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，以及将血液保持在血液中的凝血蛋白。 循环系统 凝血酶是止血的主要调节剂：它激活血小板， 与内皮细胞结合，增加前列环素的分泌， 抑制其纤溶酶原激活剂;它具有促有丝分裂作用， 敏感细胞，它酶促激活因子V，VIII和XIII， 将纤维蛋白原转化为纤维蛋白形成凝块 主要目的 这个项目的目的是合理化凝血酶的许多不同的功能， 确定其三维X射线晶体结构。 我们有 以衍射X射线的形式结晶的牛α-凝血酶， 至少3.5埃分辨率，和PMSF抑制凝血酶， 衍射到至少2.5埃的分辨率。 我们的具体目标是 这项建议的三年是实现我们公开宣布的目标的里程碑： 1. 我们将从现在的晶体形式中收集X射线衍射数据， 衍射仪和振荡器的分辨率达到极限 方法. 2. 我们将尝试用分子方法解决相位问题的捷径 使用凝血酶核心与 胰凝乳蛋白酶 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，并解决相位问题，由多个同构 更换. 4. 我们将继续努力改善凝血酶晶体， 酶的超纯化和非共价抑制剂的使用。 我们还计划启动水蛭素的结构研究，水蛭素是一种蛋白质， 凝血酶抑制剂，和血小板因子4，它参与 止血并像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.