This study will examine vitamin K-dependent proteins that display improved membrane affinity. The vitamin K-dependent plasma proteins function in both pro- and anti-coagulation pathways. Membrane contact is important for nearly all of the steps of these reactions. Calcium and membrane binding by vitamin K-dependent proteins depend on several gamma-carboxyglutamic acid (Gla) residues in amino acids 1-45 of these proteins. Gla is synthesized in the liver in a vitamin K-dependent reaction. Administration of vitamin K antagonists, which lower the level of Gla-containing proteins in the circulation, is a common biomedical application for persons subject to thrombosis. In addition, administration of intact proteins is important to treatment for some bleeding disorders. A major goal of this project is to establish the membrane contact mechanisms of the vitamin K-dependent proteins and to use that knowledge to generate proteins with improved function. This proposal presents a new hypothesis for membrane contact.
Seven specific aims i nclude: 1. Design mutants of protein C and factor VII that have improved membrane contact sites. 2. Determine the relationship between amino acids in the Gla domain and membrane binding affinity. 3. Characterize the relationship between membrane affinity and the ability of a zymogen protein to function as substrate for activation by other membrane-bound enzymes. 4. Determine the impact of membrane affinity (specific aim 1) on the enzyme activity of the activated forms of these proteins (factor VIIa and activated protein C, APC), including the impact of cofactor proteins, membrane content, and other properties. 5. Determine the mechanisms of membrane contact by vitamin K proteins. 6. Determine the role of the hydrophobic residues in the Gla domain. 7. Determine the structures responsible for greatest folding stability of the Gla domain. Methods used include expression of mutant proteins and their characterization by enzyme and coagulation assays, spectroscopic assays by fluorescence, and biophysical characterization with NMR and calorimetry. The use of carefully prepared membrane vesicles is required in most areas of this project. This study should reveal structure/function relationships of gamma-carboxyglutamic acid, and its ultimate function in creating a membrane-contact site. It will increase knowledge of the role that membranes play in enzymatic events of coagulation and in biological systems. It may ultimately provide new and/or improved materials for biomedical applications.
