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Thromb Haemost 1992; 67(04): 445-452
DOI: 10.1055/s-0038-1648468
Original Articles
Schattauer GmbH Stuttgart

Biochemical, Thrombolytic and Pharmacokinetic Properties of rt-PA P47G, K49N, a Substitution Variant of Human Tissue-Type Plasminogen Activator

L Nelles
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
X-K Li
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
I Vanlinthout
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
F De Cock
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
H R Lijnen
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
D Collen
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 15 July 1991

Accepted after revision 22 October 1991

Publication Date:
03 July 2018 (online)

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Summary

rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahem et al. (J Biol Chem 1990; 265: 5540—5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse®).

The specific fibrinolytic activities on fibrin plates were 160,000 ± 17,000, 210,000 ± 88,000 and 460,000 ± 72,000 IU/mg (mean ± SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k 2 K m) in the absence of fibrin were comparable (1.1 to 1.7 × 10-3 μM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 ± 0.14 pg/ml rt-PA P47G, K49N, 0.36 ± 0.01 pg/ml rt-PA and 0.17 ± 0.01 pg/ml alteplase, respectively (mean ± SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.

The thrombolytic properties were examined in a hamster pulmonary embolism model. The thrombolytic potency, expressed as percent lysis per mg activator administered per kg body weight, was 160 ± 28 for rt-PA P47G, K49N, 150 ± 36 for wild-type rt-PA and 310 ± 42 for alteplase. The specific thrombolytic activity, expressed as percent lysis per pg steady-state t-PA-related antigen level per ml plasma, was 49 ± 18% for rt-PA P47G, K49N, 160 ± 49 for rt-PA and 500 ± 79 for alteplase. The clearance rates following bolus injection in hamsters were 0.18 ± 0.02, 1.9 ± 0.2 and 2.1 ± 0.1 ml/min respectively.

Thus, the substitution of only two residues in wild-type rt-PA markedly reduces its clearance, but it also significantly alters its specific thrombolytic activity, resulting in a virtually unaltered thrombolytic potency.

 

  • References

  • 1 Collen D, Lijnen HR, Todd PA, Goa KL. Tissue-type plasminogen activator. A review of its pharmacology and therapeutic use as a thrombolytic agent. Drugs 1989; 38: 346-388
    CrossrefPubMedGoogle Scholar
  • 2 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
    PubMedGoogle Scholar
  • 3 Rijken DC, Emeis JJ. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. Biochem J 1986; 238: 643-646
    CrossrefPubMedGoogle Scholar
  • 4 Larsen GR, Metzger M, Henson K, Blue Y, Horgan P. Pharmacokinetics and distribution analysis of variant forms of tissue-type plasminogen activator with prolonged clearance in rat. Blood 1989; 73: 1842-1850
    PubMedGoogle Scholar
  • 5 Browne MJ, Carey JE, Chapman CG, Tyrrell AWR, Entwisle C, Lawrence GMP, 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
    PubMedGoogle Scholar
  • 6 Hotchkiss A, Refino CJ, Leonard CK, O’Connor JV, Crowley C, McCabe J, Tate K, Nakamura G, Powers D, Levinson A, Mohler M, Spellman MW. The influence of carbohydrate structure on the clearance of recombinant tissue-type plasminogen activator. Thromb Haemostas 1988; 60: 255-261
    PubMedGoogle Scholar
  • 7 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
    CrossrefPubMedGoogle Scholar
  • 8 Verheijen JH, Caspers MPM, Chang GTG, de Munk GAW, 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
    PubMedGoogle Scholar
  • 9 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
    PubMedGoogle Scholar
  • 10 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
    PubMedGoogle Scholar
  • 11 Ahern TJ, Morris GE, Barone KM, Horgan PG, Timony GA, Angus LB, Henson KS, Stoudemire JB, Langer-Safer PR, Larsen GR. Site-directed mutagenesis in human tissue-type plasminogen activator: distinguishing the sites in the amino-terminal region required for full fibrinolytic activity and rapid clearance from the circulation. J Biol Chem 1990; 265: 5540-5545
    PubMedGoogle Scholar
  • 12 Collen D, Lijnen HR, Bulens F, Van Damme A, Tulinsky A, Nelles L. Biochemical and functional characterization of human tissue-type plasminogen activator variants with mutagenized kringle domains. J Biol Chem 1990; 265: 12184-12191
    PubMedGoogle Scholar
  • 13 Zamarron C, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator. J Biol Chem 1984; 259: 2080-2083
    PubMedGoogle Scholar
  • 14 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
    PubMedGoogle Scholar
  • 15 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-5467
    CrossrefPubMedGoogle Scholar
  • 16 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
    PubMedGoogle Scholar
  • 17 Lijnen HR, Nelles L, Holmes WE, Collen D. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-5598
    PubMedGoogle Scholar
  • 18 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
    PubMedGoogle 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 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
    CrossrefPubMedGoogle Scholar
  • 21 Astrup T, Miillertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
    CrossrefPubMedGoogle Scholar
  • 22 Gaffney PJ, Curtis AD. A collaborative study to establish the 2nd international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1987; 58: 1085-1087
    PubMedGoogle Scholar
  • 23 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestim-mung des Fibrinogens. Acta Haematol 1957; 17: 237-246
    CrossrefPubMedGoogle Scholar
  • 24 Edy J, Collen D, Verstraete M. Quantitation of the plasma protease inhibitor antiplasmin with the chromogenic substrate S-2251. In: Progress in Chemical Fibrinolysis and Thrombolysis, Vol3. Davidson JF, Rowan RM, Samama MM, Desnoyers PC. (eds) Raven Press; New York: 1978. pp 315-322
    Google Scholar
  • 25 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
    PubMedGoogle Scholar
  • 26 Erlanger BF, Kokowsky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961; 95: 271-278
    CrossrefPubMedGoogle Scholar
  • 27 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
  • 28 Stassen JM, Vanlinthout I, Lijnen HR, Collen D. A hamster pulmonary embolism model for the evaluation of the thrombolytic and pharmacokinetic properties of thrombolytic agents. Fibrinolysis 1990; 4: 15-21
    PubMedGoogle Scholar
  • 29 Gibaldi M, Perrier DP. In: Pharmacokinetics. Marcel Dekker; New York: 1983. pp 45-111
  • 30 Smedsrpd B, Einarsson M, Pertoft H. Tissue plasminogen activator is endocytosed by mannose and galactose receptors of rat liver cells. Thromb Haemostas 1988; 59: 480-484
    PubMedGoogle Scholar
  • 31 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin fingerlike and/or the epidermal growth factor domains. J Biol Chem 1988; 263: 1023-1029
    PubMedGoogle Scholar