Download PDF
Thromb Haemost 1991; 65(02): 174-180
DOI: 10.1055/s-0038-1647479
Original Article
Schattauer GmbH Stuttgart

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

D Collen
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
,
I Vanlinthout
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
L Kieckens
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
L Nelles
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
J M Stassen
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received: 10 July 1990

Accepted after revision 06 September 1990

Publication Date:
02 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 μi 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs.

Recombinant t-PA (home-made rt-PA or Activase®) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in μg/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1+e−a(ex−eb)) and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = ¼ ac .eb) were determined. With rt-PA, these parameters were c = 72 ± 6% (mean ± SEM), b = 0.19 ± 0.08 mg/kg, z = 68 ± 25% lysis per mg/kg, with corresponding values of 87 ± 5%, 0.07 ± 0.03 mg/kg and 150 ± 38% lysis per mg/kg for Activase® (p = NS). Deletion of the finger and growth factor domains in rt-PA ( rt-PA-ΔFE) was not associated with marked alteration of the thrombolytic potency (c = 90 ± 30%, b = 0.34 ± 0.35 mg/kg, and z = 54 ± 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity. Substitution of the kringle 1 domain by a second copy of the kringle 2 domain in rt-PA and in rt-PA-ΔFE did not significantly alter the thrombolytic potency as characterized by a simultaneous decrease of the b value and an increase of the z value (b = 0.07 ± 0.03 mg/kg and z = 210 ± 70% per mg/kg for rt-PA-ΔK1∇K2 and b = 0.13 ± 0.02 mg/kg and z = 210 ± 29% per mg/kg for rt-PA-ΔFEK1∇K2). Deletion of either the finger or the growth factor domain in rt-PA-ΔK1∇K2 also did not significantly alter the thrombolytic potency (b = 0.08 ± 0.008 mg/kg and z = 320 ± 33% per mg/kg for rt-PA-ΔEK1∇K2 and b = 0.05 ± 0.01 mg/kg and z = 270 ± 48% per mg/kg for rt-PA-ΔFK1∇K2). Likewise, none of the variants had a higher specific thrombolytic activity than wild-type rt-PA. Plasma clearance rates were reduced 10- to 20-fold for the rt-PA mutants lacking the FE domains, and 3- to 5-fold for rt-PA-ΔK1∇K2.

 

  • 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
    PubMedSearch in Google Scholar
  • 2 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
    CrossrefPubMedSearch in Google Scholar
  • 3 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
    CrossrefPubMedSearch in Google Scholar
  • 4 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
    PubMedSearch in Google Scholar
  • 5 van Zonneveld AJ, Veerman H, Pannekoe H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4674
    CrossrefPubMedSearch in Google Scholar
  • 6 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
    PubMedSearch in Google Scholar
  • 7 Browne MJ, Carey JE, Chapman CG, Tyrrell AW, Entwisle C, Lawrence GM, Reavy B, Dodd IS, 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
    PubMedSearch in Google Scholar
  • 8 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
    PubMedSearch in Google Scholar
  • 9 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
    PubMedSearch in Google Scholar
  • 10 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 (Suppl. 02) 15-21
    PubMedSearch in Google Scholar
  • 11 Tate KM, Higgins DL, Holmes WE, Winkler ME, Heyneker HL, Vehar GA. Functional role of proteolytic cleavage at Arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. Biochemistry 1987; 26: 338-343
    CrossrefPubMedSearch in Google Scholar
  • 12 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestim-mung des Fibrinogens. Acta Haematol 1957; 17: 237-246
    CrossrefPubMedSearch in Google Scholar
  • 13 Vermylen J, De Vreker R, Verstraete M. A rapid enzymatic method for assay of fibrinogen: the fibrin polymerization test (F. P. T.). Clin Chim Acta 1963; 8: 418-424
    CrossrefPubMedSearch in Google Scholar
  • 14 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, Vol. 3 Davidson JF, Rowan RM, Samama MM, Desnoyers PC. (eds) Raven Press; New York: 1978: 315-322
  • 15 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 anticlonal antibodies to t-PA. Thromb Haemostas 1985; 54: 684-687
    PubMedSearch in Google Scholar
  • 16 Gibaldi M, Perrier D. Pharmacokinetics. Marcel Dekker New York: 1983: 45-111
    Search in Google Scholar
  • 17 Cambier P, Van de WerfF, Larsen GR, Collen D. Pharmacokinetics and thrombolytic properties of a nonglycosylated mutant of human tissue-type plasminogen activator, lacking the finger and growth factor domains, in dogs with copper coil-induced coronary thrombosis. J Cardiovasc Pharmacol 1988; 11: 468-472
    CrossrefPubMedSearch in Google Scholar
  • 18 Kominger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
    PubMedSearch in Google Scholar
  • 19 Fuchs HE, Berger H, Pizzo SV. Catabolism of human tissue plasminogen activator in mice. Blood 1985; 65: 539-544
    PubMedSearch in Google Scholar
  • 20 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
    CrossrefPubMedSearch in Google Scholar
  • 21 Flameng W, Van de Werf F, Vanhaecke J, Verstraete M, Collen D. Coronary thrombolysis and infarct size reduction after intravenous infusion of recombinant tissue-type plasminogen activator in nonhuman primates. J Clin Invest 1985; 75: 84-90
    CrossrefPubMedSearch in Google Scholar
  • 22 Verstraete M BounameauxH, De Cock F, Van de WerfF, Collen DS. Pharmacokinetics and systematic fibrinogenolytic effects of recombinant human tissue-type plasminogen activator (rt-PA) in humans. J Pharmacol Exp Therap 1985; 235: 506-512
    PubMedSearch in Google Scholar
  • 23 Verstraete M, Su CAPF, Tanswell P, Feuerer W, Collen D. Pharmacokinetics and effects on fibrinolytic and coagulation parameters of two doses of recombinant tissue-type plasminogen activator in healthy volunteers. Thromb Haemostas 1986; 56: 1-5
    PubMedSearch in Google Scholar
  • 24 Garabedian HD, Gold HK, Leinbach RC, Johns JA, Yasuda T, Kanke M, Collen D. Comparative properties of two clinical preparations of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction. J Am Coll Cardiol 1987; 9: 599-607
    CrossrefPubMedSearch in Google Scholar
  • 25 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
    PubMedSearch in Google Scholar
  • 26 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
    PubMedSearch in Google Scholar