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).

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Wadsworth Center
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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
Kimura, M; Nakamura, K; Fenton 2nd, J W et al. (1994) Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets. Am J Physiol 267:C1543-52
Gralnick, H R; Williams, S; McKeown, L P et al. (1994) High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains. Proc Natl Acad Sci U S A 91:6334-8
Salatti, J A; Fenton 2nd, J; Anton, P et al. (1994) alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood. Blood Coagul Fibrinolysis 5:561-6
Beecher, K L; Andersen, T T; Fenton 2nd, J W et al. (1994) Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture. J Neurosci Res 37:108-15

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