Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

Johann Wojta; Bernd R Binder; Kurt Huber; Richard L Hoover

doi:10.1055/s-0038-1646578

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1989; 61(02): 289-293
DOI: 10.1055/s-0038-1646578

Original Article

Schattauer GmbH Stuttgart

Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

Johann Wojta

¹The Department of Pathology and Pediatrics, Vanderbilt University, Nashville, TN, USA

²The Laboratory for Clinical Experimental Physiology, Department of Medical Physiology, Universityof Vienna, Austria

,

Bernd R Binder

²The Laboratory for Clinical Experimental Physiology, Department of Medical Physiology, Universityof Vienna, Austria

,

Kurt Huber

³The Department of Cardiology, University of Vienna, Austria

,

Richard L Hoover

¹The Department of Pathology and Pediatrics, Vanderbilt University, Nashville, TN, USA

› Institutsangaben

Abstract

als PDF herunterladen

Summary

A combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 nglml ± 0.61. Furthennore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.

Keywords

u-PA ELISA - u-PA activity - Monoclonal antibodies - scu-PA - Thrombolytic therapy monitoring

PDF (257 kb)

Referenzen

References
1 Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissues and body fluids. J Lab Clin Med 1981; 97: 477-486
2 Wun TC, Schleuning WD, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3283
3 Binder BR, Spragg J, Austen KF. Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates. J Biol Chem 1979; 254: 1998-2003
4 Rijken DC, Wijngaards G, Zaal de Jong M, Welbergen J. Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim Biophys Acta 1979; 580: 140-153
5 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 25: 7035-7041
6 Wagner O, Wojta J, Binder BR. Analysis of the plasminogen activator production by two tumor cells lines. In: Clinical Aspects of Fibrinolysis and Thrombolysis. Jespersen J, Kluft C, Korsgaard O. (eds) South Jutland University Press, Esbjerg; Denmark: 1983. pp 35-42
7 Husain SS, Gurewich V, Lipinsky B. Prourokinase. Purification and partial characterization of a single chain high molecular weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 31-38
8 Pannel R, Gurewich V. Prourokinase. A study of its stability in plasma and of its selective fibrinolytic effect. Blood 1986; 67: 1215-1223
9 Kirchheimer JC, Resch I, Christ G, Wojta J, Binder BR. Effect of cyanogen-bromide-2 fragments of fibrinogen on plasminogen activation by single chain urokinase-type plasminogen activator. Eur J Biochem 1987; 166: 393-397
10 Lijnen HR, Zamarron C, Blaber M, Winkler M, Collen D. Activation of plasminogen by pro-urokinase. I. Mechanism. J Biol Chem 1986; 261: 1253-1258
11 Collen D. Coronary thrombolysis with recombinant human tissue type plasminogen activator: a prospective, randomized, placebo controlled trial. Circulation 1984; 70: 1012-1017
12 Van de Werf F, Ludbrook PP, Bergmann SR, Tiefenbrunn AJ, Fox KA A, de Geest H, Verstraete M, Collen D, Sobel BE. Coronary thrombolysis with tissue-type plasminogen activator in patients with evolving myocardial infarction. N Engl J Med 1984; 310: 609-613
13 Van de Werf F, Nobuhara M, Collen D. Coronary thrombolysis with human single chain, urokinase-type plasminogen activator (prourokinase) in patients with acute myocardial infarction. Ann Intern Med 1986; 104: 345-348
14 Yasuno M, Saito Y, Ishida M, Suzuki K, Endo S, Takahashi M. Effects of percutaneous transluminal coronary angioplasty: Intracoronary thrombolysis with urokinase in acute myocardial infarction. Am J Cardiol 1984; 53: 1217-1220
15 Tennant SS, Dixon J, Venable TT, Page HL, Roach A, Kaiser AA, Frederiksen R, Tacogue L, Kaplan P, Babu NS, Anderson EE, Wooten E, Jennings HS, Breinig J, Campbell WB. Intracoronary thrombolysis in patients with acute myocardial infarction: comparison of the efficacyof urokinase with streptokinase. Circulation 1984; 69: 756-760
16 Vetterlein D, Bell TE, Young PL, Roblin R. Immunological quantitation and immunoadsorbtion of urokinase-like plasminogen activators secreted by human cells. J Biol Chem 1980; 255: 3665-3672
17 Herion P, Portetelle D, Franssen JD, Urbain J, Bollen A. Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies. Biosci Rep 1983; 3: 381-388
18 Stump DC, Thienpont M, Collen D. Urokinase-related proteins in human urine. Isolation and characterization of single chain urokinase (pro-urokinase) and urokinase-inhibitor complex. J Biol Chem 1986; 261: 1267-1273
19 Binnema DJ, van Iersel JJ L, Dooijewaard G. Quantitation of urokinase antigen in plasma and culture media by use of an ELISA. Thromb Res 1986; 43: 569-577
20 Darras V, Thienpont M, Stump DD, Collen D. Measurement of urokinase-type plasminogen activator (u-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies. Thromb Haemostas 1986; 56: 411-414
21 Grondahl-Hansen J, Agerlin N, Munkholm-Larsen P, Bach F, Nielsen LL, Dombemovsky P, Dano K. Sensitive and specific enzyme-linked immunosorbent assay for urokinase-type plasminogen activator and its application to plasma from patients with breast cancer. J Lab Clin Med 1988; 111: 42-51
22 Wojta J, Turcu L, Wagner OO, Kominger C, Binder BR. Evaluation of fibrinolytic capacity by a combined assay system for tissue-type plasminogen activator antigen and function using monoclonal antitissue-type plasminogen activator antibodies. J Lab Clin Med 1987; 109: 665-671
23 Wagner O, Binder BR. Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor. J Biol Chem 1986; 261: 14474-14481
24 Deutsch DG, Mertz ET. Plasminogen purification from human plasma by affinity chromatography. Science 1970; 179: 1095-1096
25 Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
26 Lowe CR, Dean PD G. Cyanogen bromide activation of agarose. In: Affinity Chromatography.. John Wiley & Sons Ltd; London: 1974. pp 250-253
27 Wojta J, Kirchheimer JC, Turcu L, Christ G, Binder BR. Monoclonal antibodies against human high molecular weight urinary urokinase: Application for affinity purification of urinary prourokinase. Thromb Haemostas 1986; 55: 347-351
28 Nakane PA, Kawaoi A. Peroxidase labeled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091
29 Huber K, Kirchheimer J, Binder BR. Characterization of a specific anti-human urokinase antibody: development of a sensitive competition radioimmunoassay for urokinase antigen. J Lab Clin Med 1984; 103: 684-694
30 Korninger C, Speiser W, Wojta J, Binder BR. Sandwich ELISA for t- PA antigen employing a monoclonal antibody. Thromb Res 1986; 41: 527-535
31 Fletcher AP, Alkjaersig N, Sherry S, Genton E, Hirsch J, Bachmann F. The development of urokinase as a thrombolytic agent. Maintenance of a sustained thrombolytic state in man by its intravenous infusion. J Lab Clin Med 1965; 65: 713-731
32 Collen D, De Cock F, Lijnen HR. Biological and thrombolytic properties of proenzyme and active forms of human urokinase - II. Turnover of natural and recombinant urokinase in rabbits and squirrel monkeys. Thromb Haemostas 1984; 52: 24-26
33 Geiger M, Heeb MJ, Binder BR, Griffin JH. Competition of activated protein C and urokinase for a heparin dependent inhibitor. FASEB J 1988; 2: 2263-2267