RSS-Feed abonnieren
DOI: 10.1160/TH10-08-0538
Molecular basis of antithrombin deficiency
Publikationsverlauf
Received:
18. August 2010
Accepted after major revision:
16. Januar 2011
Publikationsdatum:
28. November 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/201104/lookinside/thumbnails/10-1160-th10-08-0538-1.jpg)
Summary
Antithrombin (AT) is the most important physiological inhibitor of coagulation proteases. It is activated by glycosaminoglycans such as heparin. Hereditary antithrombin deficiency is a rare disease that is mainly associated with venous thromboembolism. So far, more than 200 different mutations in the antithrombin gene (SERPINC1) have been described. The aim of our study was to characterise the molecular background in a large cohort of patients with AT deficiency. Mutation analysis was performed by direct sequencing of SERPINC1 in 272 AT-deficient patients. Large deletions were identified by multiplex PCR coupled with liquid chromatography or multiplex ligation-dependent probe amplification (MLPA) analysis. To predict the effect of SERPINC1 sequence variations on the pathogenesis of AT deficiency, in silico assessments, multiple sequence alignment, and molecular graphic imaging were performed. The mutation profile consisted of 59% missense, 10% non-sense, 8% splice site mutations, 15% small deletions/insertions/duplications, and 8% large deletions. Altogether 87 different mutations, including 42 novel mutations (22 missense and 20 null mutations), were identified. Of the novel missense mutations, nine are suspected to impair the conformational changes that are needed for AT activation, two to affect the central reactive loop or the heparin binding site, and six to impair the structural integrity of the molecule. Despite the heterogeneous background of AT deficiency, 10 AT variants occurred in multiple index patients. Characterisation of the SERPINC1 mutation profile in large cohorts of patients may help to further elucidate the pathogenesis of AT deficiency and to establish genotype-phenotype associations.
-
References
- 1 Silverman GA. et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 2001; 276: 33293-33296.
- 2 Bock SC. et al. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res 1982; 10: 8113-8125.
- 3 Huber R, Carrell RW. Implications of the three-dimensional structure of α1-anti-thripsin for the structure and function of serpins. Biochemistry 1989; 28: 8951-8966.
- 4 Kurachi K. et al. Inhibition of bovine factor IXa and factor Xab by antithrombin III. Biochemistry 1976; 15: 373-377.
- 5 Buchanan MR. et al. The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin. Blood 1985; 65: 198-201.
- 6 Björk I, Olson ST. Antithrombin. A bloody important serpin. Adv Exp Med Biol 1997; 425: 17-33.
- 7 Wright HT, Scarsdale JN. Structural basis for serpin inhibitor activity. Proteins 1995; 22: 210-225.
- 8 Jin L. et al. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci USA 1997; 94: 14683-14688.
- 9 Huntington JA. et al. Mechanism of heparin activation of antithrombin: evidence for reactive center loop preinsertion with expulsion upon heparin binding. Biochemistry 1996; 35: 8495-8503.
- 10 Yang L. et al. Contribution of exosite occupancy by heparin to the regulation of coagulation proteases by antithrombin. Thromb Haemost 2010; 103: 277-283.
- 11 Van Boven HH, Lane DA. Antithrombin and its inherited deficiency states. Semin Hematol 1997; 34: 188-204.
- 12 Lijfering WM. et al. Selective testing for thrombophilia in patients with first venous thrombosis. Results from a retrospective family cohort study on absolute thrombotic risk for currently known thrombophilic defects in 2479 relatives. Blood 2009; 113: 5314-5322.
- 13 Ishiguro K. et al. Complete antithrombin deficiency in mice results in embryonic lethality. J Clin Invest 2000; 106: 873-878.
- 14 Corral J. et al. Antithrombin Cambridge II (A384S): an underestimated genetic risk factor for venous thrombosis. Blood 2007; 109: 4258-4263.
- 15 Kuhle S. et al. Homozygous antithrombin deficiency type II (99 Leu to Phe mutation) and childhood thromboembolism. Thromb Haemost 2001; 86: 1007-1011.
- 16 Rossi E. et al. Report of a novel kindred with antithrombin heparin-binding site variant (47 Arg to His): demand for an automated progressive antithrombin assay to detect molecular variants with low thrombotic risk. Thromb Haemost 2007; 98: 695-697.
- 17 Tait RC. et al. Influence of demographic factors on antithrombin III activity in a healthy population. Br J Haematol 1993; 84: 476-480.
- 18 Wells PS. et al. Prevalence of antithrombin deficiency in healthy blood donors: a cross-sectional study. Am J Hematol 1994; 45: 321-324.
- 19 Perry DJ, Carrell RW. Molecular genetics of human antithrombin deficiency. Hum Mutat 1996; 7: 7-22.
- 20 Rosendaal FR. Risks factors for venous thrombotic disease. Thromb Haemost 1999; 82: 610-619.
- 21 Rossi E. et al. The risk of symptomatic pulmonary embolism due to proximal deep venous thrombosis differs in patients with different types of inherited thrombophilia. Thromb Haemost 2008; 99: 1030-1034.
- 22 Lane DA. et al. Inherited thrombophilia: Part I. Thromb Haemost 1996; 76: 651-662.
- 23 Finazzi G. et al. Different prevalence of thromboembolism in the subtypes of congenital antithrombin deficiency: review of 404 cases. Thromb Haemost 1987; 58: 1094.
- 24 Bock SC. et al. Assignment of the human antithrombin III structural gene to chromosome 1q23–25. Cytogenet Cell Genet 1985; 39: 67-69.
- 25 Olds RJ. et al. Complete nucleotide sequence of the antithrombin gene: evidence for homologous recombination causing thrombophilia. Biochemistry 1993; 32: 4216-4224.
- 26 Lane DA. et al. Antithrombin mutation database: 2nd (1997) Update. Thromb Haemost 1997; 77: 197-211.
- 27 Bayton T, Lane D. Antithrombin Mutation Data Base. Departement of Haematology, Imperial College Faculty of Medicine, Charing Cross Hospital Campus, London, UK. 2003. Available at: http://www.med.ic.ac.uk/divisions/7/antithrombin
- 28 Beauchamp NJ. et al. Major structural defects in the antithrombin gene in four families with Type I antithrombin deficiency. Thromb Haemost 2000; 83: 715-721.
- 29 Pavlova A. et al. Detection of heterozygous large deletions in the antithrombin gene using multiplex polymerase chain reaction and denatured high performance liquid chromatography. Haematologica 2006; 91: 1264-1267.
- 30 Picard V. et al. Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene. Hum Mutat 2006; 27: 600.
- 31 Human Gene Mutation Database. Available at: http://www.hgmd.cf.ac.uk/ac/index.php accessed July and November 2010.
- 32 Miller SA. et al. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215.
- 33 Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31: 3812-3814.
- 34 Ramensky V. et al. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002; 30: 3894-3900.
- 35 Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency. A model for conformational diseases. N Engl J Med 2002; 346: 45-53.
- 36 Langdown J. et al. The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin. J Mol Biol 2009; 386: 1278-1289.
- 37 Mille B. et al. Role of N- and C-terminal amino acids in antithrombin binding to pentasaccharide. J Biol Chem 1994; 269: 29435-29443.
- 38 Hultin MB. et al. Antithrombin Oslo: type Ib classification of the first reported antithrombin-deficient family, with a review of hereditary antithrombin variants. Thromb Haemost 1988; 59: 468-473.
- 39 Meagher JL. et al. Critical role of the linker region between helix D and strand 2A in heparin activation of antithrombin. J Biol Chem 2000; 28 (275) 2698-2704.
- 40 National Center for Biotechnology Information (NCBI). dbSNP Home Page. Available at: http://www.ncbi.nlm.nih.gov/projects/SNP Accessed November 2010.
- 41 Daly M. et al. Antithrombin Dublin (- 3 Val+Glu): an N-terminal variant which has an aberrant signal peptidase cleavage site. FEBS Lett 1990; 273: 87-90.
- 42 Dürr C. et al. Genetic studies of antithrombin III with IEF and ASO hybridization. Hum Genet 1992; 90: 457-459.
- 43 Hernández-Espinosa D. et al. Factors with conformational effects on haemostatic serpins: implications in thrombosis. Thromb Haemost 2007; 98: 557-563.
- 44 Picard V. et al. Two new antithrombin variants support a role for K114 and R13 in heparin binding. J Thromb Haemost 2003; 1: 386-387.