This research has three general objectives that are related to understanding the physical and kinetic properties of assembling the blood coagulation cascade. The first is to understand the function of the vitamin K-dependent amino acid, Gamma-carboxyglutamic acid in calcium binding and protein-membrane association. The protein structures that are involved in, and change as a result of, calcium ion binding will be investigated by use of nmr and lanthanide substitution experiments utilizing the vitamin K-dependent, membrane-binding portion of prothrombin. The lanthanide experiments will also make extensive use of fluorescence techniques. The structure at the protein-membrane interface will be studied by measuring dynamic and equilibrium properties of protein-membrane association and by substitution of lanthanides for calcium and determining various distances and changes in the complex detected by fluorescence techniques. The second major area is the structure and function of membrane complexes containing more than one protein. The initial hypothesis is that the membrane-binding site of the vitamin K-dependent proteins functions as a portion of an overall binding interaction. That is, these proteins normally bind simultaneously to a membrane plus at least one other membrane-bound protein. Specific protein-protein complexes on the membrane surface to be studied include: protein S with protein C(a) and with complement C4-binding protein. Other examples include protein C(a) with factor Va, factor VIIIa or thrombomodulin. Equilibrium, dynamic and hydrodynamic aspects of these interactions will be measured to determine if the proteins associate with each other while they are membrane bound. Light scattering, quasielastic light scattering and various fluorescence techniques will be used. Protein binding to phospholipid monolayers will also be used to document the properties of the protein-lipid associations. The third area of study involves proteins of the contract phase of coagulation. At least some of these proteins bind to lipid membranes and undergo activation. The various properties of the protein-lipid associations describe above will be measured to help understand the physical forces involved in binding. Analysis of the kinetics of factor activation will be correlated with protein-lipid binding to try to understand how the surface influences this kinetic event. These studies should increase our knowledge of protein-membrane interactions in general and how such interactions are specifically responsible for enhancing the rates of the blood coagulation process.
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