The goal of this research is to understand, at the molecular level, how blood coagulation is regulated by serine protease inhibitors. The role of heparin and other glycosaminoglycans (GAGs), procoagulant cofactor proteins, divalent metal ions and GAG- binding proteins is modulating the rate of neutralization of blood coagulation-proteases by antithrombin III and other inhibitors will be investigated by rapid kinetic and equilibrium binding studies. Whether heparin and other modulators promote the initial encounter of protease and inhibitor or alter the limiting rate of protease neutralization and whether these effects are mediated by induced protein conformational changes will be evaluated. Inhibitors and proteases modified chemically or by natural mutation will serve as probes of the mechanism of action of GAG and other effectors on inhibitor/protease reactions. The enhancement by heparin and possibly other GAGs of inhibitor turnover and inactivation by proteases during their neutralization, and the modulation of this reaction by effectors, will be investigated as a potential regulatory mechanism for protection of these proteases from neutralization. Whether this reaction reflects a mechanism in which the protease is trapped by an inhibitor conformational change during turnover of the inhibitor as a normal protease substrate, will be elucidated. These studies will employ fluorescence or absorbance probes to quantitate interactions between proteins, GAGs and metal ions, as well as reactions between inhibitors and their target proteases in the presence and absence of modulators. Quantitative affinity chromatography studies of binding interactions and direct monitoring of inhibitor/protease kinetics by residual protease activity will serve to validate probes as signals for these interactions and reactions. These studies should enable us to determine what limits the rate at which inhibitors can neutralize their target proteases under physiological conditions and will provide a basis for evaluating the relative importance of in vivo regulation of each protease as well as the potential role of GAGs on the vascular endothelium or in the extravascular space in this regulation.
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| Huang, Xin; Rezaie, Alireza R; Broze Jr, George J et al. (2011) Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor. J Biol Chem 286:8740-51 |
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