Purinergic Thromboregulation

嘌呤能血栓调节

• 批准号: 7563342
• 负责人: SIMON C. ROBSON
• 金额: $ 73.75万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7563342
• 关键词: ADP Receptors; ATP Receptors; Accounting; Acute; Adenosine; Adoptive Transfer; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Apolipoprotein A-II; Apyrase; Binding; Blood Clot; Blood Platelets; Blood Vessels; Blood coagulation; Bone Marrow Transplantation; Cardiovascular Diseases; Cations; Cell Proliferation; Cell membrane; Cells; Ceramides; Cholesterol; Clinic; Coagulation Process; Coupled; Data; Deposition; Endothelial Cells; Endothelium; Enzymes; Family; Fibrin; GTP-Binding Proteins; Generations; Goals; Hemostatic function; High Density Lipoproteins; Human; Hydrolysis; Hyperlipidemia; Immune; Inflammatory; Injury; Integrins; Ischemia; Kinetics; Knockout Mice; Laboratories; Leukocytes; Life; Link; Lipids; Lipoprotein (a); Lipoproteins; Lymphocyte; Macromolecular Complexes; Mediating; Mediator of activation protein; Membrane Microdomains; Membrane Proteins; Morbidity - disease rate; Mus; Mutant Strains Mice; NTPDase2; North America; Nucleosides; Nucleotides; P2X-receptor; Pericytes; Platelet Activation; Platelet aggregation; Play; Post-Translational Protein Processing; Process; Property; RanBMP-90; Receptor Signaling; Role; Scaffolding Protein; Signal Transduction; Sphingomyelins; Stroke; Testing; Therapeutic Agents; Thromboplastin; Thrombosis; Thrombus; Time; Transgenic Mice; Vascular Diseases; Vascular Endothelium; Vascular Smooth Muscle; Video Microscopy; Work; acid sphingomyelinase; ectoADPase; ectoATPase; extracellular; immune function; in vivo; insight; killer T cell; mortality; novel therapeutics; nucleoside triphosphate; palmitoylation; prevent; receptor; reconstitution; research study; response; vascular inflammation

Extracellular nucleotides (ATP, ADP and UTP) are known to activate type-2 purinergic (P2Y and P2X) receptors on platelets, endothelium and immune cells to induce vascular inflammation and thrombosis. Cellular ectonucleotidases hydrolyze P2-mediators to down regulate P2 receptor-signaling responses. CD39 is the dominant vascular, T regulatory and Natural Killer (NK)T cell ectonucleotidase/NTPDase and plays a crucial role in the ectonucleotidase cascade, involving CD73, to generate adenosine. This nucleoside has important antithrombotic and anti-inflammatory properties. The roles of CD39L1 (a preferential ecto-ATPase), expressed by adventitial cells, and CD39L3 (functionally akin to CD39) expressed by vascular endothelium, on thrombus formation are completely unexplored in vivo. Specific Aim 1: Study functional impacts of the three vascular CD39/NTPDases on thrombus formation in vivo. a) Evaluate and compare accumulation of CD39, CD39L1 and CD39L3 in generated thrombi, with platelet sequestration, associated tissue factor and fibrin deposition. b) Contrast the impact of vascular Cd39L1 and/or Cd39L3 deletion vs. global absence of Cd39 on arteriolar thrombus formation and platelet activation in mutant mice. Specific Aim 2: Thromboregulatory functions of immune cells in vivo a) Perform adoptive transfer of T regulatory cells expressing Cd39 to determine the role of immune regulatory cells on platelet activation and thrombus formation in vivo. b) Test mice following adoptive transfer of isolated NKT cells expressing Cd39 and Cd73 to determine impact on thrombus formation in vivo. Specific Aim 3: Association of CD39 with membrane proteins and lipids. Palmitoylation at the N-terminus targets CD39 to lipid rafts within cell membranes that are linked to microparticle formation. The C-terminus of CD39 binds apo-lipoprotein A2 and incorporates CD39 into High Density Lipoproteins (HDL). a) Explore structural and functional interactions of CD39 within lipid rafts. b) Examine how palmitoylation and other post-translational modifications of CD39 impact association with cholesterol and sphingomyelin. c) Determine effects of activation of acid sphingomyelinase and ceramide generation on CD39 bioactivity in endothelial cells, lymphocytes and their derived microparticles. d) Study thrombus formation in hyperlipidemic Cd39 mutant mice. This work will provide insights into the central thromboregulatory mechanisms impacting platelet activation and vascular injury in vivo.

已知细胞外核苷酸（ATP、ADP和UTP）激活血小板、内皮和免疫细胞上的2型嘌呤能（P2 Y和P2 X）受体以诱导血管炎症和血栓形成。细胞外核苷酸酶水解P2-介质以下调P2受体信号传导反应。CD 39是主要的血管、T调节和自然杀伤（NK）T细胞外核苷酸酶/NTPD酶，并在涉及CD 73的外核苷酸酶级联中起关键作用，以产生腺苷。这种核苷具有重要的抗血栓形成和抗炎特性。由外膜细胞表达的CD 39 L1（一种优先的外膜ATP酶）和由血管内皮表达的CD 39 L3（功能上类似于CD 39）对血栓形成的作用在体内完全未被探索。具体目的1：研究三种血管CD 39/NTPD酶对体内血栓形成的功能影响。a）评估和比较所产生的血栓中的⑶ 39、⑶ 39 L1和⑶ 39 L3的积累，以及血小板隔离、相关的组织因子和纤维蛋白沉积。B）对比血管Cd 39 L1和/或Cd 39 L3缺失与Cd 39的整体缺失对突变小鼠中小动脉血栓形成和血小板活化的影响。具体目标二：a）进行表达Cd 39的T调节细胞的过继转移，以确定免疫调节细胞对体内血小板活化和血栓形成的作用。B）过继转移表达Cd 39和Cd 73的分离的NKT细胞后的测试小鼠，以确定对体内血栓形成的影响。具体目标3：CD 39与膜蛋白和脂质的结合。N-末端的棕榈酰化将CD 39靶向细胞膜内的脂筏，其与微粒形成有关。CD 39的C-末端结合载脂蛋白A2并将CD 39并入高密度脂蛋白（HDL）中。a）探索CD 39在脂筏内的结构和功能相互作用。B）检查棕榈酰化和其他CD 39的翻译后修饰如何影响与胆固醇和鞘磷脂的结合。c）确定酸性鞘磷脂酶的活化和神经酰胺生成对内皮细胞、淋巴细胞及其衍生微粒中的CD 39生物活性的影响。d）研究高脂血症Cd 39突变小鼠中的血栓形成。这项工作将提供深入了解影响血小板活化和血管损伤的中心血栓调节机制在体内。