FLUOROGENIC FIBRINOGEN AND FIBRIN FACILITATE MACROMOLECULAR ASSEMBLY AND DYNAMIC ASSAY OF PICOMOLAR LEVELS OF PLASMINOGEN ACTIVATORS UNDER WELL-MIXED CONDITIONS

Fibrinogen labeled with fluorescein isothiocyanate (FITC) was tested f or its ability to serve as a template for macromolecular assembly as w ell as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FIT C-fibrinogen or FITC-fibrin fiber suspension were degraded during lysi s, release of fluorescent fragments abolished proximity-based quenchin g and resulted in a 2.0- or 3.6-fold increase in fluorescence intensit y, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescenc e dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating th e dissociation of a reversible antiplasmin-plasmin complex. The detect ion limit of the reaction assay was 20 pM and 200 pM of recombinant tP A and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminoge n, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fi brinogen polymerization followed by dequenching due to fibrinolysis. A ddition of 10 mM epsilon-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the prese nce of active plasmin. FITC-fibrinogen could be copolymerized with rec alcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasm in was used to degrade the fibrin formed from the platelet poor plasma . Given the large signal generated upon degradation of the fluorogenic fibrin(ogen), at least 10(5) determinations can be run from 100 mg of FITC-labeled fibrinogen using a standard fluorimeter and 0.1 to 3.0 m l reaction volumes. The versatility of the fluorgenic fibrinogen subst rate allowed the configuration of assays to detect and measure the act ivity of thrombin, plasmin, tPA, uPA, and alpha(2)-antiplasmin. The ab ility to assemble blood proteins on a fluorogenic fibrinogen or fibrin template provides unique opportunities for the dynamic study of bindi ng and enzymatic events on the fibrin surface under well mixed conditi ons.