STRUCTURAL STUDIES ON PROTEINS INVOLVED IN HEMOSTASIS

止血是血管壁之间微妙的相互作用， 血小板，以及将血液保留在体内的凝血蛋白 循环系统。 凝血酶是止血的主要调节剂：它激活血小板；它 与内皮细胞结合，增加其前列环素和 抑制其纤溶酶原激活物；它对 敏感细胞，它在酶作用下激活因子V、VIII和XIII， 它将纤维蛋白原转化为纤维蛋白，形成凝块。主要目标 这个项目的目的是通过以下方式使凝血酶的多种功能合理化 确定了它的三维X射线晶体结构。我们有 结晶的牛α-凝血酶的形式使X射线衍射到At 分辨率至少为3.5埃，并且PMSF抑制的凝血酶的形式 衍射分辨率至少达到2.5埃。我们的具体目标是 这项提议的三年是我们实现既定目标的里程碑： 1.我们现在将收集晶体形态的X射线衍射数据，地址为 通过衍射仪和振荡器达到其分辨率的极限 方法：研究方法。 2.我们将尝试用分子来解决相问题的捷径 利用凝血酶核心和凝血酶核心之间的高度同源性进行置换 凝乳酶。 3.我们将找到这两种形式的重原子衍生物，收集它们的 衍射数据，并用多重同构解决了位相问题 替补。 4.我们将继续努力通过以下途径改善凝血酶晶体 酶的超纯和非共价抑制剂的使用。 我们还计划启动水飞蓟素的结构研究，水飞蓟素是一种蛋白质 凝血酶抑制物和血小板因子4，参与 止血和凝血酶一样结合肝素。

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.