SERPIN GENETICS STRUCTURE AND FUNCTION

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The long term goal of this project is to understand serpin (serine protease inhibitor) structure/function relationships by studying naturally occurring and in vitro generated serpin mutants. Improved understanding of serpin structure/function principles will lead to more effective treatment for thrombosis, bleeding, permeability and pulmonary disorders associated with serpin/protease imbalances. Specific Aim 1 is to determine why perturbations of the P12 region change a serpin from an inhibitor to a substrate. The P12 region is located on the amino terminal side of the reactive center. Analysis of naturally occurring serpin mutants indicates that the P12 region is involved in determining whether a serpin is an inhibitor or a substrate for its target protease(s). A model for the role of the P12 region in determining serpin inhibitor/substrate status is proposed and will be tested by studying the interactions of normal serpins and P12 region mutants with proteases. Specific Aim 2 is to determine why integrity of the ATIII P11' region is required to obtain normal circulating levels of a serpin. The P11' region is located on the carboxy terminal side of the reactive center. Naturally occurring serpin mutants with lesions in the P11' region are present at extremely reduced levels in the circulation, suggesting that it is important for determining intracellular degradation, secretion or clearance rates. These parameters will be studied in cells expressing normal serpins and Pll' region mutants in order to elucidate the role the Pll' region plays in determining circulating serpin levels. Specific Aim 3 is to elucidate the structural basis of antithrombin III heparin cofactor activity. A model for ATIII heparin cofactor activity is proposed and will be tested by making antithrombin mutants with defined structural abnormalities and studying their functional properties. A further test of the model will involve trying to transfer the heparin cofactor property of antithrombin III to another serpin, such as alpha 1-antitrypsin.