A FLUORESCENCE QUENCH AND DEQUENCH ASSAY OF FIBRINOGEN POLYMERIZATION, FIBRINOGENOLYSIS, OR FIBRINOLYSIS

We present a kinetic assay based on the use of fluorescein isothiocyan ate (FITC)-labeled fibrinogen as a fluoroactive substrate. The multipl e FITCs bound to fibrinogen experienced quenching due to their close p roximity. The thrombin-induced polymerization of FITC-fibrinogen led t o additional fluorescence quenching due to enhanced neighbor-neighbor interactions in protofibrils and protofibril aggregates. The initial r ate of quenching was directly dependent on the thrombin concentration at either low or high ionic strength. The final extent of quenching du ring polymerization with thrombin could be modulated by prevailing ion ic strength and thrombin concentration suggesting that the quenching w as due to fibril extension as well as aggregation, The full extent of quenching was greatly reduced by addition to the reaction of unlabeled fibrinogen or Gly-Pro-Arg-Pro, as expected for quenching due to neigh bor-neighbor interactions. In contrast to polymerization, cleavage of fibrinogen by plasmin released FITC-labeled fragments free of proximit y-based quenching that resulted in a large intensity increase as lysis proceeded-a process termed dequenching. The majority of the dequenchi ng signal during fibrinogenolysis occurred during the generation of fr agment X which proceeded as a first-order process with respect to fibr inogen-bound plasmin with k(cat) = 0.479 s(-1). The K-d of active plas min to fibrinogen was calculated to be 0.42 mu M. Addition of epsilon- aminocaproic acid (epsilon ACA)-plasmin complex to FITC-fibrinogen pro duced little dequenching, demonstrating a requirement for binding in o rder to initiate lysis. Also, addition of excess epsilon ACA after pla smin-mediated fibrinogenolysis was initiated resulted in a dose-depend ent inhibition of dequenching, indicating that plasmin can be desorbed from fibrinogen either by epsilon ACA disruption of the fibrinogen-pl asmin complex or by epsilon ACA capture of desorbing plasmin. Similar to fibrinogenolysis, dequenching occurred in a plasmin-dependent manne r during lysis of polymerized fibrin fibers in suspension. The use of fluorescently labeled fibrinogen as a fluoroactive substrate for plasm in or thrombin will allow kinetic analysis of very dilute systems wher e (i) the presence of the lysine binding sites and (ii) steric phenome na are critical-two situations where small chromogenic peptide substra tes are unsuited. (C) 1995 Academic Press, Inc.