The role of metal ions in blood coagulation has been a continuing interest of this laboratory. We have employed trivalent lanthanide ions as substitutes for Ca (II) to explore the role of metal ions in complex formation and zymogen activation during conversion of factor X to factor Xa by the coagulant protein of Russell's viper venom and the conversion of prothrombin to thrombin by factor Xa. Similar studies employing lanthanide ions will be carried out on factor Xa, factor IX and factor IXa, abnormal prothrombin, factor VIII and factor V. Where appropriate, affinity chromatographic methods of purification will be explored for these proteins using lanthanide ions to facilitate non-productive enzyme-substrate complex formation. Calcium (II) ions play an important role in many phases of blood coagulation from platelet aggregation to the cross-linking of the fibrin clot. This and the observation that activation of prothrombin by activated factor X does not obey classical Michaelis-Menten kinetics have led us to propose a possible regulatory role for Ca (II) ions in the process. This hypothesis will be tested experimentally. Although the cascade model of coagulation has been studied in chemically defined systems, these processes have not been studied in their normal milieu-plasma. Using purified radiolabelled human coagulation proteins added back to human plasma we will investigate pathways of the cascade and kinetic features for the reactions involved in the soluble phase of coagulation to determine order of activation, possible feedback processes, effects of Ca (II) concentration, possible alternate pathways of coagulation in congenitally deficient plasma, and kinetic models of the cascade. Finally, a group of derivatized benzamidines will be used to explore the chemical environment of the active site of factor Xa, the distance between the active site and the metal-binding sites of factor Xa, structural changes in factor X during zymogen activation and the varying features of the active sites of the active forms of the vitamin K-dependent proteins.