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Thromb Haemost 1992; 68(03): 331-335
DOI: 10.1055/s-0038-1656375
Original Article
Schattauer GmbH Stuttgart

Thrombolytic Activity of Two Chimeric Recombinant Plasminogen Activators (FK2tu-PA and K2tu-PA) in Rabbits

Giancarlo Agnelli
1   The Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Italy
,
Claudia Pascucci
1   The Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Italy
,
Mario Colucci
2   The Istituto di Patologia Generale, Università di Bari, Italy
,
Giuseppe G Nenci
1   The Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Italy
,
Antonio Mele
3   The Menarini Ricerche Sud, Rome, Italy
,
Rolf Bürgi
4   The Ciba Geigy, Basel, Switzerland
,
Jutta Heim
4   The Ciba Geigy, Basel, Switzerland
› Author Affiliations
Further Information

Publication History

Received 11 December 1991

Accepted after revision 16 April 1992

Publication Date:
04 July 2018 (online)

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Summary

The aim of this study was to evaluate the thrombolytic activity of two hybrid plasminogen activators (HPAs) in a rabbit jugular vein thrombosis model. In the two HPAs the kringle-2 domain (K2tu-PA) or the finger and the kringle-2 domains (FK2tu-PA) of tissue-type plasminogen activator (t-PA) are linked to the catalytic protease domain of single chain urokinase type plasminogen activator (scu-PA). The two HPAs were compared with rt-PA and scu-PA on a weight/weight basis. K2tu-PA, FK2tu-PA, rt-PA and scu-PA were infused at doses of 0.4, 0.8 and 1.2 mg/kg over 3 h. Saline served as control. Saline produced 11 ± 2% thrombolysis. The three doses of K2tu-PA led to 38 ± 4%, 66 ± 5% and 89 ± 7% thrombolysis, respectively; the three doses of FK2tu-PA: 18 ± 3%, 29 ± 5% and 33 ± 6%, respectively; the three doses of rt-PA 32 ± 2%, 49 ± 3% and 68 ± 6%, respectively; the three doses of scu-PA 16 ± 2%, 24 ± 3% and 32 ± 4%, respectively. K2tu-PA and rt-PA showed a statistically significant higher thrombolytic activity than FK2tu-PA and scu-PA at the three tested doses (p <0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p <0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, α2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses. No statistically significant differences between K2tu-Pa and rt-PA were observed. Concomitant with the lower thrombolytic activity, the systemic proteolytic effects of FK2tu-PA and scu-PA were less pronounced. We conclude that the two HPAs we tested are effective thrombolytic agents. K2tu-PA deserves particular attention in future experiments.

 

  • References

  • 1 Pennica D, Holmes WE, Kohr WH, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heynecker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E coli . Nature 1983; 301: 214-221
    CrossrefPubMedGoogle Scholar
  • 2 Holmes WE, Pennica D, Blaber M, Rey MW, Guenzler WA, Steffens GJ, Heyneker HL. Cloning and expression of the gene for pro-urokinase in Escherichia coli . Biotechnology 1985; 3: 923-929
    CrossrefPubMedGoogle Scholar
  • 3 Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardiaco (Gissi) 2 A factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12490 patients with acute myocardial infarction. Lancet 1990; 336: 65-71
    PubMedGoogle Scholar
  • 4 ISIS – 3 (Third International Study of Infarct Survival) Collaborative Group Preliminary Results presented at the 40th Congress of the American College of Cardiology, Atlanta, March 1991
    PubMed
  • 5 Rao AK, Pratt C, Berke A, Jaffe A, Ockene I, Schreiber TL, Bell WR, Knatterud G, Robertson TL, Terrin ML. Thrombolysis in myocardial infarction (Timi) Trial-Phase I: Hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol 1988; 11: 01-11
    CrossrefPubMedGoogle Scholar
  • 6 PRIMI Trial Study Group Randomised double-blind trial of recombinant pro-urokinase against streptokinase in acute myocardial infarction. Lancet 1989; 1: 863-868
    PubMedGoogle Scholar
  • 7 Lijnen TH, Collen D. Strategies for the improvement of thrombolytic agents. Thromb Haemostas 1991; 66: 088-110
    PubMedGoogle Scholar
  • 8 Colucci M, Cavallo LG, Agnelli G, Bürgi R, Heim J, Mele A, Semeraro N. In vitro properties of two chimaeric recombinant plasminogen activators with high plasminogen activator activity, fibrin specificity and prolonged in vivo half-life. Fibrinolysis 1990; Suppl 3 110
    PubMedGoogle Scholar
  • 9 Asselberg FAM, Burgi R, Heim J, van Ostrum J, Agnelli G, Lucchesi BR. Biochemical and biological characterization of K2tu-PA, a hybrid plasminogen activator. Thromb Haemostas 1991; 65: 779
    PubMedGoogle Scholar
  • 10 Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemostas 1987; 58: 1078-1084
    PubMedGoogle Scholar
  • 11 Zimmerman M, Quigley JP, Ashe B, Dorn C, Goldfarb R, Troll W. Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles. Proc Natl Acad Sci 1978; 75: 750-753
    CrossrefPubMedGoogle Scholar
  • 12 Philo RD, Gaffney PJ. Plasmin potency estimates: influence of the substrate used in assay. Thromb Haemostas 1981; 45: 107-109
    PubMedGoogle Scholar
  • 13 Agnelli G, Buchanan MR, Fernandez F, Boneu B, Van Ryn J, Hirsh J, Collen D. A comparison of the thrombolytic and hemorrhagic effects of tissue-type plasminogen activator and streptokinase in rabbits. Circulation 1985; 72: 178-182
    CrossrefPubMedGoogle Scholar
  • 14 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246
    CrossrefPubMedGoogle Scholar
  • 15 Friberger P, Knos M. Plasminogen determination in human plasma. In: Chromogenic Peptide Substances: Chemistry and Clinical Use. Scully MF, Kakkar VV. (eds) Churchill Livingstone, Edinburgh: 1979. pp 128-139
    Google Scholar
  • 16 Teger-Nielsson AC, Friberger P, Gyzander E. Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate. Scand J Lab Invest 1977; 37: 403-409
    CrossrefPubMedGoogle Scholar
  • 17 van Zonneveld AJ, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci 1986; 83: 4670-4674
    CrossrefPubMedGoogle Scholar
  • 18 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 1984; 81: 5355-5359
    CrossrefPubMedGoogle Scholar
  • 19 Holvoet P, Lijnen HR, Collen D. Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies. Eur J Biochem 1986; 158: 173-177
    CrossrefPubMedGoogle Scholar
  • 20 Lijnen HR, Nelles L, van Hoef B, De Cock F, Collen D. Biochemical characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural function domains. J Biol Chem 1990; 265: 5677-5683
    PubMedGoogle Scholar
  • 21 Zamarron C, Lijnen HR, van Hoef B, Collen D. Biological and thrombolytic properties of proenzyme and active forms of human urokinase. I. Fibrinolytic and fibrinogenolytic properties in human plasma in vitro of urokinases obtained from human urine or by recombinant DNA technology. Thromb Haemostas 1984; 52: 19-23
    PubMedGoogle Scholar
  • 22 Lijnen HR, Nelles L, Holmes WE, Collen D. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu 144 through Leu411) of recombinant single chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-5598
    PubMedGoogle Scholar
  • 23 Gheysen D, Lijnen HR, Pierard L, de Foresta F, Demarsin E, Jacobs P, De Wilde M, Bollen A, Collen D. Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator. J Biol Chem 1987; 262: 11779-11784
    PubMedGoogle Scholar
  • 24 Pierard L, Jacobs P, Gheysen D, Hoylaerts M, Adré B, Topisirovic L, Cravador A, de Foresta F, Herzog A, Collen D, De Wilde M, Bollen A. Mutant and chimeric recombinant plasminogen activators. J Biol Chem 1987; 262: 11771-11778
    PubMedGoogle Scholar
  • 25 Nelles L, Lijnen HR, Collen D, Holmes WE. Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator. J Biol Chem 1987; 262: 10855-10862
    PubMedGoogle Scholar
  • 26 Lee SG, Kalyan N, Wilhelm J, Hum WT, Rappaport R, Cheng SM, Dheer S, Urbano C, Hartzell RW, Ronchetti-Blume M, Levner M, Hung PP. Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urinokase-type plasminogen activator genes. J Biol Chem 1988; 263: 2917-2924
    PubMedGoogle Scholar
  • 27 Di 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 and u-PA hybrid proteins. Biochemistry 1988; 27: 2565-2572
    CrossrefPubMedGoogle Scholar
  • 28 Kalyan NK, Lee SG, Cheng SM, Hartzell R, Urbano C, Hung PP. Construction and expression of a hybrid plasminogen activator gene with sequences from non-protease region of tissue-type plasminogen activator (t-PA) and protease region of urokinase (u-PA). Gene 1988; 68: 205-212
    CrossrefPubMedGoogle Scholar
  • 29 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 amino acids 138 to 411 of single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 19083-19091
    PubMedGoogle Scholar
  • 30 Pierard L, Quintana LG, Reff ME, Bollen A. Production in eucaryotic cells and characterization of four hybrids of tissue-type and urokinase-type plasminogen activators. DNA 1989; 8: 321-328
    CrossrefPubMedGoogle Scholar
  • 31 Collen D, Lu HR, Lijnen HR, Nelles L, Stassen JM. Thrombolytic and pharmacokinetic properties of chimeric tissue-type and urokinase-type plasminogen activators. Circulation 1991; 84: 1216-1234
    CrossrefPubMedGoogle Scholar
  • 32 Lucchesi BR, Bürgi R, Heim J, Cronin M. The in vivo thrombolytic activity of K2tu-PA, a hybrid plasminogen activator and fibrinolytic agent. Coronary Artery Dis 1991; 2: 247-258
    CrossrefPubMedGoogle Scholar