Role of the C‐sheet in the maturation of N‐glycans on antithrombin: functional relevance of pleiotropic mutations

Abstract

To cite this article:Aguila S, Navarro-Fern andez J, Bohdan N, Guti errez-Gallego R, de la Morena-Barrio ME, Vicente V, Corral J, Mart ınez- Summary. 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.

Clinical and laboratory characteristics of antithrombin deficiencies: A large cohort study from a single diagnostic center

“…AT activity and antigen concentrations were around 50% in these heterozygous individuals, indicating that the mutant AT may not appear in the circulation. The p.Pro461Thr mutation is suggested as a type IIPE variant based on our laboratory results and on a publication, in which the p.Pro461Leu was described [40].…”

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

“…The identification and analysis of natural mutations in patients with antithrombin deficiency have assisted the description of key functional domains or residues of this anticoagulant (15)(16)(17)(18)22). Thus, mutations at the RCL, HBS, and the C-sheet are responsible for the three subtypes of antithrombin type II deficiency.…”

“…Type II antithrombin deficiencies are provoked by variants secreted to the plasma with an impaired anticoagulant activity. Type II variants have helped identify functional domains of this key anticoagulant, such as the RCL, the heparin-binding site (HBS), or the C-sheet, in which mutations can cause a variety of functional defects (15)(16)(17)(18)(19). We aimed to identify new functional domains of antithrombin by evaluating cases with type II deficiency with mutations outside these classical functional domains (RCL, HBS, or C-sheet).…”

Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

“…Therefore, alteration of heparin affinity results in a defective inhibitory function of this serpin. Several studies have reported the procoagulant state derived from mutations affecting the antithrombin heparin binding [ 12 – 15 ].…”