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Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator
Abstract:
The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.
Insights
Tissue-type plasminogen activator (t-PA) activates plasminogen efficiently with soluble fibrinogen fragments, similar to solid-phase fibrin. This cofactor significantly enhances t-PA
Area of Science:
- Biochemistry
- Molecular Biology
- Hemostasis
Background:
- Plasminogen activation is crucial for fibrinolysis.
- Tissue-type plasminogen activator (t-PA) is a key enzyme in this process.
- Fibrin acts as a cofactor, enhancing t-PA's catalytic efficiency.
Purpose of the Study:
- To investigate the kinetics of plasminogen activation by t-PA using soluble fibrinogen fragments as a cofactor.
- To compare these kinetics with those observed in the presence of solid-phase fibrin.
- To elucidate the mechanism of t-PA-mediated plasminogen activation.
Main Methods:
- Michaelis-Menten kinetic analysis.
- Enzyme kinetics studies using purified plasminogen and t-PA.
- Utilized CNBr-digested fibrinogen as a soluble cofactor.
- Investigated interactions using lysine-binding site fragments.
Main Results:
- Michaelis-Menten kinetics were observed, with parameters similar to solid-phase fibrin activation.
- Soluble fibrinogen fragments significantly increased plasminogen affinity for t-PA (KM decreased from 83 to 0.18 µM).
- The catalytic rate constant (kcat) showed a modest increase (0.07 to 0.28 s⁻¹).
- Lysine-binding sites on plasminogen competed for interaction with fibrinogen fragments, with a dissociation constant of 4.5 µM for the high-affinity site.
- No significant differences in kinetic parameters were found between melanoma-derived t-PA and recombinant glycosylated t-PA.
Conclusions:
- The findings support a mechanism where both t-PA and plasminogen bind to fibrin (or its soluble fragments) to facilitate activation.
- Soluble fibrinogen fragments effectively mimic the cofactor role of solid-phase fibrin in plasminogen activation.
- The study provides insights into the molecular interactions governing fibrinolysis and the role of t-PA.