The activation of Factor X and prothrombin are key steps in the coagulation cascade. We have devised continuous spectrophotometric rate assays to investigate the enzymology and biochemical regulatory mechanisms of the activation of these coagulation factors. The steady-state kinetic parameters of the activation of Factor X by Factor IXa in the presence of one or more effectors (i.e. Factor VIII, phospholipid, Ca) will be determined. Similar studies will be performed for the activation of prothrombin by Factor Xa, Factor V, phospholipid and Ca. The activation of prothrombin and Factor X by various snake venom enzymes will also be studied. The reactions catalyzed by the snake venom enzymes will serve as models for studying the effect of each effector ligand in the absence of the others and will allow the differentiation between the effect of ligand binding to the enzymes (thrombin, Factor IXa or Factor Xa) and ligand binding to the substrate. The quantitative effect on the reaction rate, of varying each component, will be determined and the kinetic mechanism for the reactions will be deduced.