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.
| Galis, Z S; Kranzhofer, R; Fenton 2nd, J W et al. (1997) Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 17:483-9 |
| Witting, J I; Brezniak, D V; Andersen, T T et al. (1997) Thrombin affinity mapping of its receptor tethered ligand. Blood Coagul Fibrinolysis 8:65-9 |
| Johnson, P C; Heil, B V; Parva, B et al. (1996) Thrombin activity on dissected and anastomosed human arteries. J Surg Res 60:193-8 |
| Armstrong, M T; Fenton 2nd, J W; Andersen, T T et al. (1996) Thrombin stimulation of matrix fibronectin. J Cell Physiol 166:112-20 |
| Schini-Kerth, V B; Fisslthaler, B; Andersen, T T et al. (1995) Thrombin prevents the expression of inducible nitric oxide synthase in vascular smooth muscle cells by a proteolytically-activated thrombin receptor. Thromb Haemost 74:980-6 |
| Seiler, S M; Peluso, M; Michel, I M et al. (1995) Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist. Biochem Pharmacol 49:519-28 |
| Brezniak, D V; Moon, D G; Beaver, J A et al. (1994) Haemoglobin inhibition of fibrin polymerization and clotting. Blood Coagul Fibrinolysis 5:139-43 |
| Lum, H; Andersen, T T; Fenton 2nd, J W et al. (1994) Thrombin receptor activation peptide induces pulmonary vasoconstriction. Am J Physiol 266:C448-54 |
| Liu, L; Freedman, J; Hornstein, A et al. (1994) Thrombin binding to platelets and their activation in plasma. Br J Haematol 88:592-600 |
| Rydel, T J; Yin, M; Padmanabhan, K P et al. (1994) Crystallographic structure of human gamma-thrombin. J Biol Chem 269:22000-6 |
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