A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin.
125I-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. 125I-labeled digestion products released from the 125I-fibrin-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 125I-fibrin, in probable non-cross-linked 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) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml 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.
D000614:Aminocaproates / Q000494:pharmacology; D001665:Binding Sites; D002846:Chromatography, Affinity; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000032:analysis* / Q000037:antagonists & inhibitors; D005342:Fibrinolysis; D006801:Humans; D007457:Iodine Radioisotopes; D010958:Plasminogen / Q000302:isolation & purification; D011137:Polystyrenes / Q000378:metabolism; D013300:Streptokinase / Q000032:analysis; D013917:Thrombin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis
D000614:Aminocaproates; D003509:Cyclohexanecarboxylic Acids; D007457:Iodine Radioisotopes; D011137:Polystyrenes; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D013300:Streptokinase; D013917:Thrombin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator