This proposal is based on the hypothesis that prothrombin and factor X (factor Xa) contain discrete functional domains which are involved in discrete portions of the expression of the biological activity of these complex proteins. It is proposed to identify these regions by the combined use of specific chemical modification and limited proteolysis. Prothrombin will be modified with 2-hydroxy-5-nitrobenzyl bromide or with tetranitromethane. The modified protein(s) will be analyzed with respect to the extent of modification and changes in functional properties. The two reagents selected can identify functional tryptophanyl and tyrosyl residues respectively and, in addition, provide the basis for the introduction of structural probes whose spectral and fluorescence properties can measure changes in the microenvironment around the modified residues. Limited proteolysis of the native prothrombin in the presence and absence of ligands such as divalent cations, phospholipid and other components of the prothrombinase complex will provide derivatives for comparison with native prothrombin. Similar studies are proposed for the elucidation of functional domains in factor X and factor Xa. The primary effort will be directed toward the study of factor Xa since our emphasis is directed toward the study of the prothrombinase complex. The proposed experiments will provide a molecular understanding of prothrombinase complex. Such understanding is critical to the elucidation of the mechanism of normal blood coagulation.