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Thromb Haemost 1992; 68(03): 291-296
DOI: 10.1055/s-0038-1656367
Original Article
Schattauer GmbH Stuttgart

Molecular Cloning and Cell-Free Expression of Mouse Antithrombin III

John K Wu
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
,
William P Sheffield
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
,
Morris A Blajchman
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
2   The Department of Pathology, McMaster University, Hamilton, Ontario, Canada
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Publikationsverlauf

Received 05. Dezember 1991

Accepted after revision 06. April 1992

Publikationsdatum:
04. Juli 2018 (online)

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Summary

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human α-thrombin efficiently (second order rate constant [K 2nd] 5.8 × 103 M–1 s–1) as compared to human AT-III (K 2nd 6.7 × 103 M–1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a λZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5’ untranslated region, followed by a 75 bp 3’ untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human α-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

 

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