The specific aims of the project are: (1) to elucidate the kinetic pathway and reaction mechanism for the inhibition of tPA by PAI-1; (2)to study the conformational changes and interactions which affect the stability, activity and specificity of PAI-1; (3) to determine what limits the turnover of tPA with plasminogen, its natural substrate, and how cleavage of the single chain inhibitor increases it; and (4) to evaluate structure-function aspects and conformational changes in the mechanism of action of streptokinase. The studies will depend on certain unique methods which we have utilized in the past. Rapid reaction kinetics, specifically stopped-flow fluorimetry and rapid mixing chemical quenching will be used to follow the formation and decay of intermediates. Fluorescence spectroscopy; including polarization or anisotropy and resonance energy transfer, will be used extensively to evaluate interactions and different conformational states of the proteins. At the conclusion of this work we hope to have a clearer understanding of the structure of the PAI-1 protease complex, the kinetic pathway for its formation, and the basis for its stability. This information may be relevant to all serpins. We also hope to elucidate the reaction mechanism of tPA and some of the structural features and conformational changes critical to the reaction mechanism of streptokinase. Enhanced understanding of the PAI-1 mechanism can result in development of inactivators which would provide a novel antithrombotic action. More knowledge about the tPA and streptokinase mechanisms can result in more effective thrombolytic therapy.
|Olson, S T; Swanson, R; Day, D et al. (2001) Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. Biochemistry 40:11742-56|
|Sherman, P M; Lawrence, D A; Yang, A Y et al. (1992) Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center. J Biol Chem 267:7588-95|