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

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
• 2 Verstraete M. Thrombolytic treatment in acute myocardial infarction. Circulation 1990; 82 (Suppl. 02) 1196-1109
• 3 Lijnen R, Collen D. Towards the development of improved thrombolytic agents. Br J Haematol 1991; 77: 261-266
• 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
• 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
• 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
• 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
• 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
• 9 Patthy L. Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 1985; 41: 657-663
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 21 Lijnen HR, Collen D. Strategies for the improvement of thrombolytic agents. Thromb Haemostas 1991; 66: 88-110
• 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
• 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
• 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
• 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
• 26 Astrup T, Mullertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
• 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
• 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
• 29 Loskutoff DJ, Mussoni L. Interaction between fibrin and the plasminogen activators produced by cultured endothelial cells. Blood 1983; 62: 62-68
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 37 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. J Biol Chem 1988; 263: 1023-1029
• 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
• 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
• 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
• 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
• 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
• 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
• 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