Browsing by Subject "Antithrombin"
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- PublicationRestrictedA human antithrombin isoform dampens inflammatory responses and protects from organ damage during bacterial infection(Nature Research, 2019) Papareddy, Praveen; Rossnagel, Madlen; Hollwedel, Femke; Kilic, Gülcan; Veerla, Srinivas; Naudin, Clément; Smeds, Emanuel; Westman, Johannes; Martínez-Martínez, Irene; Egesten, Arne; Morena-Barrio, María Eugenia de la; Corral, Javier; Linder, Adam; Artoni, Andrea; Abbattista, Maria; Novembrino, Cristina; Brakebusch, Cord Hebert; Martinelli, Ida; Kasetty, Gopinath; Herwarld, Heiko; MedicinaSevere 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 β-isoform (hβAT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of hβAT 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 hβAT 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 hβAT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.
- PublicationEmbargoAntithrombin p.Thr147Ala: The First Founder Mutation in People of African Origin Responsible for Inherited Antithrombin Deficiency(Thieme Gruppe, 2021) Orlando, Christelle; Morena-Barrio, Belén de la; Pareyn, Inge; Vanhoorelbeke, Karen; Martínez-Martínez, Irene; Vicente, Vicente; Corral, Javier; Jochmans, Kristin; Morena-Barrio, María Eugenia de la; MedicinaBackground: Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described. Objectives: We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated. Methods: Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation. Results: Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms. Conclusion: Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype.
- PublicationRestrictedCongenital disorder of glycosylation (PMM2-CDG) in a patient with antithrombin deficiency and severe thrombophilia(Elsevier, 2012-12) Morena-Barrio, María Eugenia de la; Sevivas, Teresa S.; Martínez-Martínez, Irene; Miñano, Antonia; Vicente, Vicente; Jaeken, Jaak; Corral, Javier; Medicina
- PublicationRestrictedHigh levels of latent antithrombin in plasma from patients with antithrombin deficiency(Thieme Gruppe, 2017) Morena-Barrio, María Eugenia de la; Sandoval, Edna; Llamas, Pilar; Wypasek, Ewa; Toderici, Mara; Navarro-Fernández, José; Rodríguez-Alen, Agustín; Revilla, Nuria; López-Gálvez, Raquel; Miñano, Antoni; Padilla, José; Morena-Barrio, Belén de la; Cuesta, Jorge; Corral, Javier; Vicente, Vicente; MedicinaAntithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.
- PublicationOpen AccessIdentification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?(Public Library of Science, 2013) Morena-Barrio, María Eugenia de la; Buil, Alfonso; Antón, Ana Isabel; Martínez-Martínez, Irene; Miñano, Antonia; Guriérrez-Gallego, Ricardo; Navarro-Fernandez, José; Águila, Sonia; Souto, Juan Carlos; Vicente, Vicente; Soria, José Manuel; Corral, Javier; MedicinaThe 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 a1-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 glycoproteins.
- PublicationRestrictedLatent and polymeric antithrombin: Clearance and potential thrombotic risk(Frontiers Media, 2007) Guerrero López, José Antonio; Corral de la Calle, Javier; Rivera Pozo, José; Miñano, Antonia; Alberca, Ignacio; Hernández Espinosa, David; Ordóñez, Adriana; Martínez, Constantino; Navarro-Núñez, Leyre; González-Conejero Hilla, Rocío; Lozano Almela, María Luisa; Vicente García, Vicente; Medicina Interna
- PublicationOpen AccessMPI-CDG with transient hypoglycosylation and antithrombin deficiency(Ferrata Storti Foundation, 2019) Morena-Barrio, María Eugenia de la; Wypasek, Ewa; Owczarek, Danuta; Miñano, Antonia; Vicente, Vicente; Corral, Javier; Undas, Anetta; Medicina
- PublicationOpen AccessN-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.(MDPI, 2021-06-06) Águila Martínez, Sonia; Noto, Rosina; Luengo-Gil, Ginés; Espín, Salvador; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Peñas, Julia; Rodenas, Maria Carmen; Vicente García, Vicente; Corral de la Calle, Javier; Manno, Mauro; Martínez-Martínez, Irene; Medicina InternaN-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an Nglycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.
- PublicationEmbargoProtective role of antithrombin in mouse models of liver injury(Elsevier, 2012) Teruel, Raul; Martínez, Constantino; Arcas, Isabel; Martínez-Martínez, Irene; Morena-Barrio, María Eugenia de la; Vicente, Vicente; Corral, Javier; Guerrero López, José Antonio; MedicinaBackground & Aims: Coagulopathy caused by an imbalance of hemostatic factors is associated with the pathophysiology of liver disease. We have investigated the role of antithrombin (AT), a key anticoagulant serpin, in the onset of liver disease. Methods: Liver injury was induced by CCl4 injection and bile duct ligation (BDL) in wild type (WT) and AT-deficient (AT+/ ) mice. Twenty-four hours after CCl4 treatment, aspartate-transaminase, alanine-transaminase, liver lesion size, leukocyte infiltration, and apoptosis were reduced in WT animals compared to AT+/ mice. Results: Administration of exogenous AT in AT+/ animals did not restore the values observed in WT mice, suggesting that intrahepatic AT might also offer protection against CCl4. In the BDL model, increased liver injury was also evident in AT+/ compared to WT mice. An 85 kDa covalent complex involving AT was identified in immunoblottings of liver lysates from CCl4-treated animals. This complex was also present in anoikis hepatocytes and H2O2-treated HepG2 cells, suggesting a role for AT in apoptosis. Expression of recombinant WT–AT by HEK-EBNA cells increased cell survival while expression of AT mutants, DR393 and R47C, did not modify viability. Finally, plasma anti-FXa activity was attenuated by liver injury, with AT+/ animals showing a greater reduction than WT mice. Conclusions: Our study reveals a protective role of AT against liver injury due to its recognized anticoagulant and antiinflammatory action. AT may also act via a previously unrecognized antiapoptotic effect. The clinical implications of AT deficiency in patients with liver disease should be further addressed. 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
- PublicationRestrictedRegulatory regions of SERPINC1 gene: identification of the first mutation associated with antithrombin deficiency(Thieme Gruppe, 2012) Morena-Barrio, Maria Eugenia de la; Antón, Ana Isabel; Martínez Martínez, Irene; Padilla, José; Miñano, Antonia; Navarro Fernández, José; Águila, Sonia; López, María Fernanda; Fontcuberta, Jordi; Vicente, Vicente; Corral, Javier; MedicinaAntithrombin is the main endogenous anticoagulant. Impaired function or deficiency of this molecule significantly increases the risk of thrombosis. We studied the genetic variability of SERPINC1 , the gene encoding antithrombin, to identify mutations affecting regulatory regions with functional effect on its levels. We sequenced 15,375 bp of this gene, including the potential promoter region, in three groups of subjects: five healthy subjects with antithrombin levels in the lowest (75%) and highest (115%) ranges of our population, 14 patients with venous thrombosis and a moderate antithrombin deficiency as the single thrombophilic defect, and two families with type I antithrombin deficiency who had neither mutations affecting exons or flanking regions, nor gross gene deletions. Our study confirmed the low genetic variability of SERPINC1 , particularly in the coding region, and its minor influence in the heterogeneity of antithrombin levels. Interestingly, in one family, we identified a g.2143 C>G transversion, located 170 bp upstream from the translation initiation codon. This mutation affected one of the four regions located in the minimal promoter that have potential regulatory activity according to previous DNase footprinting protection assays. Genotype-phenotype analysis in the affected family and reporter analysis in different hepatic cell lines demonstrated that this mutation significantly impaired, although it did not abolish, the downstream transcription. Therefore, this is the first mutation affecting a regulatory region of the SERPINC1 gene associated with antithrombin deficiency. Our results strongly sustain the inclusion of the promoter region of SERPINC1 in the molecular analysis of patients with antithrombin deficiency.