A Fluorescence Quench and Dequench Assay of Fibrinogen Polymerization, Fibrinogenolysis, or Fibrinolysis

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We present a kinetic assay based on the use of fluorescein isothiocyanate (FITC)-labeled fibrinogen as a fluoroactive substrate. The multiple FITCs bound to fibrinogen experienced quenching due to their close proximity. The thrombin-induced polymerization of FITC-fibrinogen led to additional fluorescence quenching due to enhanced neighbor-neighbor interactions in protofibrils and protofibril aggregates. The initial rate of quenching was directly dependent on the thrombin concentration at either low or high ionic strength. The final extent of quenching during polymerization with thrombin could be modulated by prevailing ionic strength and thrombin concentration suggesting that the quenching was 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 neighbor-neighbor interactions. In contrast to polymerization, cleavage of fibrinogen by plasmin released FITC-labeled fragments free of proximity-based quenching that resulted in a large intensity increase as lysis proceeded-a process termed dequenching. The majority of the dequenching signal during fibrinogenolysis occurred during the generation of fragment X which proceeded as a first-order process with respect to fibrinogen-bound plasmin with k cat = 0.479 s −1. The K d of active plasmin to fibrinogen was calculated to be 0.42 μM. Addition of ϵ-aminocaproic acid (ϵACA)-plasmin complex to FITC-fibrinogen produced little dequenching, demonstrating a requirement for binding in order to initiate lysis. Also, addition of excess ϵ ACA after plasmin-mediated fibrinogenolysis was initiated resulted in a dose-dependent inhibition of dequenching, indicating that plasmin can be desorbed from fibrinogen either by ϵACA disruption of the fibrinogen-plasmin complex or by ϵACA capture of desorbing plasmin. Similar to fibrinogenolysis, dequenching occurred in a plasmin-dependent manner during lysis of polymerized fibrin fibers in suspension. The use of fluorescently labeled fibrinogen as a fluoroactive substrate for plasmin or thrombin will allow kinetic analysis of very dilute systems where (i) the presence of the lysine binding sites and (ii) steric phenomena are critical-two situations where small chromogenic peptide substrates are unsuited.