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Thromb Haemost 1995; 74(05): 1305-1309
DOI: 10.1055/s-0038-1649931
Original Article
Fibrinolysis
Schattauer GmbH Stuttgart

Immunoassay of Murine t-PA, u-PA and PAI-1 Using Monoclonal Antibodies Raised in Gene-inactivated Mice

Paul J Declerck
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
Maria Verstreken
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
Désiré J Collen
The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 07 June 1995

Accepted after revision 14 July 1995

Publication Date:
10 July 2018 (online)

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Summary

Three enzyme-linked immunosorbent assays for the quantitation of murine tissue-type plasminogen activator (t-PA), urokinase-type plasminogen aetivator (u-PA) and plasminogen activator inhibitor I (PAI-1), were developed using monoclonal antibodies raised against the autologous proteins in gene-inactivated mice. Dose-response was linear for t-PA and PAI-1 between 5 and 0.1 ng/ml and for u-PA between 50 and 1 ng/ml, with intra-assay, inter-assay and inter-dilution coefficients of variation of 6 to 14%. Assay recoveries of proteins (5 to 100 ng/ml) added to plasma were 73 to 95% for t-PA and PAI-1. Linear correlations (r = 0.65, r = 0.91 and r = 0.92, for t-PA, u-PA and PAI-1 respectively) were found between antigen and activity in plasma, urine and tissue extracts. Levels of t-PA and PAI-1 antigen in murine plasma were 2.5 ± 1.0 ng/ml (mean ± SD, n = 9) and 1.9 ± 0.6 ng/ml (mean ± SD, n = 8), respectively, in wild-type mice and undetectable in gene-inactivated mice. Bradykinin injection in mice provoked a 12-fold increase (p <0.0002) of t-PA and endotoxin injection an 80-fold increase (p <0.005) of PAI-1 levels. u-PA antigen levels in urine from wild-type mice ranged between 0.2 and 8.2 μ;g/ml (1.8 ± 1.9 μg/ml, mean ± SD, n = 17) and were undetectable in gene-inactivated mice.

Thus, these assays may be useful for studies on the role of these proteins in tissue remodeling, atherosclerosis, embryogenesis, etc., in established mouse models. Gene-inactivated mice may constitute a general approach for the generation of monoclonal antibodies against the deficient translation products and for the development of specific immunoassays for murine proteins.

 

  • References

  • 1 Astrup T. Fibrinolysis: an overview. In: Progress in Chemical Fibrinolysis and Thrombolysis. Vol 3. Davidson JF, Rowan RM, Samania MM, Desnoyers PC. (eds) New York: Raven Press Lid; 1978. pp 1-57
    Google Scholar
  • 2 Cullen D, Lijnen MR. Basie and clinical aspeels of fibrinolysis and thrombolysis. Blood 1991; 78: 3114-3124
    PubMedGoogle Scholar
  • 3 Vassalli JD, Sappino AP, Bclin D. The plasminogen aelivator/plasmin system. J din Invest 1991; 88: 1067-1072
    PubMedGoogle Scholar
  • 4 Ohlsson M, Peng WR, Liu YX, Jia XC, Hsueh AJ W, Ny T. Hormone regulation of tissue-type plasminogen activator gene expression and plasminogen activator-mediated proteolysis. Semin Thromb Macmost 1991; 17: 286-290
    PubMedGoogle Scholar
  • 5 Tsafriri A, Biesak TA, Cajander SB, Ny T, Hsueh AJ W. Suppression of ovulation rate by antibodies to tissue-type plasminogen activator and α2antiplasmin. Endocrinology 1989; 124: 415-421
    CrossrefPubMedGoogle Scholar
  • 6 Strickland S, Reich E, Sherman ML. Plasminogen activator in early cmbryogcncsis: enzyme production by trophoblast and parietal endoderm. Cell 1976; 9: 231-240
    CrossrefPubMedGoogle Scholar
  • 7 Qian Z, Gilbert ME, Colieos MA, Kandel HR, Kuhl D. Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation. Nature (Loud.) 1993; 361: 453-457
    CrossrefPubMedGoogle Scholar
  • 8 Lala PK, Graham CH. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev 1990; 9: 369-379
    CrossrefPubMedGoogle Scholar
  • 9 Rifkin DB, Moscatelli D, Bizik J, Quarto N, Blei E, Dennis P, Elaumcnhaft R, Mignati P. Growth factor control of extracellular proteolysis. Cell Differ Dev 1990; 32: 313-318
    CrossrefPubMedGoogle Scholar
  • 10 Blasi F, Verde P. Urokinase-dependent cell surface proteolysis and cancer. Semin Cancer Biol 1990; 1: 117-126
    PubMedGoogle Scholar
  • 11 Schneiderman J, Loskutoff DJ. Plasminogen activator inhibitors. Semin Trends Cardiovasc Med 1991; 1: 99-102
    PubMedGoogle Scholar
  • 12 Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381-387
    PubMedGoogle Scholar
  • 13 Kruifhof EK O, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activator in plasma. Blood 1984; 64: 907-913
    PubMedGoogle Scholar
  • 14 Declerck PJ, Juhan-Vague I, Felez J, Wiman B. Pathophysiology of fibrinolysis. J Int Med 1994; 236: 425-432
    CrossrefPubMedGoogle Scholar
  • 15 Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature (Lond) 1990; 346: 74-76
    CrossrefPubMedGoogle Scholar
  • 16 Francis RB Jr, Liebman H, Koehler S, Feinstein DI. Accelerated fibrinolysis in amyloidosis, specific binding of tissue plasminogen activator inhibitor by an amyloidogenic monoclonal IgG. Blood 1986; 68 Suppl (Suppl. 01) 333 (Abstr)
    PubMedGoogle Scholar
  • 17 Dièval J, Nguyen G, Gross S, Delobel J, Kruithof EK O. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type-1. Blood 1991; 77: 528-532
    PubMedGoogle Scholar
  • 18 Lee MH, Vosburgh E, Anderson K, McDonagh J. Deficiency of plasma plasminogen activator inhibitor 1 results in hyperfibrinolytic bleeding. Blood 1993; 81: 2357-2362
    PubMedGoogle Scholar
  • 19 Schleef RR, Higgins DL, Pillemer E, Levitt LJ. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. J Clin Invest 1989; 83: 1747-1752
    CrossrefPubMedGoogle Scholar
  • 20 Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Complete deficiency of plasminogen-activator inhibitor type 1 due to a frame-shift mutation. N Engl J Med 1992; 327: 1729-1733
    CrossrefPubMedGoogle Scholar
  • 21 Juhan-Vague J, Collen D. On the role of coagulation and fibrinolysis in atherosclerosis. Ann Epidemiol 1992; 2: 427-438
    CrossrefPubMedGoogle Scholar
  • 22 Tomooka S, Border WA, Marshall BO, Nobel NA. Glomerular matrix accumulation is linked to inhibition of the plasmin protease system. Kidney Int 1992; 42: 1462-1469
    CrossrefPubMedGoogle Scholar
  • 23 Idell S, James KK, Levin HG, Schwartz IL S, Manchada N, Maunder RJ, Martin TR, McLarly J, Pair DS. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989; 84: 695-705
    CrossrefPubMedGoogle Scholar
  • 24 Wagner EF. On transferring genes into stem cells and mice. EMBO J 1990; 9: 3024-32
    PubMedGoogle Scholar
  • 25 Danø K, Andrcascn PA, Grondahl-Hansen J, Kristcnscn P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 1985; 44: 139-266
    PubMedGoogle Scholar
  • 26 Carmeliet P, Schoonjans L, Kieckcns L, Ream B, Degen J, Bronson R, De Vos R, Van Den Oord JJ, Collen D, Mulligan R. Physiological consequences of loss of plasminogen activator gene function in mice. Nature 1994; 368: 419-424
    CrossrefPubMedGoogle Scholar
  • 27 Carmeliet P, Kieckcns L, Schoonjans L, Ream B, Van Nuffelen A, Pren- dergast G, Cole M, Bronson R, Collen D, Mulligan RC. Plasminogen activator inhibitor-1 gene-deficient mice. I.Generation by homologous recombination and characterization J Clin Invest 1993; 92: 2746-2755
    CrossrefPubMedGoogle Scholar
  • 28 Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis and thrombolysis J Clin Invest 1993; 92: 2756-2760
    CrossrefPubMedGoogle Scholar
  • 29 Declerck PJ, Carmeliet P, Verstreken M, De Cock F, Collen D. Generation of monoclonal antibodies against autologous proteins in gene-inactivated mice. J Biol Chem 1995; 270: 8397-8400
    CrossrefPubMedGoogle Scholar
  • 30 Galfrè G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Meth Enzymol 1981; 73: 3-46
    PubMedGoogle Scholar
  • 31 Anderson PM, Potter M. Induction of plasma cell tumors in Balb/c mice with 2,6,10,4 tetramethyl-pentadecane (pristane). Nature 1969; 222: 994-995
    CrossrefPubMedGoogle Scholar
  • 32 Ey PL, Prowse SJ, Jenkins CR. Isolation of pure IgG1IgG2aand IgG2bimmunoglobulin from mouse serum using protein A-Sepharose. Immunochemistry 1978; 15: 429-436
    CrossrefPubMedGoogle Scholar
  • 33 Nakane PA, Kawaoi A. Peroxidase labeled antibody. A new method for conjugation J Histochem Cytochem 1974; 22: 1084-1091
    CrossrefPubMedGoogle Scholar
  • 34 Astrup T, Miillertz S. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
    CrossrefPubMedGoogle Scholar
  • 35 Verheijen JH, Mullaart E, Chang GT G, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemost 1982; 48: 266-269
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 Declerck PJ, Verstreken M, Collen D. An immunofunctional assay for active plasminogen activator inhibitor-1 (PAI-1). Fibrinolysis 1988; 2 (Suppl) 77-78
    PubMedGoogle Scholar
  • 37 Thomas KR, Capecchi MR. Site-directed mutagenesis by gene-targeting in mouse embryo-derived stem cells. Cell 1987; 51: 503-512
    CrossrefPubMedGoogle Scholar
  • 38 Carmeliet P, Collen D. Evaluation of the plasminogen/plasmin system in transgenic mice. Fibrinolysis 1994; 8 Suppl (Suppl. 01) 269-276
    PubMedGoogle Scholar
  • 39 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
    CrossrefPubMedGoogle Scholar