Chromogenic substrate assays: Present and potential applications

Abstract

Chromogenic substrates are now available for the study of many aspects of blood coagulation and related enzyme reactions in blood. These substrates have opened up new vistas, including ease of control of routine anticoagulant therapy. The overall impact on routine haemostatic testing will probably be immense since automated systems may be used as an initial screen for patients with haemostasis problems. This applies not only to the coagulation but also for related systems such as the fibrinolytic pathway. The major point one must remember in their use is that activity directed against these substrates may not be identical with the biological activity one wishes to examine. This must make one cautious in their interpretation in the same way as one would be cautious in interpreting the results employing antibodies for antithrombin III levels. A great advantage of chromogen substrate techniques is their accuracy and reproducibility. Some of these points will be illustrated with investigations in our laboratory with the chromogenic substrate S2238. This substrate can assay thrombin formation in a thrombin generation system consisting of recalcified platelet rich plasma. Early in these studies it was shown that S2238 recognized thrombin activity long after the biological activity of the enzyme had disappeared against fibrinogen. The reason for this is because thrombin is inactivated in plasma initially by binding α₂ microglobulin, and that thrombin so bound can still attack S2238. This is a good example of the possible lack of correlation between biologic and substrate activity in certain circumstances. Such property however, would not exclude these agents from being very useful in a general automated screen of clotting problems in large numbers of patients coming through teaching hospitals. One could identify patients with haemostatic problems which might then be investigated by standard coagulation techniques.