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Thromb Haemost 1990; 64(03): 455-463
DOI: 10.1055/s-0038-1647336
Original Article
Schattauer GmbH Stuttgart

A Precise and Rapid Microtitre Plate Clot Lysis Assay: Methodology, Kinetic Modeling and Measurement of Catalytic Constants for Plasminogen Activation during Fibrinolysis

A J S Jones
The Department of Pharmaceutical Research and Development, Genentech, Inc, South San Francisco, California, U.S.A
,
A M Meunier
The Department of Pharmaceutical Research and Development, Genentech, Inc, South San Francisco, California, U.S.A
› Author Affiliations
Further Information

Publication History

Received 20 February 1990

Accepted after revision 02 July 1990

Publication Date:
25 July 2018 (online)

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Summary

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear toinfluence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and0.1 s-1, respectively, in excellent agreement with published values The assay should therefore prove useful in quantitative evaluationsof the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen. When the data were re-analysed using published values ofthe affinities of the two forms, t-PA was found to activate both forms indistinguishably on the fibrin surface, consistent with suggestions that the conformation of glu-plasminogen changes to one more like that of lys-plasminogen upon binding to fibrin.

 

  • References

  • 1 Collen D, Tytgat G, Verstraete M. Semi-automatic method for routine evaluation of fibrinolytic components. J Clin Pathol 1968; 21: 705-707
    CrossrefPubMedGoogle Scholar
  • 2 Carlson RH, Garnick RL, Jones AJ S, Meunier AM. The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer. Anal Biochem 1988; 168: 428-435
    CrossrefPubMedGoogle Scholar
  • 3 Gaffney PJ, Curtis AD. A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1985; 53: 134-136
    PubMedGoogle Scholar
  • 4 Gaffney PJ, Curtis AD. A collaborative study to establish the second international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1987; 58: 1085-1087
    PubMedGoogle Scholar
  • 5 Urano S, Metzger AR, Castellino FJ. Plasmin mediated fibrinolysis by various recombinant tissue plasminogen activators. Proc Natl Acad Sci (USA) 1989; 86: 2568-2571
    CrossrefPubMedGoogle Scholar
  • 6 Jones AJ S. Automated clot lysis assays for measurement of the potency of recombinant tissue plasminogen activator. Eastern Analytical Symposium 1988; 27: 180 Abstract #201
    PubMedGoogle Scholar
  • 7 Jones AJ S, Meunier AM. Utility of a microtitre plate assay for analysis of molecular parameters during clot lysis mediated by tissue plasminogen activator. Thromb Haemostas 1989; 62: 19 Abstract #20
    PubMedGoogle Scholar
  • 8 Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
    CrossrefPubMedGoogle Scholar
  • 9 Rodriguez H, Kohr WJ, Harkins RN. Design and operation of a completely automated Beckman microsequencer. Anal Biochem 1984; 140: 538-547
    CrossrefPubMedGoogle Scholar
  • 10 Laemmli UK. Cleavage of structural proteins during the assambly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 11 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Bennett WE, Yelvertofl E, Seeburg PH, Heyneker HL, Goeddel DY, Collen D. Cloning and expression of human tissue-type plasminogen activator in E. coli. Nature 1983; 301: 214-221
    CrossrefPubMedGoogle Scholar
  • 12 Blombiick B. On the properties of fibrinogen and fibrin. Ark Kemi 1958; 12: 99-113
    PubMedGoogle Scholar
  • 13 Robbins KC, Summaria L. Human plasminogen and plasmin. Meth Enzymol 1970; 19: 184-199
    PubMedGoogle Scholar
  • 14 Shaw E, Mares-Guia M, Cohen W. Evidence for an active-center histidine in trypsin through use of a specific reagent, 1-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethyl ketone derived from N-alpha-tosyl-lysine. Biochemistry 1965; 4: 2219-2224
    CrossrefPubMedGoogle Scholar
  • 15 Westlund LE, Andersson L-O. Studies on the influence of reactants and buffer environment on clot lysis induced by human plasminogen activators. Thromb Res 1985; 37: 213-223
    CrossrefPubMedGoogle Scholar
  • 16 Urano T, Chibber BA K, Castellino FJ. The reciprocal effects of epsilon-aminohexanoic acid and chloride ion on the activation of human [Glu-1]plasminogen by human urokinase. Proc Natl Acad Sci (USA) 1987; 84: 4031-4034
    CrossrefPubMedGoogle Scholar
  • 17 Keyt BA, Higgins D, Meunier AM. Conversion of single chain t-PA to the two chain form during fibrinolysis. Fibrinolysis 1988; 2 (Suppl. 01) Suppl 107
    PubMedGoogle Scholar
  • 18 Philo RD, Gaffney PJ. Plasmin potency estimates: influence of the substrate used in assay. Thromb Haemostas 1981; 45: 107-109
    PubMedGoogle Scholar
  • 19 Beebe DR, Aronson DL. An automated fibrinolytic assay performed in microtiter plates. Thromb Res 1987; 47: 123-128
    CrossrefPubMedGoogle Scholar
  • 20 Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem 1984; 140: 513-522
    CrossrefPubMedGoogle Scholar
  • 21 Carr Jr ME, Hermans J. Size and density of fibrin fibres from turbidity. Macromolecules 1978; 11: 46-50
    CrossrefPubMedGoogle Scholar
  • 22 Hantgan RR, Hermans J. Assembly of fibrin: A light scattering study. J Biol Chem 1979; 254: 11272-11281
    PubMedGoogle Scholar
  • 23 Stewart UA, Bradley MS, Johnson Jr CS, Gabriel DA. Tiansport of probe molecules through fibrin gels as observed by means of holographic relaxation methods. Biopolymers 1988; 27: 173-185
    CrossrefPubMedGoogle Scholar
  • 24 Drapier JC, Tenu JP, Lemaire G, Petit JE. Regulation of plasminogen activator secretion in mouse peritoneal macrophages I Role of serum studied by a new spectrophotometric assay for plasminogen activators. Biochimie 1979; 6l: 463-671
    PubMedGoogle Scholar
  • 25 Garman AJ, Smith RA G. The binding of plasminogen to fibrin: evidence for plasminogen-bridging. Thromb Res 1982; 27: 311-320
    CrossrefPubMedGoogle Scholar
  • 26 Norrman B, Wallen P, Rflnby M. Fibrinolysis mediated by tissue plasminogen activator: Disclosure of a kinetic transition. Eur J Biochem 1985; 149: 193-200
    CrossrefPubMedGoogle Scholar
  • 27 Ralnby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
    CrossrefPubMedGoogle Scholar
  • 28 Urano T, de Serrano VS, Chibber BA K, Castellino FJ. The control of the urokinase-catalyzed activation of human glutamic acid I-plasminogen by positive and negative effectors. J Biol Chem 1987; 262: 15959-15964
    PubMedGoogle Scholar
  • 29 Lucas MA, Fretto LJ, McKee PA. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258: 4249-4256
    PubMedGoogle Scholar
  • 30 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 2912-2919
    PubMedGoogle Scholar
  • 31 Bok RA, Mangel WE. Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin. Biochemistry 1985; 24: 3279-3286
    CrossrefPubMedGoogle Scholar
  • 32 Higgins DL, Bennett WE. Tissue Plasminogen activator: the biochemistry and pharmacology of variants produced by mutagenesis. Ann Rev Pharmacol Toxicol 1990; 30: 9l-121
    PubMedGoogle Scholar
  • 33 Higgins D, Young S, Wong A, Marks C, Powers D, Anderson S. Site specific mutagenesis of the second kringle and protease domains of tissue plasminogen activator. Thromb Haemostas 1989; 62: 322 Abstract
    PubMedGoogle Scholar
  • 34 Nieuwenhuizen W, Voskuilen M, Vermond A, Hoegee-de Nobel B, Tiaas DW. The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue-type plasminogen-activator-mediated activation of different forms of plasminogen. Eur J Biochem 1988; 174: 163-169
    CrossrefPubMedGoogle Scholar
  • 35 Yonekawa O, Vermond A, Nieuwenhuizen W. Localization of a new site in fibrin, involved in the acceleration of t-PA mediated activation of plasminogen. Thromb Haemostas 1989; 62-71 Abstract
    PubMedGoogle Scholar
  • 36 Takada A, Thkada Y. Inhibition by tranexamic acid of the conversion of single chain tissue plasminogen activator to its two chain form by plasmin: the presence on tissue plasminogen activator of a site to bind with lysine binding sites of plasmin. Thromb Res 1989; 55: 717-725
    CrossrefPubMedGoogle Scholar
  • 37 Homandberg GA, Wai T. Comparison of affinities of urokinase and tissue plasminogen activator for fibrin clots. Thromb Res 1989; 55: 493-501
    CrossrefPubMedGoogle Scholar
  • 38 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
  • 39 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of onechain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
    PubMedGoogle Scholar
  • 40 Beckmatr R, Geiger M, Binder BR. Plasminogen activation by tissue plasminogen activator in the presence of stimulating CNBr fragment FCB-2 of tibrinogen is a two-phase reaction. J Biol Chem 1988; 263: 7176-7190
    PubMedGoogle Scholar
  • 41 Ralnby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
    CrossrefPubMedGoogle Scholar
  • 42 Ralnby M. On the properties of the international standards for tissue plasminogen activator. Thromb Haemostas 1990; 63: 139
    PubMedGoogle Scholar