Thromb Haemost 1990; 64(01): 047-052
DOI: 10.1055/s-0038-1647252
Original Article
Schattauer GmbH Stuttgart

Plasminogen Activation at Low Temperatures in Plasma Samples Gontaining Therapeutic Concentrations of Tissue-Type Plasm i nogen Activator or Other Thrombolytic Agents[*]

D C Rijken
1   The Gaubius Institute TNO, Leiden, The Netherlands
,
E Seifried
2   Medizinische Klinik und Poliklinik, Abteilung III, Universität Ulm, F.R.G.
,
M M Barrett-Bergshoeffi
1   The Gaubius Institute TNO, Leiden, The Netherlands
,
G Dooijewaard
1   The Gaubius Institute TNO, Leiden, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 10 October 1989

Accepted after revision 12 March 1990

Publication Date:
25 July 2018 (online)

Summary

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and α2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 μg/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and α2-antiplasmin consumption were most pronounced at 37° C, were less pronounced at 25° C, but surprisingly, did not further decrease at 10° C, 0° C or −8° C. In contrast, when plasma was supplemented with 10° IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and α2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10° C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogeo, α2- antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0° C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples.

In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 μg/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0° C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy

Dedicated to Professor M. Verstraete on the occasion of his 65th birthday.


 
  • References

  • 1 Collen D, Stump DC, Gold HK. Thrombolytic therapy. Annu Rev Med 1988; 39: 405-423
  • 2 Marder VJ, Sherry S. Thrombolytic therapy: current status. Part I. N Engl J Med 1988; 318: 1512-1520
  • 3 Marder VJ, Sherry S. Thrombolytic therapy: current status. Part II. N Engl J Med 1988; 318: 1585-1595
  • 4 Gold HK, Fallon JT, Yasuda T, Leinbach RC, Khaw BA, Newell JB, Guerrero JL, Vislosky FM, Hoyng CF, Grossbard E, Collen D. Coronary thrombolysis wih recombinant human tissue-type plasminogen activator. Circulation 1984; 70: 700-707
  • 5 Verstraete M, Bounameaux H, De Cock F, Van de Werf F, Collen D. Pharmacokinetics and systemic fibrinogenolytic effects of recombinant human tissue-type plasminogen activator (rt-PA) in man. J Pharmacol Exp Ther 1985; 235: 506-512
  • 6 Holvoet P, Lijnen HR, Collen D. A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion. Blood 1986; 67: 1482-1487
  • 7 MacGregor IR, Prowse CV, Micklem LR, James K. A monoclonal antibody which prevents reductions in assayed fibrinogen and α2-antiplasmin in plasma samples containing therapeutic concentrations of tissue plasminogen activator (t-PA). Thromb Res 1986; 44: 241-245
  • 8 Seifried E, Tanswell P. Comparison of specific antibody, D-Phe-Pro-Arg-CH2C1 and aprotinin for prevention of in vitro effects of recombinant tissue-type plasminogen activator on haemostasis parameters. Thromb Haemostas 1987; 58: 921-926
  • 9 Mohler MA, Refino CJ, Chen SA, Chen AB, Hotchkiss AJ. D-Phe-Pro-Arg-Chloromethylketone: its potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemostas 1986; 56: 160-164
  • 10 Tiefenbrunn AJ, Graor RA, Robison AK, Lucas FV, Hotchkiss A, Sobel BE. Pharmacodynamics of tissue-type plasminogen activator characterized by computer-assisted simulation. Circulation 1986; 73: 1291-1299
  • 11 Garabedian HD, Gold HK, Leinbach RC, Yasuda T, Johns JA, Thornton D, Collen D. Laboratory monitoring of hemostasis during thrombolytic therapy with recombinant human tissue-type plasminogen activator. Thromb Res 1988; 50: 121-133
  • 12 Fears R, Ferres H, Greenwood H. An investigation of methods to prevent fibrinogen degradation during processing of plasma samples containing the fibrinolytic agents, APS AC and t-PA. Fibrinolysis 1989; 3: 45-49
  • 13 Rijken DC, Seifried E, Barrett-Bergshoeff MM, Kluft C. Mechanism of plasminogen activation at low temperature in plasma samples containing therapeutic concentrations of t-PA. XIth International Congress on Thrombosis and Haemostasis. Thromb Haemostas. 1987; 58: 428 (Abstr 1574)
  • 14 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
  • 15 Kluft C, Van Wezel A, Van der Velden CA M, Emeis JJ, Verheijen JH, Wijngaards G. Large-scale production of extrinsic (tissue-type) plasminogen activator from human melanoma cells. In: Mizrahi A, Van Wezel AL. (eds). Advances in Biotechnological Processes. Alan R. Liss Inc; New York: 1983. 2 98-110
  • 16 Van Ruijven-Vermeer IA M, Nieuwenhuizen W. Purification of rat fibrinogen and its constituent chains. Biochem J 1978; 169: 653-658
  • 17 Vuento M, Vaheri A. Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions. Biochem J 1979; 183: 331-337
  • 18 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
  • 19 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 Haemostas 1982; 48: 266-269
  • 20 Kluft C, Vellenga E, Brommer EJ P, Wijngaards G. A familial hemorrhagic diathesis in a Dutch Family: an inherited deficiency of μ2-antiplasmin. Blood 1982; 59: 1169-1180
  • 21 Friberger P, Knos M, Gustavsson S. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis 1978; 7: 138-145
  • 22 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246
  • 23 Soria J, Soria C, Bertrand O, Dunn F, Samama M, Bachmann F. The amidolytic activity of the SK-plasminogen complex is enhanced by a potentiator which is generated in the presence of vascular plasminogen activator - Role of fibrin degradation products. Thromb Haemostas 1982; 47: 193-196
  • 24 Gram J, Jespersen J. A functional plasminogen assay utilizing the potentiating effect of fibrinogen to correct for the overestimation of plasminogen in pathological plasma samples. Thromb Haemostas 1985; 53: 255-259
  • 25 Mullertz S, Clemmensen I. The primary inhibitor of plasmin in human plasma. Biochem J 1976; 159: 545-553
  • 26 Booth NA, Bennett B, Wijngaards G, Grieve JH K. A new life-long hemorrhagic disorder due to excess plasminogen activator. Blood 1983; 61: 267-275
  • 27 Kluft C, Jie AF H, Los P, Dooijewaard G, Traas DW. Expression of the two forms of α2-antiplasmin in functional and immunochemical assays. In: Davidson JF, Bachmann F, Bouvier CA. (eds) Progress in Fibrinolysis; Churchill Livingstone, Edinburgh: 1983. Vol6 385-387
  • 28 Kominger C, Collen D. Neutralization of human extrinsic (tissue-type) plasminogen activator in human plasma: no evidence for a specific inhibitor. Thromb Haemostas 1981; 46: 662-665
  • 29 Kondo M, Hosokawa K, Masuda M. Cold activation of complement. I. Presence of coagulation-related activator. J Immunol 1976; 117: 486-490
  • 30 Gjønnaess H. Cold promoted activation of F VII. III Relation to the kallikrein system. Thromb Diath Haemorrh 1972 28: 182-193
  • 31 Czendlik C, Lammle B, Duckert F. Cold promoted activation and factor XII, prekallikrein and Q-inhibitor. Thromb Haemostas 1985; 53: 242-245
  • 32 Radcliffe R, Heinze T. Stimulation of tissue plasminogen activator by denatured protein and fibrin clots: a possible additional role for plasminogen activator?. Arch Biochem Biophys 1981; 211: 750-761
  • 33 Wiman B, Ranby M. Determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay. Thromb Haemostas 1986; 55: 189-193
  • 34 Seifried E, Tanswell P, Rijken DC, Barrett-Bergshoeff MM, Su CA P F, Kluft C. Pharmacokinetics of antigen and activity of recombinant tissue-type plasminogen activator after infusion in healthy volunteers. Arzneim-Forsch/Drug Res 1988; 38: 418-422