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.
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