KINETIC ANALYSES OF THE ACTIVATION OF GLU-PLASMINOGEN BY TISSUE PLASMINOGEN ACTIVATOR IN THE PRESENCE OF FIBRIN, FIBRINOGEN, FRAGMENTS D AND E

Abstract

Kinetic analyses were made for the activation of Glu-plg (Glu-plasminogen) by tissue plasminogen activator (t-PA: two chain t-PA obtained from Bowes melanoma cell line) in the presence of fibrin, fibrinogen, fragment D or E. Glu-plg was activated by t-PA in the presence of various concentrations of fibrinogen, fibrin, fragment D or E, and the activation rate was measured by the hydrolysis of S-2251 with plasmin generated. Straight Line-weaver-Burk plots were obtained by changing the concentration of Glu-plg. In the absence of fibrin, t-PA activated Glu-plg with Km of 1.11 μM, kcat of 0.005 sec-1, kcat/Km of 0.0045 μM-1,sec-1. The presence of catalytic amounts (0.0001 μM) of fibrin resulted in 11.4 fold decrease in Km, thus 15.6 fold increase in kcat/Km. The presence of larger amounts of fibrin resulted in increase in kcat and decrease in Km, which resulted in 142 fold increase in kcat/Km. There seems to be two phases in the enhancement of the activation of Glu-plg by t-PA, one induced in the presence of catalytic amounts of fibrin, and the other induced in the presence of higher concentration of fibrin (up to 0.05/0.2 of the molar ratio of fibrin to plasminogen). When effects of fragment D or E were examined for the enhanced activation of Glu-plg by t-PA, the presence of increasing amounts of E resulted in increase in kcat. Presence of D resulted in increase in kcat and decrease in Km. Since plasminogen was shown to bind to both D and E domains of fibrin, and t-PA was shown to bind to D domain, we hypothesize that the role of D in the activation of Glu-plg by t-PA was to provide a site to t-PA and plasminogen to form a ternary complex, which facillitates the activation of plasminogen by t-PA, and that the role of E was to provide a site to bind to plasminogen, where t-PA activates plasminogen more effectively. The results suggest that roles of D and E domains of fibrin may be different in the enhanced activation of plasminogen by t-PA.