STRUCTURAL STUDIES ON PROTEINS INVOLVED IN HEMOSTASIS

止血相关蛋白质的结构研究

• 批准号: 3282599
• 负责人: Brian Francis Edwards
• 金额: $ 13.81万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-02-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282599
• 关键词: X ray crystallography; affinity chromatography; chymotrypsin; cow; crystallization; hemostasis; hirudins; platelet factor 4; protein structure; thrombin

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.

止血是血管壁之间微妙的相互作用， 血小板和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，增加前列环素的输出 抑制纤溶酶原激活剂；它具有促有丝分裂作用 易感细胞，它通过酶促激活因子 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 该项目的目的是合理化凝血酶的多种功能 确定其三维 X 射线晶体结构。 我们有 结晶牛 α-凝血酶，其 X 射线衍射至 至少 3.5 埃的分辨率，以及 PMSF 抑制的凝血酶，其形式为 衍射分辨率至少为 2.5 埃。 我们的具体目标是 该提案的三年是实现我们公开目标的里程碑： 1. 我们现在将收集晶型的 X 射线衍射数据 通过衍射仪和振荡达到其分辨率的极限 方法。 2、尝试用分子解决相位问题的捷径 利用凝血酶核心与凝血酶核心之间的强同源性进行替换 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 衍射数据，并通过多重同晶解决相位问题 替代品。 4. 我们将继续努力改进凝血酶晶体 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 凝血酶和血小板因子 4 的抑制剂，参与 像凝血酶一样止血并结合肝素。