A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin

degradation products from an unlabeled conventional fibrin clot. The ‘25l-fibrin, in probable non-crosslinked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng ± 1.2 (SD) fibnn (1% of the total bound ‘251-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125l-fibrin, which exceeds published sensitivities (1000-5000 ng) using 1251 or fiuorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 zg/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 zg/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/mI and 0.04 CTA U/mI, respectively (sensitivity of fibrin-plate method, 0.5 U/mI for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system. P LASMINOGEN IS THE proencyme in plasma which, upon conversion to its active form, plasmin (E.C. 3.4.4.14), plays a central role in interrelationships among coagulation, fibrinolytic, and inflammatory processes.’ Examination of physiologic factors which modulate the function of plasmin either directly or by influencing the activation of its proenzyme requires methodology which monitors its enzymatic activity on its major physiologic substrate, fibrin. Available methods, including those based on lysis of radioactively or fluorochrome-labeled fibrin clots2� or of fibrin gels (fibrin plate method),5-6 are often insufficiently sensitive for this purpose, are excessively time consuming, fail to monitor activators/inhibitors which enter the substrate poorly (in the case of gels or clots), or do not provide adequate separation of reaction products from residual substrate. Although the mechanism is unexplained, the firm adsorption of protein (in the form of antibody or antigen) to polystyrene at low ionic strength is an established feature of solid-phase radioimmunoassay technology.7’8 We have used this binding as the basis for a solid-phase radioassay method. ‘25I-fibrinogen is adsorbed to polystyrene tubes and converted to fibrin by thrombin. The result