PDl: Function in thrombus formation and antithrombotic action of inhibitors in m

PDl：m 中抑制剂的血栓形成功能和抗血栓作用

• 批准号: 8401639
• 负责人: Bruce Furie
• 金额: $ 40.98万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401639
• 关键词: Active Sites; Amino Acids; Antibodies; Antigens; Antiphospholipid Syndrome; Bacitracin; Binding; Blood Circulation; Blood Coagulation Disorders; Blood Platelets; Blood Vessels; Cells; Cessation of life; Cloning; Complex; Cysteine; Deep Vein Thrombosis; Disulfides; Endoplasmic Reticulum; Enzymes; Extracellular Protein; Family; Fibrin; Fibrinolytic Agents; Generations; Glycoproteins; Human; Imagery; In Vitro; Infusion procedures; Injury; Instruction; Integrin beta3; Integrins; Isomerase; Lasers; Link; Location; Mentors; Methods; Modeling; Monitor; Monoclonal Antibodies; Mus; Myocardial Infarction; Nature; Nobel Prize; Oxidoreductase; Paralysed; Perinatal; Pharmacology; Platelet Activation; Platelet aggregation; Play; Property; Protein Biosynthesis; Protein C Deficiency; Protein Disulfide Isomerase; Proteins; Pulmonary Embolism; Quercetin; Rest; Roentgen Rays; Role; Rutin; Shapes; Stroke; Structure; Sulfhydryl Compounds; Surface; TFPI; Testing; Thioredoxin; Thromboembolism; Thromboplastin; Thrombosis; Thrombus; Time; United States; Venous; cremaster muscle; design; disulfide bond; factor V Leiden; fetal; in vivo; inhibitor/antagonist; intravital fluorescence microscopy; intravital microscopy; mortality; mouse model; mutant; neonatal death; novel; prevent; prototype; yeast protein

PROJECT SUMMARY (See instructions): To determine whether extracellular PDl plays a role in thrombus formation, PDl expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice following laser-induced arteriolar injury. A time-dependent increase in PDl antigen was observed in the thrombus following injury. Infusion of bacitracin or a blocking monoclonal antibody to PDl into the circulation completely inhibited platelet thrombus formation and fibrin generation. These results indicate that PDl is required in vivo for both fibrin generation and platelet thrombus formation. The objective in this project is to determine whether inhibitors of PDl represent a new class of antithrombotic agents. In Aim #1, the current project proposes to Identify the substrates of PDl that participate in the initiation of thrombus formation by the expression of mutant forms of thiol isomerases into experimental thrombi ex vivo and the trapping of disulfide-linked complexes of thiol isomerases and their substrates. In Aim #2, the xray crystal structure of human PDl and PDl in complex will be determined. These structures will include PDl and PDl bound to an anfi-PDI Fab, PDl complexed to quercetin 3-rutinoside and to quercetin, and PDl complexed to beta3 integrin. In Aim #3, antithrombotic properties of quercetin will be tested in mice. The pharmacology of quercetin as an antithrombotic agent will be initially studied by direct visualization of thrombus inhibifion using the laserinduced thrombus formation model in the cremaster muscle of the mouse using intravital microscopy. The inhibition of thrombosis-initiated fetal/neonatal demise by quercetin will be tested in two mouse models: perinatal thrombosis with homozygous Factor V Leiden and heterozygous TFPI deficiency, and consumptive coagulopathy with homozygous protein C deficiency. If quercetin is an active antithrombotic, we will determine whether quercetin, which inhibits both platelet aggregation and fibrin generation, is superior to standard antithrombotic agents. PDl inhibitors will be compared to conventional antithrombotic agents in mouse models of thrombosis. These will include death or paralysis induced by pulmonary embolism, and by intravital study of thrombus formation associated with acquired anti-phospholipid syndrome.

项目总结(见说明)： 为了确定细胞外PDL是否在血栓形成中起作用，监测了激光诱导的小动脉损伤后小鼠血管中PDL的表达、血小板聚集和纤维蛋白生成。损伤后血栓中PDL抗原的表达呈时间依赖性增加。 向循环中注入杆菌肽或阻断PDL的单抗可完全抑制血小板血栓形成和纤维蛋白生成。这些结果表明PDL在体内对于纤维蛋白的产生和血小板血栓的形成都是必需的。本项目的目的是确定PDL的抑制剂是否代表了一类新的抗血栓药物。在目标#1中，本项目建议通过在体外将硫醇异构酶的突变形式表达到实验性血栓中并捕获硫醇异构酶及其底物的二硫键连接的络合物来识别参与血栓形成的PDL底物。在目标2中，将确定人的PDL和络合物中的PDL的X射线晶体结构。这些结构将包括PDL和绑定到ANFI-PDI Fab、PDL的PDL PDL与β3整合素形成络合物，与3-芦丁糖苷和槲皮素络合。在目标3中，将在小鼠身上测试槲皮素的抗血栓特性。利用激光诱导的小鼠提睾肌血栓形成模型，通过活体显微镜直接观察血栓抑制作用，初步研究了作为抗血栓药的栎素的药理作用。在两种小鼠模型中，将测试槲皮素对血栓引发的胎儿/新生儿死亡的抑制作用：纯合子因子V Leiden和杂合子TFPI缺乏的围产期血栓形成和纯合子蛋白C缺乏的消耗性凝血障碍。如果Quercetin是一种有效的抗血栓药，我们将确定同时抑制血小板聚集和纤维蛋白生成的Quercetin是否优于标准的抗血栓药。PDL抑制剂将与传统的抗血栓药物在 血栓形成的小鼠模型。这些将包括肺栓塞导致的死亡或瘫痪，以及与获得性抗磷脂综合征相关的血栓形成的活体研究。