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，由血小板膜结合酶（因子 Xa）及其相关辅因子（因子 Va）通过凝血酶原（II）蛋白水解产生。该膜可将 II 的激活加速 105 倍。激活后，血小板会释放出表面含有磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）的囊泡。与广泛支持的范例相反，我们的工作表明，PS 分子，而不是膜表面，引发了这种显着的加速。这一直困扰着研究人员，因为 PS 位于膜中，蛋白质结构和相互作用很难研究。为了证明这一点，我们使用了短链可溶形式的 PS (C6PS)，它不会在体内发生，但却是揭示被膜掩盖的效应的宝贵工具。一旦揭示出来，这些效应就可以通过仔细的膜实验来证实。我们现在提出三个问题来扩展我们对人体血液凝固的脂质调节的理解。 PS 在涉及凝血的其他关键蛋白水解反应中是否也发挥类似的调节作用？因子 IXa 及其辅因子 VIIIa 和因子 VIIa 及其辅因子组织因子将因子 X 激活为 Xa。因子 IXa 和 VIIa 在结构上与 Xa 相似，辅因子 VIIIa 与 Va 也相似。初步结果表明，IXa 和 VIIa 均对 C6PS 有反应。活化蛋白 C (APC) 是一种关键的下调酶，对 PS 膜敏感，在初步研究中，它对 C6PS 也敏感。我们假设PS像调节Xa一样调节IXa、VIIa和APC，像调节Va一样调节VIIIa。如果我们证实我们的假设，则意味着PS暴露在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 也会影响 II 激活复合物的活性。我们问如何？最近的结果表明可溶性 C6PE 调节 X​​a 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，并且它通过与 PS 位点相连的 PE 调节位点来实现这一点。 PS 与膜附近的调节域结合影响远离膜的事件的结构机制是什么？我们对 Xa-Va 复合物或 Xa 和 Va 与 PS 结合时的原子结构的信息有限。我们已经交联了由 C6PS 组装的 Xa-Va 复合物，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 Xa 和 Va 的现有模型，并测试我们的假设，即 PS 结合产生内部结构域重排，从而为 PS 调节提供结构基础。

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.