Lipid Regulation of Thrombin Generation

说明(申请人提供)：凝血酶原激活是凝血的关键反应。该反应的酶(凝血因子Xa)、辅因子(凝血因子Va)和底物都与膜结合，将凝血酶原激活速度提高15万倍。凝血酶原激活的产物凝血酶是凝血的中心酶。不能调节它的产生和失活可能会导致中风和心脏病发作。这一反应所需的小的膜泡是从激活的血小板中释放出来的，并含有带负电荷的磷脂，磷脂酰丝氨酸(PS)。只有当血小板被激活时，PS才会出现在血小板膜表面。凝血酶原激活的所有三种成分都与这些带负电的膜结合。流行的观点认为，血小板囊泡的主要作用是将辅因子、酶和底物结合到二维囊泡表面，在那里反应速度比在三维溶液(血浆)中快得多。我们的工作表明，血小板膜发挥了更核心的作用。 我们已经证明了PS(C6PS)的可溶性形式与Xa和Va上的调节位点结合。C6PS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，在C6PS存在下，凝血因子Va与Xa结合，在溶液中形成全功能的“凝血酶原酶”复合体，没有任何形式的表面。由于这些观察，我们认为暴露在血小板囊泡上的PS调节部分凝血过程。另一个关键的调节事件是从血小板释放Va因子。我们的假设是，在血浆中形成的Xa以非活性二聚体形式存在于血小板囊泡上，直到Va因子在PS的影响下与Xa结合形成活性的凝血酶原酶。AIM 1在溶液中测试这一假设的关键元素，在溶液中测试这些元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体是如何在膜上组装和调节的。由于体内组装是在离散的囊泡上进行的，而且由于Xa与这些囊泡结合可能形成二聚体，这一过程是复杂的。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体的结构几乎一无所知。这是因为它聚集在膜上，膜蛋白或其复合体的晶体很难获得。目的3描述了通过分析由C6PS组装的单个络合物的电子显微图像来获得该络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的了解很少，除了它与C6PS的四个分子结合外。我们已经定位了一个对膜结合有很大贡献的C6PS位点。其他网站在哪里？它们对膜结合和调节Va有贡献吗？PS如何绑定到这些站点？结合是否有助于Va内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？这些问题在目标2中得到了解决。

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.