Thromb Haemost 2020; 120(07): 1045-1055
DOI: 10.1055/s-0040-1710590
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Thr90Ser Mutation in Antithrombin is Associated with Recurrent Thrombosis in a Heterozygous Carrier

Yeling Lu
1  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
2  Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
Bruno O. Villoutreix
3  Drugs and Molecules for Living Systems, Inserm, Institut Pasteur de Lille, University of Lille, Lille, France
,
Indranil Biswas
2  Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
Qiulan Ding
1  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Xuefeng Wang
1  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Alireza R. Rezaie
2  Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
4  Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
› Institutsangaben
Funding This study was supported by an institutional fund from OMRF and grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health HL101917 and HL062565 to A.R.R.; and The General Program of National Natural Science Foundation of China (81570114) to Q.D. and (81870107) to Y.L.
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Publikationsverlauf

27. Februar 2020

08. April 2020

Publikationsdatum:
18. Mai 2020 (online)

Abstract

Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.

Authors' Contributions

L.Y. designed experiments and performed research; B.O.V. performed molecular modeling and contributed to writing of the related section; I.B. conducted the permeability assays; X.W. and Q.D. designed experiments, supervised studies conducted in Ruijin Hospital with the subjects' plasma; A.R.R. designed experiments, analyzed data, wrote the manuscript, and supervised the project. All authors approved the final version of this manuscript.


Supplementary Material