15 mg SC q12h for as long as clinically required) in 245 surgical and medically-ill subjects enrolled in DESIRABLE, a multicenter, open-label, clinical trial of hospitalized patients requiring VTE prophylaxis. Sera obtained before and 30 days after desirudin administration were analyzed for IgG anti-desirudin by immunoenzymetric assay using immobilized desirudin to bind desirudin-reactive antibody and peroxidase conjugated monoclonal-anti-human IgG Fc to detect bound IgG antibody. Of 245 study subjects, 19 (7.7%) were antibody "responders" (>2-fold increase in IgG antibody levels with >50% inhibition by desirudin 30 days post-treatment). There were no differences between responders and non-responders in incidence of clinical outcomes or bleeding-related adverse events. Forty-six patients had detectable desirudin-reactive IgG antibody prior to treatment, with no significant increase in antibody levels after exposure and no increase in clinical events. The origin of pre-existing hirudin-reactive IgG antibody requires further investigation involving suspected anti-thrombin-thrombin interactions. These results indicate a low potential for immunogenicity, with <8% of patients developing IgG antibodies after desirudin administration for VTE prophylaxis. In contrast to reports on lepirudin, production of anti-hirudin antibodies to desirudin has no apparent effect on clinical events.
Asunto(s)
Anticuerpos/inmunología , Hirudinas/inmunología , Técnicas para Inmunoenzimas/métodos , Trombina/inmunología , Anciano , Anticuerpos/sangre , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antitrombinas/administración & dosificación , Antitrombinas/inmunología , Femenino , Heparina/efectos adversos , Terapia con Hirudina , Hirudinas/administración & dosificación , Hirudinas/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Trombocitopenia/inducido químicamente , Trombocitopenia/prevención & control , Factores de Tiempo , Tromboembolia Venosa/inducido químicamente , Tromboembolia Venosa/prevención & controlRESUMEN
BACKGROUND: Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor. We evaluated the ability of Gla-domainless factor Xa, a truncated form of factor Xa devoid of procoagulant properties, to bind to tissue factor pathway inhibitor and to alleviate the physiological inhibition of the extrinsic tenase. DESIGN AND METHODS: Using a thrombin generation assay triggered by a low concentration of tissue factor, we evaluated the ability of Gla-domainless factor Xa to restore blood coagulation in plasma from hemophilia A and B patients without and with inhibitors. We then compared its efficacy to generate thrombin to depletion of antithrombin or tissue factor pathway inhibitor by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and Gla-domainless factor Xa by antithrombin and tissue factor pathway inhibitor. RESULTS: Gla-domainless factor Xa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. Gla-domainless factor Xa had a lower affinity than factor Xa for tissue factor pathway inhibitor whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of Gla-domainless factor Xa on thrombin generation was sustained for at least 1 hour. CONCLUSIONS: As Gla-domainless factor Xa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without an inhibitor.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factor Xa/metabolismo , Proteínas de Neoplasias/metabolismo , Anticuerpos/inmunología , Anticuerpos/farmacología , Antitrombinas/inmunología , Antitrombinas/metabolismo , Factor Xa/química , Semivida , Hemofilia A/sangre , Hemofilia A/metabolismo , Humanos , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Complejos Multiproteicos/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Trombina/metabolismoRESUMEN
BACKGROUND: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. METHODS: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. RESULTS: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. CONCLUSIONS: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.
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Anticuerpos Antifosfolípidos/análisis , Afinidad de Anticuerpos , Antitrombinas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Fosfatidilserinas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antifosfolípidos/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Protrombina/inmunología , Adulto JovenRESUMEN
The effect of protamine sulfate on factor Xa (FXa) and the factor Xa-antithrombin complex was studied via SDS-PAGE and Western blot analysis. Human factor Xa [(FXaα) â¼52 kDa, FXaß â¼47 kDa] and human antithrombin (AT â¼55 kDa) form a primary (1°) complex at 109 and 104 kDa, a secondary (2°) complex at 99 and 95 kDa, and a tertiary (3°) complex at 66 and 62 kDa, as detected with polyclonal anti-FXa and anti-AT antibodies. Addition of protamine sulfate stimulates a transformation from free FXaα to FXaß and degradation thence to inactive FXaγ (â¼30 kDa), as well as transformation of FXaα-AT complexes to FXaß-AT complexes. Additionally, protamine sulfate promotes digestive degradation from 1° to 3° complexes and FXaγ by the active enzyme. It further promotes reduction in total complex formation as a function of stimulation of hydrolysis of FXa moieties from the complex, thereby generating ATM. Protamine sulfate effects are proportional to the protamine sulfate concentration.
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Antitrombinas/metabolismo , Factor Xa/metabolismo , Protaminas/farmacología , Anticuerpos/inmunología , Anticuerpos/metabolismo , Antitrombinas/inmunología , Western Blotting , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Factor Xa/inmunología , Humanos , Hidrólisis/efectos de los fármacos , Peso MolecularRESUMEN
Citrullination is a post-translational modification that plays essential roles in both physiological processes and disease. Recent studies have found increased levels of citrullinated antithrombin in patients with rheumatoid arthritis and in different malignant tumours. Antithrombin, the main haemostatic serpin, loses its anticoagulant function via citrullination, which might contribute to the pathogenesis or thrombotic side effects of these disorders. We have developed a specific monoclonal antibody against citrullinated antithrombin. We determined the levels of citrullinated antithrombin and anti-FXa activity in plasma from 66 donors, 17 patients with rheumatoid arthritis and 77 patients with colorectal adenocarcinoma (42 suffering from venous thrombosis). Healthy subjects had negligible amounts of citrullinated antithrombin in plasma (7.9 ± 22.1 ng/ml), while it significantly increased in patients with rheumatoid arthritis or adenocarcinoma (159.7 ± 237.6 ng/ml and 36.8 ± 66.1 ng/ml), levels that, however, did not modify the plasma anticoagulant activity. Moreover, we did not find association between citrullinated antithrombin and the thrombotic risk in patients with adenocarcinoma. In conclusion, we have developed an antibody specific for citrullinated antithrombin that allows its quantification in biological samples, offering a new tool for the analysis of citrullination in different diseases. We confirm increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and adenocarcinoma. This modification, probably local, could have pathological consequences in both disorders, but only affects a minor fraction of plasma antithrombin, resulting in no significant reduction of global anticoagulant activity. This result explains the absence of association of this marker with an increased risk of thrombosis in patients with colorectal adenocarcinoma.
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Adenocarcinoma/sangre , Anticuerpos Monoclonales/inmunología , Antitrombinas/sangre , Artritis Reumatoide/sangre , Citrulina/sangre , Neoplasias Colorrectales/sangre , Ensayo de Inmunoadsorción Enzimática , Adenocarcinoma/complicaciones , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Antitrombinas/inmunología , Biomarcadores/sangre , Coagulación Sanguínea , Citrulina/inmunología , Neoplasias Colorrectales/complicaciones , Inhibidores del Factor Xa , Humanos , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Trombosis de la Vena/sangre , Trombosis de la Vena/etiologíaRESUMEN
Ischaemia/reperfusion (I/R) injury is central to a number of pathologies including myocardial infarction and stroke. Several cellular processes are involved in the progress of I/R injury, involving complex interactions between coagulation and inflammatory or apoptotic processes. Besides for their anti-coagulant function, anticoagulant proteins such as activated protein C (APC), active site inhibited factor VIIa (ASIS), tissue factor pathway inhibitor (TFPI), and antithrombin (AT) are also known for their anti-inflammatory or cell protective effects. This review gives an overview of the application of these anti-coagulants in several animal models of I/R injury in critical organs and describes the effects of these proteins on cellular processes including inflammation and apoptosis. The future testing of mutant forms of some of these inhibitors including APC in a clinical setting should be actively explored.
Asunto(s)
Antiinflamatorios/metabolismo , Anticoagulantes/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/inmunología , Animales , Antiinflamatorios/inmunología , Antiinflamatorios/uso terapéutico , Anticoagulantes/inmunología , Anticoagulantes/uso terapéutico , Antitrombinas/inmunología , Antitrombinas/metabolismo , Apoptosis , Modelos Animales de Enfermedad , Factor VIIa/inmunología , Factor VIIa/metabolismo , Humanos , Inflamación , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Proteína C/inmunología , Proteína C/metabolismoRESUMEN
BACKGROUND: Complement 5a factor (C5a) elicits a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. C5a is also linked to the coagulant activity in autoimmune diseases. Therefore, C5a most likely plays a crucial role in the instant blood-mediated inflammatory reaction. METHODS: Intraportal transplantation of 2.5 islet equivalents/g of syngeneic rat islet grafts was performed in two groups of streptozotocin-induced diabetic rats: controls and C5a inhibitory peptide (C5aIP)-treated group. RESULTS: The thrombin-antithrombin complex was significantly suppressed in the C5aIP group (P=0.003), and both the curative rate and the glucose tolerance were significantly improved in the C5aIP group (P<0.05 and P<0.005, respectively). Expression of tissue factor on granulocytes in recipient livers was up-regulated 1 h after islet infusion (P<0.0001), which was significantly suppressed by C5aIP (P<0.005). However, C5aIP was unable to regulate tissue factor expression on isolated islets. Furthermore, no differences were detected between the groups, regarding infiltration of CD11b-positive cells and deposition of C5b-9 on the islet grafts. CONCLUSIONS: These data suggest that C5aIP attenuates cross-talk between the complement and coagulation cascades through suppressing up-regulation of tissue factor expression on leukocytes in recipient livers but not on islet grafts, a process leading to improvement in islet engraftment. Therefore, C5aIP in combination with conventional anticoagulants could be a strong candidate strategy to control the instant blood-mediated inflammatory reaction induced in clinical islet transplantation.
Asunto(s)
Complemento C5a/antagonistas & inhibidores , Trasplante de Islotes Pancreáticos/métodos , Sistema Porta/fisiología , Animales , Anticoagulantes/uso terapéutico , Antitrombinas/inmunología , Coagulación Sanguínea/fisiología , Complemento C5a/fisiología , Proteínas del Sistema Complemento/fisiología , Diabetes Mellitus Experimental/cirugía , Granulocitos/fisiología , Inflamación/prevención & control , Trasplante de Islotes Pancreáticos/fisiología , Hígado/fisiología , Ratas , Ratas Endogámicas Lew , Trombina/inmunología , Tromboplastina/genética , Trasplante Isogénico/fisiología , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be approximately 4.8 mg L(-1) in healthy individuals. The K(D) of the interaction between native and latent AT was found to be 51 mum, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood.
Asunto(s)
Antitrombinas/análisis , Inmunoensayo/métodos , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antitrombinas/química , Antitrombinas/inmunología , Análisis Químico de la Sangre/métodos , Dimerización , Femenino , Humanos , Técnicas In Vitro , Masculino , Ratones , Persona de Mediana Edad , Isoformas de Proteínas/análisis , Valores de Referencia , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Antiphospholipid Ab have been shown to promote thrombosis and fetal loss in the antiphospholipid syndrome (APS). Previously, we found IgG anti-thrombin Ab in some APS patients that could interfere with inactivation of thrombin by antithrombin (AT). Considering that activated coagulation factor X (FXa) is homologous to thrombin in the catalytic domains and is also regulated primarily by AT, we hypothesized that some thrombin-reactive Ab may bind to FXa and interfere with AT inactivation of FXa. To test these hypotheses, we studied reactivity of eight patient-derived monoclonal IgG antiphospholipid Ab with FXa and the presence of IgG anti-FXa Ab in APS patients and investigated the effects of FXa-reactive mAb on AT inactivation of FXa. The results revealed that six of six thrombin-reactive IgG mAb bound to FXa and that the levels of plasma IgG anti-FXa Ab in 38 APS patients were significantly higher than those in 30 normal controls (p < 0.001). When the mean plus 3 SDs of the 30 normal controls was used as the cutoff, 5 of 38 APS patients (13.2%) had IgG anti-FXa Ab. Importantly, three of six FXa-reactive mAb significantly inhibited AT inactivation of FXa. Combined, these results indicate that anti-FXa Ab may contribute to thrombosis by interfering with the anticoagulant function of AT on FXa in some APS patients.
Asunto(s)
Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , Antitrombinas/inmunología , Autoanticuerpos/sangre , Factor Xa/inmunología , Anticuerpos Monoclonales/sangre , Factor X/inmunología , Humanos , Inmunoglobulina G/sangre , Trombosis/etiología , Trombosis/inmunologíaRESUMEN
Sepsis is a complex disease and coagulation derangements are part of this context. The inflammatory storm is ultimately responsible for coagulation derangements. It is characterized by exacerbated coagulation, impaired anticoagulation and decreased fibrin removal. These derangements are implicated in the generation of microcirculation thrombosis, with deposition of microclots and obstruction of circulation, impairing blood flow and contributing to tissue hypoperfusion and consequently, organ dysfunction. This review will address the main issues regarding coagulation disorders in the context of sepsis.
Asunto(s)
Coagulación Sanguínea/inmunología , Fibrinólisis/fisiología , Sepsis/sangre , Antitrombinas/inmunología , Antitrombinas/metabolismo , Coagulación Sanguínea/fisiología , Factores de Coagulación Sanguínea/inmunología , Factores de Coagulación Sanguínea/metabolismo , Humanos , Lipoproteínas/sangre , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Receptores de Superficie Celular/sangre , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Sepsis/inmunología , Sepsis/metabolismoRESUMEN
Antithrombin (AT), which is mainly synthesized in the liver, is an acute-phase plasma protein in mammalian species. Here, we demonstrated that sheep anti-human AT antibody cross-reacted with the humoral fluids in amphioxus Branchiostoma belcheri tsingtauense as well as human serum. The concentration of AT in the humoral fluids in amphioxus decreased slightly at first and then increased after the acute challenge with lipopolysaccharide, while the level of total proteins remained unchanged. These suggest the presence of the same acute-phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AT was localized in the hepatic diverticulum. It is clear that the hepatic diverticulum in amphioxus is homologous to the vertebrate liver with respect to AT synthesis. This lends support to the hypothesis originally suggested by Müller that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.
Asunto(s)
Antitrombinas/metabolismo , Cordados no Vertebrados/metabolismo , Lipopolisacáridos/farmacología , Hígado/metabolismo , Animales , Anticuerpos/inmunología , Antitrombinas/inmunología , Líquidos Corporales/metabolismo , Humanos , Notocorda/metabolismo , SueroRESUMEN
Immunoglobulin G (IgG) isolated from the blood plasma of a patient with secondary antiphospholipid syndrome (APS) expresses fibrinogen-clotting and amidolytic activity (the thrombin activity in 20 micromole IgG is equivalent to approximately 5 nmole pure thrombin), and activates factor XIII. Hirudin (1 microM) decreases the intrinsic thrombin activity of the APS IgG by only 25%, whereas it inhibits completely pure thrombin with equivalent activity. Under conditions, when antithrombin inactivates 60% of the thrombin activity in the presence of normal IgG, the APS IgG protects almost completely the added thrombin against inactivation by antithrombin. Heparin, however, partially relieves this protective effect and at the same time it facilitates the inhibition of the intrinsic thrombin activity by antithrombin. The APS IgG reduces the thrombin activity in protein C activation assay by 50% compared to the activity in the presence of normal IgG. All described properties are related to the Fab fragment of the antibody. The IgG preserving the fibrin-generating activity of thrombin with concomitant protection against inhibitors unravels a new aspect of the thrombotic mechanism in APS. This condition is probably rare: only one out of 23 examined patients with primary or secondary APS expresses IgG with the described properties.