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Thromb Haemost 2004; 92(01): 47-53
DOI: 10.1160/TH04-01-0047
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide

Christoph E. Hagemeyer   *
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Ivo Tomic   *
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Patrick Jaminet   *
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Uta Weirich
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Nicole Bassler
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Meike Schwarz
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Marschall S. Runge
2   Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
,
Christoph Bode
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
,
Karlheinz Peter
1   Department of Cardiology, University of Freiburg, Freiburg, Germany
2   Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
› Author Affiliations
Further Information

Publication History

Received 25 January 2004

Accepted after revision 12 April 2004

Publication Date:
29 November 2017 (online)

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Summary

We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv59D8). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clotbound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications. TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for antifXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni2+ and β15-22-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv59D8-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv59D8-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

Keywords

Coagulation inhibitors - drug design - fibrinogen / fibrin

* These authors contributed equally to this study


 

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