Release Pattern of the Vascular Plasminogen Activator and Its Inbibitor in Human Postvenous Occlusion Plasma as Assessed by a Spectrophotometric Solid-Phase Fibrin-tPA Activity Assay^[1]

Summary

Vascular or tissue-type plasminogen activatbr(plasma t-PA) is the circulating physiological fibrinolytic enzyme of endothelial cell origin which function is regulated by fibrin and a specific inhibitor (PAI). To study the pattern of release of t-PA and the behavior of t-PA-PAI complexes in plasma. we determined t-PA activity in 44 healthy subjects before and after 10 min offorearm venous occlusion using a new spectrophotmnetrio solid-phase fibrin-tPA activity assay. The assay is based on 1) the high affinity binding of t-PA tofibrin, and 2) the detection of fibrin-bound t-PA by measuring the release of pNA from a chromogenic substrate in the presence of plasminogeu. Values at.rest were rather undetectable in plasma (0.05 ± 0.03 IU/ml, in 23 out of 44 samples) but were positively detected in all the euglobulins: 0.88 ± 0.68 IU/ml. After venous occlusion the majority ofplasmas (36 out of 44) shoWed a slight increase in t-PA activity (0.65 ± 0.63 IU/ml) as compared to the important level observed in all the euglobulins (9.78 ± 9.58 IU/ml). So, the ratio plasma/euglobulin t-PA activity was very low (0.06) and remained identical in both pre- and postocclusion samples. However, when diluted plasmas were tested the inhibitory effect disappeared and t-PA activity increased indicating that although t-PA circulates in a neutralized state it can be available for fibrinolysis. Since 1) no binding of α₂ antiplasmin to fibrin in solid-phase could be demonstrated, 2) there was no correlation between α₂ antiplasmin and t-PA activity, and 3) a PAI-depleted plasma with a normal content of α₂ antiplasmin did not suppress the activity of t-PA, the inhibitory effect was attributed to PAL Our findings suggest that both t-PA and PAI are released. by venous occlusion and circulates in plasma as a t-PA-PAI complex.

Keywords

t-PA activity - PA inhibitor - Venous occlusion - Solid-phase fibrin - Spectrophotometric assay

• References

• 1 Todd AS. The histological localization of fibrinolysin activator. J Pathol Bacteriol 1959; 78: 281-283
• 2 Camiolo SM, Thorsen S, Astrup T. Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin, and plasmin. Proc Soc Exp Biol Med 1971; 38: 277-281
• 3 Thorsen S, Glas-Greenwalt P, Astrup T. Differences in the binding to fibrin of urokinase and tissue plasminogen activator. Thromb Diathes Haemorrh 1972; 28: 65-74
• 4 Astrup T. Fibrinolysis in the organism. Blood 1956; 11: 781-806
• 5 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
• 6 Rijken DC, Wijngaards G, Welbergen J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb Res 1980; 18: 815-830
• 7 Angles-Cano E, Balaton A, Le Bonniec B, Genot E, Elion J, Sultan Y. Production of monoclonal antibodies to the high fibrin affinity tissue-plasminogen activator of human plasma. Demonstration of its endothelial origin by immunolocalization. Blood 1985; 66: 913-920
• 8 Wun TC, Schleuning WD, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3282
• 9 Niewiarowski S, Prou-Waftelle O. Role du facteur contact (facteur Hageman) dans la fibrinolyse. Thromb Diath Haemorrh 1959; 3: 593-603
• 10 Biggs R, Macfarlane RG, Pilling J. Observations on fibrinolysis. Experimental production by exercise or adrenaline. Lancet 1947; I: 402-404
• 11 Clarke RL, Urandi A, Chilton LL. Induction of fibrinolysis by venous obstruction. Angiology 1960; 11: 367-370
• 12 Ilolemans R. Increase in fibiiuulytie activity by venous occlusion. J Applied Physiol 1963; 18: 1123-1129
• 13 Cash JD. Short-term enhancement of plasminogen activator in vivo by drugs. Thromb Diath Haemorrh 1975; 34: 648-651
• 14 Wiman B, Chmielewska J, Ranby M. Inactivation of tissue plasminogen activator in plasma. J Biol Chem 1984; 259: 3644-3647
• 15 Wiman B, Mellbring G, Ranby M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin Chim Acta 1983; 127: 279-288
• 16 Verheijen JH, Mullaart E, Chang G TG, Kluft C, Wijngaards G. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1983; 48: 266-269
• 17 Allen RA. An enhancing effect of poly-lysine on the activation of plasminogen. Thromb Haemostas 1982; 47: 41-45
• 18 Angles-Cano E. A spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA) for high fibrin affinity tissue plasminogen activators. Anal Biochem 1986; 153: 201-210
• 19 Boutiere B, Arnoux D, Benchimol P, Sampol J, Angles-Cano E. Solid-phase fibrin-tissue-plasminogen-activator activity assay (SOFIA-tPA): tPA but not UK is specifically detected. Thromb Res 1986; 43: 129-132
• 20 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
• 21 Teger-Nilsson AC, Friberger P, Gysander E. Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate. Scand J Clin Clin Lab Invest 1977; 37: 403-409
• 22 Kazal LA, Amsel S, Miller OP, Tocantis LM. The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc Soc Exp Biol Med 1963; 113: 989-994
• 23 Engvall E, Ruoslahti E. Binding of soluble forms of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 1977; 20: 1-5
• 24 Blomback B, Blomback M. Purification of human and bovine fibrinogen. Arkiv Kemi 1956; 10: 415-443
• 25 Wallen P, Wiman B. Characterization of human plasminogen. 11. Separation and partial characterization of different molecular forms of human plasminogen. Biochim Biophys Acta 1972; 257: 122-134
• 26 Gaffney PJ, Curtis AD. A collaborative study of a proposed International Standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1985; 53: 134-136
• 27 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
• 28 Angles-Cano E, Sultan Y. A solid-phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue plasminogen activators. J Immunol Meth 1984; 69: 115-127
• 29 Carrell R, Travis J. α₁-antitrypsin and the serpins: variation and countervariation. TIBS 1985; 10: 20-24
• 30 Angles-Cano E, Rondeau E, Delarue F, Hagege J, Sultan Y, Sraer JD. Identification and cellular localization of plasminogen activators from human glomeruli. Thromb Haemostas 1985; 54: 688-692
• 31 Angles-Cano E, Pannell R, Gurewich V. Pro-urokinase, the singlechain urokinase-type plasminogen activator does not bind to fibrin. Blood 1986; 68 suppl. (01) 343a
• 32 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
• 33 Laidler KJ, Bunting PS. The kinetics of immobilized enzyme systems. Meth Enzymol 1980; 64: 227-248
• 34 Godstein L. Kinetic behavior of immobilized enzyme systems. Meth Enzymol 1980; 64: 397-443
• 35 Angles-Cano E, Le Bonniec B. Plasminogen (Pg) activation kinetics by crude or pure tissue-type Pg-activator (t-PA) using a spec- trophotometric solid-phase fibrin-tPA activity assay as determined by computer analysis. Thromb Haemost 1985; 54: 163 (abstr.)
• 36 Kosow DP. Kinetic mechanism of the activation of human plasminogen by streptokinase. Biochemistry 1975; 14: 4459-4464
• 37 Drapier JC, Tenu JP, Lemaire G, Petit JF. Regulation of plasminogen activator secretion in mouse peritoneal macrophages. Biochimie 1979; 61: 463-471
• 38 Petersen LCh, Brender J, Suenson E. Zymogen activation kinetics. Biochem J 1985; 225: 149-158
• 39 Angles-Cano E, Masson C, Boutiere B. The activation pathway of plasminogen by solid-phase fibrin-bound tissue plasminogen activator does not involve lys-plasminogen intermediates. Fibrinolysis 1987; 1 (suppl.) 77 (abstr.)
• 40 Wiman B, Ljungberg B, Chmielewska J, Urden G, Blomback M, Johnsson H. The role of the fibrinolytic system in deep vein thrombosis. J Lab Clin Med 1985; 105: 265-270
• 41 Korninger C, Wagner O, Binder BR. Tissue plasminogen activator inhibitor in human plasma: development of a functional assay system and demonstration of a correlating Mr = 50000 antiactivator. J Lab Clin Med 1985; 105: 718-724
• 42 Nilsson IM, Ljungner 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: 1453-1456
• 43 Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzymatic-inhibitor complex. Proc Natl Acad Sci USA 1983; 80: 6804-6808
• 44 Philips M, Juul AG, Thorsen S. Human endothelial cells produce a plasminogen activator-inhibitor complex. Biochim Biophys Acta 1984; 802: 99-110
• 45 Miillertz S. Mechanism of activation and effect of plasmin in blood. Acta Physiol Scand 1956; 38 Suppl. (130) 1-66
• 46 Gurewich V, Hyde E, Lipinski B. The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs. Blood 1975; 46: 555-565
• 47 Kluft C. Occurrence of Cl inactivator and other proteinase inhibitors in euglobulin fractions and their influence on fibrinolytic activity. Haemostasis 1976; 5: 136-146
• 48 Verheijen JH, Chang G TG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
• 49 Thorsen S, Philips M. Isolation of tissue-type plasminogen activator- inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor. Biochim Biophys Acta 1984; 802: 111-118
• 50 Kruithof E KO, Tran-Thang Ch, Ransijn A, Bachman F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
• 51 Fearnley GR, Tweed JM. Evidence of an active fibrinolytic enzyme in the plasma of normal people with observations on inhibition associated with the presence of calcium. Clin Sci 1953; 12: 81-89
• 52 Fearnley GR, Lackner R. The fibrinolytic activity of normal blood. Br J Haematol 1955; 1: 189-198