ID: 243 The Kinetics of Glu- and Lys-Plasminogen Activation on Untreated and TAFIa Treated High Molecular Weight Fibrin Degradation Products

Abstract

Plasminogen (Pg) activation to plasmin (Pn) by tissue-type Pg activator (tPA) requires fibrin as a cofactor. Intact fibrin up regulates tPA mediated Glu-Pg activation by approximately 1000-fold compared to no fibrin. Before lysis of the fibrin clot, Pn modifies the fibrin surface resulting in the exposure of carboxyl-terminal lysine and arginine residues. This Pn modified form of fibrin further up regulates Glu-Pg activation by approximately 3-fold over intact fibrin, creating a positive feedback loop for Pn generation. The carboxypeptidase B-like enzyme, activated thrombin activatable fibrinolysis inhibitor (TAFIa), removes these exposed lysine and arginine residues, decreasing the cofactor activity of fibrin to approximately 100-fold less than Pn modified fibrin, thereby attenuating fibrinolysis. While Glu-Pg activation by tPA on plasmin and TAFIa modified fibrin has been characterized, no such analysis has been done with Lys-Pg. Because a fibrin clot is insoluble, soluble high molecular weight fibrin degradation products (HMW-FDPs) were used as a model for plasmin modified fibrin. A fluorescently labeled recombinant Pg mutant, 5IAF-Pg, was used to simplify the kinetic analysis. The active site serine in this Pg derivative has been mutated to a cysteine (S741C), to which 5-iodoacetamidofluorescein has been covalently attached. 5IAF-Pg cleavage does not generate active Pn, thus eliminating the feedback cleavages. In addition, cleavage of this derivative results in an approximate 50% decrease in fluorescence intensity, which can be used to monitor the kinetics of cleavage. Both 5IAF-Glu-Pg and 5IAF-Lys-Pg cleavage by tPA were studied on untreated and TAFIa treated HMW-FDPs. The initial rates of cleavage were fit to a previously derived steady-state template model for tPA mediated Pg activation on fibrin. The catalytic efficiency (kcat/Km) for 5IAF-Glu-Pg cleavage decreased by 94% upon TAFIa treatment of the HMW-FDPs (0.90 microM^-1s^-1 to 0.05 microM^-1s^-1), whereas a 36% decrease in kcat/Km was observed for 5IAF-Lys-Pg (1.38 microM^-1s^-1 to 0.84 microM^-1s^-1). Thus, TAFIa treated HMW-FDPs are an approximately 17-fold better cofactor for 5IAF-Lys-Pg cleavage than 5IAF-Glu-Pg cleavage. This increase in cofactor activity is due to the higher affinity of 5IAF-Lys-Pg for HMW-FDPs than 5IAF-Glu-Pg. TAFIa treatment of the HMW-FDPs resulted in a 62-fold increase in the Kd for 5IAF-Glu-Pg (0.356 ± 0.251 microM to 22.120 ± 472.731 microM). However, TAFIa treatment resulted in only a 2-fold increase in the Kd for 5IAF-Lys-Pg binding to HMW-FDPs (0.356 ± 0.251 microM to 0.721 ± 2.311 microM). Furthermore, a 5-fold increase in the Kd for 5IAF-Glu-Pg binding to HMW-FDPs with bound tPA was observed upon TAFIa treatment (0.079 ± 0.022 microM to 0.387 ± 0.076 microM), but no change was observed for 5IAF-Lys-Pg (0.033 ± 0.017 microM to 0.030 ± 0.029 microM). These data rationalize previous results showing that although TAFIa increases the lysis time of purified clots in the presence of Glu-Pg, it does not do so in the presence...