application) There have been many advances in understanding structure/function relationships in vitamin K-dependent proteins including the Gla domains in the ten years since the last renewal of this application. The current frontier in understanding blood coagulation on a molecular basis is determining how the functional macromolecular complexes which facilitate blood coagulation assemble. This application explores the phospholipid binding specificity of prothrombin and the nature of the prothrombin-membrane binding site. In addition, studies are proposed to determine sites of interaction between factor Va and factor Xa and between factor Va and prothrombin. A long-term goal is to determine the structure of the factor Va heavy chain-prothrombin complex. Elucidating the determinants of prothrombinase complex assembly may provide insights into methods of regulating complex formation in pathologic states such as thrombosis. We will determine the potential role of phosphatidylethanolamine in prothrombin activation by prothrombinase. We will also use soluble phospholipids and phospholipid head group analogs to determine the important structural determinants on the phospholipid that mediate prothrombin binding. We will establish the affinity and stoichiometry of soluble phospholipid binding to prothrombin. Data from these studies will assist in design of X-ray crystallographic experiments to determine the mode of interaction of prothrombin fragment 1 with phospholipid. We will proceed step-wise, first determining the structure of the Gla domain of metal free prothrombin fragment 1 at high resolution. Using cryocrystallography we believe we will be able to determine the structure of most of the Gla domain of apo-fragment 1. This portion of the apo-fragment 1 structure has not been previously defined. Next we will determine the structure of prothrombin fragment 1 bound to magnesium ions. Finally we will determine the structure of prothrombin fragment 1 bound to calcium ions and glycerophosphoserine and/or a short chain phospholipid bearing the phosphoserine head group. To localize interaction sites among enzyme, cofactor and substrate in the prothrombinase complex we will use two novel cross-linking strategies. We will determine if the Gla domains of factor X and prothrombin or the EGF domains of factor X interact with factor Va using synthetic peptides with benzoylphenylalanine placed at different positions within the peptide. To determine if the factor X Gla-EGF 1 module or the prothrombin kringle domains interact with factor Va we will use a fibronectin derived transamidation site in conjunction with factor XIIIa. A long-term goal of these studies is to determine, by X-ray crystallography, the structure of factor Va heavy chain-prothrombin fragment 1-calcium complex.
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