A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.

SummaryAn International Collaborative Study involving 12 laboratories in 7 different countries was undertaken in order to replace the 1st International Standard (IS) for Fibrinogen, Plasma (89/644). The candidate replacement standard was the ampouled and freeze-dried residue of solvent/detergent treated plasma and was calibrated as coded duplicates (A and B) versus the 1st IS Fibrinogen, Plasma by automated Clauss assay and by a recommended clot collection (gravimetric) assay. This latter method had been used to calibrate the 1st IS Fibrinogen, Plasma.Comparing the ratios of the potency estimates of sample A to sample B (the coded duplicates), all of the laboratories obtained a ratio within 5% of the expected value of 1.0 by automated Clauss assay, which suggests that the laboratories were able to perform this assay well. Scrutiny of the data obtained from the gravimetric assays revealed that in almost all cases the results were invalid. The results of these assays are included in this report but clearly should be treated with caution and indeed produced significantly lower mean estimates of potency than the other assay methods. The overall geometric mean of all estimates of potency of the proposed 2nd IS Fibrinogen, Plasma (98/612) is 2.19 mg/ampoule by the automated Clauss assay. These data have been presented to the Fibrinogen Sub-Committee of the Standardisation and Scientific Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) (Washington, DC, August 1999), which recommended the establishment of 98/612 as the 2nd IS Fibrinogen, Plasma. This report has been presented to the Expert Committee on Biological Standardisation of the World Health Organisation (ECBS-WHO) at their 1999 session and 98/612 was established as the 2nd IS Fibrinogen, Plasma with a potency of 2.2 mg/ ampoule.

Summary. An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in-house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

Prof. Tage Astrup first elaborated the notion that blood fluidity involved a balance between the tendency of blood to clot and for such clots to lyse. It would seem that, at that time, this haemostatic balance involved the notion that forming fibrin orchestrated its own destruction by stimulating fibrinolytic activity. In this review, we have clarified the detail of this balance and developed the thesis that Astrup’s far-sighted balance notions involve a variety of control mechanisms. These involve the notion that thrombin, being at first sight a procoagulant, can also, in conjunction with thrombomodulin, act as a stimulus of anticoagulant activity by the generation of activated protein C. The thrombin-activatable fibrinolytic inhibitor (TAFI) is also involved in this balance since the generation of thrombin provokes the neutralisation of fibrinolysis by the TAFI pathway. The kallikrein/factor XII/urokinase pathway is discussed indicating yet another aspect of balance between the generation of coagulation and fibrinolysis. The overall theme of this review, apart from an insight into various aspects of the haemostatic balance, is that blood has a strong tendency to clot when tissue is damaged, and the intact vasculature requires major anticoagulant systems to prevent clots adhering to and stabilising in the vasculature.

Four antibodies used routinely in-house for the assessment of antiglobulin reagents (anti-Fyb, anti-Jka, anti-S) were tested in parallel using tube and antiglobulin 'gel-test' low ionic strength antiglobulin techniques. In the latter the red cells are centrifuged following incubation through a dextran matrix incorporating an anti-human globulin reagent. The results show that the antiglobulin 'gel-test' was less sensitive than the tube technique in the detection of these difficult antibodies.