Lipid Regulation of Thrombin Generation

描述（由申请人提供）：凝血酶是血液凝固的中心调节分子，由凝血酶原（II）蛋白水解血小板膜结合酶（Xa因子）及其相关辅助因子（Va因子）产生。该膜将II的激活速度提高了105倍。活化后，血小板表面释放出含有磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）的囊泡。与广泛的帮助范例相反，我们的工作表明，PS分子，而不是膜表面，触发了这种显著的加速。研究人员一直没有发现这一点，因为PS位于膜上，很难研究膜上的蛋白质结构和相互作用。为了证明这一点，我们使用了短链可溶性形式的PS (C6PS)，它不会在体内发生，但它是揭示被膜掩盖的作用的宝贵工具。一旦发现，这些效应可以通过仔细的膜实验来证实。我们现在问三个问题来扩展我们对人类血液凝固的脂质调节的理解。PS是否在其他涉及凝血的关键蛋白水解反应中发挥类似的调节作用？因子IXa及其辅因子VIIa和因子VIIa及其辅因子组织因子激活因子X到Xa。因子IXa和VIIa与Xa结构相似，辅因子VIIa与Va结构相似，初步结果表明IXa和VIIa对C6PS均有响应。活化蛋白C （Activated Protein C， APC）是一种关键的下调酶，对ps膜敏感，在初步研究中也对C6PS敏感。我们假设PS对IXa、VIIa和APC的调节作用与Xa一样，对VIIa的调节作用与Va一样。如果我们的假设得到证实，这将意味着PS暴露于活化的血小板膜上是开启凝血放大阶段的关键调节事件。膜中的PE也影响ii活化复合物的活性。我们问怎么做？最近的研究结果表明可溶性C6PE调节Xa和Va。我们假设分子PE在II激活中具有类似于PS的调节作用，并且通过PE调节位点连接到PS位点来实现这一目标。PS结合到膜附近的调控域影响远离膜的事件的结构机制是什么？我们对Xa-Va配合物或Xa和Va结合到PS时的原子结构的信息有限。我们已经交联了一个由C6PS组装的Xa-Va配合物，并将确定它是否适合结晶试验。我们还将使用突变FRET标记，以及交联和修饰蛋白的质谱分析来提供实验约束，以改进现有的Xa和Va模型，并验证我们的假设，即PS结合产生内部结构域重排，为PS调控提供结构基础。

DESCRIPTION (provided by applicant): Thrombin, the central regulatory molecule of blood coagulation, is produced by prothrombin (II) proteolysis by a platelet-membrane-bound enzyme (factor Xa) and its associated cofactor (factor Va). The membrane accelerates II activation by 105-fold. Upon activation, platelets give off vesicles with phosphatidylserine (PS) and phosphatidylethanolamine (PE) on their surface. Contrary to a widely help paradigm, our work shows that PS molecules, not a membrane surface, trigger this remarkable acceleration. This has eluded researchers because PS is located in membranes where protein structure and interactions are very difficult to study. To show this, we used a short-chain soluble form of PS (C6PS), which does not occur in vivo, but is an invaluable tool for revealing effects that are masked by membranes. Once revealed, these effects can be confirmed by careful experiments with membranes. We now ask three questions to extend our understanding of lipid regulation of human blood coagulation. Does PS plays a similar regulatory role in other key proteolytic reactions involved in blood coagulation? Factor IXa with its cofactor VIIIa and factor VIIa with its cofactor tissue factor activate factor X to Xa. Factors IXa and VIIa are structurally similar to Xa, as is cofactor VIIIa to Va. Preliminary results show that IXa and VIIa both respond to C6PS. Activated Protein C (APC) is a key down-regulating enzyme that is sensitive to PS-membranes and, in preliminary studies, to C6PS as well. We hypothesize that PS regulates IXa, VIIa, and APC as it does Xa, and regulates VIIIa as it does Va. If we confirm our hypotheses, it will mean that exposure of PS on activated platelet membranes is the key regulatory event that turns on the amplification stage of blood coagulation. PE in membranes also influences the activity of the II-activating complex. We ask how? Recent results show soluble C6PE regulates Xa and Va. We hypothesize that molecular PE has a regulatory role similar to that of PS in II activation and it accomplishes this though PE regulatory sites linked to PS sites. What are the structural mechanisms by which PS binding to regulatory domains near the membrane influence events far removed from the membrane? We have limited information about the atomic structure of the Xa-Va complex or of Xa and Va when bound to PS. We have cross-linked a Xa-Va complex assembled by C6PS and will determine if it would be appropriate for crystallization trials. We will also use mutational FRET labeling, and mass spectroscopic analysis of cross-linked and modified proteins to provide experimental constraints to improve existing models for Xa and Va, and test our hypothesis that PS binding produces internal domain rearrangements that provide the structura basis of PS regulation.