Lipid Regulation of Thrombin Generation

凝血酶生成的脂质调节

• 批准号: 8077274
• 负责人: Barry R Lentz
• 金额: $ 37.65万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8077274
• 关键词: Acceleration; Address; Affect; Antithrombin III; Apoptosis; Apoptotic; Binding; Binding Sites; Biological Assay; Blood Coagulation Factor; Blood Platelets; Blood coagulation; C2 Domain; Catalytic Domain; Cell Death; Cell membrane; Cells; Clinical; Coagulation Process; Coenzymes; Complex; Computing Methodologies; Crystallization; DNA Sequence Rearrangement; Data; Development; Dialysis procedure; Dimerization; Endothelial Cells; Enzymes; Erythrocytes; Event; Exposure to; Face; Factor IXa; Factor V; Factor VIIIa; Factor VIIa; Factor Va; Factor Xa; Failure; Fluorescence; Fluorescence Resonance Energy Transfer; Generations; Goals; Health; Homologous Protein; Human; Label; Lead; Legal patent; Link; Lipid Binding; Lipids; Location; Masks; Mass Spectrum Analysis; Measurement; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Methods; Modeling; Molecular; Myocardial Infarction; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phosphatidylethanolamine; Phosphatidylserines; Phospholipids; Physiology; Plasma; Platelet Activation; Play; Procedures; Production; Property; Protein C; Proteins; Proteolysis; Prothrombin; Publishing; Pump; Reaction; Recombinants; Regulation; Reporting; Research Personnel; Rest; Roentgen Rays; Role; Scheme; Second Messenger Systems; Serine; Signal Transduction; Signaling Molecule; Site; Solutions; Spleen; Staging; Stream; Stroke; Structure; Surface; Testing; Thrombin; Thromboplastin; Time; Vesicle; Water; Work; activated Protein C; base; cell growth regulation; cell injury; cofactor; crosslink; dimer; flexibility; improved; in vivo; inhibitor/antagonist; insight; membrane activity; membrane model; molecular dynamics; mutant; phosphatidylethanolamine; protein structure; prothrombinase complex; receptor; research study; response; second messenger; success; tool

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.

描述（由申请方提供）：凝血酶是凝血的中心调节分子，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。膜加速II激活105倍。在活化时，血小板在其表面上释放具有磷脂酰丝氨酸（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，触发了这种显着的加速。这一直困扰着研究人员，因为PS位于蛋白质结构和相互作用非常难以研究的膜中。为了证明这一点，我们使用了一种短链可溶性形式的PS（C6 PS），它在体内不会发生，但它是揭示被膜掩盖的效应的宝贵工具。一旦发现，这些效应可以通过膜的仔细实验来证实。我们现在提出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VIIIa和因子VIIa及其辅因子组织因子将因子X活化为Xa。因子IXa和VIIa在结构上类似于Xa，辅因子VIIIa至Va也是如此。初步结果表明，IXa和VIIa都对C6 PS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，对C6 PS也敏感。我们假设PS调节IXa、VIIa和APC，就像调节Xa一样，调节VIIIa，就像调节Va一样。如果我们证实我们的假设，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C6 PE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，并且它通过与PS位点连接的PE调节位点来实现这一点。PS与膜附近的调节结构域的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C6 PS组装的Xa-Va复合物，并将确定它是否适合于结晶试验。我们还将使用突变的FRET标记，和质谱分析的交联和修饰的蛋白质，以提供实验的限制，以改善现有的模型Xa和Va，并测试我们的假设，PS结合产生内部结构域重排，提供PS调节的结构基础。公共卫生相关性：凝血酶原（II）活化为凝血酶是血液凝固的关键，因为凝血酶是血液凝固的中心调节分子。如果不能调节其产生和失活，可能导致中风和心脏病发作。来自活化血小板的膜显示两种在静息血小板中不暴露于血浆的磷脂：磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）。我们发现，分子PS调节所有凝血因子参与凝血酶的生产。我们现在问：PS是否在其他涉及血液凝固的关键蛋白水解反应中发挥类似的调节作用？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，为什么第二个调节分子是必要的？PS（PE？）与靠近膜的凝血蛋白的结合影响远离膜的结合和催化事件？我们希望表明，PS（和PE？）血小板膜上的暴露是调节凝血过程的新的信号基序。迄今为止的结果已经产生了一项改进的凝血因子测定的专利，并有望产生用于在临床环境中管理凝血的药物/程序。