Tissue factor, a protein found on the surfaces of certain cell types, is the primary triggering agent of blood clotting. Its ligand, factor VII/VIIa, is a serine protease that can exist as an inert zymogen (factor VII) or active enzyme (factor VIIa), both of which circulate in the plasma. Assembly of tissue factor/factor VIIa complexes on cell surfaces is a critical step in controlling the clotting cascade both in hemostasis and in thrombosis. Thrombotic diseases (heart attack, thrombotic stroke, and so forth) constitute the major cause of premature death and disability in the United States and other industrialized countries. Accordingly, a long-term goal of this laboratory is to understand how the tissue factor/factor VIIa system functions and how it is regulated. This project focuses on how tissue factor allosterically activates factor VIIa and positions it for optimal attack on membrane-bound protein substrates.
The specific aims of this proposal are to: (1) Elucidate the role of substrate binding site(s) on tissue factor in recognition of macromolecular substrates; (2) Investigate the role of the 4-carboxyglutamate-rich domains of factors VII, IX, and X in the activity of the tissue factor-factor VIIa complex; (3) Investigate the function of membrane- interactive regions of tissue factor and map the topography of the tissue factor-factor VIIa-phospholipid complex; and (4) Probe the molecular basis for substrate and inhibitor specificity of factor VIIa. These studies will employ site-directed mutagenesis and domain-swapping approaches, and will analyze the functional consequences of these alterations using enzyme kinetics and measurements of protein-protein and protein-phospholipid interactions. Achieving the goals of this project will provide detailed knowledge of the assembly and function of critical components of the initiator complex of blood coagulation. Such knowledge will ultimately be useful in designing and applying novel antithrombotic agents targeted at the initiation phase of the clotting system.
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