Lipid Regulation of Thrombin Generation

凝血酶生成的脂质调节

• 批准号: 8113689
• 负责人: Barry R Lentz
• 金额: $ 3.42万
• 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/8113689
• 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; Complement component C1s; 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; Homologous Protein; Human; Knowledge; 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; public health relevance; 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.

描述（由申请人提供）：凝血酶，血液凝结的中央调节分子，由凝血酶原（II）蛋白水解由血小板 - 膜结合酶（因子XA）及其相关辅助因子（因子VA）产生。膜使II激活加速105倍。激活后，血小板在其表面上用磷脂酰丝氨酸（PS）（PS）和磷脂酰乙醇胺（PE）释放囊泡。与广泛帮助范式相反，我们的工作表明，PS分子而非膜表面会触发这种显着的加速度。这是因为PS位于蛋白质结构和相互作用很难研究的膜中，因此避免了研究人员。为了证明这一点，我们使用了PS（C6P）的短链可溶性形式，该形式不在体内发生，但是揭示膜掩盖效果的宝贵工具。一旦揭示，这些效果就可以通过对膜进行仔细的实验​​来确认。现在，我们提出三个问题，以扩展我们对人类血液凝血的脂质调节的理解。 PS在涉及血液凝血的其他关键蛋白水解反应中起类似的调节作用？ IXA及其辅因子VIIIA和VIIA及其辅因子组织因子因子因子X激活Xa的因子IXA。 IXA和VIIA因子在结构上与XA相似，辅助因子VIIIA也对VA。初步结果表明，IXA和VIIA都对C6P响应。活化的蛋白C（APC）是对PS-膜敏感的关键下调酶，在初步研究中也对C6P敏感。我们假设PS可以像进行XA一样调节IXA，VIIA和APC，并调节VIIIA的作用。膜中的PE还会影响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XA和VA。我们假设分子PE具有与II激活中PS相似的调节作用，并且可以实现与PS位点相关的PE调节位点。 PS与膜附近的调节结构结合的结构机制是什么影响与膜远离膜的事件的？与PS绑定时，我们对XA-VA复合物或XA和VA的原子结构的信息有限。我们已经交联了由C6P组装的XA-VA复合物，并将确定是否适合结晶试验。我们还将使用突变的FRET标记，以及对交联蛋白和修饰蛋白的质谱分析来提供实验性约束，以改善XA和VA的现有模型，并测试我们的假设，即PS结合会产生内部结构域的重排，从而提供PS调节的结构基础。