Lipid Regulation of Thrombin Generation

描述（由申请人提供）：凝血酶原活化是血液凝固的关键反应。该反应的酶（Xa因子）、辅因子（Va因子）和底物都与膜结合，将凝血酶原的激活速度提高了15万倍。凝血酶原活化的产物，凝血酶，是血液凝固的中心酶。不能调节其产生和失活可导致中风和心脏病发作。这个反应所需的小膜泡从活化的血小板中释放出来，含有带负电荷的磷脂，磷脂酰丝氨酸（PS）。PS仅在血小板被激活时出现在血小板膜表面。凝血酶原活化的所有三种成分都与这些带负电荷的膜结合。普遍的观点是，血小板囊泡的主要作用是将辅助因子、酶和底物聚集到一个二维囊泡表面，在那里反应将比在三维溶液（等离子体）中快得多。我们的工作表明血小板膜具有更重要的作用。

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.