Águila Martínez, Sonia

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Águila Martínez, Sonia

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Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations
(2014-04-15) Águila Martínez, Sonia; Navarro Fernández, José Luis; Bohdan, N.; Gutiérrez Gallego, R.; Morena Barrio, María Eugenia de la; Vicente García, Vicente; Corral de la Calle, Javier; Martínez-Martínez, I.; Medicina Interna; Facultad de Medicina
Open Access
von Willebrand factor links primary hemostasis to innate immunity.
(Nature Research, 2022-11-03) Drakeford, Clive; Águila Martínez, Sonia; Roche, Fiona; Hokamp, Karsten; Fazavana, Judicael; Cervantes, Mariana P.; Curtis, Annie M.; Hawerkamp, Heike C.; Dhami, Sukhraj Pal Singh; Charles-Messance, Hugo; Hackett, Emer E.; Chion, Alain; Ward, Soracha; Ahmad, Azaz; Schoen, Ingmar; Breen, Eamon; Keane, Joe; Murphy,Ross; Preston, Roger J. S.; O’Sullivan, Jamie M.; Sheedy, Frederick J.; Fallon, Padraic; O’Donnell, James S.; Medicina Interna
The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles forVWF, and in particular suggest that VWFmay play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWFbinding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of proinflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.
Open Access
Neutrophil extracellular traps and von Willebrand factor are allies that negatively influence COVID-19 outcomes
(Wiley, 2021-01) Águila Martínez, Sonia; Fernández-Pérez, M. P.; Reguilón-Gallego, L.; de Los Reyes-García, A. M.; Miñano, A.; Bravo-Pérez, C.; García-Barberá, N.; Gómez-Verdú, J. M.; Martínez, C.; Morena Barrio, María Eugenia de la; Corral de la Calle, Javier; Bernal Morell, Enrique; Herranz Marín, María Teresa; Vicente García, Vicente; González-Conejero Hilla, Rocío; Lozano Almela, María Luisa; Medicina Interna
Open Access
Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease
(Elsevier; American Society for Biochemistry and Molecular Biology, 2014-12-05) Águila Martínez, Sonia; Izaguirre, Gonzalo; Qi, Lixin; Swanson, Richard; Roth, Ryan; Rezaie, Alireza R.; Gettins, Peter G. W.; Olson, Steven T.; Medicina Interna
Background: Exosites are known to mediate heparin allosteric activation of antithrombin. Results: Mutagenesis revealed that an exosite differentially contributes to antithrombin reactivity with factors Xa/IXa in unactivated and heparin-activated states. Conclusion: Heparin allosteric activation of antithrombin results from alterations in an exosite interaction with protease induced by core conformational changes. Significance: The findings support our recently proposed model of antithrombin allosteric activation.
Open Access
N-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 Interna
N-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.
Open Access
Disease-causing mutations in the serpin antithrombin reveal a keydomaincritical for inhibiting protease activities
(Elsevier, American Society for Biochemistry and Molecular Biology , 2017-10-06) Águila Martínez, Sonia; Izaguirre, G.; Vicente García, Vicente; Martínez-Martínez, I.; Olson, S. T.; Corral de la Calle, Javier; Medicina Interna
Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin–protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin–protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.
Open Access
Amelioration 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; Medicina
The 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
Open Access
Compound heterozygosity involving Antithrombin Cambridge II (p.Ala416Ser) in antithrombin deficiency.
(Elsevier, 2013-03-10) Águila Martínez, Sonia; Martínez-Martínez, Irene; Collado, Miriam; Llamas, Pilar; Antón, Ana I.; Martínez-Redondo, Consuelo; Padilla, José; Miñano, Antonia; Morena Barrio, María Eugenia de la; García-Avello, Ángel; Vicente García, Vicente; Corral de la Calle, Javier; Medicina Interna
Background: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. Objectives: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. Methods: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. Results and Conclusions: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity
Open Access
Increased galactose expression and enhanced clearance in patients with low von Willebrand factor
(American Society of Hematology (ASH Publications), 2019-04-04) Águila Martínez, Sonia; Lavin, M.; Dalton, N.; Patmore, S.; Chion, A.; Trahan, G.D.; Jones, K.L.; Keenan, C.; Brophy, T.M.; O'Connell, N.M.; Ryan, K.; Byrne, M.; Nolan, M.; Patel, A.; Preston, R.J.S.; James, P.; Di Paola, J.; O'Sullivan, J.M.; O'Donnell, J.S.; Medicina Interna
Glycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment ofVWF glycan composition, the importance of carbohydrates in humanVWDpathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood groupmatched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal a(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population.
Open Access
Nuevos mecanismos implicados en la deficiencia de antitrombina : relevancia de la N-glicosilación= new mechanisms involved in antithrombin deficiency: relevance of N-glycosylation
(2014-11-25) Águila Martínez, Sonia; Corral de la Calle, Javier; Martínez Martínez, Irene; Facultad de Medicina
Introducción La antitrombina es el principal anticoagulante endógeno. Sus principales dianas son el factor Xa (FXa) y la trombina (FIIa). Dicha serpina se sintetiza en el hígado y presenta cuatro sitios de N-glicosilación (N96, N135, N155, N192) . Este proceso ocurre sobre las asparraginas que se encuentran en contexto de esta secuencia consenso, Asn-X-Ser/Thr/Cys. La N135 no es eficientemente glicosilada, sólo en el 50% de las ocasiones y por lo tanto en plasma hay dos glicoformas: α-antitrombina y β-antitrombina, con cuatro y tres glicanos, respectivamente. La pérdida del glicano en posción 135 produce que la β-antitrombina tenga mayor afinidad por heparina y como consecuencia un mayor aclaramiento. Por esta razón en plasma sólose detecta un 10% de esta glicoforma. La función de este anticoagulante puede estar potenciada por la unión de glicosaminoglicanos al sitio de unión a heparina. Además, su mecanismo inhibitorio también depende la secuencia del centro reactivo, mediante el cual la proteína interacciona con la proteasa diana. Así, la antitrombina es muy sensible incluso a pequeñas modificaciones, como mutaciones puntales. El papel clave de la antitrombina queda reflejado en el alto riesgo de trombosis asociado a su deficiencia (OR: 10-20). Las consecuencias de estas mutaciones definen dos tipos de deficiencia: tipo I (no se detecta forma mutada en el plasma), o tipo II (la forma mutada secretada tiene afectada la actividad inhibitoria). Dentro de las deficiencias tipo II hay tres subgrupos: i) tipo IIa, afectan a la reactividad , ii) tipo IIb, disminuyen la afinidad por heparina, iii) tipo IIc o pleiotrópicas, localizadas en la lámina C y con múltiples efectos. Objetivos 1. Caracterización de nuevos factores de riesgo trombótico asociados con la deficiencia de antitrombina. 2. Identificación de nuevas regiones implicadas en la funcionalidad de la antitrombina. 3. Evaluación del proceso y papel de la N-glicosilación de antitrombina. Métodos Para llevar a cabo estos objetivos estudiamos mutaciones naturales en sujetos con deficiencia de antitrombina. Posteriormente, empleamos un sistema recombinante para producir estos mutantes y otros, con mutaciones no descritas previamente y los purificamos por cromatografía de afinidad por heparina e intercambio aniónico. A continuación, realizamos su caracterización profunda mediante estudios bioquímicos: afinidad por heparina, cinéticas de inhibición, temperatura de desnaturalización, glicómica, proteómica, SDS-PAGE y geles en condiciones no desnaturalizantes. Los resultados obtenidos a partir de estos ensayos generaron las siguientes conclusiones: Conclusiones 1. La heterocigosis compuesta que implica a la mutación A384S, es relativamente frecuente en pacientes con deficiencia de antitrombina (2/96, 2%). Este estado afecta significativamente la capacidad anticoagulante e incrementa tanto el riesgo trombótico como la severidad clínica. Estos datos junto a la ineficacia de los test trombofílicos empleados, para detectar la mutación A384S, animan a evaluar esta alteración mediante métodos moleculares en estudios familiares de pacientes con deficiencia de antitrombina, incluso si el probando no presenta esta alteración genética. 2. Identificamos una nueva región en antitrombina (la puerta de salida del loop reactivo internalizado en la lámina A central) relevante en el mecanismo inhibitorio de esta serpina, concretamente afecta a la inserción del loop reactivo durante la traslocación de la proteasa al polo opuesto de la antitrombina para su completa inhibición. 3. La lámina C es una región relevante en la correcta maduración de los N-glicanos de antitrombina. Mutaciones en esta región provocan la aparición de una variante con glicosilación aberrante que provoca la reducción de la afinidad por heparina. 4. La introducción de una secuencia aromática (Phe-X-Asn-Y-Thr) en posición 135 de la antitrombina consigue una glicosilación completamente eficaz, dificultando la activación inducida por la unión de heparina. Estos resultados apoyan que las secuencias aromáticas podrían ser empleadas para favorecer una glicosilación más eficiente en diferentes contextos estructurales. 5. Las mutaciones que generan nuevas secuencias de glicosilación en el RCL son correctamente glicosiladas y plegadas, pero el nuevo glicano interfiere con la reactividad de la serpina por diferentes mecanismos dependiendo de la localización de glicano. El mutante G392N presenta especificidad de proteasa diana, ya que puede inhibir al FXa, pero no al FIIa, lo que podría tener implicaciones terapéuticas. Introducction Antithrombin plays a key role in the haemostatic system as it is the most important inhibitor of proteases, such as thrombin (FIIa) and factor Xa (FXa). This serpin presents four N-glycosylation sites (N96, N135, N155, N192). N-glycosylation of proteins takes place on the asparagines located in the context of a tripeptide consensus sequence (Asn-X-Ser/Thr/Cys). However, N135 is not efficiently glycosylated and only 50% of the molecules are glycosylated at this position. Therefore, there are two glycoforms in plasma: α-antithrombin and β-antithrombin, with four and three N-glycans, respectively. The lack of carbohydrate in N135 causes an increased heparin affinity and faster clearance of β-antithrombin (10% detected in plasma) in comparison with the α-glycoform. The anticoagulant function of antithrombin is accelerated by the binding of glycosaminoglycans on the heparin binding site. Moreover, the inhibitory mechanism is also dependent on the sequence of the reactive center loop because it interacts with the target proteases. This mechanism is very sensitive and even point mutations may provoke different anithrombin deficiencies. Antithrombin deficiency is a high thrombotic risk (OR: 10-20) and is classified in different types: type I (the mutant antithrombin is not detected in plasma) and type II (the mutant protein is secreted and it has affected the inhibitory activity). Type II antithrombin deficiencies are further subclassified in three groups: i) type IIa, mutations affecting the reactivity, ii) type IIb, mutations decreasing the heparin affinity and iii) type IIc or pleiotropic, mutations located in the C-sheet with multiple effects. Aims 1. Characterization of new thrombotic risk factors associated with antithrombin deficiency. 2. Identification of new regions relevant for the antithrombin function. 3. Evaluation of the role and process of glycosylation in antithrombin. Methods To perform these aims we studied natural mutations in subject with antithrombin deficiency. Then, we produced these mutants and other mutations not describe previously by a recombinant system. We purified different variants by heparin affinity and anion exchange chromatography in order to perform their biochemical characterization. The assays carried out were: heparin affinity by measuring the intrinsic fluorescence during heparin titration and stopped flow, kinetic studies with FIIa and FXa, determination of the melting temperature, glycomic, proteomic, non denaturing and SDS-PAGE. The results obtained after these studies rendered these conclusions: Conclusions 1. Compound heterozygosity involving the A384S mutation is relatively frequent among patients with antithrombin deficiency. This state significantly impairs the anticoagulant capacity and increases the risk of venous thrombosis and the severity of clinical manifestations. Moreover, the failure of usual thrombophilic tests to detect A384S mutation, encourage the evaluation of potential compound heterozygosis involving A384S in familial studies, even if the proband does not carry this mutation. 2. The opening for the internalized RCL to exit from the central A-sheet is a key new region for the reactive center loop insertion. Mutations in this region affect to the dragged of the covalently bound protease to the opposite end of antithrombin for the complete inhibition. 3. The C-sheet is a relevant region for the correct maturation of N-glycans on antithrombin. The aberrant glycosylation induced by mutations on residues of the C-sheet reduces the heparin affinity. 4. The introduction of the aromatic sequon (Phe-X-Asn-Y-Thr) on N135 of antithrombin improves the efficiency of N-glycosylation hindering the changes induced by heparin. These results support that this aromatic sequon may be used for an efficient glycosylation in structural contexts other than reverse turns. 5. Mutations introducing new glycans on the reactive center loop of antithrombin are correctly glycosylated and the variants are secreted, but interfere in the reactivity. The N392 variant determines specificity of protease, since this variant properly inhibits FXa but not FIIa.