The event that triggers the blood clotting system in thrombotic disease and normal hemostasis is the assembly of the cell surface complex of tissue factor (an integral membrane protein) and factor VIIa (a plasma serine protease). The resulting tissue factor-factor VIIa-phospholipid complex activates factors IX and X by limited proteolysis. The long-term goals of these studies are to understand how the this two-subunit enzyme assembles on membrane surfaces, how tissue factor allosterically activates factor VIIa, and how the membrane participates in catalysis. This proposal will address the following three questions. (1) What governs the topography of the tissue factor-factor VIIa-phospholipid complex? Tissue factor not only allosterically activates factor VIIa, but is also thought to position and orient factor VIIa's active site (relative to the membrane surface) for optimum attack on the scissile bonds of membrane-bound substrates. How it does this is not well understood. This project will employ a number of approaches to understanding how the tissue factor-factor VIIa complex assembles and functions on membrane surfaces. (2) How does the putative macromolecular substrate-binding site on tissue factor contribute to catalysis? A region on tissue factor has been proposed to contribute to catalysis by providing an additional binding/recognition site for macromolecular substrates. This will be studied in detail in this proposal. (3) How do zinc ions inhibit factor VIIa? Zinc ions may be a physiological regulator of factor VIIa function, but the binding site on factor VIIa for zinc is not known, nor is its mechanism of action. This project will study the mechanism of action of zinc and determine the location of the zinc binding site on factor VIIa. These studies are designed to provide new insights in the process by which the blood clotting system is triggered in thrombotic disease, the major cause of disability and death in the United States.
Showing the most recent 10 out of 84 publications
