ROLE OF Y-CARBOXYGLUTAMIC ACID IN BLOOD COAGULATION

The calcium ion binding and phospholipid binding behavior of prothrombin necessary for optimal rates of thrombin production in the blood coagulation process depend on the presence of gamma-carboxyglutamic acid (Gla) residues. This vitamin K-dependent amino acid is located at ten positions within the first 33 residues of prothromibin and at similar positions in the other vitamin K-dependent coagulation proteins factors VII, IX, X and Protein C. A related gamma-carboxyglutamix-containing region also occurs in bone proteins. We suggest that the 1-40 region of prothrombin, containing the 18-23 cystine loop, is critical to the functional behavior of prothrombin as well as related structures of the other vitamin K-dependent coagulation factors and perhaps other proteins, such as the bone protein, containing Gla and a similar cystine loop. We intend to prepare segments of the 1-40 region by chemical synthesis; we will also utilize the peptides representing the 1-39 and 12-44 sequences obtained from prothrombin. We intend to establish; (a) the chemical structure of the smallest unit of the amino terminal region of prothrombin that will exhibit the metal ion-induced characteristics of the protein; (b) the role of the cystine loop; (c) the nature of the ligand system in the Gla region that is required for metal ion binding; and, (d) the minimum number and location of Gla residues in the 1-33 sequence necessary for prothrombin-phospholipid interaction. Experimental techniques that will be employed to study the chemical and physical properties of these synthetic and natural peptides include nuclear magnetic resonance, laser Raman and Emu3+ luminescence spectroscopy; metal ion binding; interaction with antibodies specific to the calcium ion-induced conformation; and peptide; solvent and peptide:lipid partition experiments. A method for chemical modification of Gla residues is also proposed.