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DOI: 10.1055/s-0038-1647473
An Analysis of the Activators of Single-Chain Urokinase-Type Plasminogen Activator (scu-PA) in the Dextran Sulphate Euglobulin Fraction of Normal Plasma and of Plasmas Deficient in Factor XII and Prekallikrein
Publication History
Received: 12 July 1990
Accepted after revision 12 September 1990
Publication Date:
02 July 2018 (online)
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Summary
An analysis was made of the various possible activators of single-chain urokinase-type plasminogen activator (scu-PA) in the dextran sulphate euglobulin fraction (DEF) of human plasma. scu-PA activators were detected in an assay system in which the substrate scu-PA, in physiological concentration (50 pM), was immuno-immobilized. After activation of the immobilized scu-PA for a certain period of time the activity of the generated amount of immuno-immobilized two-chain u-PA was determined with plasminogen and the chromogenic substrate S-2251. The scu-PA activator activity (scuPA-AA) in the DEF of plasmas deficient in factor XII or prekallikrein was about half of that in the DEF of normal plasma. Separation of scuPA-AA in the DEF by gel chromatography showed to major peaks, one eluting with an apparent Mr of 500,000 and the other around Mr 100,000. The former peak, which coincided with the activity peak of the kallikrein-kininogen complex, was absent in the DEF of plasma depleted of prekallikrein and therefore was identified as kalli-krein. The latter peak was still present in the depleted plasma and most likely represents plasmin, because its scuPA-AA coincided with the activity peak of plasmin and could be fully inhibited by antibodies raised against human plasminogen. It is concluded that plasmin and the contact-activation factor kallikrein each contribute for about 50% to the scuPA-AA in the DEF. Compared on a molar basis, however, plasmin was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo plasmin is the primary activator of scu-PA and the role of the contact system is of secondary importance.
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REFERENCES
- 1 Bachmann F. Fibrinolysis. In: Thrombosis and Haemostasis Ver-straete M, Vermijlen J, Lijnen HR, Amout J. (eds) International Society on Thrombosis and Haemostasis and Leuven University Press; Leuven: 1987: 227-263
- 2 Wijngaards G, Kluft C, Groeneveld E. Demonstration of urokinase-related fibrinolytic activity in human plasma. Br J Haematol 1982; 51: 165-169
- 3 Wun T-C, Schleuning WD, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3283
- 4 Tissot JD, Schneider P, Hauert J, Ruegg M, Kruithof EKO, Bachmann F. Isolation from human plasma of a plasminogen activator identical to urinary high molecular weight urokinase. J Clin Invest 1982; 70: 1320-1323
- 5 Wun T-C, Ossowski L, Reich E. A proenzyme form of human urokinase. J Biol Chem 1982; 257: 7262-7268
- 6 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 31-38
- 7 Dooijewaard G, Van Iersel JJL, Brommer EJP. Quantitation of pro-UK, and UK. Inhibitor levels in plasma of patients and healthy man. In: Abstracts of the Eighth International Congress on Fibrinolysis Binder BR. (ed) Vienna 1986. Churchill Livingstone; Edinburgh: 1986: 142
- 8 Nielsen LS, Hansen JG, Skriver L, Wilson EL, Kaltoft K, Zeuthen J, Danϕ K. Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody. Biochemistry 1982; 21: 6410-6415
- 9 Eaton DL, Scott RW, Baker JB. Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. J Biol Chem 1984; 259: 6241-6247
- 10 Gurewich V, Panell R. Pro-urokinase (PUK) – Characterization of its enzymatic activation. Thromb Haemostas 1985; 54: 102
- 11 Kasai S, Arimura H, Nishida M, Suyama T. Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin. J Biol Chem 1985; 260: 12377-12381
- 12 Ichinose A, Fujikawa K, Suyama T. The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 3486-3489
- 13 Miles LA. Mechanisms of intrinsic fibrinolysis. Thesis, University of California; San Diego, CA: 1982
- 14 Miles LA, Rothschild ZS, Griffin JH. Dextran sulphate-dependent fibrinolytic activity in whole human plasma. In: Progress in Fibrinolysis Davidson JF. et al. (eds) Churchill Livingstone; Edinburgh: 1983. 6 58-61
- 15 Huisveld IA, Hospers JEH, Van Heeswijk GM, Bemick MJE, Erich WBM, Bouma BN. Contribution of contact activation factors to urokinase-related fibrinolytic activity in whole human plasma. Thromb Haemostas 1985; 54: 102
- 16 Hauert J, Nicoloso G, Schleunig W-D, Bachmann F, Schapira M. Plasminogen activators in dextran sulphate-activated euglobulin fractions: a molecular analysis of factor XII- and prekallikrein-dependent fibrinolysis. Blood 1989; 73: 994-999
- 17 Binnema DJ, Dooijewaard G, Van IerselJJL, Turion PNC, Kluft C. The contact system-dependent plasminogen activator from human plasma: identification and characterization. Thromb Haemostas 1990; 64: 390-397
- 18 Kluft C, Wijngaards G, Jie AFH. Intrinsic plasma fibrinolysis: Involvement of urokinase-related activity in the factor XH-indepen-dent plasminogen proactivator pathway. J Lab Clin Med 1984; 103: 408-419
- 19 Binnema DJ, Dooijewaard G. Involvement of factor XII (F XII) and prekallikrein (PKK) in the activation of urokinase (UK)-related proteins in human plasma. Thromb Haemostas 1987; 58: 137
- 20 Kluft C, Dooijewaard GS, Emeis JJ. 1987 Role of the contact system in fibrinolysis. Semin Thromb Hemostas 1987; 13: 50-68
- 21 Binnema DJ, Dooijewaard G, Turion PNC. Inventory of prourokinase activating components in the dextran sulphate euglobulin fraction of plasma. Fibrinolysis 1988; 2 (Suppl. 01) 48 Abstr 107
- 22 Kluft C, Brakman P, Veldhuyzen-Stolk EC. 1976 Screening of fibrinolytic activity in plasma euglobulin fractions on the fibrin plate. In: Progress in Chemical Fibrinolysis and Thrombolysis Davidson JF. et al. (eds) Raven Press; New York: 1976. 2 57-65
- 23 Kluft C. Studies on the fibrinolytic system in human plasma: Quantitative determination of plasminogen activators and proactivators. Thromb Haemostas 1979; 41: 365-383
- 24 Kluft C. Synthetic substrates for the assay of prekallikrein and factor XII. In: Methods in Enzymology Di Sabato G. (ed) Academic Press; San Diego, CA: 1988. 163 170-179
- 25 Kluft C, Trumpi-Kalshoven MM, Jie AFH, Veldhuyzen-Stolk EC. Factor XH-dependent fibrinolysis: a double function of plasma kallikrein and the occurrence of a previously undescribed factor XII-and kallikrein-dependent plasminogen proactivator. Thromb Haemostas 1979; 41: 756-773
- 26 Binnema DJ, Van IerselJJL, Dooijewaard G. Quantitation of urokinase antigen in plasma and culture media by use of an ELISA. Thromb Res 1986; 43: 569-577
- 27 Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards G. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
- 28 Friberger P, Knos M, Gustavsson S. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis 1978; 7: 138-45
- 29 Exner T, Cotton B, Howden M. Detection of specific proenzyme activators in snake venoms by a new immunoadsorbent-chromogenic substrate method. Biochim Biophys Acta 1985; 832: 351-356
- 30 Wojta J, Binder BR, Huber K, Hoover RL. 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. Thromb Haemostas. 1989; 61: 289-293
- 31 Darras V, Thienpont M, Stump DC, 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
- 32 Grϕndahl-Hansen J, Agerlin NS, Munkholm-Larsen P, Bach F, Nielsen LS, Dombemovsky P, Danϕ K. Sensitive and specific enzyme-linked immuno-sorbent assay for urokinase-type plasminogen activator and its application to plasma from patients with breast cancer. J Lab Clin Med 1988; 111: 42-51
- 33 Blasi F, Vassalli J-D, Danϕ K. Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. J Cell Biol 1987; 104: 801-804
- 34 Miles LA, Levin EG, Plescia J, Collen D, Plow EF. Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cell. Blood 1988; 72: 628-635
- 35 Barnathan ES, Kuo A, Rosenfeld L, Kariko K, Leski M, Robbiati F, Nolli ML, Henkin J, Cines DB. Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells. J Biol Chem 1990; 265: 2685-2872
- 36 Ellis V, Scully MF, Kakkar W. Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by u937 monocytes. J Biol Chem 1989; 264: 2185-2188
- 37 Stephens RW, Pöllänen J, Tapiovaara H, Leung K-C, Sim P-S, Salonen E-M, Rϕnne E, Behrendt N, Danϕ K, Vaheri A. Activation of prourokinase and plasminogen on human sarcoma cells: A proteolytic system with surface-bound reactants. J Cell Biol 1989; 108: 1987-1995