Download PDF
Thromb Haemost 1992; 68(01): 060-063
DOI: 10.1055/s-0038-1656318
Original Article
Schattauer GmbH Stuttgart

Reactivated Recombinant Plasminogen Activator Inhibitor-1 (rPAI-1) Effectively Prevents Thrombolysis In Vivo

Douglas E Vaughan
The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
,
Paul J Declerck
The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
,
Elizabeth Van Houtte
The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
,
Maria De Mol
The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
,
Désiré Collen
The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 10 October 1991

Accepted after revision 04 February 1992

Publication Date:
03 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

The effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (~2 units of t-PA neutralizing activity per µg protein) and reactivated rPAI-1 (~150 units/µg).

Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity ~0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus. We propose that the present model may be a valuable tool in monitoring and evaluating the in vivo thrombolytic efficacy of various t-PA mutants designed to be less sensitive to the inhibitory effects of PAI-1.

 

  • References

  • 1 Hunt LT, Dayhoff MO. A surprising new protein superfamily containing ovalbumin, antithrombin III and alpha 1-protease inhibitor. Biochem Biophys Res Commun 1980; 95: 864-871
    CrossrefPubMedGoogle Scholar
  • 2 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: 2539-2544
    PubMedGoogle Scholar
  • 3 Andreasen PA, Riccio A, Welinder KG, Douglas R, Sartorio R, Nielsen LS, Oppenheimer C, Blasi F, Dano K. Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. FEBS Lett 1986; 209: 213-218
    CrossrefPubMedGoogle Scholar
  • 4 Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ. Cloning and sequence of a cDNA coding for the human β-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 1986; 83: 6776-6780
    CrossrefPubMedGoogle Scholar
  • 5 Ginsburg D, Zeheb R, Yang AY, Rafferty UM, Andreasen PA, Nielsen L, Danø K, Lebo RV, Gelehrter TD. cDNA cloning of human plasminogen activator inhibitor from endothelial cells. J Clin Invest 1986; 78: 1673-1680
    CrossrefPubMedGoogle Scholar
  • 6 Kruithof EKO, Ransijn A, Bachmann F. Inhibition of tissue plasminogen activator by human plasma. In: Progress in Fibrinolysis, Vol 5. Churchill Livingstone; Edinburgh: 1983: 365-369
    Google Scholar
  • 7 Kruithof EKO, Tran-Thang C, Bachmann F. The fast-acting inhibitor of tissue-type plasminogen activator in plasma is also the primary plasma inhibitor of urokinase. Thromb Haemostas 1986; 55: 65-69
    PubMedGoogle Scholar
  • 8 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
    CrossrefPubMedGoogle Scholar
  • 9 Wiman B, Chmielewska J, Ranby M. Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor. J Biol Chem 1984; 259: 3644-3647
    PubMedGoogle Scholar
  • 10 Kruithof EKO, Nicolosa G, Bachmann F. Plasminogen activator inhibitor-1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation. Blood 1987; 70: 1645-1653
    PubMedGoogle Scholar
  • 11 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; 71: 220-225
    PubMedGoogle Scholar
  • 12 Levin EG. Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells. Blood 1986; 67: 1309-1313
    PubMedGoogle Scholar
  • 13 Sprengers ED, Akkerman JWN, Jansen BG. Blood platelet plasminogen activator inhibitor: two different pools of endothelial cell type plasminogen activator in human blood. Thromb Haemostas 1986; 55: 325-329
    PubMedGoogle Scholar
  • 14 Kooistra T, Sprengers ED, Van Hinsbergh VWM. Rapid inactivation of the plasminogen-activator inhibitor upon secretion from cultures of human endothelial cells. Biochem J 1986; 239: 497-503
    CrossrefPubMedGoogle Scholar
  • 15 Declerck P, De Mol M, Alessi M-C, Baudner S, Paques EP, Preissner KT, Muller-Berghaus G, Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. J Biol Chem 1988; 263: 15454-15461
    CrossrefPubMedGoogle Scholar
  • 16 Katagiri K, Okada K, Hattori H, Yano M. Bovine endothelial cell plasminogen activator inhibitor. Eur J Biochem 1988; 176: 81-87
    CrossrefPubMedGoogle Scholar
  • 17 Lambers JWJ, Cammenga M, Koenig BW, Mertens K, Pannekoek H, van Mourik JA. Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. J Biol Chem 1988; 262: 17492-17496
    PubMedGoogle Scholar
  • 18 Vaughan DE, Declerck PJ, Van Houtte E, De Mol M, Collen D. Studies of recombinant plasminogen activator (rPAI-1) in rabbits: Pharmacokinetics and evidence for reactivation of latent PAI-1 in vivo. Circ Res 1990; 64: 1281-1286
    PubMedGoogle Scholar
  • 19 Kruithof EKO, Tran-Thang C, Ransijn A, Bachmann F. Demonstrations of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
    PubMedGoogle Scholar
  • 20 Chmielewska J, Ranby M, Wiman B. Kinetics of the inhibition of plasminogen activators by the plasminogen activator system. Biochem J 1988; 251: 327-332
    CrossrefPubMedGoogle Scholar
  • 21 Masson C, Angeles-Cano E. Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator. Biochem J 1988; 256: 237-244
    CrossrefPubMedGoogle Scholar
  • 22 Hajjar KA, Hamel NM, Harpel PC, Nachman RL. Binding of tissue type plasminogen activator to cultured human endothelial cells. J Clin Invest 1987; 80: 1712-1719
    CrossrefPubMedGoogle Scholar
  • 23 Colucci M, Paramo JA, Stassen JM, Collen D. Influence of the fast-acting inhibitor of plasminogen activator on in vivo thrombolysis induced by tissue-type plasminogen activator in rabbits. Interference of tissue-derived components. J Clin Invest 1986; 78: 138-144
    PubMedGoogle Scholar
  • 24 Alessi MC, Declerck PJ, De Mol M, 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
    CrossrefPubMedGoogle Scholar
  • 25 Collen D, Stassen JM, Verstraete M. Thrombolysis with human intrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration. J Clin Invest 1983; 71: 368-376
    PubMedGoogle Scholar
  • 26 Vaughan DE, Mendelsohn ME, Declerck PJ, Van Houtte E, Collen D, Loscalzo J. Characterization of the binding of tissue-type plasminogen activator to platelets. J Biol Chem 1989; 264: 15869-15874
    PubMedGoogle Scholar
  • 27 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 Haemostas 1985; 54: 684-687
    PubMedGoogle Scholar
  • 28 Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
    PubMedGoogle Scholar
  • 29 Declerck PJ, Verstreken M, Collen D. An immunofunctional assay for active plasminogen activator inhibitor-1 (PAI-1). Fibrinolysis 1988; 2 (Suppl 2) 77-78
    PubMedGoogle Scholar
  • 30 Verheijen JH, Mullaert E, Chang GTG, Kluft C, Wijngaards G. A simple, sensitive spectophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurement in plasma. Thromb Haemostas 1982; 48: 266-269
    PubMedGoogle Scholar
  • 31 Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T 4. Nature (London) 1970; 227: 680-682
    CrossrefPubMedGoogle Scholar
  • 32 Vaughan DE, Declerck PJ, De Mol M, Collen D. Recombinant plasminogen activator inhibitor-1 reverses the bleeding tendency associated with the combined administration of tissue-type plasminogen activator and aspirin in rabbits. J Clin Invest 1989; 84: 586-591
    CrossrefPubMedGoogle Scholar
  • 33 Madison EL, Goldsmith EJ, Gerard RD, Gething M-HJ, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature 1989; 339: 721-724
    CrossrefPubMedGoogle Scholar