The antithrombin (AT)-anticoagulant heparan sulfate proteoglycan (HSPGact) interaction serves as the basis for a major anticoagulant mechanism of the blood vessel wall. However, the regulation of endothelial cell HSPGact generation and the importance of AT-HSPGact interactions in different organs remain to be defined. The investigator has cloned 3-0-sulfotransferase (3-0-ST) and provided evidence that this enzyme controls HSPGact generation. This proposal will use a combined biochemical/immunologic approach to determine whether a single 3-0-ST form exists in endothelial cells and whether endothelial cell HSPGact precursor is present in excess amounts. It will infect endothelial cells with retroviral vectors expressing 3-0-ST, document the elevated concentrations of enzyme, and quantitate the augmentation in HSPGact generation. Mutated 3-0STs will also be produced with greatly reduced biologic activity and the defects characterized by kinetic analyses. Other studies will determine the structures of HSPGact precursor and HSPGinact precursor which produce upon 3-0-sulfation either the AT binding site or a variant inactive sequence. This is designed to delineate early biochemical events in HSPGact generation. Gene targeting will be employed to create mice with 3-0-ST gene knockouts. If no viable homozygous mice are born, embryonic lethality will be bypassed by generating chimeric mice with localized absence of 3-0-ST in different vascular beds, producing mice with mutated enzyme possessing the greatest reduction in biologic activity compatible with survival, and creating mice with a homozygous endothelial cell specific knockout of the 3-0-ST gene with the Cre/Lox system. Genetically engineered mice will be examined in the ambient state, after a thrombogenic challenge, and in backgrounds with suppressed fibrinolytic activity. The study will include measurement of 3-0-ST and HSPGact, quantitation of thrombin generation, inhibition by activation peptide/enzyme immunocapture assays, and determination of thrombotic events by detection of crosslinked fibrin deposition, histologic examination, as well as evaluation of life span. These studies will establish the importance of AT - HSPGact interactions vis a vis other natural anticoagulant mechanisms in different vascular beds.
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