Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor

Background and objective: As activated thrombin‐activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid‐derived sin...

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Published in:Journal of thrombosis and haemostasis 2010-06, Vol.8 (6), p.1302-1312
Main Authors: BUELENS, K., HASSANZADEH‐GHASSABEH, G., MUYLDERMANS, S., GILS, A., DECLERCK, P. J.
Format: Article
Language:English
Subjects:
Biocatalysis
Carboxypeptidase B2 - antagonists & inhibitors
fibrinolysis
inhibition
nanobodies
Nanotechnology
profibrinolytic
Recombinant Proteins - antagonists & inhibitors
TAFI
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Summary:Background and objective: As activated thrombin‐activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid‐derived single‐domain antibodies towards TAFI. Methods and results: This study reports the generation and characterization of a panel of 22 inhibitory nanobodies. This panel represents a wide diversity in mechanisms for interference with the functional properties of TAFI as the nanobodies interfere with various modes of TAFI activation, TAFIa activity and/or TAFI zymogen activity. Nanobodies inhibiting TAFIa activity and thrombin/thrombomodulin‐mediated TAFI activation revealed profibrinolytic properties in a clot lysis experiment with exogenously added thrombomodulin (TM), whereas nanobodies inhibiting plasmin‐mediated TAFI activation only revealed profibrinolytic properties in a clot lysis experiment without TM. The results of in vitro clot lysis experiments provided evidence that inhibitory nanobodies penetrate the clot better compared with inhibitory monoclonal antibodies. Conclusions: These data suggest that the generated nanobodies are potent TAFI inhibitors and are a step forward in the development of a profibrinolytic drug. They might also be an excellent tool to unravel the role of the physiological activators of TAFI in various pathophysiological processes.
ISSN:1538-7933
1538-7836
1538-7836
DOI:10.1111/j.1538-7836.2010.03816.x