Human Alpha-thrombin is being produced (approximately 5g/yr) for detailed studies on the protein structure, enzyme specificity and biological functions of this central, bioregulatory serine protease in hemostasis. In addition to this form with high procoagulant and all other thrombin-ascribed activities, forms with minimal clotting yet high synthetic-substrate activities are being prepared by either controlled proteolytic cleavage (e.g., Gamma-thrombin) or selective chemical modification (e.g., nitrated-tyrosine; oxidized-methionine thrombins). Catalytically inactivated forms are also being made which retain the noncovalent-protein binding or recognition site (e.g., various affinity-labeled; active-site serine derivatized thrombins). These modified thrombins are being used for mapping active-site regions (e.g., within the 3-dimensional B-chain model) and bracketing the binding site for fibrin(ogen) recognition and most other biological activities (e.g., platelet, fibroblast receptors). Binding interactions are being examined by stationary phase partitioning with immobilized proteins (e.g., nonpolymerized fibrin binding of various thrombin forms). Sialic acid derivatization of the single carbohydrate moiety on the thrombin B chain does not affect fibrin(ogen) interactions (e.g., binding, clotting activity) and enables new approaches for labeling Alpha-thrombin (e.g., carbohydrate conjugated fluorescent, radioactive, biotin-derivative, or immunochemical groups) to follow it during clotting or receptor processing events (e.g., clot entrapped thrombin). Complexing of biotin or hapten conjugates with avidin or antibodies, respectively, not only provides interactive probes of thrombin functions but also approaches for purification (e.g., receptors). Enzyme-linked double-antibody immunoassays should allow monitoring of various functional activities (e.g., thrombin enhancement of Factor VIII Ag:C complexing), whereas stabilized control plasma should permit external standardization and redefinition of the thrombin clotting unit (e.g., percent plasma prothrombin). Such approaches are multi-purpose designed for our own in-house, various collaborative, and numerous noncollaborative projects (e.g., Protein Chemistry to Cell Biology; Clinical Hematology to Cancer Research), which utilize materials originating from our laboratory (est. greater than $1-million/yr of human thrombin). Our research will continue to focus mainly on the fibrin(ogen) recognition site of Alpha-thrombin.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL013160-15
Application #
3334576
Study Section
(EH)
Project Start
1978-07-01
Project End
1986-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
15
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204
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