Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene

Picard, Véronique; Bura, Alessandra; Emmerich, Joseph; Alhenc‐Gelas, Martine; Biron, C.; Houbouyan-Reveillard, Lucie L.; Molho, P; Labatide-Alanore, Agnès; Sié, Pièrre; Toulon, Pierre; Verdy, E; Aiach, Martine

doi:10.1046/j.1365-2141.2000.02245.x

Cited by 16 publications

(12 citation statements)

References 11 publications

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“…The S365L carrier is a 59-year-old woman, who developed recurrent deep venous thrombosis (first episode at the age of 43). This mutation is not described, although another mutation also changing the same residue to proline has been described in a patient with type I deficiency (20). Contrasting the type I deficiency profile, our patient showed heparin cofactor anti-Xa and anti-IIa activity values severely reduced by 54 and 50%, respectively, but only slightly reduced antigen levels (71%).…”

Section: Identification Of Patients With Type II Antithrombin Deficiementioning

confidence: 74%

“…Characterization of antithrombin deficiency was done as described elsewhere (19,20). Briefly, (i) plasma antithrombin activity (anti-FXa and anti-FIIa) was determined by chromogenic assays in the presence of heparin; (ii) antigen levels were determined by immunodiffusion and ELISA; (iii) heparin affinity of plasma antithrombin was evaluated by crossed-immunoelectrophoresis; (iv) electrophoretic features of plasma antithrombin were assayed using native-PAGE (in the presence and absence of 6 M urea) and SDS-PAGE under reducing and nonreducing conditions; and (v) molecular analysis of SERPINC1, the gene encoding antithrombin, was performed by PCR amplification and sequencing.…”

Section: Functional and Genetic Analysis Of Patients And Family Membersmentioning

confidence: 99%

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Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Águila

Izaguirre

Martínez‐Martínez

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellAntithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.Serine protease inhibitors (serpins) are a superfamily of proteins that control proteases involved in inflammation, complement, coagulation, and fibrinolytic pathways (1). Inhibitory serpins exert control over their target proteases by an unusual branched pathway suicide substrate mechanism. Antithrombin inhibits its target proteases by this branched pathway in a manner that preferentially promotes the formation of a stable inhibitory covalent complex with the target protease instead of allowing the proteolytic cleavage of the inhibitor (2). This serpin requires activation by heparin to achieve a physiologically significant rate of inhibition of its target proteases. Heparin activates antithrombin by two mechanisms. In one mechanism, heparin serves as a polysaccharide bridge to promote the encounter between protease and antithrombin, whereas in the second mechanism, the serpin is activated through conformational changes, which enhance reactivity with protease and involve several exosite interactions (3-6). The first mechanism is dominant with the protease, thrombin, and the second selectively enhances antithrombin reactivity with factor Xa and factor IXa. In the serpin inhibitory mechanism, target proteases initially recognize and bind a substrate amino acid sequence in the reactive center loop (RCL) 3 of the...

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Section: Identification Of Patients With Type II Antithrombin Deficiementioning

confidence: 74%

Section: Functional and Genetic Analysis Of Patients And Family Membersmentioning

confidence: 99%

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Águila

Izaguirre

Martínez‐Martínez

et al. 2017

Journal of Biological Chemistry

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“…DNA was isolated by the Miller method 23 and stored at 4°C. The 7 AT exons and intron-exon junctions were amplified by polymerase chain reaction (PCR) as previously described 24 (primer sequences and amplification program available on request). PCR products (8 L) were submitted to digestion with exonuclease I (10 U) and shrimp alkaline phosphatase (2 U) for 15 minutes at 37°C, using a PCR product presequencing kit (USB; Amersham Biosciences, Freiburg, Germany).…”

Section: Dna Studiesmentioning

confidence: 99%

Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis

Picard

Dautzenberg

Villoutreix

et al. 2003

Blood

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There is increasing evidence that serpin conformational alteration caused by single point mutations can be responsible for protein deficiency associated with human diseases. A typical example is the 1-antitrypsin deficiency caused by the Z variant carrying a Glu342Lys substitution. Only a few cases of "conforma-tional disease" involving other serpins have been described so far. We investigated a severe antithrombin deficiency in a 13-month-old child with fever and cere-bral venous thrombosis. The infant was found to be homozygous for a new anti-thrombin gene mutation (7396T>C, predicting a Phe229Leu antithrombin variant), and heterozygous for the factor V Leiden mutation. Mild atypical antithrom-bin deficiency was found in both parents, who were first cousins, asymptomatic, and heterozygous for the same antithrom-bin gene mutation. The Phe229Leu variant , which does not readily fit into the current classification of antithrombin deficiency , was shown to be a thermolabile antithrombin that spontaneously polymer-ized in the proband's circulation. This points to a key role for the conserved Phe at position 229, which is near the reactive site loop in a region critical for serpin function and stability. Molecular model-ing suggested how the mutation might destabilize this region of the protein and thereby favor reactive site loop insertion and polymerization. This study provides the first direct evidence of antithrombin polymerization in vivo causing antithrom-bin deficiency and severe thrombotic disease. (Blood. 2003;102:919-925)

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“…Since Koide et al [10] reported the molecular defect of AT deficiency (AT Toyama, Arg47Cys) for the first time in 1984, more than 150 different AT gene mutations have been identified, about 70% of which belong to type I deficiency [5,[11][12][13][14][15]. Identification of gene mutations occurring in AT-deficient pedigrees and further molecular characterization would give important information on the structure-function relationships of this major hemostatic protein.…”

Section: Discussionmentioning

confidence: 99%

Characterization of molecular defect of 13387-9delG mutated antithrombin in inherited type I antithrombin deficiency

Wang

Ding

et al. 2005

Blood Coagulation &Amp; Fibrinolysis

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As a major physiological inhibitor of thrombin and other coagulation proteases, antithrombin (AT) plays an important role in the maintenance of normal hemostasis and its deficiency is associated with a predisposition for familial venous thromboembolic disease. Recently, we found a novel mutation (13387-9delG) in the antithrombin gene that is associated with type I AT deficiency. To examine the molecular pathologic mechanism of this mutation causing type I AT deficiency, the wild-type and the mutant AT constructs were expressed in COS-7 cells or Chinese Hamster Ovary cells. No AT antigen could be detected by enzyme-linked immunosorbent assay in the conditioned media of cells expressing the mutant protein, and the AT antigen level was reduced in cell lysates. The mutant AT-expressing cells did not have less intracellular mRNA levels than the wild-type transfectants as estimated by quantitative reverse transcriptase-polymerase chain reaction. Metabolic and pulse-chase experiments showed the newly synthesized wild-type AT protein was gradually secreted into the media, whereas no labeled mutant AT protein was detected in the media and the total amount of radioactivity was significantly reduced in the cells during the chase periods. By immunofluorescence analysis, the staining of the mutant AT was weaker than that of the wild type, and was predominantly diffuse without perinuclear enhancement. These results indicate that the 13387-9delG mutation, which disrupts the disulfide bridge Cys247-Cys430, impairs the secretion and stability of the truncated AT protein associated with intracellular degradation.

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Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene

Cited by 16 publications

References 11 publications

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis

Characterization of molecular defect of 13387-9delG mutated antithrombin in inherited type I antithrombin deficiency

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