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

Fred A M Asselbergs; Rolf Bürgi; Jessica Hamerman; Jutta Heim; Jan van Oostrum; Giancarlo Agnelli

doi:10.1055/s-0038-1651547

Thrombosis and Haemostasis, Inhaltsverzeichnis

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

Authors

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

¹The Instituto di Semiotica Medica, University of Perugia, Perugia, Italy

Abstract

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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.

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Referenzen

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
2 Miles LA, Plow EF. Plasminogen receptors: ubiquitous sites for cellular regulation of fibrinolysis. Fibrinolysis 1988; 2: 73-84
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
20 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246
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
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
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
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
25 Herion P, Glineur C, Franssen J-D, Urbain J, Bollen A. Monoclonal antibodies against urokinase. Biosci Rep 1981; 1: 885-892
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
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
28 Friezner-Degen S, Rajput B, Reich E. The human plasminogen activator gene. J Biol Chem 1986; 261: 6972-6985
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
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
31 Krause J. Catabolism of tissue-type plasminogen activator (t-PA), its variants, mutants and hybrids. Fibrinolysis 1988; 2: 133-142
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
48 Nykjaer A, Petersen CM, Christensen EI, Davidsen O, Gliemann J. Urokinase receptors in human monocytes. Biochim Biophys Acta 1990; 1052: 399-407
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
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
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
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
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
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
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
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