REGULATION OF EXTRACELLULAR PROTEOLYSIS BY SERPINS

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DESCRIPTION: The proposal is focused on the mechanisms of protease control by serine protease inhibitors (SERPINS). Two different model SERPINs will be employed to investigate the mechanism underlying SERPIN activation by heparin, and the mechanism by which SERPIN: Protease complexes are bound, internalized, and processed by cells. The first long range goal is to gain insight into the molecular basis of antithrombotic therapy. The thrombin inhibitory activity of ATIII is accelerated 1,000 to 10,000 fold by heparin, but the structural and molecular basis underlying this important clinical application of heparin and related compounds, remains to be elucidated. This level of understanding is requisite to the development of alternatives to heparin therapy. This will be approached by a combination of site-directed mutagenesis within the identified heparin binding region of ATIII, followed by a quantitative biochemical assessment of the effects of these changes on heparin activation in the engineered proteins. These studies will be extended to characterize the interactions of these variants with vascular endothelial cells which represent the physiological source of heparin for ATIII-thrombin interactions. In addition, SERPIN levels are regulated by the internalization and processing of SERPIN: Protease complexes by specific cell types. This aspect of the present studies will focus on a combined biochemical and site directed-mutagenesis analysis of PN1 to probe the mechanism of SERPIN:Protease binding to cells, and the mechanism that mediates their internalization, which is requisite to processing. Based on previously obtained data, genetically engineered variants of PN1 and biologically active synthetic peptides will be used to probe the molecules responsible for the entry of Th:PN1 complexes into the cells that process them.