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

项目总结（见说明）： 为了确定细胞外PD 1是否在血栓形成中起作用，在激光诱导的小动脉损伤后，在小鼠的血管中监测PD 1表达、血小板积累和纤维蛋白产生。在损伤后的血栓中观察到PD 1抗原的时间依赖性增加。 将杆菌肽或PD 1的阻断性单克隆抗体输注到循环中完全抑制了血小板血栓形成和纤维蛋白生成。这些结果表明，PD 1是体内纤维蛋白生成和血小板血栓形成所需的。本项目的目的是确定PD 1抑制剂是否代表一类新的抗血栓形成剂。在目标#1中，本项目提出通过将硫醇异构酶的突变形式表达到离体实验血栓中并捕获硫醇异构酶及其底物的二硫键连接的复合物来鉴定参与血栓形成起始的PDl底物。在目标#2中，将确定人PD 1和复合物中的PD 1的X射线晶体结构。这些结构将包括PD 1和结合至抗-PDI Fab的PD 1，PD 1 与槲皮素3-芸香糖苷和与槲皮素复合的PD 1，以及与β 3整联蛋白复合的PD 1。在目标#3中，将在小鼠中测试槲皮素的抗血栓形成特性。槲皮素作为一种抗血栓药物的药理学最初将通过使用活体显微镜在小鼠提睾肌中使用激光诱导血栓形成模型直接观察血栓抑制来研究。将在两种小鼠模型中测试槲皮素对血栓形成引发的胎儿/新生儿死亡的抑制作用：纯合因子V Leiden和杂合TFPI缺乏的围产期血栓形成，以及纯合蛋白C缺乏的消耗性凝血病。如果槲皮素是一种有效的抗血栓药物，我们将确定槲皮素，它抑制血小板聚集和纤维蛋白的产生，是否优于标准的抗血栓药物上级。PD 1抑制剂将与常规抗血栓形成剂进行比较，以确定PD 1抑制剂的用途。 血栓形成的小鼠模型。这些将包括肺栓塞引起的死亡或瘫痪，以及与获得性抗磷脂综合征相关的血栓形成的活体研究。

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.