. The protein thrombin initiates blood clotting by cleaving a set of circulating proteins (fibrinogen and Factors V, VIII and XIII) and activating platelets. Its activity must be controlled to prevent catalytic amounts of thrombin from leading to a rampant, systemic thrombotic state. The chief control step occurs when thrombin binds to thrombomodulin (TM), a transmembrane, endothelial cell protein of the blood vessel wall. On binding to thrombomodulin, thrombin loses all procoagulant activities and acquires up to a 20,000-fold increased activity toward protein C (an anticoagulant when activated), while it maintains activity toward endogenous inhibitors. The long term aims of this research are to elucidate the structural features of thrombin that enable it to react with this diverse array of macromolecular substrates, and hopefully, from this knowledge, to understand how these remarkable changes in thrombin specificity arise. The knowledge is crucial in understanding the role of thrombin in the pathogenesis of cardiovascular diseases, which collectively are the largest leading cause of death in this country. The approach used will be to raise polyclonal and cloned Fab antibody fragments to synthetic peptides derived from the 3D structure of thrombin. The peptides to be synthesized are insertion sequence in thrombin, compared to the homologous proteases trypsin and chymotrypsin. The insertions sequences form loops on the surface of thrombin and are likely candidates for binding sites on thrombin that determine and restrict the macromolecular substrate specificity of the enzyme. The epitope mapping will also be extended to the production of cloned Fab fragments recognizing protein C, and thrombomodulin, to determine features of these proteins required for their mutual interactions in the protein C activation complex. Additionally, the Fab fragments will be screened to determine if any possess protein C cofactor activity. Both kinetic measurements of binding (use of the antibodies as inhibitors of thrombin and thrombin-thrombomodulin activities) and direct binding measurements (using ELISA systems) will be performed. Molecular graphics depictions of thrombin will be utilized as a guide for the national selection of peptides for synthesis.
