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Thromb Haemost 1993; 69(01): 050-055
DOI: 10.1055/s-0038-1651547
Original Article
Fibrinolysis
Schattauer GmbH Stuttgart

A Hybrid Plasminogen Activator Binds to the u-PA Receptor and Has a Reduced Thrombolytic Potency In Vivo

Fred A M Asselbergs
The Biotechnology Department, CIBA-GEIGY Ltd. K681.4.42, Basle, Switzerland
,
Rolf Bürgi
The Biotechnology Department, CIBA-GEIGY Ltd. K681.4.42, Basle, Switzerland
,
Jessica Hamerman
The Biotechnology Department, CIBA-GEIGY Ltd. K681.4.42, Basle, Switzerland
,
Jutta Heim
The Biotechnology Department, CIBA-GEIGY Ltd. K681.4.42, Basle, Switzerland
,
Jan van Oostrum
The Biotechnology Department, CIBA-GEIGY Ltd. K681.4.42, Basle, Switzerland
,
Giancarlo Agnelli
1   The Instituto di Semiotica Medica, University of Perugia, Perugia, Italy
› Author Affiliations
Further Information

Publication History

Received 16 April 1992

Accepted after revision 11 September 1992

Publication Date:
04 July 2018 (online)

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Summary

Fibrinolytic properties of four hybrids of u-PA and t-PA, all containing the u-PA growth factor domain and binding to recombinant human u-PA receptor expressed in CHO cells, were compared. Highest fibrin stimulation was observed with uK2tPA which when compared to t-PA in the rabbit system, had a considerably prolonged circulatory half-life in vivo. Compared to an equimolar dose of t-PA, 0.4 mg/kg uK2tPA caused a similar consumption of α2-antiplasmin and fibrinogen and a considerably greater prolongation of the ex-vivo blood clotting time. Nevertheless, this dose of uK2tPA was inactive in the jugular vein thrombosis assay. This lack of thrombolytic activity is presumably due to the presence of a functional u-PA growth factor domain, which in binding uK2tPA to cellular blood elements possibly retards its penetration into the blood clot and in this manner could neutralize the potential thrombolytic activity of the t-PA kringle 2 and protease domains in uK2tPA.

 

  • References

  • 1 Plow EF, Freaney D, Plescia J, Miles LA. The plasminogen system and cell surfaces: Evidence for plasminogen and urokinase receptors on the same cell type. J Cell Biol 1986; 103: 2411-2420
    CrossrefPubMedGoogle Scholar
  • 2 Miles LA, Plow EF. Plasminogen receptors: ubiquitous sites for cellular regulation of fibrinolysis. Fibrinolysis 1988; 2: 73-84
    PubMedGoogle Scholar
  • 3 Stephens RW, Pollanen J, Tapiovaara H, Leung KG, Sim PS, Salonen EM, Ronne E, Behrendt N, Dano K, Vaheri A. Activation of prourokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants. J Cell Biol 1989; 108: 1987-1995
    CrossrefPubMedGoogle Scholar
  • 4 Ellis V, Wun T-Z, Behrendt N, Ronne E, Dano K. Inhibition of receptor-bound urokinase by plasminogen activator inhibitors. J Biol Chem 1990; 265: 9904-9908
    PubMedGoogle Scholar
  • 5 Hall SW, VandenBerg SR, Gonias SL. Plasminogen carbohydrate side chains in receptor binding and enzyme activation: a study of C6 glioma cells and primary cultures of rat hepatocytes. J Cell Biochem 1990; 43: 213-227
    CrossrefPubMedGoogle Scholar
  • 6 Harpel PC, Silverstein RL Pannell R, Gurewich V, Nachman RL. Thrombospondin forms complexes with single-chain and two-chain forms of urokinase. J Biol Chem 1990; 265: 11289-11294
    PubMedGoogle Scholar
  • 7 Estreicher A, Wohlwend A, Belin D, Schleuning WD, Vassalli J-D. Characterization of the cellular binding site for the urokinase-type plasminogen activator. J Biol Chem 1989; 264: 1180-1189
    PubMedGoogle Scholar
  • 8 Barnathan Es, Kuo A, Kariko K, Rosenfeld L, Murray SC, Behrendt N, Ronne E, Wiener D, Henkin J, Cines DB. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA. Blood 1990; 76: 1795-1806
    PubMedGoogle Scholar
  • 9 Roldan AL, Cubellis MV, Massucci MT, Behrendt N, Lund LR, Danø K, Appella E, Blasi F. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in the cell surface, plasmin-dependent proteolysis. EMBO J 1990; 9: 467-474
    PubMedGoogle Scholar
  • 10 Ploug M, Rønne E, Behrendt N, Jensen AL, Blasi F, Danø K. Cellular receptor for urokinase plasminogen activator. Carboxylterminal processing and membrane-anchoring by glycosyl-phosphatidyl-inositol. J Biol Chem 1991; 266: 1926-1933
    PubMedGoogle Scholar
  • 11 Cubellis MV, Andreasen P, Ragno P, Mayer M, Danø K, Blasi F. Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor. Proc Natl Acad Sci USA 1989; 86: 4828-4832
    CrossrefPubMedGoogle Scholar
  • 12 Chapman HA, Bertozzi P, Sailkor LZ, Nusrat AR. Alveolar macrophage urokinase receptors localize enzyme activity to the cell surface. Am J Physiol 1990; 259: L432-L438
    CrossrefPubMedGoogle Scholar
  • 13 Kirchheimer JC, Remold HG. Functional characteristics of receptor-bound urokinase on human monocytes: catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors. Blood 1989; 74: 1396-1402
    PubMedGoogle Scholar
  • 14 Schlechte JW, Boyd DD. Insensitivity of laminin degradation directed by receptor bound urokinase to PAI-1 in cultured colon cancer. Cancer Comm 1990; 2: 261-269
    PubMedGoogle Scholar
  • 15 Asselbergs FAM, Bürgi R, Chaudhuri F, 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. 1992 in press
    PubMedGoogle Scholar
  • 16 Bradford M. A rapid and sensitive method for the determination of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976; 62: 248-254
    PubMedGoogle Scholar
  • 17 Verheijen JH, Mullaart E, Chang GT, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
    PubMedGoogle Scholar
  • 18 Agnelli G, Pascucci C, Cosmi B, Nenci GG. Effects of therapeutic doses of heparin on thrombolysis with tissue-type plasminogen activator in rabbits. Blood 1990; 76: 2030-2036
    PubMedGoogle Scholar
  • 19 Agnelli G, Buchanan MR, Fernandez F, Boneu B, van Rijn J, Hirsch 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
  • 20 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246
    CrossrefPubMedGoogle Scholar
  • 21 Friberger P, Knos M. Plasminogen determination in human plasma. In: Chromogenic Peptide Substances. Scully M, Kakkar V. (eds). Churchill-Livingston; New York: 1979: 128-139
    Google Scholar
  • 22 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
  • 23 Southern P, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1982; 1: 327-341
    PubMedGoogle Scholar
  • 24 Asselbergs FAM, Will H, Wingfield P, Hirschi M. A recombinant Chinese Hamster Ovary cell line containing a 300-fold amplified tetramer of the Hepatitis B-genome together with a double selection marker expresses high levels of viral protein. J Mol Biol 1986; 189: 401-411
    CrossrefPubMedGoogle Scholar
  • 25 Herion P, Glineur C, Franssen J-D, Urbain J, Bollen A. Monoclonal antibodies against urokinase. Biosci Rep 1981; 1: 885-892
    CrossrefPubMedGoogle Scholar
  • 26 MacGregor IR, Micklem LR, James K, Pepper DS. Characterization of the epitopes on tissue-type plasminogen activator recognized by a group of monoclonal antibodies. Thromb Haemostas 1985; 53: 45-50
    PubMedGoogle Scholar
  • 27 Pannekoek H, de Vries C, van Zonneveld A-J. Mutants of human tissue-type plasminogen activator (t-PA): structural aspects and functional properties. Fibrinolysis 1988; 2: 123-132
    PubMedGoogle Scholar
  • 28 Friezner-Degen S, Rajput B, Reich E. The human plasminogen activator gene. J Biol Chem 1986; 261: 6972-6985
    PubMedGoogle Scholar
  • 29 Liu J, Song A, Zhu D, Gurewich V. The inhibition of fibrin stimulated t-PA-induced plasminogen activation by the A-chain fragment 149-157 of urokinase. In: Peptides, Chemistry and Biology. Smith J, Rivier JE. (eds). ESCOM; Leiden: 1992: 810-811
    Google Scholar
  • 30 Kaufman RJ, Wasley LC, Spiliotes AJ, Gosseld SD, Latt SA, Larsen GR, Kay RM. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol 1985; 5: 1750-1759
    CrossrefPubMedGoogle Scholar
  • 31 Krause J. Catabolism of tissue-type plasminogen activator (t-PA), its variants, mutants and hybrids. Fibrinolysis 1988; 2: 133-142
    PubMedGoogle Scholar
  • 32 Krause J, Seydel W, Heinzel G, Tanswell P. Different receptors mediate the hepatic catabolism of tissue-type plasminogen activator and urokinase. Biochem J 1990; 267: 647-652
    CrossrefPubMedGoogle Scholar
  • 33 Marks GJ, Hart TK, Rush GF, Hoffstein ST, Fong KL, Bugelski PJ. Internalization of recombinant tissue-type plasminogen activator by isolated rat hepatocytes is via coated pits. Thromb Haemostas 1990; 63: 251-258
    PubMedGoogle Scholar
  • 34 Marshall JM, Ballance DJ, Courtney M, Cederholm-Williams SA. Binding to the urokinase growth factor-PAI-2 hybrid to the urokinase receptor. Thromb Haemostas 1991; 65: 880
    PubMedGoogle Scholar
  • 35 Robbiati F, Nolli ML, Soffientini A, Sarubbi E, Stoppeli MP, Cassani G, Parenti F, Blasi F. A recombinant prourokinase derived mutant missing the growth factor-like domain does not bind to its receptor. Fibrinolysis 1990; 4: 53-60
    PubMedGoogle Scholar
  • 36 Cubellis MV, Wun T-Z, Blasi F. Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1. EMBO J 1990; 9: 1079-1085
    PubMedGoogle Scholar
  • 37 Estreicher A, Muhlhauser J, Carpentier JL, Orci L, Vassalli J-D. The receptor for urokinase-type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes. J Cell Biol 1990; 111: 783-792
    CrossrefPubMedGoogle Scholar
  • 38 DelRosso M, Fibbi G, Pucci M, Dini G, Grappone C, Nolli ML. Modulation of surface-associated urokinase: binding interiorization, delivery to lysosomes and degradation in human keratinocytes. Exp Cell Res 1991; 193: 346-355
    CrossrefPubMedGoogle Scholar
  • 39 Heim H, Asselbergs F, Bürgi R, Chaudhuri B, Küenzi M, Meyhack B, Rajput B, van Oostrum J. Chimeric recombinant plasminogen activators - expression and characterization. Thromb Haemostas 1989; 62: 337
    PubMedGoogle Scholar
  • 40 Wikström K, Mattson C, Sterky C, Pohl G. Tissue plasminogen activator mutants lacking the growth factor domain and the first kringle domain II. Enzymatic properties in plasma and in vivo thrombolytic activity and clearance rates in rabbits. Fibrinolysis 1991; 5: 31-41
    PubMedGoogle Scholar
  • 41 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
    PubMedGoogle Scholar
  • 42 Martin U, Fischer S, Kohnert U, Opitz U, Rudolph R, Sponer G, Stern A, Strein K. Thrombolysis with an E. coli-produced recombinant plasminogen activator in the rabbit model of jugular vein thrombosis. Thromb Haemostas 1991; 65: 560-564
    PubMedGoogle Scholar
  • 43 Hiramatsu R, Kasai S, Amatsuji Y, Kawai T, Hirose M, Morita M, Tanabe T, Kawabe H, Arimura H, Yokoyama K. Effect of deletion of epidermal growth factor-like domain on plasma clearance of prourokinase. Fibrinolysis 1989; 3: 147-151
    PubMedGoogle Scholar
  • 44 Mattson C, Wikstrom K, Sterky C, Pohl G. Synergism between tissue-type plasminogen activator and genetically engineered variant lacking the finger domain, the growth factor domain and the first kringle domain. Thromb Haemostas 1991; 65: 286-290
    PubMedGoogle Scholar
  • 45 Saito Y, Sasaki H, Hayashi M, Suzuki S, Ishii Y, Koyama S, Notani J, Kobayashi M, Asada T, Horiai H, Niwa M, Shibayama F. Novel recombinant tissue plasminogen activators produced in Escherichia coli. Ann NY Acad Sci 1990; 613: 452-454
    CrossrefPubMedGoogle Scholar
  • 46 Jackson CV, Crowe VG, Craft TJ, Sundboom JL, Grinnell BW, Bobbitt JL, Burck PJ, Quay JF, Smith GF. Thrombolytic activity of a new plasminogen activator, LY210825, compared to recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis. Circulation 1990; 82: 930-940
    CrossrefPubMedGoogle Scholar
  • 47 Fibbi G, Ziche M, Morbidelli L, Magnelli L, DelRosso M. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells. Exp Cell Res 1990; 186: 196
    CrossrefPubMedGoogle Scholar
  • 48 Nykjaer A, Petersen CM, Christensen EI, Davidsen O, Gliemann J. Urokinase receptors in human monocytes. Biochim Biophys Acta 1990; 1052: 399-407
    CrossrefPubMedGoogle Scholar
  • 49 Zacharias U, Handschank W, Schneider F, Loster K, Kleitke C, Noll F, Will H. Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies. J Immunol Methods 1990; 130: 81-90
    CrossrefPubMedGoogle Scholar
  • 50 Park S, Harker LA, Marzek UM, Levin EG. Demonstration of single-chain urokinase-type plasminogen activator on human platelet membrane. Blood 1989; 73: 1421-1425
    PubMedGoogle Scholar
  • 51 Miles LA, Levin EG, Plescia J, Collen D, Plow ED. Plasminogen receptors, urokinase receptors and their modulation on human endothelial cells. Blood 1988; 72: 628-635
    PubMedGoogle Scholar
  • 52 Hajjar KA, Hamel NM. Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase. J Biol Chem 1990; 265: 2908-2916
    PubMedGoogle Scholar
  • 53 Russell ME, Quartemous T, Declerck PJ, Collen D, Haber E, Homey CJ. Binding of tissue-type plasminogen activator with human endothelial cell monolayers. J Biol Chem 1990; 263: 2569-2575
    PubMedGoogle Scholar
  • 54 Mignatti P, Mazzieri R, Rifkin DB. Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J Cell Biol 1991; 113: 1193-1201
    CrossrefPubMedGoogle Scholar
  • 55 Holmes WE, Pennica D, Blaber M, Rey MW, Guenzler WA, Steffens GJ, Heyneker HL. Cloning and expression the the gene for prourokinase in Escherichia coli . Bio/technology 1985; 3: 923-929
    CrossrefPubMedGoogle Scholar
  • 56 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg P, Heyneker 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