Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics.

Sara Reda,Behnaz Pezeshkpoor,Heiko Rühl,Bernd Pötzsch,Anna Pavlova,Jens Müller,Johannes Oldenburg

doi:10.3390/ijms22042119

Sara Reda, Behnaz Pezeshkpoor + Show 5 more

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https://doi.org/10.3390/ijms22042119

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Abstract

Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.

Highlights

The serine protease inhibitor (SERPIN) antithrombin (AT) is an important physiological anticoagulant protein
In a previous study we have presented a novel approach to measure thrombin inactivation kinetics at sub-threshold thrombin concentrations [11]; plasma levels that are too low to induce fibrin clot formation but are high enough to induce a prothrombotic phenotype by activation of factors V, VIII, and XI [12]
In the present study we extended this approach to carriers of AT mutations in whom the inhibition kinetics of human and bovine thrombin were assessed

Summary

Introduction

The serine protease inhibitor (SERPIN) antithrombin (AT) is an important physiological anticoagulant protein It inhibits clot formation by forming enzymatically inactive complexes with the active coagulation factors thrombin, activated factor X (FXa) and, less importantly, activated factors IX and XI [1,2]. The mechanism by which AT exerts its inhibitory function requires the interplay of two different regions of the protein: a reactive site that binds to the enzymatically active site of the target serine protease and a heparinbinding site. Through this heparin-binding site a conformational change is induced in the presence of heparin leading to a more than 1000-fold acceleration of AT activity [3]. Reflecting the key role of AT in the regulation of clot formation, hereditary AT deficiency is considered a “classical” thrombophilic risk factor and is associated with the highest risk of venous thromboembolism (VTE) among the hereditary thrombophilias [4].

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