A Collaborative Study to Establish a Standard for High Molecular Weight Urinary-Type Plasminogen Activator (HMW/u-PA)[*]
Received: 20 February 1990
Accepted after revision28 May 1990
25 July 2018 (online)
An international collaborative study involving eleven laboratories located in eight countries was undertaken to establish an International Standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA). The current International Reference Preparation (IRP code numbered 66146) for urinarytype plasminogen activator (u-PA) ot urokinase (see Nomenclature footnote) is a 66/34 molar ratio mixture of low molecular weight (LMW) - and high molecular weight (HMW) - u-PA’s and is considered unsuitable as a standard for homogeneous preparations of HMW/u-PA. The putative standard for HMWu-PA (code number, 87/594) was compared for potency in a clot lysis assay with the current IRP for u-PA (code numbet, 66146) and a lyophilised preparation of single chain urinary-type plasminogen activator (SCuPA), the latter being used in the assay without prior activation by plasmin to its active two chain form (TCuPA).
Both the proposed standard for HMWu-PA (871594) and the SCuPA compared in a statistically satisfactory manner in parallel line bioassays with the current IRP for u-PA (66146), thus allowing potency estimates to be obtained for these two materials in relation to defined international units. Data from the eleven laboratories indicated that each ampoule of the proposed standard for HMWu-PA contained 4,300 i. u. of activity and was stable for over 1 year at 4° C. Most participants indicated that SCuPA expressed only a small amount of its activity without a prior activator step and this suggests that SCuPA assays need to be preceded by a plasmin activation step
The Expert Committee on Biological Standardization of the World Health Organisation (Geneva, Oct 1989) established 871 594 as the International Standard for high molecular weight two chain urinary type plasminogen activator (HMW/TCuPA, of HMWu-PA), with an assigned unitage of 4,300 international units per ampoule.
Nomenclature: The conventional name for the plasminogen activator found in and isolated from urine is Urokinase (UK). The Fibrinolysis Sub-Committee of the International Committee for Thrombosis and Haemostasis meeting in San Diego, CA, U.S.A. in 1985, adopted a nomenclature which at that time more adequately described the source and molecular composition of the urokinases as members of a group of plasminogen activators. Thus UK was named urinary-type plasminogen activator (u-PA) while prourokinase or single chain urokinase was described as single chain urinary type plasminogen activator (SCuPA). Thus two chain urokinase would be described as two chain urinary-type plasminogen activator (TCuPA); however, whenever the term urokinase (UK) is used in the text it is assumed to be active and thus in the two chain (TC) form. The respective high and low molecular weight forms of TCuPA were identified by the prefix, LMW or HMW respectively.
- 1 Urano T, Sator de Serrano V, Gaffney PJ, Castellino FJ. Activation of human (GLU) plasminogen by single chain urokinase. Arch Biochem Biophys 1988; 264: 222-30
- 2 Philo RD, Gaffngy PJ. Relative potencies of different molecular weight forms of urokinase. In: Progress in Fibrinolysis and Thrombosis Davidson JF, Nilsson IM, Samama MM, Desnoyers pC. eds Churchill-Livingstone; Edinburgh: 1987: 220-2
- 3 Philo RD, Gaffney PJ. Assay methodology for urokinase. Its use in assessing the composition of mixtures of high- and low-molecular weight urokinases. Thromb Res 1981 21. 81-8
- 4 World Health Organisation. Twenty First Meeting. Expert Committee on Biological Standardization. WHO Tech Rep Ser No 413 1968
- 5 Gaffney PJ, Curtis AD. A collaborative study to establish the 2nd International Standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1987; 58: 1085-1087
- 6 Campbell PJ. International biological standards and reference preparations. I. Preparation and presentation of materials to serve as standards and reference preparations. J Biol Stand 1974 2. 249-58
- 7 Gurewich Y, Pannell R, Louie S, Kelly R, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species J Clin Invest 1984; 73: 173l-9
- 8 Beebe DP, Aronson DL. An automated fibrinolytic assay performed in microtitre plates. Thromb Res 1987; 47: 123-8
- 9 Finney DJ. Statistical Method in Biological Assay. 3rd edition. Griffin, London 1978
- 10 Gaffney PJ, Tydeman MS, Kirkwood TB L, Aronson D, Murano G. International collaborative study on the assay of commercially available high and low molecular weight urokinase. Thromb Haemostas 1981; 45: 34-7
- 11 Kirkwood TB L, Tydeman MS. Design and analysis of accelerated degradation tests for the stability of biological standards. J Biol Stand 1984; 12: 207-14