Download PDF
Thromb Haemost 1992; 68(05): 486-494
DOI: 10.1055/s-0038-1646305
Original Article
Schattauer GmbH Stuttgart

A Specific Immunologic Assay for Functional Plasminogen Activator Inhibitor 1 in Plasma – Standardized Measurements of the Inhibitor and Related Parameters in Patients with Venous Thromboembolic Disease

Malou Philips
1   The Department of Clinical Chemistry, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark
2   The Coagulation Section, Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
,
Anne-Grethe Juul
3   The Novo Nordisk Research Institute, Bagsværd, Denmark
,
Johan Selmer
3   The Novo Nordisk Research Institute, Bagsværd, Denmark
,
Bent Lind
2   The Coagulation Section, Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
,
Sixtus Thorsen
2   The Coagulation Section, Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
› Author Affiliations
Further Information

Publication History

Received 05 February 1992

Accepted after revision 09 June 1992

Publication Date:
04 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

A new assay for functional plasminogen activator inhibitor 1 (PAI-1) in plasma was developed. The assay is based on the quantitative conversion of PAI-1 to urokinase-type plasminogen activator (u-PA)-PAI-l complex the concentration of which is then determined by an ELISA employing monoclonal anti-PAI-1 as catching antibody and monoclonal anti-u-PA as detecting antibody. The assay exhibits high sensitivity, specificity, accuracy, and precision. The level of functional PAI-1, tissue-type plasminogen activator (t-PA) activity and t-PA-PAI-1 complex was measured in normal subjects and in patients with venous thromboembolism in a silent phase. Blood collection procedures and calibration of the respective assays were rigorously standardized. It was found that the patients had a decreased fibrinolytic capacity. This could be ascribed to high plasma levels of PAI-1. The release of t-PA during venous occlusion of an arm for 10 min expressed as the increase in t-PA + t-PA-PAI-1 complex exhibited great variation and no significant difference could be demonstrated between the patients with a thrombotic tendency and the normal subjects.

 

  • REFERENCES

  • 1 Bachmann E. Fibrinolysis. In: Thrombosis and Haemostasis. Verstraete M, Vermylen J, Lijnen R, Arnout J. (eds) International Society on Thrombosis and Haemostasis. Leuven University Press, Leuven: 1987. pp 227-265
    Google Scholar
  • 2 Müllertz S. Fibrinolysis. General aspects, characteristic features and perspectives. Fibrinolysis 1987; 1: 03-12
    PubMedGoogle Scholar
  • 3 Wiman B, Hamsten A. The fibrinolytic enzyme system and its role in the etiology of thromboembolic disease. Semin Thromb Hemostas 1990; 16: 207-216
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Chmielewska J, Wiman B. Determination of tissue plasminogen activator and its “fast” inhibitor in plasma. Clin Chem 1986; 32: 482-485
    PubMedGoogle Scholar
  • 5 Nilsson K, Rosén S, Friberger P. A new kit for the determination of tissue plasminogen activator and its inhibitor in blood. Fibrinolysis 1987; 1: 163-168
    PubMedGoogle Scholar
  • 6 Petersen LC, Handest P, Brender J, Selmer J, Jørgensen M, Thorsen S. A sensitive solid-phase immunosorbent assay for tissue-type plasminogen activator activity in plasma using trinitrobenzoylated poly-D-lysine as a stimulator for plasminogen activation. Thromb Haemostas 1987; 57: 205-211
    PubMedGoogle Scholar
  • 7 Chandler WL, Schmer G, Stratton JR. Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma. J Lab Clin Med 1989; 113: 362-371
    PubMedGoogle Scholar
  • 8 Bergsdorf N, Nilsson T, Wallén P. An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb Haemostas 1983; 50: 740-744
    PubMedGoogle Scholar
  • 9 MacGregor IR, Prowse CV. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thromb Res 1983; 31: 461-474
    CrossrefPubMedGoogle Scholar
  • 10 Matsuo O, Kato K, Matsuo C, Matsuo T. Determination of tissue plasminogen activator by an enzyme-immunoassay method. Anal Biochem 1983; 135: 58-63
    CrossrefPubMedGoogle Scholar
  • 11 Urdén G, Blombäck M. Determination of tissue plasminogen activator in plasma samples by means of a radioimmunoassay. Scand J Clin Lab Invest 1984; 44: 495-502
    CrossrefPubMedGoogle Scholar
  • 12 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
  • 13 Rånby M, Bergsdorf N, Nilsson T, Mellbring G, Winblad B, Bucht G. Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay. Clin Chem 1986; 32: 2160-2165
    PubMedGoogle Scholar
  • 14 Rånby M, Nguyen G, Scarabin PY, Samama MM. Immunoreactivity of tissue plasminogen activator and of its inhibitor complexes. Biochemical and multicenter validation of a two site immunosorbent assay. Thromb Haemostas 1989; 61: 409-414
    PubMedGoogle Scholar
  • 15 Korninger C, Wagner O, Binder BR. Tissue plasminogen activator inhibitor in human plasma. Development of a functional assay system and demonstration of a correlating M r = 50,000 antiactivator. J Lab Clin Med. 1985; 105: 718-724
    PubMedGoogle Scholar
  • 16 Speiser W, Bowry S, Anders E, Binder BR, Müller-Berghaus Q. Method for the determination of fast acting plasminogen activator inhibitor capacity (PAI-CAP) in plasma, platelets and endothelial cells. Thromb Res 1986; 44: 503-515
    CrossrefPubMedGoogle Scholar
  • 17 Sprengers ED. A sensitive assay, specific for endothelial cell type plasminogen activator inhibitor in blood plasma. Thromb Haemostas 1986; 55: 74-77
    PubMedGoogle Scholar
  • 18 Eriksson E, Rånby M, Gyzander E, Risberg B. Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay. Thromb Res 1988; 50: 091-101
    CrossrefPubMedGoogle Scholar
  • 19 Chandler WL, Loo S-C, Nguyen SV, Schmer G, Stratton JR. Standardization of methods for measuring plasminogen activator inhibitor activity in human plasma. Clin Chem 1989; 35: 787-793
    PubMedGoogle Scholar
  • 20 Kruithof EKO, Nicolosa G, Bachmann F. Plasminogen activator inhibitor 1: Development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation. Blood 1987; 70: 1645-1653
    PubMedGoogle Scholar
  • 21 MacGregor IR, Booth NA. An enzyme-linked immunosorbent assay (ELISA) used to study the cellular secretion of endothelial plasminogen activator inhibitor (PAI-1). Thromb Haemostas 1988; 59: 68-72
    PubMedGoogle Scholar
  • 22 Urdén G, Chmielewska J, Carlsson T, Wiman B. Immunological relationship between plasminogen activator inhibitors from different sources. Thromb Haemostas 1987; 57: 29-34
    PubMedGoogle Scholar
  • 23 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Br J Haematol 1988; 70: 327-333
    CrossrefPubMedGoogle Scholar
  • 24 Holvoet P, Boes J, Collen D. Measurement of free, one-chain tissue-type plasminogen activator in human plasma with an enzyme-linked immunosorbent assay based on an active site-specific murine monoclonal antibody. Blood 1987; 69: 284-289
    PubMedGoogle Scholar
  • 25 Declerck PJ, Alessi M-C, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D. Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 1988; 71: 220-225
    PubMedGoogle Scholar
  • 26 Declerck PJ, Collen D. Measurement of plasminogen activator inhibitor 1 (PAI-1) in plasma with various monoclonal antibody-based enzyme-linked immunosorbent assays. Thromb Res 1990; SupplX: 3-9
    PubMedGoogle Scholar
  • 27 Takada Y, Takada A. Measurements of the concentration of free plasminogen activator inhibitor (PAI-1) and its complex with tissue plasminogen activator in human plasma. Thromb Res 1988; Suppl VIII: 15-22
    PubMedGoogle Scholar
  • 28 Chandler WL, Trimble SL, Loo S-C, Mornin D. Effect of PAI-1 levels on the molar concentrations of active tissue plasminogen activator (t-PA) and t-PA/PAI-1 complex in plasma. Blood 1990; 76: 930-937
    PubMedGoogle Scholar
  • 29 Philips M, Juul A-G, Thorsen S, Selmer J. Enzyme-linked immunosorbent assay (ELISA) of reactive plasminogen activator inhibitor 1 (PAI-1) in plasma after complex formation with urokinase (u-PA). Thromb Haemostas 1989; 62 abstract 746
    PubMedGoogle Scholar
  • 30 Thorsen S, Philips M, Lind B, Selmer J. Enzyme-linked immunosorbent assays (ELISA) of reactive plasminogen activator inhibitor 1 (PAI-1) and the complex between tissue-type plasminogen activator (t-PA) and PAI-1 in plasma. Fibrinolysis 1990; 4 (Suppl 3): abstract 18
    PubMedGoogle Scholar
  • 31 Thorsen S, Philips M, Selmer J, Lecander I, Åstedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen activator inhibitor type 1 and type 2. Eur J Biochem 1988; 175: 33-39
    CrossrefPubMedGoogle Scholar
  • 32 Philips M, Juul A-G, Thorsen S, Selmer J, Zeuthen J. Immunological relationship between the fast-acting plasminogen activator inhibitors from plasma, blood platelets an endothelial cells demonstrated with a monoclonal antibody against and inhibitor from placenta. Thromb Haemostas 1986; 55: 213-217
    PubMedGoogle Scholar
  • 33 Contant G, Gouault-Heilmann M, Martinoli JL. Heparin inactivation during blood storage: Its prevention by blood collection in citric acid, theophylline, adenosine, dipyridamole - C.T.A.D. mixture. Thromb Res 1983; 31: 365-374
    CrossrefPubMedGoogle Scholar
  • 34 Rånby M, Sundell B, Nilsson TK. Blood collection in strong acidic citrate anticoagulant used in the study of dietary influence on basal tPA activity. Thromb Haemostas 1989; 62: 917-922
    PubMedGoogle Scholar
  • 35 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry. 1978; 15: 429-436
    PubMedGoogle Scholar
  • 36 Ishikawa E, Imagawa M, Hashida S, Yoshitake S, Hamaguchi Y, Ueno T. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay 1983; 4: 209-327
    PubMedGoogle Scholar
  • 37 Jørgensen M, Philips M, Thorsen S, Selmer J, Zeuthen J. Plasminogen activator inhibitor-1 is the primary inhibitor of tissue-type plasminogen activator in pregnancy plasma. Thromb Haemostas 1987; 58: 872-878
    PubMedGoogle Scholar
  • 38 Declerck PJ, De Mol M, Alessi M-C, Baudner S, Pâques E-P, Preissner KT, Müller-Berghaus G, Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. J Biol Chem 1988; 263: 15454-15461
    PubMedGoogle Scholar
  • 39 Lindahl TL, Sigurdardottir O, Wiman B. Stability of plasminogen activator inhibitor 1 (PAI-1). Thromb Haemostas 1989; 62: 748-751
    PubMedGoogle Scholar
  • 40 Kruithof EKO, Tran-Thang C, Bachmann F. Studies on the release of a plasminogen activator inhibitor by human platelets. Thromb Haemostas 1986; 55: 201-205
    PubMedGoogle Scholar
  • 41 Juhan-Vague I, Alessi MC, Fossat C, Declerck PJ, Kruithof EKO. Plasma determination of plasminogen activator inhibitor 1 antigen must be performed in blood collected on antiplatelet/anticoagulant mixture. Thromb Haemostas 1987; 58: 1096
    PubMedGoogle Scholar
  • 42 Krishnamurti C, Tang DB, Barr CF, Alving BM. Plasminogen activator and plasminogen activator inhibitor activities in a reference population. Am J Clin Pathol 1988; 89: 747-752
    CrossrefPubMedGoogle Scholar
  • 43 Takada Y, Takada A. Plasma levels of t-PA, free PAI-1 and a complex of t-PA with PAI-1 in human males and females at various ages. Thromb Res 1989; 55: 601-609
    CrossrefPubMedGoogle Scholar
  • 44 Nguyen G, Horellou MH, Kruithof EKO, Conard J, Samama MM. Residual plasminogen activator inhibitor activity after venous stasis as a criterion for hypofibrinolysis: A study in 83 patients with confirmed deep vein thrombosis. Blood 1988; 72: 601-605
    CrossrefPubMedGoogle Scholar
  • 45 Nilsson IM, Ljungnér H, Tengborn L. Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor. Br Med J 1985; 290: 1453-1455
    CrossrefPubMedGoogle Scholar
  • 46 Wiman B, Ljungberg B, Chmielewska J, Urdén G, Blombäck M, Johnsson H. The role of the fibrinolytic system in deep vein thrombosis. J Lab Clin Med 1985; 105: 265-270
    PubMedGoogle Scholar
  • 47 Juhan-Vague I, Valadier J, Alessi MC, Aillaud MF, Ansaldi J, Philip-Joet C, Holvoet P, Serradimigni A, Collen D. Deficient t-PA release and elevated PA inhibitor levels in patients with spontaneous or recurrent deep venous thrombosis. Thromb Haemostas 1987; 57: 67-72
    PubMedGoogle Scholar
  • 48 Petäjä J. Fibrinolytic response to venous occlusion for 10 and 20 minutes in healthy subjects and in patients with deep vein thrombosis. Thromb Res 1989; 56: 251-263
    CrossrefPubMedGoogle Scholar