Functional, biochemical, and clinical characterization of natural mutations affecting arginine residues in the heparin-binding site of antithrombin

Abstract

Arginine residues in antithrombin are essential for heparin binding and for the activation of this key anticoagulant serpin. Mutations affecting these residues may cause antithrombin deficiency, specifically Type II heparin-binding site (HBS) defects, and increase the risk of thrombosis. However, previous alanine-scanning mutagenesis and crystallographic studies have yielded conflicting results regarding the specific residues involved in heparin binding. Our aim was to characterize natural variants affecting arginine residues in antithrombin. Genetic, biochemical, and functional characterization of antithrombin was done in 663 unrelated patients, most with antithrombin deficiency. Recombinant expression of the variants was performed in two different N-glycosylation backgrounds. We identified nine SERPINC1 missense variants affecting eight arginine residues located at or near the HBS, four of them novel. Two variants, the most distant from the HBS (p.R291H and p.R177C), were found to be benign. In contrast, p.R45W, p.R56C, p.R79C, p.R79H, and p.R161Q caused Type II HBS deficiency. p.R78Q, causing a mild Type II HBS defect, was identified in a patient with compound heterozygosity with other HBS mutations. N-glycosylation at N167 modulated heparin affinity and influenced the clinical severity of mutations affecting arginine residues involved in heparin interaction. Finally, p.R89S caused Type I deficiency by creating a novel N-glycosylation motif, leading to hyperglycosylation and intracellular retention. Natural variants provide valuable insights into the functional contribution of arginine residues to antithrombin-heparin interaction. Our findings highlight the biochemical, functional, and clinical heterogeneity of these mutations and underscore the importance of comprehensive characterization for accurate diagnosis and prognosis in affected individuals.