In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody

Maarten L. V. Hendrickx; Monika Zatloukalova; Gholamreza Hassanzadeh-Ghassabeh; Serge Muyldermans; Ann Gils; Paul J. Declerck

doi:10.1160/TH13-08-0645

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(05): 824-832
DOI: 10.1160/TH13-08-0645

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody

Authors

Maarten L. V. Hendrickx

¹Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
Monika Zatloukalova

¹Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
Gholamreza Hassanzadeh-Ghassabeh

²Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium

³Nanobody Service facility (NSF), VIB, Brussels, Belgium
Serge Muyldermans

²Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium

⁴VIB Department of Structural Biology, Brussels, Belgium
Ann Gils

¹Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
Paul J. Declerck

¹Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium

Abstract

Summary

One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC₅₀ of 0.46 (molar ratio nanobody over TAFI). The affinity (K_A) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 × 10¹⁰ M⁻¹ and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys³⁹² is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr¹⁷⁵ and Glu¹⁸³. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.

Keywords

TAFI - nanobody - fibrinolysis - TAFI inhibition - profibrinolytic - thromboembolism

Full Text

References

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