Identification of antithrombin-modulating genes. Role of LARGE, a gene encoding a bifunctional

The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteinsThis study was supported partially by 04515/GERM/06 (Fundación Séneca de la Región de Murcia), SAF2009-08993 and SAF2008/01859 (Spanish Ministerio de Ciencia y Tecnología & Fondo Europeo de Desarrollo Regional de la Unión Europea FEDER), PI-08/0756, PI-11/0184 and RECAVA RD06/0014/0039 & RD06/0014/0016 (Spanish Instituto de Salud Carlos III & Fondo Europeo de Desarrollo Regional de la Unión Europea FEDER), and Centre National du Genotypage (Evry, France). MEMB is a holder of a predoctoral research grant from Spanish Instituto de Salud Carlos III (FI09/00190). IMM is a researcher from Fundació n para la Formación e Investigación Sanitarias. JNF is a postdoctoral researcher of the University of Murcia. JM Soria was supported by ‘‘Programa d’ Estabilització d’Investigadors de la Direcció d’Estrategia i Coordinació del Departament de Salut’’ (Generalitat de Catalunya)

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation.

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10 000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins