Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

A. P. Bijnens; T. H. Ngo; A. Gils; J. Dewaele; I. Knockaert; J. M. Stassen; P. J. Declerck

doi:10.1055/s-0037-1615761

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2001; 85(05): 866-874
DOI: 10.1055/s-0037-1615761

Review Article

Schattauer GmbH

Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

Autoren

A. P. Bijnens

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
T. H. Ngo

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
A. Gils

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
J. Dewaele

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
I. Knockaert

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
J. M. Stassen

²Cardiovascular Pharmacology, Boehringer Ingelheim, Pharma KG, Biberach a.d. Riss, Germany
P. J. Declerck

¹Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium

Abstract

Summary

Increased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore, eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising -helices hD, hE and hF, -strands s4C, s3A, s2A and s1A and the loops connecting these elements), (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data, together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.

Keywords

PAI-1 - epitope - chimera - antibody - serpin

Volltext

Referenzen

References
1 Kruithof EK, Tran Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-13.
2 Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 1986; 83: 6776-80.
3 Pannekoek H, Veerman H, Lambers H, Diergaarde P, Verweij CL, van Zonneveld AJ, van Mourik JA. Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. EMBO J 1986; 5: 2539-44.
4 Vaughan DE, Declerck PJ, Van Houtte E, De Mol M, Collen D. Reactivated recombinant plasminogen activator inhibitor-1 (rPAI-1) effectively prevents thrombolysis in vivo . Thromb Haemost 1992; 68: 60-3.
5 Booth NA, Robbie LA, Croll AM, Bennett B. Lysis of platelet-rich thrombi: the role of PAI-1. Ann N Y Acad Sci 1992; 667: 70-80.
6 Juhan Vague I, Alessi MC, Declerck PJ. Pathophysiology of fibrinolysis. Baillieres Clin Haematol 1995; 8: 329-43.
7 Prins MH, Hirsh J. A critical review of the evidence supporting a relationship between impaired fibrinolytic activity and venous thromboembolism. Arch Intern Med. 1991; 151: 1721-31.
8 Wiman B. Predictive value of fibrinolytic factors in coronary heart disease. Scand J Clin Lab Invest 1999; 59: 23-31.
9 Declerck PJ, De Mol M, Alessi MC, Baudner S, Paques EP, Preissner KT, Müller-Berghaus G, Collen D. Purification and characterization of a plasminogen activator inhibitor-1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin). J Biol Chem 1988; 263: 15454-61.
10 Bajou K, Noel A, Gerard RD, Masson V, Brunner N, HolstHansen C, Skobe M, Fusenig NE, Carmeliet P, Collen D, Foidart JM. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nature Medicine 1998; 4: 923-28.
11 Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 gene deficient mice. II. Effects on hemostasis, thrombosis and thrombolysis. J Clin Invest 1993; 92: 2756-60.
12 Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature 1990; 346: 74-6.
13 Levi M, Biemond BJ, van Zonneveld AJ, ten Cate JW, Pannekoek H. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85: 305-12.
14 Biemond BJ, Levi M, Coronel R, Janse MJ, ten Cate JW, Pannekoek H. Thrombolysis and reocclusion in experimental jugular vein and coronary artery thrombosis. Effects of a plasminogen activator inhibitor type 1-neutralizing monoclonal antibody. Circulation 1995; 91: 1175-81.
15 Berry CN, Lunven C, Lechaire I, Girardot C, O’Connor SE. Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis. Br J Pharmacology 1998; 125: 29-34.
16 Eitzman DT, Fay WP, Lawrence DA. et al. Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro . J Clin Invest 1995; 95: 2416-20.
17 Kvassman JO, Verhamme I, Shore JD. Inhibitory mechanism of serpins: loop insertion forces acylation of plasminogen activator by plasminogen activator inhibitor-1. Biochemistry 1998; 37: 15491-502.
18 Xue Y, Björquist P, Inghardt T, Linschoten M, Musil D, Sjölin L, Deinum J. Interfering with the inhibitory meachnism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide. Structure 1998; 6: 627-36.
19 Bryans J, Charlton P, Chicarelli-Robinson I, Collins M, Faint R, Latham C, Shaw I, Trew S. Inhibition of plasminogen activator inhibitor 1 activity by two diketopiperazines, XR330 and XR334 produced by Streptomyces species. J Antibiot 1996; 49: 1014-21.
20 Charlton PA, Faint RW, Bent F, Bryans J, Chicarelli-Robinson I, Mackie I, Machin S, Bevan P. Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. Thromb Haemost 1996; 75: 808-15.
21 Friederich PW, Levi MR, Biemond BJ, Charlton PA, Templeton D, van Zonneveld AJ, Bevan P, Pannekoek H, Tencate J. Novel low molecular weight inhibitor of PAI-1 (XR5118) promotes endogenous fibrinolysis and reduces postthrombolysis thrombus growth in rabbits. Circulation 1997; 96: 916-21.
22 Charlton PA, Faint RW, Barnes C, Bent F, Folkes A, Templeton D, Mackie I, Machin S, Bevan P. XR5118, a novel modulator of plasminogen activator inhibitor 1 (PAI-1), increases endogenous tpa activity in the rat. Fibrinolysis and Proteolysis 1997; 11: 51-6.
23 Bjorquist P, Ehnebom J, Inghardt T, Hansson L, Lindberg M, Linschoten M, Stromqvist M, Deinum J. Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site- directed mutagenesis. Biochemistry 1998; 37: 1227-34.
24 Neve J, de Almeida Leone P, Carroll AR, Moni R, Paczkowski N, Pierens G, Bjorquist P, Deinum J, Ehnebom J, Inghardt T, Guymer G, Grimshaw P, Quinn R. Sideroxylonal C a new inhibitor of human plasminogen activator inhibitor type-1, from the flowers of Eucalyptus albens . J Nat Prod 1999; 62: 324-6.
25 Gils A, Stassen JM, Kley JT, Wienen W, Ries UJ, Declerck PJ. Comparative analysis of PAI-1 inhibitors. Fibrinolysis and Proteolysis 2000; 14: 72.
26 Debrock S, Declerck PJ. Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms. Biochim Biophys Acta 1997; 1337: 257-66.
27 Gils A, Declerck PJ. Structure-function relationship in serpins: current concepts and controversies. Thromb Haemost 1998; 80: 531-41.
28 Verhamme L, Kvassman JQ, Day DE, Debrock S, Vleugels N, Declerck PJ, Shore JD. J Biol Chem 1999; 274: 17511-17.
29 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 1985; 260: 11581-7.
30 Declerck PJ, De Mol M, Vaughan DE, Collen D. Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator. J Biol Chem 1992; 267: 11693-6.
31 Urano T, Strandberg L, Johansson LB, Ny T. A substrate-like form of plasminogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate. Eur J Biochem 1992; 209: 985-92.
32 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.
33 Perrie AM, MacGregor IR, Booth NA. Definition of epitopes within plasminogen activator inhibitor type-1 (PAI-1) using multiple peptide synthesis. Fibrinolysis 1993; 7: 257-63.
34 van Zonneveld AJ, van den Berg BM, van Meijer M, Pannekoek H. Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries. Gene 1995; 167: 49-52.
35 Debrock S, Declerck PJ. Identification of a functional epitope in plasminogen activator inhibitor-1, not localized in the reactive center loop. Thromb Haemost 1998; 79: 597-601.
36 Keijer J, Linders M, van Zonneveld AJ, Ehrlich HJ, de Boer JP, Pannekoek H. The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance. Blood 1991; 78: 401-9.
37 Bijnens AP, Gils A, Knockaert I, Declerck PJ. Importance of the hinge region between a-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: 6375-80.
38 Padmanabhan J, Sane DC. Localization of a vitronectin binding region of plasminogen activator inhibitor-1. Thromb Haemost 1995; 73: 829-34.
39 Muehlenweg B, Guthaus E, de Prada N, Schmitt M, Schmiedeberg N, Kotsch M, Creutzburg S, Kramer M, Kessler H, Wilhelm O, Magdole V. Thromb Res 2000; 98: 73-81.
40 Gils A, Knockaert I, Declerck PJ. Substrate behavior of plasminogen activator inhibitor-1 is not associated with a lack of insertion of the reactive site loop. Biochemistry 1996; 35: 7474-81.
41 Ngo TH, Bijnens AP, Knockaert I, Declerck PJ. Expression, purification and characterization of recombinant rat plasminogen inhibitor-1. Fibrinolys Proteolys 1997; 11: 37-43.
42 Verheijen JH, Chang GT, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost 1984; 51: 392-5.
43 Cunningham BC, Jhurani P, Ng P, Wells JA. Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science 1989; 243: 1330-6.
44 Lawrence DA, Strandberg L, Ericson J, Ny T. Structure-function studies of the serpin plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants. J Biol Chem 1990; 265: 20293-301.
45 Aertgeerts K, De Ranter CJ, Booth NA, Declerck PJ. Rational design of complex formation between plasminogen activator inhibitor-1 and its target proteinases. J Struct Biol 1997; 118: 236-42.
46 Nar H, Bauer M, Stassen JM, Lang D, Gils A, Declerck PJ. Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation. J Mol Biol 2000; 297: 683-95.
47 Stoop AA, Jespers L, Lasters I, Eldering E, Pannekoek H. High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: applications to study structure-function of plasminogen activator inhibitor 1 (PAI-1). J Mol Biol 2000; 301: 1135-47.
48 Bjorquist P, Ehnebom J, Inghardt T, Deinum JR. Epitopes on plasminogen activator inhibitor type 1 important for binding to tissue plasminogen activator. Biochim Biophys Acta 1997; 1341: 87-98.
49 Ngo TH, Debrock S, Declerck P. Identification of functional synergism between monoclonal antibodies. Application to the enhancement of plasminogen activator inhibitor-1 neutralizing effects. FEBS Lett 1997; 416: 373-6.