A Rapid and Sensitive ‘251-Fibrin Solid-Phase Fibrinolytic Assay for Plasmin

‘251-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 1251-labeled digestion products released from the ‘25lfibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated 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.