The activity of coagulation proteases is regulated primarily by the serine protease inhibitors (serpins) in plasma. Antithrombin (AT) is a major serpin that regulates the activity of proteases of both intrinsic and extrinsic pathways. AT, however, is a weak inhibitor of coagulation proteases unless it is activated by heparin- like glycosaminoglycans that line the microvasculature. This is the basis for the extensive use of heparin for prophylaxis and treatment of venous thrombosis. Heparin activates AT by binding to a basic exosite on the serpin and inducing a conformational change on the reactive center loop, thereby facilitating the optimal recognition of AT by the coagulation proteases. Heparin can also bind to the basic exosite of a coagulation protease to hold AT and the protease in one complex, thereby facilitating the recognition by a bridging mechanism. AT also exhibits anti-inflammatory activity, however, heparin eliminates this activity of the serpin. Protein Z (PZ)-dependent protease inhibitor (ZPI) is another serpin which, unlike AT, has a narrower target specificity, thus being capable of inhibiting only factors IXa (fIXa), Xa (fXa) and XIa (fXIa). While the ZPI inhibition of both fIXa and fXIa is PZ-independent, inhibition of fXa requires PZ as the cofactor. Unlike the relatively well-studied mechanism of the cofactor function of heparin, the mechanism by which PZ functions as a cofactor to dramatically promote the ZPI inhibition of fXa has not been investigated. The overall objective of this proposal is to understand how these two serpins regulate the catalytic activities of coagulation proteases in the absence and presence of their respective cofactors. Thus, we propose to prepare several AT, ZPI, PZ and coagulation protease mutants to investigate 1) the mechanism by which heparin promotes the AT inactivation of coagulation proteases;2) the mechanism of anti-inflammatory activities of wild-type AT and an AT mutant that can inhibit fXa but not other coagulation proteases using cellular models, 3) the mechanism by which ZPI specifically inhibits its target proteases;and 4) the mechanism by which PZ functions as a cofactor to promote the inactivation of fXa by ZPI. These studies will utilize enzyme kinetics, fluorescence spectroscopy, plasmon resonance, and competitive binding methods to provide critical information about kinetic and thermodynamic constants that define the biological properties and the mechanism of interaction of the two serpins with their target molecules. These studies can lead to design and development of specific therapeutic drugs that may potentially be useful for treatment of thrombotic and inflammatory disorders. The proposed studies in this application will utilize enzyme kinetics, fluorescence spectroscopy, plasmon resonance, and competitive binding methods to provide critical information about the kinetic and thermodynamic constants that define the specific interaction of plasma inhibitors with their target clotting enzymes. Understanding the mechanism by which the natural plasma inhibitors regulate the activity of the clotting enzymes can lead to design and development of specific therapeutic drugs and inhibitors that may potentially be useful for treatment of abnormalities (thrombosis and hemorrhage) in blood.
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