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

TitleIn vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody.
Publication TypeJournal Article
Year of Publication2014
AuthorsHendrickx, M. L. V., M. Zatloukalova, G. Hassanzadeh-Ghassabeh, S. Muyldermans, A. Gils, and P. J. Declerck
JournalThromb Haemost
Volume111
Issue5
Pagination824-32
Date Published2014 May 5
ISSN0340-6245
Abstract

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 (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) 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.

DOI10.1160/TH13-08-0645
Alternate JournalThromb. Haemost.
PubMed ID24402608
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