The proteolytic activity of coagulation proteases is regulated by the serine protease inhibitors (serpins) in plasma. Anti-thrombin (AT) is a major serpin that regulates the proteolytic activity of coagulation proteases of both intrinsic and extrinsic pathways. AT is a relatively weak inhibitor of coagulation proteases unless it binds to the heparin-like glycosaminoglycans lining the microvasculature or therapeutically administered for prophylaxis and treatment of venous thrombosis. In addition to its anticoagulant function through the direct inhibition of procoagulant coagulation proteases, AT also elicits potent anti-inflammatory activities when it binds to vessel wall glycosaminoglycans, thereby eliciting intracellular signaling responses in vascular endothelial cells. Protein Z (PZ)-dependent protease inhibitor (ZPI) is another serpin which, unlike AT, exhibits a narrower protease specificity, thus capable of inhibiting only factors Xa (fXa) and XIa (fXIa). While the ZPI inhibiton of fXIa is independent of a protein cofactor, its inhibition of fXa requires PZ as the cofactor. Recent results have indicated that heparin can bind to an unknown site on ZPI to promote its reactivity with both fXa and fXIa. Unlike the relatively well-studied mechanism of the cofactor function of heparin in enhancing the AT inhibition of coagulation proteases, less is known about mechanisms through which PZ and heparin function as cofactors to accelerate the ZPI inhibition of its target proteases. The overall objective of this project is to understand how these two serpins regulate the catalytic activities of coagulation proteases in the absence and presence of their respective cofactors and how AT elicits protective intracellular signaling responses. We propose to prepare several AT, ZPI, PZ and coagulation protease mutants 1) to investigate the mechanism by which AT elicits anti-inflammatory signaling activities in response to proinflammatory cytokines using cellular models;2) to investigate the anti-inflammatory and cardioprotective properties of AT derivatives having altered anticoagulant and signaling properties in a mouse left anterior descending coronary artery (LAD) ischemia/reperfusion injury model;3) to investigate the mechanism by which ZPI interacts with its target proteases;and 4) to investigate the mechanism by which PZ and heparin function as cofactors to promote the inactivation of fXa and fXIa by ZPI. These studies will utilize cellular and animal models as well as basic biochemistry, kinetics and molecular biology methods to provide critical information as to how AT elicits protective intracellular signaling responses and how ZPI interacts with protein and polysaccharide cofactors to inhibit its target proteases.
The proposed studies of this application will provide new insight into mechanisms through which the two plasma inhibitors, anti-thrombin and protein Z-dependent protease inhibitor, regulate the anticoagulant and anti-inflammatory pathways. The use of anti-thrombin as an anti-inflammatory molecule is associated with increased risk of bleeding. The studies of this application can lead to design and development of specific anti-thrombin molecules with improved therapeutic profiles which may be useful for treatment of thrombotic, inflammatory and cardiovascular disorders.
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