The long-term goal of this proposal is to provide a complete understanding of how blood clotting proteinases are regulated by the plasma serine proteinase inhibitor, antithrombin, and its glycosaminoglycan cofactor, heparin, at the molecular level.
The specific aims are: 1) to elucidate the structural determinants of the target proteinase specificity of antithrombin and to determine how this specificity is expressed; ii) to map the heparin binding site in antithrombin and to determine the molecular events involved in heparin activation of antithrombin; and iii) to determine the extent to which antithrombin inhibition of thrombin and factor Xa is modulated by the binding of these proteinases in physiologic complexes which localize their action and to elucidate the consequences of this inhibition on the bound proteinase. These studies will utilize site-directed mutagenesis to map the functional sites of antithrombin which mediate its interactions with proteinases and heparin and to test hypotheses which define the relationship between antithrombin structure and function. Thermodynamic and kinetic approaches will be used to quantitate the interactions between antithrombin, proteinases, heparin and other regulatory components and the effects of mutation on these interactions. These studies are anticipated to provide new insights into the structural basis of antithrombin s specificity for clotting proteinases, its ability to trap clotting proteinases in stable inactive complexes and its activation by heparin. They should further elaborate a new model of dynamic regulation of clotting proteinases by antithrombin in which proteinases bound to their receptor sites of action are inhibited by antithrombin at these sites with concomitant release of the inhibited proteinase. These studies should also provide a rational basis for the design of recombinant antithrombins or synthetic heparins with tailor-made antithrombotic activity.
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