Publication: Compound heterozygosity involving Antithrombin Cambridge II (p.Ala416Ser) in antithrombin deficiency.
Authors
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
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Publisher
Elsevier
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DOI
10.1160/TH12-09-0707
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info:eu-repo/semantics/article
Description
Abstract
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
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Citation
Journal of Thrombosis and Haemostasis, 12: 1131–1140
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