Abstract

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10−6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10−7 M, 2.15 × 10−8 M and 6.4 × 10−10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.

Highlights

  • Antithrombin (AT) is a single-chain glycoprotein belonging to the serpin family, synthesized in the liver, the molecular weight of it is 58,200 Da [1]

  • Venous thrombosis was registered in almost all AT Budapest 3 (ATBp3) homozygotes (88.8%) and it was relatively frequent in ATBp3 heterozygotes, while a great proportion of AT Basel was free from venous thrombotic symptoms at the time of data collection

  • ATBp3 and AT Padua groups suffered from pulmonary embolism without having obvious embolic source, as investigated by standard imaging methods

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Summary

Introduction

Antithrombin (AT) is a single-chain glycoprotein belonging to the serpin family, synthesized in the liver, the molecular weight of it is 58,200 Da [1]. After the cleavage of the propeptide (32 amino acids), the mature protein is composed of 432 amino acids. Two glycoforms of AT are present in the circulation, the majority as α-glycoform (90–95%). Β-glycoform is present to a lesser extent (

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