Thrombosis is a major cause of morbidity and mortality in the United States. Most patients with thrombotic disorders require long-term therapy with oral anticoagulants, which have numerous side effects and present clinical challenges in situations of surgery or other medical interventions. Targeting a drug to the vascular endothelium, which is a primary site of action for hemostatic proteins, could avoid these systemic side effects. The overall goal of this project is to create a new antithrombotic molecule that we have termed """"""""glycosylphosphatidylinositol(GPI)- anchored antithrombin"""""""" (AT-GPI). When expressed in cells, this antithrombotic protein should be tethered from the cell surface, concentrating it at the primary site of thrombotic activity. We propose to: i) Generate AT-GPI cDNA using standard molecular biology techniques; ii) Establish in vitro endothelial cell lines that express AT-GPI and assay this system for the antithrombotic activity of AT-GPI; iii) Use gene therapy technology to establish AT-GPI in an in vivo animal model and test for antithrombotic activity'; iv) Engineer the tissue factor promoter upstream of the AT-GPI cDNA and determine whether it can regulate AT-GPI expression in response to inflammatory signals. This project will provide the Principal Investigator with new training using the techniques of molecular biology, cell culture, adenoviral gene therapy and animal models to complement his earlier training in protein biochemistry. This training will increase the number of tools and techniques available to the Principal Investigator in his future study of the normal and pathologic biology of the vascular system and thereby greatly enhance his chances of success in pursuing a career in academic medicine. In addition, gene therapy using these types of proteins could be a powerful new approach in the treatment of thrombotic disorders and in the prevention of thrombus formation on highly susceptible tissues, such as atherosclerotic arteries and venous grafts.
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