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Thromb Haemost 2005; 93(03): 430-436
DOI: 10.1160/TH04-08-0480
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Improvement of the specificity of dipetarudin by site directed mutagenesis

Mercedes López
1   Instituto Venezolano de Investigaciones Científicas, Laboratorio de Trombosis Experimental, Centro de Biofísica y Bioquímica, Caracas, Venezuela
,
Katrin Mende
2   Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany
,
Goetz Nowak
2   Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany
› InstitutsangabenFinancial support: This work was partly supported by Fresenius Medical Care GmbH, Germany.
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Publikationsverlauf

Received 05. August 2004

Accepted after resubmission 07. Februar 2004

Publikationsdatum:
14. Dezember 2017 (online)

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Summary

Protease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudin R10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudin R10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).

Keywords

Dipetarudin - specificity - P1 position - site directed mutagenesis - thrombin
 

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