In vitro exploration of latent prothrombin mutants conveying antithrombin resistance

Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR...

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Bibliographic Details
Published in:Thrombosis research 2017-11, Vol.159, p.33-38
Main Authors: Tamura, Shogo, Murata-Kawakami, Moe, Takagi, Yuki, Suzuki, Sachiko, Katsumi, Akira, Takagi, Akira, Kojima, Tetsuhito
Format: Article
Language:English
Subjects:
Antithrombin
Antithrombin resistance
Antithrombins - pharmacology
Antithrombins - therapeutic use
Drug Resistance, Neoplasm - genetics
Humans
Mutant prothrombin
Mutation
Prothrombin
Prothrombin - pharmacology
Prothrombin - therapeutic use
Residual clotting activity score
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Summary:Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade. •Mutations of prothrombin at the sodium-binding domain in thrombin cause ATR phenotypes.•RCA score is a useful index to estimate thrombosis risk of ATR-prothrombin mutants.•Prothrombins Lys599Arg and Glu592Gln potentially increase the risk of thrombosis.
ISSN:0049-3848
1879-2472
DOI:10.1016/j.thromres.2017.09.020