Expression and Characterization of Recombinant Porcine Plasminogen Activator Inhibitor-1

A P Bijnens; I Knockaert; E Cousin; E K O Kruithof; P J Declerck

doi:10.1055/s-0038-1655967

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1997; 77(02): 350-356
DOI: 10.1055/s-0038-1655967

Original Article

Schattauer GmbH Stuttgart

Expression and Characterization of Recombinant Porcine Plasminogen Activator Inhibitor-1

A P Bijnens

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

,

E Cousin

²Division of Hematology, University Hospital of Lausanne, Switzerland

,

E K O Kruithof

³Division of Angiology and Hemostasis, University Hospital of Geneva, Switzerland

,

P J Declerck

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

› Author Affiliations

Abstract

PDF Download

Summary

Porcine models are, among other animal models, very suitable for in vivo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis. In order to use this model to define the in vivo role of PAI-1, the characterization of porcine PAI-1 and its availability for the generation of immunological tools are a prerequisite. Porcine plasminogen activator inhibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared from cultured porcine aortic cells and characterized in comparison with PAI-1 cDNA’s from other species including human, bovine, rabbit, rat and murine. Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli and recombinant porcine PAI-1 was purified and characterized. On SDS-PAGE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1. The purified recombinant porcine PAI-1 (rpoPAI-1) had a specific activity of 508,800 ± 800 U/mg (mean ± SD, n = 3) towards human tissue-type plasminogen activator (ht-PA) and a functional half-life in vitro of 2.1 ± 0.8 h (n = 3). Incubation with a two fold molar excess of ht-PA (n = 3) or human urokinase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 ± 7% resp. 96 ± 3.6%), latent (12 ± 0.4% resp. 2.6 ± 2.4%) and substrate (16.6 ± 6.8% resp. 1.5 ± 1.3) forms. Inactivated samples of porcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA. The second order rate constant of inhibition of ht-PA was 1.64 ± 0.37 1 0⁷M^-1 s^-1 (n = 9). In gel filtration rpoPAI-1 in buffer eluted at a volume corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corresponding to 330 kDa, presumably as a consequence of binding of active PAI-1 to vitronectin.

Taken together, these data demonstrate that no obvious functional differences exist between human and porcine PAI-1.

PDF (606 kb)

References

References
1 Kruithof EKo, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
2 Kruithof EKO. Plasminogen activator inhibitor type 1:biochemical, biological and clinical aspects. Fibrinolysis 1988; 02 (Suppl. 02) 59-70
3 Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381-387
4 Keijer J, Linders M, Wegman JJ, Ehrlich HJ, Mertens K, Pannekoek H. On the target specificity of plasminogen activator inhibitor 1:the role of heparin, vitronectin, and the reactive site. Blood 1991; 78: 1254-1261
5 Ginsburg D, Zeheb R, Yang AY, Rafferty UM, Andreasen PA, Nielsen L, DanøK LeboRV, Gelehrter TD. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells. J Clin Invest 1986; 78: 1673-1680
6 Pannekoek H, Veerman H, Lambers H, Diergaarde P, Verweij CL, van ZonneveldAJ, Van MourikJA. Endothelial plasminogen activator inhibitor (PAI):a new member of the serpin gene family. EMBO J 1986; 05: 2539-2544
7 Ny T, Sawdey M, Lawrence D, Milan 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: 6776-6780
8 Andreasen PA, Riccio A, Welinder KG, Douglas R, Sartorio R, Nielsen LS, Oppenheimer C, Blasi F, Danø K. Plasminogen activator inhibitor type 1:reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. FEBS Lett 1986; 209: 213-218
9 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 1985; 260: 11581-11587
10 Declerck PJ, De MolM, Vaughan DE, Collen D. 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
11 Declerck PJ, De MolM, Alessi M-C, Baudner S, pâques E-P, Preissner KT, Müller-Berghaus KT, Collen D. Purification and characterization of a plasminogen activator l binding protein from human plasma. J Biol Chem 1988; 263: 5454-5461
12 Mimuro J, Loskutoff DJ. Purification of a protein from bovine plasma that binds to plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin. J Biol Chem 1989; 264: 936-939
13 Vaughan DE, Declerck PJ, Van HoutteE, De MolM, Collen D. Reactivated recombinant plasminogen activator I (rPAI-1) effectively prevents thrombolysis in vivo. Thromb Haemost 1992; 68: 60-63
14 Booth NA, Robbie LA, Croll AM, Bennett B. Lysis of platelet-rich thrombi:the role of PAI-1. Ann NY Acad Sci 1992; 667: 70-80
15 Robbie LA, Booth NA, Croll AM, Bennett B. The roles of α₂-antiplasmin and plasminogen activator inhibitor-1 (PAI-1) in the inhibition of clot lysis. Thromb Haemost 1993; 70 (02) 301-306
16 Krishnamurti C, Barr CF, Hassett MA. Plasminogen activator inhibitor:a regulator of ancrod induced fibrin deposition in rabbits. Blood 1987; 69: 798-803
17 Levi M, Biemond BJ, van ZonneveldAJ, ten CateJW, Pannekoek H. Inhibition of plasminogen activator inhibitor-l activity results in promotion of endogenous thrombolysis and in inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85: 305-312
18 Juhan-Vague I, Alessi M-C. Plasminogen activator inhibitor l and athero-thrombosis. Thromb Haemost 1993; 70: 138-143
19 Prins MH, Hirsch J. A critical review of the relationship between impaired fibrinolysis and myocardial infarction. American Heart Journal 1991; 122: 545-551
20 Barbash GI, Hod H, Rotha A, Miller HI, Rath S, Zahav YH, Modan M, Zivelin A, Laniado S, Seligsohn U. Correlation of baseline plasminogen activator inhibitor activity with patency of the infarct artery after thrombolytic therapy in acute myocardial infarction. Am J Cardiol 1989; 64: 1231-1235
21 Lupu F, Bergonzelli GE, Heim DA, Cousin E, Genton CY, Bachmann F, Kruithof EKO. Localization and production of plasminogen activator-l in human healthy and atherosclerotic arteries. Arterioscler Thromb 1993; 13: 1090-1100
22 Schneidermann J, Sawdey MS, Keeton MR, Bordin GM, Bernstein EF, Dilley RB, Loskutoff DJ. Increased type l plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc Natl Acad Sci USA 1992; 89: 6998-7002
23 Amman V, Nilsson A, Stenne S, Risberg B, Rymo L. Expression of plasminogen activator-l mRNA in healthy, atherosclerotic and thrombotic human arteries and veins. Thromb Res 1994; 76: 487-499
24 Wissler RW, Vesselinovich D. Experimental models of human atherosclerosis. Ann NY Acad Sci 1968; 149: 907-922
25 Buja LM, Kita T, Goldstein JL, Watanabe Y, Brown MS. Cellular pathology of progressive atherosclerosis in the WHHL rabbit:an original model of familial hypercholesterolemia. Arteriosclerosis 1983; 03: 87-101
26 Fuster V, Ip JH, Badimon L, Badimon JJ, Stein B, Chesebro JH. Importance of experimental models for the development of clinical trials on thrombo-atherosclerosis. Circulation 1991; 83 (06) 1415-1425
27 Ratcliff HL, Luginbuhl H. The domestic pig:a model for experimental atherosclerosis. Atherosclerosis 1971; 13: 133-136
28 Moreland AF, Clarckson TB, Hofland HB. Atherosclerosis in miniature swine. Arch Pathol 1963; 76: 203-208
29 Swindle MM, Smith AC. Swine as model in experimental surgery. J Invest Surg 1988; 01: 65-79
30 Badimon L, Steele P, Badimon J-J, Bowie EJW, Fuster V. Aortic atherosclerosis in pigs with heterozygous von Willebrand disease:comparison with homologous von Willebrand and normal pigs. Arteriosclerosis 1985; 05: 366-370
31 Fuster V, Fass DN, Kaye MP, Josa M, Zinsmeister AR, Bowie EJW. Arteriosclerosis in normal and von Willebrand pigs:long term prospective study and aortic transplantation study. Circ Res 1982; 51: 587-593
32 Audenaert A-M, Knockaert I, Collen D, Declerck PJ. Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop. J Biol Chem 1994; 269: 19559-19564
33 Holvoet P, Cleemput H, Collen D. Assay of human tissue-type plasminogen activator (t-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies to t-PA. Thromb Haemost 1985; 54: 685-687
34 Declerck PJ, Verstreken M, Collen D. An immunofunctional assay for active plasminogen activator inhibitor-1 (PAI-1). Fibrinolysis 1988; 02 (Suppl. 02) 77-78
35 Debrock S, Declerck PJ. Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor-1 after cleavage of the reactive center loop of after complex formation with various serine proteinases. FEBS Lett 1995; 376: 243-246
36 Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost 1984; 51: 392-395
37 Declerck PJ, Alessi MC, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D. Measurement of plasminogen activator inhibitor 1 in biological fluids with a murine monoclonal antibody-based enzyme linked immunosorbent assay. Blood 1988; 07 (01) 220-225
38 Bosma PJ, van denBerg EA, Kooistra T, Siemieniak DR, Slightom JL. Human plasminogen activator inhibitor-1 gene. J Biol Chem 1988; 263 (19) 9129-9141
39 Mimuro J, Sawdey M, Hattori M, Loskutoff DJ. cDNA for bovine type 1 plasminogen activator inhibitor (PAI-1). Nucleic Acids Res 1989; 17: 8872
40 Prendergast GC, Diamond LE, Dahl D, Cole MD. The c-myc-regulated genemrlencodes plasminogen activator inhibitor 1. Mol Cell Biol 1990; 10: 1265-1269
41 Zeheb R, Gelehrter TD. Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1. Gene 1988; 73: 459-468
42 Hofmann KJ, Mayer EJ, Schultz LD, Socher SH, Reilly CF. Purification and characterisation of recombinant rabbit plasminogen activator inhibitor-1 expressed inSaccharomyces cerevisiae . Fibrinolysis 1992; 06: 263-272
43 Aertgeerts K, De BondtHL, De RanterCJ, Declerck PJ. Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1. Nature Structural Biology 1995; 02 (10) 891-897
44 Kvassman J-O, Lawrence DA, Shore JD. The acid stabilisation of plasminogen activator inhibitor-1 depends on protonation of a single group that affects loop insertion into β-sheet A. J Biol Chem 1995; 270: 27942-27947
45 Huber R, Carrell RW. Implications of the three-dimensional structure of α₁-antitrypsin for structure and function of serpins. Biochemistry 1989; 28 (23) 8951-8966
46 Sancho E, Tonge DW, Hockney RC, Booth NA. Purification and characterization of active and stable recombinant plasminogen activator inhibitor accumulated at high levels in E. coli . Eur J Biochem 1994; 224: 125-134
47 Alessi M-C, Declerck PJ, De MolM, Nelles L, Collen D. Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1). Eur J Biochem 1988; 175: 531-540
48 Lawrence D, Strandberg L, Grundström T, Ny T. Purification of active human plasminogen activator inhibitor 1 fromEscherichia coli . Eur J Biochem 1989; 186: 523-533
49 Lang IM, Marsh JJ, Moser KM, Schleef RR. Presence of active and latent type 1 plasminogen activator inhibitor associated with porcine platelets. Blood 1992; 80: 2269-2274
50 Thorsen S, Philips M, Selmer J, Lecander I, Astedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator inhibitor type 1 and type 2. Eur J Biochem 1988; 175: 33-39
51 Sigurdardóttir O, Wiman B. Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma. Biochem Biophys Acta 1991; 1035: 56-61