This project takes advantage of new understanding about membrane association by vitamin K-dependent proteins. Key proteins of the coagulation cascade (factors VII and IX) display low affinity for membranes. This affinity can be increased to that of high affinity vitamin K-dependent proteins by site directed mutations. In vitro assays show that these changes have a large impact on activity of factor VII and VIIa in vitro, using assays that mimic biological conditions. Enhancement is realized in both tissue factor dependent as well as tissue factor- independent systems.
Six specific aims i nclude: 1. Identify the site- directed mutant forms of human factor VIIa that have the highest activity, in vitro. Express these proteins in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in the hemophilic mouse, that provides the ability to test efficacy of factor VIIa mutants. 3. Test Efficacy of human factor VIIa mutants in the mouse model, using mutants identified in specific aim 1 and methods identified in specific aim 2. 4. Express site-directed mutants of mouse factor VIIa which correlate with the best human factor VIIa mutations. Test these in the mouse by procedures developed for the human protein molecules described in specific aims 2-3. 5. Test in vitro anti-coagulant properties of human active site-blocked factor VIIa molecules described in specific aim 1, to determine the best mutants to be tested in the rabbit model. 6. Obtain preliminary evidence for potential uses of other vitamin K-dependent proteins which have mutations in the Gla domain and increased affinity for membranes. These may include especially factors IX or X. The project uses site-directed protein mutation, protein expression, purification, and characterization by protein chemistry and numerous coagulation tests. In vivo testing will be carried out in mice and rabbits.
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