HUMAN THROMBINS

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Thrombin has central bioregulatory functions in hemostasis and wound healing. It also is a major participant in cardiovascular diseases (e.g., coronary thrombosis, stroke, pulmonary embolism) and has been implicated in other disease processes (e.g., atherosclerosis, cancer, inflammatory responses). Unlike other serine proteinases, procoagulant alpha-thrombin is multi-functional possessing enzymic as well as nonenzymic (hormone-like) activities. Methods developed over 10 years ago by our laboratory for preparing human alpha-thrombin have enabled some 700 publications and the rapid exploration of the chemistry and biology of thrombin. Our research has focused primarily on structure-function relationships; we have identified 3 (possibly 5) structural regions accounting for thrombin specificity and biological functions. Our objectives for the next 5-year period are to: i) produce 5 to 10 g/year of human alpha-thrombin and other forms (e.g., DIP- alpha,gamma,epsilon-thrombins), ii) further topographical mapping of thrombin structural/functional regions by specific cleavages (e.g., xi-thrombin) and antibodies synthetic-peptides corresponding to such regions or protein domains, iii) use erecombinant hirudins for standardizing alpha-thrombin activities (no simple, absolute method to date) and assess binding requirements with synthetic and genetically engineered analogues, iv) develop biologically-active tagged thrombins (e.g., fluorescent alpha-thrombin, biotin conjugates) for biological probes, and V) continue major collaborative studies (e.g., x-ray crystallography of cell biology). Our overall approach is cost-effective science, working throughout many disciplines in order to maximize our contributions (e.g., 42 publications over the preceding 3-year period).