Hemostasis is the delicate interplay between the blood vessel walls, platelets, and the coagulation proteins that retains blood within the circulatory system. Thrombin is the prime regulator of hemostasis: it activates platelets; it binds to endothelial cells, increasing their output of prostacyclin and inhibiting their plasminogen activators; it has mitogenic effects on susceptible cells, it enzymatically activates factors V, VIII, and XIII, and it transforms fibrinogen to fibrin to form a clot. The major objective of this project is to rationalize the many diverse functions of thrombin by determining its three-dimensional x-ray crystal structure. We have crystallized bovine alpha-thrombin in a form that diffracts x-rays to at least 3.5 angstroms resolution, and PMSF-inhibited thrombin in a form that diffracts to at least 2.5 angstroms resolution.
Our specific aims for the three years of this proposal are mileposts towards our avowed goal: 1. We shall collect x-ray diffraction data from the crystal forms now at hand to the limit of their resolution by diffractometer and oscillation methods. 2. We shall attempt the shortcut of solving the phase problem by molecular replacement using the strong homology between the core of thrombin and that of chymotrypsin. 3. We shall find heavy atom derivatives of both forms, collect their diffraction data, and solve the phase problem by multiple isomorphous replacement. 4. We shall continue the efforts to improve the thrombin crystals through ultrapurification of the enzyme and the use of noncovalent inhibitors. We also plan to initiate structural studies on hirudin, which is a protein inhibitor of thrombin, and on platelet factor 4, which participates in hemostasis and binds heparin as thrombin does.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysics and Biophysical Chemistry B Study Section (BBCB)
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Wayne State University
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Martin, P D; Malkowski, M G; DiMaio, J et al. (1996) Bovine thrombin complexed with an uncleavable analog of residues 7-19 of fibrinogen A alpha: geometry of the catalytic triad and interactions of the P1', P2', and P3' substrate residues. Biochemistry 35:13030-9
Martin, P D; Eisele, K L; Doyle, M A et al. (1996) Molecular symmetry of the dodecamer subunit of Lumbricus terrestris hemoglobin. J Mol Biol 255:170-5
Malkowski, M G; Wu, J Y; Lazar, J B et al. (1995) The crystal structure of recombinant human neutrophil-activating peptide-2 (M6L) at 1.9-A resolution. J Biol Chem 270:7077-87
de Mel, V S; Doyle, M A; Gladysheva, T B et al. (1994) Crystallization and preliminary X-ray diffraction analysis of the ArsC protein from the Escherichia coli arsenical resistance plasmid, R773. J Mol Biol 242:701-2
de Mel, V S; Kamberov, E S; Martin, P D et al. (1994) Preliminary X-ray diffraction analysis of crystals of the PII protein from Escherichia coli. J Mol Biol 243:796-8
Zdanov, A; Wu, S; DiMaio, J et al. (1993) Crystal structure of the complex of human alpha-thrombin and nonhydrolyzable bifunctional inhibitors, hirutonin-2 and hirutonin-6. Proteins 17:252-65
Vitali, J; Martin, P D; Malkowski, M G et al. (1992) The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution. J Biol Chem 267:17670-8
Stuckey, J A; St Charles, R; Edwards, B F (1992) A model of the platelet factor 4 complex with heparin. Proteins 14:277-87
Martin, P D; Robertson, W; Turk, D et al. (1992) The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution. J Biol Chem 267:7911-20
Ni, F; Ripoll, D R; Martin, P D et al. (1992) Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin. Biochemistry 31:11551-7

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