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

Xiaohua Zhou; Maarten L. V. Hendrickx; Gholamreza Hassanzadeh-Ghassabeh; Serge Muyldermans; Paul J. Declerck

doi:10.1160/TH16-04-0306

Thrombosis and Haemostasis, Table of Contents

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

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

,

Maarten L. V. Hendrickx

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

,

Gholamreza Hassanzadeh-Ghassabeh

²Cellular and Molecular Immunology, Faculty of Science and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium

³Nanobody Service Facility, VIB, Brussels, Belgium

,

Serge Muyldermans

²Cellular and Molecular Immunology, Faculty of Science and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium

,

Paul J. Declerck

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

› Author Affiliations

Abstract

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

Full Text

References

References
1 Kruithof EKO, Tran-Thang C, Ransijn A. et al. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913.
2 Simpson AJ, Booth NA, Moore NR. et al. Distribution of plasminogen activator inhibitor (PAI-1) in tissues. J Clin Pathol 1991; 44: 139-143.
3 Sancho E, Declerck PJ, Price NC. et al. Conformational studies on plasminogen activator inhibitor (PAI-1) in active, latent, substrate, and cleaved forms. Biochemistry 1995; 34: 1064-1069.
4 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000; 407: 923-926.
5 Mottonen J, Strand A, Symersky J. et al. Structural basis of latency in plasmi-nogen activator inhibitor-1. Nature 1992; 355: 270-273.
6 Kjøller L, Martensen PM, Sottrup-Jensen L. et al. Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1. Eur J Biochem 1996; 241: 38-46.
7 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plas-minogen activators that can be activated by denaturants. J Biol Chem 1985; 260: 11581-11587.
8 Katagiri K, Hattori H, Yano M. Bovine endothelial cell plasminogen activator inhibitor: purification and heat activation. Eur J Biochem 1988; 87: 81-87.
9 Declerck PJ, De Mol M, Vaughan DE. et al. Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a non-inhibitory substrate for tissue-type plasminogen activator. J Biol Chem 1992; 267: 11693-11696.
10 Urano T, Strandberg L, Johansson LB. et al. A substrate-like form of plasmi-nogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate. Eur J Biochem 1992; 209: 985-992.
11 Munch M, Heegaard CW, Andreasen PA. Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor. Biochim Biophys Acta 1993; 1202: 29-37.
12 De Taeye B, Compernolle G, Dewilde M. et al. Immobilisation of the distal hinge in the labile serpin plasminogen activator inhibitor 1: Identification of a transition state with distinct conformational and functional properties. J Biol Chem 2003; 278: 23899-23905.
13 Ehrlich HJ, Gebbink RK, Keijer J. et al. Alteration of serpin specificity by a protein cofactor: Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties. J Biol Chem 1990; 265: 13029-13035.
14 Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1 and coronary artery disease. N Engl J Med 2000; 342: 1792-1801.
15 Lindgren A, Lindoff C, Norrving B. et al. Tissue plasminogen activator and plas-minogen activator inhibitor-1 in stroke patients. Stroke 1996; 27: 1066-1071.
16 Hamsten A, De Faire U, Walldius G. et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; 02: 3-9.
17 Van De Craen B, Declerck PJ, Gils A. The biochemistry, physiology and pathological roles of PAI-1 and the requirements for PAI-1 inhibition in vivo. Thromb Res 2012; 130: 576-585.
18 Declerck PJ, Gils A. Three decades of research on plasminogen activator in-hibitor-1: A multifaceted serpin. Semin Thromb Hemost 2013; 39: 356-364.
19 Hamers-Casterman C, Atarhouch T, Muyldermans S. et al. Naturally-occurring antibodies devoid of light chains. Nature 1993; 363: 446-448.
20 Rasmussen SGF, Choi H-J, Fung JJ. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 2011; 469: 175-180.
21 Lauwereys M, Ghahroudi MA, Desmyter A. et al. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 1998; 17: 3512-3520.
22 Zhou X, Weeks SD, Ameloot P. et al. Elucidation of the molecular mechanisms of two nanobodies that inhibit TAFI activation and TAFIa activity. J Thromb Haemost 2016 in print.
23 Buelens K, Hassanzadeh-Ghassabeh G, Muyldermans S. et al. Generation and characterisation of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor. J Thromb Haemost 2010; 08: 1302-1312.
24 Bijnens A, Gils A, Knockaert I. et al. Importance of the hinge region between alpha-helix F and the main part of serpins, based upon identification of the epitope of plasminogen activator inhibitor type 1 neutralizing antibodies. J Biol Chem 2000; 275: 6375-6380.
25 Gils A, Pedersen KE, Skottrup P. et al. Biochemical importance of glycosylation of plasminogen activator inhibitor-1. Thromb Haemost 2003; 90: 206-217.
26 Nar H, Bauer M, Stassen JM. et al. Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation. J Mol Biol 2000; 297: 683-695.
27 Van De Craen B, Declerck PJ, Gils A. Glycosylation influences the stability of human plasminogen activator inhibitor-1. Blood Coagul Fibrinolysis 2012; 23: 570-572.
28 Verheijen JH, Chang GT, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Hae-most 1984; 51: 392-395.
29 Dewilde M, Van De Craen B, Compernolle G. et al. Subtle structural differences between human and mouse PAI-1 reveal the basis for biochemical differences. J Struct Biol 2010; 171: 95-101.
30 Ngo TH, Hoylaerts MF, Knockaert I. et al. Identification of a target site in plas-minogen activator inhibitor-1 that allows neutralisation of its inhibitory properties concomitant with an allosteric up-regulation of its antiadhesive properties. J Biol Chem 2001; 276: 26243-26248.
31 De Taeye B, Gils A, Declerck PJ. The story of the serpin plasminogen activator inhibitor I: Is there any need for another mutant?. Thromb Haemost 2004; 92: 898-924.
32 Mutch NJ, Thomas L, Moore NR. et al. TAFIa, PAI-1 and alpha2-antiplasmin: Complementary roles in regulating lysis of thrombi and plasma clots. J Thromb Haemost 2007; 05: 812-817.
33 Juhan-Vague I, Alessi MC. PAI-1, obesity, insulin resistance and risk of cardiovascular events. Thromb Haemost 1997; 78: 656-660.
34 Bijnens AP, Gils A, Stassen JM. et al. The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity. J Biol Chem 2001; 276: 44912-44918.
35 Debrock S, Declerck PJ. Identification of a functional epitope in plasminogen activator inhibitor-1, not localized in the reactive centre loop. Thromb Haemost 1998; 79: 597-601.
36 Naessens D, Gils A, Compernolle G. et al. Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1. J Thromb Haemost 2003; 01: 1028-1033.
37 Madison EL, Goldsmith EJ, Gerard RD. et al. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature 1989; 339: 721-724.
38 Madison EL, Goldsmith EJ, Gethings M-JH. et al. Restoration of serine pro-tease-inhibitor interaction by protein engineering. J Biol Chem 1990; 265: 21423-21426.
39 Gong L, Liu M, Zeng T. et al. Crystal structure of the Michaelis complex between tissue-type plasminogen activator and plasminogen activators in-hibitor-1. J Biol Chem 2015; 290: 25795-25804.