Lipid Regulation of Thrombin Generation

凝血酶生成的脂质调节

• 批准号: 7079288
• 负责人: Barry R Lentz
• 金额: $ 32.08万
• 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/7079288
• 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（由申请人提供）：凝血酶原激活是血液凝血的关键反应。该反应的酶，辅因子（因子VA）和底物均与膜结合，以使凝血酶原激活加速150,000倍。凝血酶原活化的产物凝血酶是血液凝结的中心酶。无法调节其生产和失活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，并包含带负电荷的磷脂磷脂酰丝氨酸（PS）。仅当血小板激活时，PS才会出现在血小板膜的表面上。凝血酶原激活的所有三个成分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助因子，酶和底物一起放在二维囊泡表面上，在二维囊泡表面中，反应的速度比三维溶液（等离子体）快得多。我们的工作暗示了血小板膜的更中心作用。 我们已经表明，PS（C6P）的可溶形式与XA和VA。C6P上的调节位点结合，可诱导溶液中XA-XA二聚体的结构变化，活性增强和形成。最值得注意的是，VA因子在C6P的存在下与XA结合，在溶液中形成完全功能的“凝血酶酶”复合物，而没有任何形式的表面。由于这些观察结果，我们认为PS在血小板囊泡上的暴露会调节血液凝结过程的一部分。另一个关键的调节事件是从血小板中释放VA因子。我们的假设是，在血浆中形成的Xa作为血小板囊泡上的非活性二聚体发生，直到在PS的影响下VA与XA结合以形成活性凝血酶酶。 AIM 1测试该假设的关键要素，其中这些假设比在膜表面更容易测试。在此目标中，我们使用C6P代替膜。但是，我们的最终目标是了解复合物如何在膜上组装并受到调节。由于体内组装发生在离散囊泡上，并且由于Xa与这些囊泡结合，因此可能形成二聚体，因此此过程很复杂。处理这些复杂性是目标4的主题。 尽管它很重要，但我们几乎对凝血酶酶络合物的结构一无所知。这是因为它在膜上组装，很难获得膜蛋白或其复合物的晶体。 AIM 3描述了通过分析由C6P组装的单个复合物的电子显微照片图像来获得复合物的中分辨率结构的计划。 虽然我们对PS的XA调节非常了解，但在VA的情况下，我们几乎不知道它结合了C6P的四个分子。我们找到了一个C6PS位点，该位点对膜结合有很大贡献。其他网站在哪里？它们是否有助于VA的膜结合和调节？ PS如何与这些站点结合？结合是否有助于VA内的结构域之间的相互作用，或者有助于VA与XA或底物之间的相互作用？这些问题在AIM 2中解决。