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Thromb Haemost 2016; 116(06): 1032-1040
DOI: 10.1160/TH16-04-0306
Coagulation and Fibrinolysis
Schattauer Publishers Schattauer

Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1

Xiaohua Zhou
1   Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
,
Maarten L. V. Hendrickx
1   Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
,
Gholamreza Hassanzadeh-Ghassabeh
2   Cellular and Molecular Immunology, Faculty of Science and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium
3   Nanobody Service Facility, VIB, Brussels, Belgium
,
Serge Muyldermans
2   Cellular and Molecular Immunology, Faculty of Science and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium
,
Paul J. Declerck
1   Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium
› Author Affiliations Financial support: This study was supported by the Fund for Scientific Research–Flanders (FWO-Vlaanderen) (grant G.0594.13 to P. J. D.)
Further Information

Publication History

Received 18 April 2016

Accepted after major revision: 15 August 2016

Publication Date:
09 March 2018 (online)

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Summary

Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1•t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

Keywords

Cardiovascular diseases - fibrinolysis - nanobodies - neutralising antibody fragments - plasminogen activator inhibitor 1
 

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