Investigation of the Differences in Antithrombin to Heparin Binding among Antithrombin Budapest 3, Basel, and Padua Mutations by Biochemical and In Silico Methods

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.

Unraveling a borderline antithrombin deficiency case with quantitative mass spectrometry

“…A heterozygous mutation within this peptide was thus evident and additional analysis identified the mutated peptide ILEATNR, concordant with a p.Pro73Leu mutation, also known as the “Basel” mutation 14 . This proteoform has a heparin‐binding site (HBS) defect reducing the AT activity and studies indicate high percentages of obstetric complications (34.7%–37.5%) in women with this mutation, matching the miscarriages of the patients 10,11 . The difficulty in diagnosing this mutation has been reported, for example by Orlando et al, 15 who tested AT activity tests from four manufacturers on samples from seven patients with a known p.Pro73Leu mutation.…”

“…Current larger studies, upon which guidelines are based, often group all types of AT deficiency to generate sufficient power for statistical analysis, leading to underestimation of RPL risk of specific molecular AT forms. Smaller studies and case reports describing specific well‐characterized AT deficiencies do show a link with obstetric complications 10,11 . We here present a case report that illustrates the need for molecularly defined AT deficiency screening at the protein level.…”

“…14 This proteoform has a heparinbinding site (HBS) defect reducing the AT activity and studies indicate high percentages of obstetric complications (34.7%-37.5%) in women with this mutation, matching the miscarriages of the patients. 10,11 The difficulty in diagnosing this mutation has been reported, for example by Orlando et al, 15 who tested AT activity tests from four manufacturers on samples from seven patients with a known p.Pro73Leu mutation. Only one test identified all patients as deficient, whereas the other tests reported normal activities for almost all samples.…”

“…Smaller studies and case reports describing specific well-characterized AT deficiencies do show a link with obstetric complications. 10,11 We here present a case report that illustrates the need for molecularly defined AT deficiency screening at the protein level.…”

Computational analyses reveal fundamental properties of the AT structure related to thrombosis

“…Type II deficiencies have normal or near-normal antigen levels but reduced activity ( Kuhle et al , 2001 ; Smith et al , 2021 )]. Interestingly, some groups started using computational techniques to study the effects of mutations in the AT protein using a variety of techniques, including molecular dynamics ( Gindele et al , 2021 ), the prediction of mutation effects ( Luxembourg et al , 2011 , 2015 ; Mulder et al , 2017 ) and explored possible heparin substitutes with higher AT affinities ( Navarro-Fernández et al , 2012 ). Although these studies represent an important step toward the digitalization and potential simulation of the AT protein features in silico , their agreement with in vitro and clinical data was not satisfactory; their findings were often restricted to a few representative variants of the AT protein, could not be generalized to other known mutations and neither be used to predict the effect of novel mutations.…”