Dexamethasone induces a heat-stress response that ameliorates the conformational consequences on antithrombin of L-asparaginase treatment.

BACKGROUND: L-asparaginase (L-ASP) treatment of patients with acute lymphoblastic leukemia causes a severe antithrombin deficiency by intracellular retention of this serpin within the endoplasmic reticulum (ER) of hepatic cells, and a subsequent risk of thrombosis. Interestingly, co-administration of dexamethasone with L-ASP seems to reduce the risk of thrombosis. OBJECTIVES: We have investigated the effect of two corticoids, dexamethasone and prednisone, on the conformational consequences of L-ASP treatment on antithrombin. PATIENTS/METHODS: Levels, activity, conformation and immunohistological features of antithrombin were studied in patients, cell and mice models. Because of the importance of the steroid receptor-heat stress response (HSR) axis, and the role of unfolded protein response (UPR) in conformational diseases, we also evaluated Hsp27, Hsp70, Hsp90, HSF-1 and ER chaperons (Grp78 and Grp94). RESULTS: In all models, L-ASP alone or in combination with prednisone caused the intracellular retention of antithrombin associated with a severe deficiency. In contrast, the combination of L-ASP with dexamethasone ameliorated both the deficiency and intracellular retention of the serpin, which is associated with increased expression of heat shock proteins and ER-chaperons. CONCLUSIONS: These results suggest a protective effect of dexamethasone on the conformational consequences of L-ASP on antithrombin as a result of exacerbated HSR and UPR that help to explain the reduced risk of thrombosis reported in patients that follow this scheme of treatment.

Caracterización de tres mutaciones en el gen de la antitrombina / Identification of three mutations in the antithrombin gene

La antitrombina es el principal anticoagulante hemostático. Desde que el hematólogo Egeber descubriera en 1965 la primera deficiencia de antitrombina asociada a un aumento del riesgo trombótico, numerosos trabajos han identificado familias con deficiencia de antitrombina asociada a una significativa incidencia de trombosis venosa. Este trabajo recoge el estudio de 3 familias trombofílicas españolas con deficienciacongénita de antitrombina. En dos identificamos mutaciones en el gen de la antitrombina no descritas hasta la fecha: InsT7429-30 y Lys125Arg. En la tercera detectamos una variante, la antitrombina Cambridge II (Ala384Ser) identificada con elevada frecuencia en población británica. Valoramos su frecuencia en población española y su papel en el desarrollode trombosis venosa mediante un estudio de asociación caso-control en 1018 pacientes de trombosis venosa y 1018controles. Comprobamos que la antitrombina Cambridge II es una alteración no restringida a población británica, estando presente en el 0.2% de la población española pero en un 1.7% de los pacientes con trombosis venosa. Así, el alelo mutado incrementó 9.75 veces el riesgo trombótico. Además, esta mutación es la principal causa de deficiencia de antitrombina en pacientes con trombosis venosa. Finalmente destacamos que la antitrombina Cambridge II únicamente puede ser detectada por métodos moleculares. Todos estos resultados aconsejan el estudio de la antitrombina Cambridge II en los estudios trombofílicos rutinarios

Antithrombin is the most important haemostatic anticoagulant. Since, in 1965 Egeber described the first antithrombin deficiency associated with an increase of thrombotic risk, many studies have identified families with antithrombin deficiency that caused a significantly increase in the risk of venous thrombosis. We studied three thrombofilic families with congenital antithrombin deficiency. In two of them, we identified mutations in antithrombin gene not described previously: InsT7429-30 and Lys125Arg. In the third family, we detected a variant, antithrombin Cambridge II (Ala384Ser) that had a high frequency in British population. We have evaluated the prevalence of this mutation in Spanish population, and its role in venous thrombosis in a case-control study including 1018 patients with venous thrombosis and 1018 healthy controls. We confirmed that antithrombin Cambridge II is not restricted to British population, as we observed this mutation in 0.2% of Spanish population and in 1.7% of patients with venous thrombosis. The mutated allele increased 9.75 fold the risk of venous thrombosis. Moreover, this mutation was the main cause of antithrombin deficiency among patients with venous thrombosis. Finally, we point that this variant can only be detected by molecular methods. All our data support the study of antithrombin Cambridge II in routine thrombophilic tests

Therapeutic treatment with poly(ADP-ribose) polymerase inhibitors attenuates the severity of acute pancreatitis and associated liver and lung injury.

BACKGROUND AND PURPOSE: The mortality associated with acute pancreatitis (AP) is largely attributable to abnormalities that occur in distant organs and supportive care remains the only treatment for patients with these complications. Recently, prophylactic pharmacological blockade of poly(ADP-ribose) polymerase (PARP) enzymes has been shown to attenuate the severity of the disease. However, the clinical relevance of PARP inhibitors administered after the onset of AP remains uncertain. The aim of the present study was to investigate the therapeutic effects of PARP inhibitors in established AP. EXPERIMENTAL APPROACH: Mice were fed a choline/methionine-deficient/ethionine-supplemented (CMDE) diet to induce AP. PARP inhibitors were given at 36 h after the onset of CMDE diet. Severity of pancreatitis was assessed by measurements of serum amylase, lipase, IL-1beta and IL-6, and histological grading. Serum hepatic enzymes, myeloperoxidase (MPO) activity and morphological changes were measured as indicators of hepatic insult. Lung injury was evaluated by MPO activity and morphological changes. Survival rates of mice were monitored for 7 days. KEY RESULTS: CMDE diet administration resulted in a significant increase in serum amylase, lipase, IL-1beta, IL-6, alanine aminotransferase and aspartate aminotranferase levels, indicating AP and associated liver injury. Analysis of the histopathological changes in pancreas, liver and lung revealed extensive tissue damage. Treatment of mice with PARP-inhibitors after the onset of AP was associated with a reduction in the severity of AP and, accordingly, with a reduced mortality rate. CONCLUSIONS AND IMPLICATIONS: Our results support the therapeutic application of PARP inhibitors in the treatment of established AP.

In vivo effects of hyperthermia on the functional and conformational characteristics of antithrombin.

BACKGROUND: High temperatures produce in vitro transitions of antithrombin to its inactive latent and polymeric forms. Accordingly, high body temperatures might contribute in vivo to conformational changes in antithrombin associated with increased thrombotic risk. METHODS: We assessed the in vivo effects of different hyperthermic stimuli on antithrombin. We studied two mouse models of hyperthermia. (i) Febrile syndrome induced by turpentine. (ii) Heat stroke generated by exposure to 42 degrees C. Body temperatures were measured. Antigen, anti-factor Xa activity and conformational features of plasma antithrombin were studied. Furthermore, structural and ultrastructural features from livers were analyzed. Intracellular retention of serpins (antithrombin and alpha1-antitrypsin) was studied by western-blotting, immunohistochemistry, and immunogold-labeling-electron microscopy. RESULTS: Hyperthermic stimuli caused a moderate deficiency of circulating antithrombin and a slight increase in its latent form. Moreover, hyperthermia caused intracellular retention of antithrombin into aggregates within the lumen of the endoplasmic reticulum of hepatocytes. This effect was similar for alpha1-antitrypsin. CONCLUSION: Hyperthermia causes minor conformational changes on circulating antithrombin in vivo, although it has severe consequences for intracellular antithrombin and other hepatic serpins, inducing the intracellular retention of the nascent protein. These effects may contribute to the moderate plasma deficiency of antithrombin and the increased thrombotic risk detected in hyperthermic conditions.

Glucación no enzimática de la antitrombina. Implicaciones en el riesgo trombótico de pacientes con diabetes mellitus / Non-enzymatic antithrombin glycation. Association with atherothrombotic risk in diabetic patients

Introducción/objetivos. La glucación no enzimática incorpora azúcares a los residuos lisina y arginina de proteínas, lo que puede alterar su función. La glucación de la antitrombina, un potente anticoagulante natural, podría asociarse con el riesgo trombótico observado en situaciones de hiperglucemia, como la diabetes mellitus. Nuestro objetivo fue estudiar el efecto de la glucación de la antitrombina y determinar si es relevante en las complicaciones trombóticas de la diabetes mellitus. Métodos. 1. Glucación no enzimática in vitro de antitrombina plasmática y purificada con metilglioxal y glucosa. 2. Se analizó el efecto de diferentes compuestos sobre la glucación no enzimática de la antitrombina in vitro. 3. Estudio de 101 pacientes diabéticos. En todas las muestras se analizaron los valores antigénicos, la actividad anti-FXa, las características conformacionales y la afinidad a la heparina de la antitrombina. Resultados. La glucación no enzimática in vitro de la antitrombina con metilglioxal o glucosa no ocasiona modificaciones conformacionales significativas en la molécula, pero induce su transformación a una forma con baja afinidad por la heparina, que explica la pérdida significativa de su actividad (valor < 40% del basal). Este efecto se previene con heparina, aminoguanidina y catequina. Los pacientes diabéticos muestran menores valores antigénicos y funcionales de antitrombina (80%) que los sujetos controles, pero esta disminución no se correlaciona con la glucemia ni con los valores de hemoglobina glucosilada. Conclusiones. La glucación de residuos lisina y arginina localizados en el sitio de unión a la heparina de la antitrombina reduce significativamente su actividad anticoagulante, aunque puede ser protegida por la heparina, la aminoguanidina y la catequina. Sin embargo, la relevancia de la glucación de la antitrombina en pacientes diabéticos es apenas perceptible debido a la lenta acción glucante de la glucosa, y a la reducida vida media de la antitrombina (AU)

Introduction/Aims. Non-enzymatic glycation of proteins can impair their function by incorporating sugars into their lysine and arginine residues. Glycation of antithrombin, a powerful anticoagulant, might be associated with the thrombotic risk observed in hyperglycemic conditions such as diabetes mellitus. Our aim was to study the effects of antithrombin glycation and determine its significance in the thrombotic complications observed in diabetes. Methods. 1) In vitro study of non-enzymatic glycation of purified and plasma antithrombin by their incubation with methylglyoxal and glucose. 2) The effect of different compounds on the in vitro glycation of antithrombin was analyzed. 3) We studied 101 diabetic patients. Antigen levels, anti-FXa activity, conformational features and antithrombin affinity to heparin were determined. Results. In vitro non-enzymatic glycation of antithrombin with methylglyoxal or glucose caused no significant conformational change in the molecule, but induced the transformation to a low heparin-affinity form, which explains the significant loss of activity observed (< 40% of basal). This effect was prevented by heparin, aminoguanidine and catechin. Diabetic patients presented lower antigenic and antithrombin functional levels (80%) than controls. However, no correlation between activity or antigen levels of antithrombin and glycemia or glycosylated hemoglobin was found in diabetic patients. Conclusions. In vitro glycation of lysine and arginine residues located in the heparin-binding site of antithrombin significantly reduces its anticoagulant activity. Interestingly, heparin, aminoguanidine and catechin prevented this effect. However, the non-enzymatic glycation of antithrombin in diabetic patients seems to be mild, since the action of glucose is very slow and the half life of antithrombin in plasma is short (AU)