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Comparison of kinetic parameters of the activation of Glu-plasminogen by tissue plasminogen activator obtained from
A Takada1, Y Sugawara, Y Takada
1Department of Physiology, Hamamatsu University, School of Medicine, Shizuoka-ken, Japan.
Abstract:
Kinetic analyses were made for the activation of Glu-plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) obtained from various sources. We compared kinetic parameters for three t-PA preparations. t-PA(M) was obtained from cultured melanoma cell line, and t-PA(W) and t-PA(TD) were obtained from cells with recombinant DNA. t-PA(W) had two chains whereas t-PA(TD) had one chain. Although values of kinetic parameters were different in the three t-PA preparations, there were following general tendencies. The presence of catalytic amounts of fibrin resulted in a decrease in Km, whereas the presence of larger amounts of fibrin resulted in an increase in kcat with a slight decrease in Km. The presence of fibrinogen also enhanced the activation of Glu-Plg by t-PA with an increase in kcat and without change in Km. When the effects of fragment D or E were compared, the presence of increasing amounts of E resulted in an increase in kcat, but the presence of increasing amounts of D resulted in an increase in kcat and a decrease in Km. The results suggest that roles of D and E domains of fibrin may be different in the enhanced activation of plasminogen by t-PA.
Insights
Tissue plasminogen activator (t-PA) kinetic analysis reveals fibrinogen and fibrin influence Glu-plasminogen activation. Fibrinogen and fibrin fragments differentially affect t-PA activity, suggesting distinct roles in fibrinolysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Tissue plasminogen activator (t-PA) is crucial for fibrinolysis.
- Understanding t-PA kinetics is vital for thrombolytic therapies.
- Glu-plasminogen (Glu-Plg) is the precursor to active plasmin.
Purpose of the Study:
- To analyze the kinetics of Glu-Plg activation by different t-PA preparations.
- To investigate the influence of fibrin, fibrinogen, and fibrin fragments (D and E) on t-PA activity.
Main Methods:
- Kinetic parameter analysis (Km, kcat) of t-PA.
- Comparison of three t-PA sources: melanoma cell line (t-PA(M)) and recombinant DNA sources (t-PA(W), t-PA(TD)).
- Assessment of t-PA activity in the presence of varying concentrations of fibrin, fibrinogen, and fibrin fragments D and E.
Main Results:
- Fibrinogen enhanced Glu-Plg activation by increasing kcat without altering Km.
- Low fibrin concentrations decreased Km, while high concentrations increased kcat and slightly decreased Km.
- Fibrin fragment E increased kcat, whereas fragment D increased both kcat and decreased Km.
Conclusions:
- Fibrinogen and fibrin differentially modulate t-PA-mediated Glu-Plg activation.
- Fibrin fragments D and E play distinct roles in enhancing plasminogen activation by t-PA.
- These findings provide insights into the complex regulation of fibrinolysis.