Lipid Regulation of Thrombin Generation

说明(申请人提供)：凝血酶是凝血的中央调节分子，由凝血酶原(II)由血小板膜结合酶(凝血因子Xa)及其相关辅因子(凝血因子Va)进行蛋白分解而产生。这种膜将II的激活速度提高了105倍。激活后，血小板表面会释放带有磷脂酰丝氨酸(PS)和磷脂酰乙醇胺(PE)的囊泡。与广泛帮助的范式相反，我们的工作表明，触发这种显著加速的是PS分子，而不是膜表面。由于PS位于膜中，蛋白质的结构和相互作用很难研究，所以研究人员一直未能做到这一点。为了证明这一点，我们使用了一种短链可溶形式的PS(C6PS)，这种形式在体内不存在，但对于揭示被膜掩盖的效应来说是一个无价的工具。一旦发现，这些效应可以通过仔细的膜实验来证实。我们现在提出三个问题，以扩大我们对人体血液凝固中脂质调节的理解。PS在其他与凝血有关的关键蛋白分解反应中是否起到类似的调节作用？因子IXa及其辅因子VIIIa和因子VIIa及其辅因子组织因子激活因子X至Xa。因子IXa和VIIa在结构上类似于Xa，辅因子VIIa到Va也是如此。初步结果表明，IXa和VIIa对C6PS均有反应。活化蛋白C(APC)是一种关键的下调酶，对PS膜敏感，在初步研究中也对C6PS敏感。我们假设PS像调控Xa一样调控IXa、VIIa和APC，像调控Va一样调控VIIa。如果我们证实我们的假设，这将意味着PS暴露在活化的血小板膜上是启动凝血放大阶段的关键调节事件。膜中的PE也影响II-激活复合体的活性。我们问如何做到这一点？最近的研究表明，可溶性C6PE对Xa和Va有调节作用。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络合物的原子结构或Xa和Va与PS结合时的原子结构的信息有限。我们已经交联化了由C6PS组装的Xa-Va络合物，并将确定它是否适合用于结晶试验。我们还将使用突变FRET标记以及对交联和修饰蛋白质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，从而为PS调控提供结构基础。公共卫生相关性：凝血酶原(II)对凝血酶的激活是凝血的关键，因为凝血酶是凝血的中枢调节分子。不能调节它的产生和失活可能会导致中风和心脏病发作。激活的血小板膜显示两种不暴露于静息血小板内血浆的磷脂：磷脂酰丝氨酸(PS)和磷脂酰乙醇胺(PE)。我们发现PS分子可以调节所有参与凝血酶产生的凝血因子。我们现在问：PS在其他涉及凝血的关键蛋白分解反应中是否起到类似的调节作用？分子PE是否影响PS调节的任何凝血因子的结构和活性？如果是这样，为什么第二个调控分子是必要的？PS(PE？)结合到膜附近的凝血蛋白会影响远离膜的结合和催化事件吗？我们希望证明PS(和PE？)血小板膜上的暴露是调节凝血过程的一个新的信号基序。到目前为止，结果已经为一种改进的凝血因子测定获得了专利，并有望产生在临床环境中管理凝血的药物/程序。

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. PUBLIC HEALTH RELEVANCE: Prothrombin (II) activation to thrombin is key to blood coagulation, since thrombin is the central regulatory molecule of blood coagulation. Failure to regulate its production and inactivation can lead to stroke and heart attack. Membranes from activated platelets display two phospholipids not exposed to plasma in resting platelets: phosphatidylserine (PS) and phosphatidylethanolamine (PE). We showed that molecular PS regulates all the clotting factors involved in thrombin production. We ask now: Does PS plays a similar regulatory role in other key proteolytic reactions involved in blood coagulation? Does molecular PE influence the structure and activity of any of the coagulation factors regulated by PS? If so, why is this second regulatory molecule necessary? What are the structural mechanisms by which PS (PE?) binding to clotting proteins near the membrane influence binding and catalytic events far removed from the membrane? We expect to show that PS (and PE?) exposure on platelet membranes is a new signaling motif that regulates the coagulation process. Results to date have produced a patent for an improved coagulation factor assay and are expected to yield drugs/procedures for managing coagulation in a clinical setting.