Browsing by Subject "Thrombosis"
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- PublicationOpen AccessAmelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism(American Society of Hematology, 2012-04-12) Martínez-Martínez, Irene; Navarro-Fernández, José; Ostergaad, Alice; Gutierrez-Gallego, Ricardo; Padilla, José; Miñano, Antonia; Pascual, Cristina; Martínez, Constantino; Morena-Barrio, María Eugenia de la; Pedersen, Shona; Kristensen, Soren Risom; Corral, Javier; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Águila Martínez, Sonia; Vicente García, Vicente; Corral de la Calle, Javier; MedicinaThe balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the β-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin
- 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
- PublicationOpen AccessContact pathway in surgical and transcatheter aortic valve replacement(Frontiers Media, 2022-07-22) Morena Barrio, María Eugenia de la; Corral, Javier; López García, Cecilia; Jiménez Díaz, Víctor Alonso; Miñano, Antonia; Juan-Salvadores, Pablo; Esteve Pastor, María Asunción; Baz Alonso, José Antonio; Rubio, Ana María; Sarabia Tirado, Francisco; García Navarro, Miguel; García Lara, Juan; Marín, Francisco; Vicente, Vicente; Pinar, Eduardo; Cánovas López, Sergio; Morena, Gonzalo de la; Cirugía, Pediatría y Obstetricia y GinecologíaBackground: Aortic valve replacement is the gold standard treatment for severe symptomatic aortic stenosis, but thrombosis of bioprosthetic valves (PVT) remains a concern. Objective: To analyze the factors involved in the contact pathway during aortic valve replacement and to assess their impact on the development of thromboembolic complications. Methods: The study was conducted in 232 consecutive patients who underwent: transcatheter aortic valve replacement (TAVR, N = 155), and surgical valve replacement (SAVR, N = 77) (MUVITAVI project). Demographic and clinical data, outcomes including a combined end point (CEP) of thrombotic events, and imaging controls were recruited. Samples were collected 24 h before and 48 h after valve replacement. FXII, FXI and (pre)kallikrein were evaluated by Western Blot and specific ELISA with nanobodies. Results: The CEP of thrombotic events was reached by 19 patients: 13 patients presented systemic embolic events and 6 patients subclinical PVT. Valve replacement did not cause FXII activation or generation of kallikrein. There was a significant reduction of FXI levels associated with the procedure, which was statistically more pronounced in SAVR than in TAVR. Cases with reductions of FXI below 80% of basal values had a lower incidence of embolic events during the procedure than patients in whom FXI increased above 150%: 2.7 vs. 16.7%; p: 0.04. Conclusion: TAVR or SAVR did not significantly activate the contact pathway. A significant reduction of FXI, was observed, particularly in SAVR, associated with lower incidence of thrombotic events. These results encourage evaluating the usefulness and safety of FXI-directed antithrombotic treatments in these patients.
- 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 AccessImplication of hepsin from primary tumor in the prognosis of colorectal cancer patients(MDPI, 2022-06-24) Zaragoza-Huesca, David; Nieto-Olivares, Andrés; García-Molina, Francisco; Ricote, Guillermo; Montenegro, Sofía; Sánchez-Cánovas, Manuel; Garrido-Rodríguez, Pedro; Peñas-Martínez, Julia; Vicente, Vicente; Martínez Díaz, Francisco; Lozano, María Luisa; Carmona-Bayona, Alberto; Martínez-Martínez, Irene; Oftalmología, Optometría, Otorrinolaringología y Anatomía PatológicaHepsin is a type II transmembrane serine protease whose deregulation promotes tumor invasion by proteolysis of the pericellular components. In colorectal cancer, the implication of hepsin is unknown. Consequently, we aimed to study the correlations between hepsin expression and different clinical-histopathological variables in 169 patients with localized colorectal cancer and 118 with metastases. Tissue microarrays were produced from samples at diagnosis of primary tumors and stained with an anti-hepsin antibody. Hepsin expression was correlated with clinical histopathological variables by using the chi-square and Kruskal–Wallis tests, Kaplan–Meier and Aalen–Johansen estimators, and Cox and Fine and Gray multivariate models. In localized cancer patients, high-intensity hepsin staining was associated with reduced 5-year disease-free survival (p-value = 0.16). Medium and high intensity of hepsin expression versus low expression was asso ciated with an increased risk of metastatic relapse (hazard ratio 2.83, p-value = 0.035 and hazard ratio 3.30, p-value = 0.012, respectively), being a better prognostic factor than classic histological variables. Additionally, in patients with localized tumor, 5-year thrombosis cumulative incidence increased with the increment of hepsin expression (p-value = 0.038). Medium and high intensities of hepsin with respect to low intensity were associated with an increase in thrombotic risk (hazard ratio 7.71, p-value = 0.043 and hazard ratio 9.02, p-value = 0.028, respectively). This relationship was independent of previous tumor relapse (p-value = 0.036). Among metastatic patients, low hepsin expression was associated with a low degree of tumor differentiation (p-value < 0.001) and with major metastatic dissemination (p-value = 0.023). Hepsin is a potential thrombotic and metastatic biomarker in patients with localized colorectal cancer. In metastatic patients, hepsin behaves in a paradoxical waywith respect to differentiation and invasion processes.
- PublicationRestrictedIn vivo relevance for platelet glycoprotein Ibα residue Tyr276 in thrombus formation(Elsevier, 2008) Guerrero López, José Antonio; Shafirstein, G.; Russell, S.; Varughese, K. I.; Kanaji, T.; Liu, J.; Gartner, T. K.; Bäumler, W.; Jarvis, G. E.; Ware, J.; Medicina Interna; Facultad de Medicina
- 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.
- PublicationOpen AccessPlatelet ad hesion receptors and (patho)physiological thrombus formation(Murcia : F. Hernández, 2001) Andrews, R.K.; Shen, Y.; Gardiner, E.E.; Berndt, M.C.In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, plateletendothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, aIIbB3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.
- 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.
- PublicationOpen AccessRecomendaciones sobre el tratamiento antitrombótico durante la pandemia COVID-19. Posicionamiento del Grupo de Trabajo de Trombosis Cardiovascular de la Sociedad Española de Cardiología(Sociedad Española de Cardiología, 2020-04-22) Vivas, David; Roldán, Vanessa; Esteve-Pastor, María Asunción; Roldan, Inmaculada; Tello-Montoliu, Antonio; Ruiz-Nodar, Juan Miguel; Cosín-Sales, Juan; Gámez, José María; Consuegra, Luciano; Ferreiro, José Luis; Marín Ortuño, Francisco; MedicinaLa pandemia producida por la infección del nuevo coronavirus SARS-CoV-2, que da lugar a una enfermedad altamente contagiosa (COVID-19), ha producido un colapso de los sistemas sanitarios de todo el mundo. Se ha descrito que estos pacientes sufren un estado inflamatorio que condiciona un alto riesgo trombótico. Sin embargo, apenas hay información sobre cómo abordar el riesgo trombótico, la coagulopatía y el tratamiento anticoagulante de estos pacientes. Por otra parte, incluso los pacientes no infectados por COVID-19 sufren una tremenda influencia en su abordaje habitual por la situación sanitaria actual. El objetivo del presente documento, elaborado por el Grupo de Trabajo de Trombosis Cardiovascular de la Sociedad Española de Cardiología, es presentar la información disponible y dar unas pautas sencillas de tratamiento con fármacos antitrombóticos.
- 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.