Lipid Regulation of Thrombin Generation

凝血酶生成的脂质调节

• 批准号: 6826058
• 负责人: Barry R Lentz
• 金额: $ 38.18万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6826058
• 关键词: active sites; blood coagulation; chemical kinetics; coagulation factor V; coagulation factor X; dimer; electron microscopy; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; fluorescent dye /probe; membrane; membrane structure; microscopy; molecular assembly /self assembly; phosphatidylserines; platelets; prothrombin; thermodynamics; thrombin; vesicle /vacuole

DESCRIPTION (provided by applicant): Prothrombin activation is a key reaction of blood coagulation. The enzyme (factor Xa), the cofactor (factor Va), and substrate of this reaction all bind to membranes to accelerate prothrombin activation by 150,000-fold. The product of prothrombin activation, thrombin, is the central enzyme of blood coagulation. Failure to regulate its production and inactivation can lead to stroke and heart attack. Small membrane vesicles required for this reaction are released from activated platelets and contain a negatively charged phospholipid, phosphatidylserine (PS). PS appears on the surface of platelet membranes only when platelets are activated. All three components of prothrombin activation bind to these negatively charged membranes. Popular opinion is that the main role of platelet vesicles is to bring cofactor, enzyme and substrate together onto a two-dimensional vesicle surface where reaction will be much faster than in a three-dimensional solution (plasma). Our work suggests a more central role for platelet membranes. We have shown that a soluble form of PS (C6PS) binds to regulatory sites on Xa and Va. C6PS induces structure changes, activity enhancement, and formation of Xa-Xa dimers in solution. Most remarkably, factor Va binds to Xa in the presence of C6PS to form fully functional "prothrombinase" complex in solution, without a surface of any kind. Because of these observations, we believe that exposure of PS on platelet vesicles regulates part of the blood coagulation process. Another key regulatory event is release of factor Va from platelets. Our hypothesis is that Xa formed in plasma occurs as an inactive dimer on a platelet vesicle until factor Va, under the influence of PS, binds to Xa to form the active prothrombinase. Aim 1 tests key elements of this hypothesis in solution, where these are easier to test than on a membrane surface. In this Aim, we use C6PS to replace membranes. However, our ultimate goal is to understand how the complex assembles and is regulated on a membrane. Because assembly in vivo takes place on discrete vesicles and because Xa bound to these vesicles likely forms dimers, this process is complex. Dealing with these complexities is the subject of Aim 4. Despite its importance, we know almost nothing about the structure of the prothrombinase complex. This is because it assembles on membranes, and crystals of membrane proteins or their complexes are difficult to obtain. Aim 3 describes plans to obtain a medium-resolution structure of the complex by analysis of electron micrograph images of individual complexes assembled by C6PS. While we know a great deal about the regulation of Xa by PS, we know little in the case of Va save that it binds four molecules of C6PS. We have located one C6PS site that contributes strongly to membrane binding. Where are other sites? Do they contribute to membrane binding and regulation of Va? How does PS bind to these sites? Does binding contribute to interactions between structural domains within Va, or to interactions between Va and Xa or substrate? These questions are addressed in Aim 2.

描述（申请人提供）：凝血酶原激活是血液凝固的关键反应。该反应的酶（因子 Xa）、辅因子（因子 Va）和底物均与膜结合，将凝血酶原激活加速 150,000 倍。凝血酶原激活的产物凝血酶是血液凝固的核心酶。如果不能调节其产生和失活，可能会导致中风和心脏病发作。该反应所需的小膜囊泡从活化的血小板中释放出来，并含有带负电荷的磷脂、磷脂酰丝氨酸（PS）。仅当血小板被激活时，PS才会出现在血小板膜表面。凝血酶原激活的所有三个成分都与这些带负电荷的膜结合。流行的观点是，血小板囊泡的主要作用是将辅因子、酶和底物聚集到二维囊泡表面上，其中反应将比三维溶液（血浆）中快得多。我们的工作表明血小板膜具有更重要的作用。 我们已经证明，可溶形式的 PS (C6PS) 与 Xa 和 Va 上的调节位点结合。C6PS 在溶液中诱导结构变化、活性增强和 Xa-Xa 二聚体的形成。最值得注意的是，在 C6PS 存在的情况下，因子 Va 与 Xa 结合，在溶液中形成功能齐全的“凝血酶原”复合物，而无需任何类型的表面。由于这些观察结果，我们相信血小板囊泡上的 PS 的暴露调节了部分凝血过程。另一个关键的调控事件是血小板释放因子 Va。我们的假设是，血浆中形成的 Xa 作为血小板囊泡上的无活性二聚体出现，直到因子 Va 在 PS 的影响下与 Xa 结合形成活性凝血酶原酶。目标 1 在溶液中测试该假设的关键要素，这些要素比在膜表面上更容易测试。在这个目标中，我们使用C6PS来代替膜。然而，我们的最终目标是了解复合物如何在膜上组装和调​​节。因为体内组装发生在离散的囊泡上，并且因为与这些囊泡结合的 Xa 可能形成二聚体，所以这个过程很复杂。处理这些复杂性是目标 4 的主题。 尽管它很重要，但我们对凝血酶原酶复合物的结构几乎一无所知。这是因为它在膜上组装，而膜蛋白或其复合物的晶体很难获得。目标 3 描述了通过分析 C6PS 组装的各个复合物的电子显微图像来获得复合物的中等分辨率结构的计划。 虽然我们对 PS 对 Xa 的调节了解很多，但我们对 Va 知之甚少，只知道它与四个 C6PS 分子结合。我们找到了一个对膜结合有强烈贡献的 C6PS 位点。其他网站在哪里？它们是否有助于膜结合和 Va 调节？ PS 如何绑定到这些网站？结合是否有助于 Va 内结构域之间的相互作用，或 Va 与 Xa 或底物之间的相互作用？这些问题在目标 2 中得到解决。