The specific aim of this project is to identify within the amino acid sequence of the thrombin-like venom enzyme, crotalase, residues that are likely to be involved in its narrowly specific fibrinogen-clotting activity. Once such residues are identified, the same functional significance can be attributed to homologous residues within the thrombin molecule. This will require first that, using Edman degradation, we complete the determination of the amino acid sequences of crotalase (from the Eastern diamondback rattlesnake) and of an enzyme from the venom of the Western diamondback rattlesnake (WDR), which has a highly homologous partial amino acid sequence but which lacks thrombin-like activity. Then, the residues of potential functional significance will be narrowed down in three successively selective steps: (1) Compare the amino acid sequence of crotalase (thrombin-like) with the sequence of the highly homologous WDR enzyme which lacks thrombin-like activity, selecting the relatively few residues which differ as possible structural grounds for their difference in thrombin-like activity; (2) Compare the amino acid sequence of crotalase with thrombin, selecting from among the differences in step 1 the residues which are the same in crotalase and thrombin as possible structural grounds for their shared thrombin-like activity; (3) Locate within three-dimensional models generated by a comparative approach using interactive computer graphics the residues which survive selective steps 1 and 2, in order to select those residues whose chemical nature and spatial setting make them more likely contributors to the thrombin-like specificity of crotalase and thrombin. Residues so identified as potential determinants of the proteolytic specificity of thrombin for fibrinogen will thus become candidates for definitive testing of their functional significance (e.g. chemical or biological modification) in future experiments. This work is especially directed toward an understanding of the role of blood coagulation in thrombotic cardiovascular disease and toward intervention in this process using rationally designed synthetic inhibitors.
