PDF herunterladen
Thromb Haemost 1993; 70(02): 307-312
DOI: 10.1055/s-0038-1649571
Original Articles
Fibrinolysis
Schattauer GmbH Stuttgart

A Slow Clearing, Fibrin-Specific, PAI-1 Resistant Variant of t-PA (T103N, KHRR 296-299 AAAA)

Nicholas F Paoni
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Bruce A Keyt
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Canio J Refino
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Alice M Chow
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Hung V Nguyen
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Lea T Berleau
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Julie Badillo
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Luis C Peña
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Kevin Brady
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
,
Florian M Wurm
2   The Department of Cell Culture R & D, Genentech., Inc., South San Francisco, CA, USA
,
John Ogez
3   The Department of Recovery Process R & D, Genentech., Inc., South San Francisco, CA, USA
,
William F Bennett
1   The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 12. Dezember 1992

Accepted after revision 17. Februar 1993

Publikationsdatum:
04. Juli 2018 (online)

Auch verfügbar auf
  als PDF herunterladen  Lizenzen und Reprints

Summary

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.

 

  • References

  • 1 Collen D, Topol EJ, Tiefenbrunn AJ, Gold HK, Weisfeldt ML, Sobel BE, Leinbach RC, Brinker JA, Ludbrook PA, Yasuda I, Bulkley BH, Robison AK, Hutter Jr AM, Bell WR, Spadard Jr JJ, Khaw BA, Grossbard EB. Coronary thrombolysis with recombinant human tissue-type plasminogen activator: a prospective, randomized, placebo controlled trial. Circulation 1984; 70: 1012-1017
    CrossrefPubMedSuche in Google Scholar
  • 2 Topol EJ, Califf RM, George BS, Keriakes DJ, Lee KL. for the TAMI Study Group. Insights derived from the thrombolysis and angioplasty in myocardial infarction (TAMI) trials. J Am Coll Cardiol 1988; 12: 24A-31A
    CrossrefPubMedSuche in Google Scholar
  • 3 Stump DC, Califf RM, Topol EJ, Sigmon K, Thornton D, Masek R, Anderson L, Collen D. the TAMI Study Group. Pharmacodynamics of thrombolysis with recombinant tissue-type plasminogen activator. Correlation with characteristics of and clinical outcomes in patients with acute myocardial infarction. Circulation 1989; 80: 1222-1230
    CrossrefPubMedSuche in Google Scholar
  • 4 TIMI Study Group. Comparison of invasive and conservative strategies after treatment with intravenous tissue plasminogen activator in acute myocardial infarction. Results of the Thrombolysis in Myocardial Infarction (TIMI) Phase II Trial. N Engl J Med 1989; 320: 618-627
    CrossrefPubMedSuche in Google Scholar
  • 5 Goldhaber SZ, Kessler CM, Heit J, Markis J, Sharma GVRK, Dawley D, Nagel JS, Meyerovitz M, Kim D, Vaughan D, Parker JA, Tumeh SS, Drum D, Loscalzo J, Reagan K, Selwyn AP, Anderson J, Braunwald E. Randomised controlled trial of recombinant tissue plasminogen activator versus urokinase in the treatment of acute pulmonary embolism. Lancet 1988; 2: 293-298
    CrossrefPubMedSuche in Google Scholar
  • 6 Parker JA, Markis JE, Palla A, Goldhaber SZ, Royal HD, Tumeh S, Kim D, Rustgi AK, Holman BL, Kolodny GM, Braunwald E. Pulmonary perfusion after rt-PA therapy for acute embolism: early improvement assessed with segmental perfusion scanning. Radiology 1988; 166: 441-445
    CrossrefPubMedSuche in Google Scholar
  • 7 Booyse FM, Scheinbuks J, Radek J, Osikowicz G, Fedor S, Quarfoot AJ. Immunological identification and comparison of plasminogen activator forms in cultured normal human endothelial cells and smooth muscle cells. Thromb Res 1981; 24: 495-504
    CrossrefPubMedSuche in Google Scholar
  • 8 Goldsmith GH, Ziats NP, Robertson AL. Studies on plasminogen activator and other proteases in subcultured human vascular cells. Exp Mol Pathol 1981; 35: 257-264
    CrossrefPubMedSuche in Google Scholar
  • 9 Levin EG, Loskutoff DJ. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol 1982; 94: 631-636
    CrossrefPubMedSuche in Google Scholar
  • 10 Baughman RA. In: The pharmacokinetics of tissue plasminogen activator. Tissue Plasminogen Activator in Thrombolytic Therapy. Sobel BE, Collen D, Grossbard EB. (eds) New York: Marcel Dekker; 1987: 41-56
  • 11 Collen D, Stassen J, Larsen G. Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits. Blood 1988; 71: 216-219
    PubMedSuche in Google Scholar
  • 12 Kalyan NK, Lee SG, Wilhelm J, Fu KP, Hum W-T, Rappaport R, Hartzell RW, Urbano C, Hung PP. Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties. J Biol Chem 1988; 263: 3971-3978
    PubMedSuche in Google Scholar
  • 13 Fu KP, Lee S, Hum WT, Kalyan N, Rappaport R, Hetzel N, Hung PP. Disposition of a novel recombinant tissue plasminogen activator, Δ2-89 tPA, in mice. Thromb Res 1988; 50: 33-41
    PubMedSuche in Google Scholar
  • 14 Refino CJ, Hotchkiss AJ, Higgins DL, Mohler MA. The pharmacokinetics and circulatory metabolism of a long half-life mutant of rt-PA. Fibrinolysis 1988; 2: 30
    PubMedSuche in Google Scholar
  • 15 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
    PubMedSuche in Google Scholar
  • 16 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. J Biol Chem 1988; 263: 1599-1602
    PubMedSuche in Google Scholar
  • 17 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
    PubMedSuche in Google Scholar
  • 18 Lau D, Kuzma G, Wei C, Livingston DJ, Hsiung N. A modified human tissue plasminogen activator with extended half-life in vivo. Bio/Technology 1987; 5: 953-958
    CrossrefPubMedSuche in Google Scholar
  • 19 Lau D, Kuzma G, Wei C, Livingston DJ, Hsiung N. Erratum. Bio/ Technology 1988; 6: 734-311
    PubMedSuche in Google Scholar
  • 20 Martin U, von Möllendorf E, Akpan W, Kientsch-Engel R, Kaufmann B, Neugebauer G. Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator BM 06.022 in healthy volunteers. Thromb Haemostas 1991; 66: 569-574
    PubMedSuche in Google Scholar
  • 21 Anderson S, Keyt B. Variants of plasminogen activators and processes for their production. 1989, International Patent Application No. PCT-US89-01947, Publication No W089-11531
    PubMed
  • 22 Bennett WF, Paoni NF, Keyt BA, Botstein D, Jones AJS, Presta L, Wurm FM, Zoller MJ. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem 1991; 266: 5191-5201
    PubMedSuche in Google Scholar
  • 23 Eastman D, Wurm FM, van Reis R, Higgins DL. A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators. Biochemistry 1992; 31: 419-422
    CrossrefPubMedSuche in Google Scholar
  • 24 Paoni NF, Refino CJ, Brady K, Peña LC, Nguyen HV, Kerr EM, Johnson AC, Wurm FM, van Reis R, Botstein D, Bennett WF. Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator. Protein Engineering 1992; 5: 259-266
    CrossrefPubMedSuche in Google Scholar
  • 25 Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 1980; 77: 4216-4220
    CrossrefPubMedSuche in Google Scholar
  • 26 Graham FL, van der EbAJ. A new technique for the assay of infectivity of human Adenovirus 5 DNA. Virology 1973; 52: 456-457
    CrossrefPubMedSuche in Google Scholar
  • 27 Hunter WM, Greenwood FC. Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature 1962; 194: 495-496
    CrossrefPubMedSuche in Google Scholar
  • 28 Keyt BA, Berleau LT, Nguyen HV, Bennett WF. Radioiodination of the active site of tissue plasminogen activator: a method for radiolabeling serine proteases with tyrosylprolylarginyl chloromethyl ketone. Anal Biochem 1992; 206: 73-83
    CrossrefPubMedSuche in Google Scholar
  • 29 Hekman CM, Loskutoff DJ. Bovine plasminogen activator inhibitor 1: specificity determinations and comparison of the active, latent, and guanidine-activated forms. Biochemistry 1988; 27: 2911-2918
    CrossrefPubMedSuche in Google Scholar
  • 30 Wun T-C, Palmier MO, Siegel NR, Smith CE. Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. J Biol Chem 1989; 264: 7862-7868
    PubMedSuche in Google Scholar
  • 31 Chmielewska J, Ranby M, Wiman B. Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Biochem J 1988; 251: 327-332
    CrossrefPubMedSuche in Google Scholar
  • 32 De Serrano VS, Castellino FJ. Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1. J Biol Chem 1990; 265: 10473-10478
    PubMedSuche in Google Scholar
  • 33 Wilhelm J, Lee SG, Kalyan NK, Cheng SM, Wiener F, Pierzchala W, Hung PP. Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation. Bio/ technology 1990; 8: 321-325
    CrossrefPubMedSuche in Google Scholar
  • 34 Madison EL, Goldsmith EJ, Gerard RD, Gething M-JH, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature 1989; 339: 721-724
    CrossrefPubMedSuche in Google Scholar
  • 35 Madison EL, Goldsmith EJ, Gerard RD, Gething M-JH, Sambrook JF, Bassel-Duby RS. Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. Proc Natl Acad Sci USA 1990; 87: 3530-3533
    CrossrefPubMedSuche in Google Scholar
  • 36 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol (Basel) 1957; 17: 237-240
    CrossrefPubMedSuche in Google Scholar