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

Journal: Thrombosis and Haemostasis
ISSN: 0340-6245
Issue: 2016: 116/6 (Dec) pp. 1003–1181
Pages: 1032-1040
Ahead of Print: 2016-09-08

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

X. Zhou (1), M. L. V. Hendrickx (1), G. Hassanzadeh-Ghassabeh (2, 3), S. Muyldermans (2), P. J. Declerck (1)

(1) Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium; (2) Cellular and Molecular Immunology, Faculty of Science and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium; (3) Nanobody Service Facility, VIB, Brussels, Belgium


fibrinolysis, cardiovascular diseases, nanobodies, neutralising antibody fragments, plasminogen activator inhibitor 1


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-1t-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.

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