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-PAI)

L Nelles; H R Lijnen; A Van Nuffelen; E Demarsin; D Collen

doi:10.1055/s-0038-1647253

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1990; 64(01): 053-060
DOI: 10.1055/s-0038-1647253

Original Article

Schattauer GmbH Stuttgart

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-PAI)^[*]

L Nelles

The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

H R Lijnen

The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

A Van Nuffelen

The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

E Demarsin

The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

D Collen

The Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

› Institutsangaben

Abstract

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Summary

Chimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, but only partial fibrin-binding properties of t-PA (Nelles et al., J Biol Chem 1987; 262: 10855–62). The following domain deletion and/or duplication mutants of such a t-PA/u-PA chimera were constructed, purified and charactertzed: rt-PA-ΔFE∇/u-PA, with deletion of the finger-like (F) and epidermal growth factor-like (E) domains, rt-PA-ΔK₁∇K₂/u-PA, with kringle 1 (K₁) replaced by a second copy of kringle 2 (K₂), and rt-PA-ΔFEK₁∇K₂/u-PA, with F and E domain deletions in rt-PAΔK₁∇K₂/u-PA.

The specific activities on fibrin plates of the single-chain (sc) chimeras ranged between 68,000 IU/mg for rt-PA-ΔK₁∇K₂/scu-PA and 200,000 IU/mg for rt-PA-ΔFEK₁∇K₂/scu-PA, as compared to L20,000 IU/mg for rscu-PA. The specific activities of their plasmin-generated two-chain (tc) derivatives ranged between 120,000 IU/mg for rt-PA-ΔK₁∇K₂/tcu-PA and 240,000 IU/mg for rt-PA-ΔFEK₁∇K₂/tcu-PA, as compared to 100,000 IU/mg for rtcu-PA. All two-chain chimeras activated plasminogen following Michaelis-Menten kinetics, with catalytic efficiencies between 0.072 μM⁻¹s⁻¹ for rt-PA-ΔK₁∇K₂/tcu-pA and 0.081 pM⁻¹ s⁻¹ for rt-PA-ΔFEK₁∇K₂/tcu-PA, as compared to 0.088 μM⁻¹ s⁻¹ for rtcu-PA. CNBr-digested fibrinogen enhanced the initial rate of plasminogen activation by a factor 2.2 to 6.2, as compared to 4.9 for rtcu-PA. The fibrin-affinity of the chimeras decreased in the order rt-PA > rt-PA-ΔK₁∇K₂/u-PA > u-PA and that for lysine in the order rt-PA-ΔFEK₁∇K₂/u-PA > > t-PA/u-PA ⩽ st-PA > rt-PA-ΔFE/u-PA > u-PA. All single-chain plasminogen activators caused a time and concentration-dependent clot lysis in an in vitro plasma clot lysis system, with equi-effective doses (causin g 50% clot lysis in 2 h) ranging between 0.53 and 0.90 μg/ml, as compared to 1 .7 μg/ml for rscu-PA, and were associated with comparable residual fibrinogen levels of approximately 80%.

Thus, substitution of K₁ by a second copy of K₂ in the chimeric protein t-PA/u-PA enhances the affinity for both fibrin and lysine significantly and improves the fibrinolytic potency in an in vitro clot lysis system marginally.

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Referenzen

References
1 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
2 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
3 Rijken DC, Groeneveld E. Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator. J Biol Chem 1986; 261: 3098-3102
4 van Zonneveld AJ, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4674
5 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
6 Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species. J Clin Invest 1984; 73: 1731-1739
7 Stump DC, Lijnen HR, Collen D. Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator. J Biol Chem 1986; 261: 17120-17126
8 Collen D, Stump DC, Gold HK. Thrombolytic therapy. Annu Rev Med 1988; 39: 405-423
9 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
10 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
11 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett 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
12 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
13 Lijnen HR, Nelles L, Van Hoef B, De Cock F, Collen D. Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains. J Biol Chem 1990; 265: 5677-5683
14 Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973; 52: 456-467
15 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
16 Nelles L, Lijnen HR, Collen D, Holmes WE. Characterization of recombinant human single-chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158. J Biol Chem 1987; 262: 5682-5689
17 Holvoet P, Cleemput 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
18 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
19 Boshart M, Weber F, Jahn G, Dorsch-Haesler K, Fleckenstein B, Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalolvirus. Cell 1985; 41: 521-530
20 Van Heuvel M, Bosveld IJ, Mooren AT A, Trapman J, Zwarthoff EC. Properties of natural and hybrid murine alpha interferons. J Gen Virol 1986; 67: 2215-2222
21 Collen D, Stassen JM, Larsen G. Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits. Blood 1988; 71: 216-219
22 Kalyan NK, Lee SG, Wilhelm 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 NH 2-terminal domains on its biochemical and biological properties. J Biol Chem 1988; 263: 3971-3978
23 Browne MJ, Carey JE, Chapman CG, Tyrrell AW R, Entwisle C, Lawrence GM P, Reavy B, Dodd I, Esmail A, Robinson JH. A tissue-type plasminogen activator mutant with prolonged clearance in vivo. Effect of removal of the growth factor domain. J Biol Chem 1988; 263: 1599-1602
24 Lijnen HR, Nelles L, Holmes WE, Collen D. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 to leu411) of recombinant single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-5598
25 de Vries C, Veerman H, Blasi F, Pannekoek H. Artificial 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
26 Collen D, Stassen JM, Demarsin E, Kieckens L, Lijnen HR, Nelles L. Pharmacokinetics and thrombolytic properties of chimaeric plasminogen activators consisting of the NH2-terminal region of human tissue-type plasminogen activator and the COOH-terminal region of human single-chain urokinase-type plasminogen activator. J Vase Med Biol 1989; 1: 234-240
27 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenoly-tic characterization of genetic variants lacking the fibronectin fingerlike and/or the epidermal growth factor domains. J Biol Chem 1988; 263: 1023-1029
28 Verheijen JH, Caspers MP M, Chang GT G, De Munk GA W, 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
29 van Zonneveld AJ, Veerman H, Pannekoek H. On the involvement of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin: inhibition of kringle-2 binding to fibrin by e-amino caproic acid. J Biol Chem 1986; 261: 14214-14218
30 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

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-PAI)[*]

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-PAI)^[*]