Download PDF
Thromb Haemost 1993; 69(05): 466-472
DOI: 10.1055/s-0038-1651634
Original Article
Fibrinolysis
Schattauer GmbH Stuttgart

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

M Colucci
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
L G Cavallo
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
G Agnelli
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
A Mele
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
R Bürgi
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
J Heim
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
,
N Semeraro
The Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale e Oncologia Sperimentale, Università di Bari, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Menarini Ricerche Sud, Pomezia, Italy, and Ciba Geigy, Basel, Switzerland
› Author Affiliations
Further Information

Publication History

Received 23 April 1992

Accepted after revision 25 January 1993

Publication Date:
25 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

 

  • References

  • 1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
    PubMedSearch in Google Scholar
  • 2 Verstraete M. Thrombolytic treatment in acute myocardial infarction. Circulation 1990; 82 (Suppl. 02) 1196-1109
    PubMedSearch in Google Scholar
  • 3 Lijnen R, Collen D. Towards the development of improved thrombolytic agents. Br J Haematol 1991; 77: 261-266
    CrossrefPubMedSearch in Google Scholar
  • 4 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
    PubMedSearch in Google Scholar
  • 5 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennet WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E.coli . Nature 1983; 301: 214-221
    CrossrefPubMedSearch in Google Scholar
  • 6 Banyai L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
    CrossrefPubMedSearch in Google Scholar
  • 7 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 1984; 81: 5355-5359
    CrossrefPubMedSearch in Google Scholar
  • 8 Pannekoek H, de Vries C, van Zonneveld AJ. Mutants of human tissue-type plasminogen activator (t-PA): structural aspects and functional properties. Fibrinolysis 1988; 2: 123-132
    PubMedSearch in Google Scholar
  • 9 Patthy L. Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 1985; 41: 657-663
    CrossrefPubMedSearch in Google Scholar
  • 10 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem 1982; 257: 2912-2919
    PubMedSearch in Google Scholar
  • 11 Holmes WE, Pennica D, Blaber M, Rey MW, Guenzler WA, Steffens GJ, Heyneker HL. Cloning and expression of the gene for prourokinase in Escherichia coli . Biotechnology 1985; 3: 923-929
    CrossrefPubMedSearch in Google Scholar
  • 12 Pannell R, Gurewich V. Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect. Blood 1986; 67: 1215-1223
    PubMedSearch in Google Scholar
  • 13 Lijnen HR, Van Hoef B, De Cock F, Collen D. The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro. Blood 1989; 73: 1864-1872
    PubMedSearch in Google Scholar
  • 14 Lijnen HR, Nelles L, Holmes W, Collen D. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu 144 through Leu 411) of recombinant single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-5598
    PubMedSearch in Google Scholar
  • 15 Piérard L, Jacobs P, Gheysen D, Hoylaerts M, André B, Topisirovic L, Cravador A, de Foresta F, Herzog A, Collen D, De Wilde M, Bollen A. Mutant and chimeric recombinant plasminogen activators: Production in eukaryotic cells and preliminary characterization. J Biol Chem 1987; 262: 11771-11778
    PubMedSearch in Google Scholar
  • 16 de Vries C, Veerman H, Blasi F, Pannekoek H. Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins. Biochemistry 1988; 27: 2565-2572
    CrossrefPubMedSearch in Google Scholar
  • 17 Lee SG, Kalyan N, Wilhelm J, Hum WT, Rapaport R, Cheng SM, Dheer S, Urbano C, Hartzell RW, Rochetti-Blume M, Levner M, Hung PP. Construction and expression of hybrid plasminogen activator prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes. J Biol Chem 1988; 263: 2917-2924
    PubMedSearch in Google Scholar
  • 18 Lijnen HR, Nelles L, Van Hoef B, Demarsin E, Collen D. Characterization of a chimeric plasminogen activator consisting of amino acids 1 to 274 of tissue-type plasminogen activator and aminoacids 138 to 411 of single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 19083-19091
    PubMedSearch in Google Scholar
  • 19 Devlin JJ, Devlin PE, Clark R, O’Rourke EC, Levenson C, Mark DF. Novel expression of chimeric plasminogen activators in insect cells. Biotechnology 1989; 7: 286-293
    CrossrefPubMedSearch in Google Scholar
  • 20 Nelles L, Lijnen HR, Van Nuffelen A, Demarsin E, Collen D. Characterization of domain deletion and/or duplication mutants of a recombinant chimera of tissue-type plasminogen activator and urokinase-type plasminogen activator (rt-PA/u-PA). Thromb Haemostas 1990; 64: 53-60
    PubMedSearch in Google Scholar
  • 21 Lijnen HR, Collen D. Strategies for the improvement of thrombolytic agents. Thromb Haemostas 1991; 66: 88-110
    PubMedSearch in Google Scholar
  • 22 Stump DC, Lijnen HR, Collen D. Purification and characterization of a low molecular weight form of single-chain urokinase-type plasminogen activator. J Biol Chem 1986; 261: 17120-17126
    PubMedSearch in Google Scholar
  • 23 Ichinose A, Fujikawa K, Suyama T. The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 3486-3489
    PubMedSearch in Google Scholar
  • 24 Asselbergs FAM, Bürgi R, Chaudhuri B, Heim J, Meyhack B, Rajput B, van Oostrum J, Alkan S. Localization of epitopes on tissue-type and urokinase-type plasminogen activators by monoclonal antibodies using recombinant hybrid enzymes. Fibrinolysis 1993; 7: 1-16
    PubMedSearch in Google Scholar
  • 25 Urden G, Hamsten A, Wiman B. Comparison of plasminogen activator inhibitor activity and antigen in plasma samples. Clin Chim Acta 1987; 169: 189-196
    CrossrefPubMedSearch in Google Scholar
  • 26 Astrup T, Mullertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
    CrossrefPubMedSearch in Google Scholar
  • 27 Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards F. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
    PubMedSearch in Google Scholar
  • 28 Holvoet P, Cleenput 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
    PubMedSearch in Google Scholar
  • 29 Loskutoff DJ, Mussoni L. Interaction between fibrin and the plasminogen activators produced by cultured endothelial cells. Blood 1983; 62: 62-68
    PubMedSearch in Google Scholar
  • 30 Colucci M, Paramo JA, Collen D. Inhibition of one-chain and two-chain forms of human tissue-type plasminogen activator by the fast-acting inhibitor of plasminogen activator in vitro and in vivo. J Lab Clin Med 1986; 108: 53-59
    PubMedSearch in Google Scholar
  • 31 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 31-38
    CrossrefPubMedSearch in Google Scholar
  • 32 Larsen GR, Metzger M, Henson K, Blue Y, Horgan P. Pharmacokinetic and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat. Blood 1989; 73: 1842-1850
    PubMedSearch in Google Scholar
  • 33 Kalyan NK, Lee SG, Wilhlem J, Fu KP, Hum WT, Rappaport R, Hartzell RW, Urbano C, Hung PP. Structure-function analysis with tissue-type plasminogen activator.Effect of deletion of NH2-termmal domains on its biochemical and biological properties. J Biol Chem 1988; 263: 3971-3978
    PubMedSearch in Google Scholar
  • 34 Wu Z, Van de Werf F, Stassen JM, Matsson C, Pohl G, Collen D. Pharmacokinetics and coronary thrombolytic properties of two human tissue-type plasminogen activator variants lacking the finger-like, growth factor-like, and first kringle domains (amino acids 6–173) in a canine model. J Cardiovasc Pharmacol 1990; 16: 197-203
    CrossrefPubMedSearch in Google Scholar
  • 35 Collen D, Lijnen HR, Vanlinthout I, Kieckens L, Nelles L, Stassen JM. Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model. Thromb Haemostas 1991; 65: 174-180
    PubMedSearch in Google Scholar
  • 36 Verheijen JH, Caspers MPM, Chang GTG, de Munk JAW, Pouwels PH, Enger-Valk BE. Involvement of finger domain and kringle 2 domain of tissue type plasminogen activator in fibrin binding and stimulation of activity by fibrin. EMBO J 1986; 5: 3525-3530
    PubMedSearch in Google Scholar
  • 37 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. J Biol Chem 1988; 263: 1023-1029
    PubMedSearch in Google Scholar
  • 38 Johannessen M, Diness V, Pingel K, Petersen LC, Rao D, Lioubin P, O’Hara P, Mulvihill E. Fibrin affinity and clearance of t-PA deletion and substitution analogues. Thromb Haemostas 1990; 63: 54-59
    PubMedSearch in Google Scholar
  • 39 Lijnen HR, Nelles L, Van Hoef B, De Cock F, Collen D. Biochemical and functional characterization of human plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains. J Biol Chem 1990; 265: 5677-5683
    PubMedSearch in Google Scholar
  • 40 Colucci M, Cavallo LG, Agnelli G, Burgi R, Heim J, Mele A, Semeraro N. In vitro properties of two chimeric recombinant plasminogen activators with high plasminogen activator activity, fibrin specificity and prolonged in vivo half-life. Fibrinolysis 1990; 4 (Suppl. 03) 110
    PubMedSearch in Google Scholar
  • 41 Colucci M, Mele A, Semeraro N. Pharmacokinetics in monkeys of a chimeric plasminogen activator (K2tu-PA) linking the kringle 2 domain of t-PA to the catalytic domain of scu-PA. Fibrinolysis 1992; 6 (Suppl. 02) 140
    PubMedSearch in Google Scholar
  • 42 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin independent inhibitors. J Clin Invest 1990; 86: 385-391
    CrossrefPubMedSearch in Google Scholar
  • 43 Agnelli G, Pascucci C, Colucci M, Nenci GG, Mele A, Bürgi R, Heim J. Thrombolytic activity of two chimeric recombinant plasminogen activators (FK2tu-PA and K2tu-PA) in rabbits. Thromb Haemostas 1992; 68: 331-335
    PubMedSearch in Google Scholar
  • 44 Collen D, Stassen JM, Verstraete M. Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. J Clin Invest 1983; 71: 368-376
    CrossrefPubMedSearch in Google Scholar