International Collaborative Study for the Establishment of the Second International Reference Preparation of Plasmin

Summary

An international collaborative study involving seven laboratories was undertaken to assess the suitability of a freeze- dried preparation of human plasmin to replace the current International Reference Preparation (IRP) for plasmin. Chromogenic and fibrinolytic assays were used by all participating laboratories to assess the potencies of the Proposed International Reference Preparation (PIRP) and two other freeze-dried plasmins, one of human and one of porcine origin

The data suggest that the PIRP is a more suitable standard for plasmin than the IRP in that the former binds to fibrin whereas only 50% of the latter binds. The PIRP compared well to other plasmin preparations and the potency assays were independent of the assay procedure and substrate used. Degradation studies indicated that the PIRP was far more stable than the glycerol solution of the IRP, surviving for 12 months at 37° C with no significant loss in either amidolytic or fibrinolytic activity. The International Committee for Thrombosis and Haemostasis (Bergamo, 1982) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the second International Reference Preparation for Plasmin with a defined potency of 10 International Units of Plasmin per ampoule.

Keywords

Plasmin - International standard - Fibrinolytic assay - Amidolytic assay

• References

• 1 Kirkwood TB L, Campbell PJ, Gaffney PJ. A standard for human plasmin. Thrombos Diathes Haemorrh 1975; 34: 20-31
• 2 28th Report, WHO Expert Committee on Biological Standardization. WHO Tech, Rep Ser No 610 1977; pp 13
• 3 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
• 4 Thorsen S. Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation. Influence of omega-amino-carboxylic acids Biochim Biophys Acta 1975; 393: 55-65
• 5 Whitaker AN, Rowe EA, Masci PP, Joe F, Gaffney PJ. The binding of glu- and lys-plasminogens to fibrin and their subsequent effects on lysis rates. Thromb Res 1980; 19: 381-391
• 6 Friberger P, Knos M, Gustavsson S, Aurell L, Claeson G. Methods for the determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis 1978; 7: 138-145
• 7 Johnson AJ, Kline DL, Alkjaersig N. Assay methods and standard preparations for plasmin, plasminogen and urokinase in purified systems. Thrombos Diathes Haemorrh 1969; 21: 259-272
• 8 Philo RD, Gaffney PJ. Plasmin potency estimates: influence of the substrate used in assay. Thromb Haemostas 1981; 45: 107-109
• 9 Wiman B, Lijnen HR, Collen D. On the specific interaction between the lysine-binding sites in plasmin and complementary sites in α₂- antiplasmin and in fibrinogen. Biochim Biophys Acta 1979; 579: 142-154
• 10 Kluft C, Traas DW, Jie AF H, Hoegee-de Nobel E. The suitability of various plasmin preparations for the functional assay of α₂-antiplasmin. Thromb Haemostas 1982; 48: 320-324
• 11 31st Report, WHO Expert Committee on Biological Standardization. WHO Tech, Rep Ser No 658. 1981; pp 19
• 12 Campbell PJ. International biological standards and reference preparations. 1: Preparation and presentation of materials to serve as standards and reference preparations. J Biol Stand 1974; 2: 249-258
• 13 Finney DJ. Statistical Method in Biological Assay. 3. Griffin & Co; London: 1978
• 14 32nd Report, WHO Expert Committee on Biological Standardization. WHO Tech Rep Ser No 673. 1982; pp 24
• 15 Gaffney PJ, Philo RD. A commentary on new methodology in haemostasis using synthetic substrates. In Perspectives in Haemostasis. Fareed J, Mesmore H, Brinkhous K. (Eds) Pergamon Press; New York: 1981. pp 405-417
• 16 Gaffney PJ, Lord K, Brasher M, Kirkwood TB L. Problems in the assay of thrombin using synthetic peptides as substrates. Thromb Res 1977; 10: 549-556
• 17 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
• 18 Rickli EE. The activation mechanism of human plasminogen. Thrombos Diathes Haemorrh 1975; 34: 386-395