Dichotomous Roles of Thrombin in Acetaminophen Hepatotoxicity

描述（由申请方提供）：对乙酰氨基酚（APAP）过量是人类急性肝衰竭的最常见原因。APAP的代谢生物活化启动了一系列导致肝细胞坏死的事件，在此进展阶段，解毒治疗最有可能成功。在人类患者中，凝血级联激活和凝血酶生成伴随APAP肝毒性的进展。在APAP处理的小鼠中，组织因子（TF）激活凝血级联反应，导致凝血酶受体、蛋白酶激活受体-1（PAR-1）激活，并导致纤维蛋白在肝脏中沉积。初步结果表明，凝血系统激活在APAP肝毒性中具有双重作用。TF依赖性凝血酶生成和PAR-1的刺激似乎有助于肝损伤的早期进展，因为TF或PAR-1的缺乏减少了早期APAP诱导的肝细胞损伤。一氧化氮和细胞因子（如肿瘤坏死因子-α和白细胞介素1-β）的产生与APAP肝毒性的早期进展有关，其他组织中非实质细胞的PAR-1活化已显示刺激这些因子中每一种的产生。相反，凝血酶介导的纤维蛋白沉积限制了后来的肝细胞损伤和出血。纤溶酶原激活物抑制剂-1（PAI-1）的抗纤溶活性增强纤维蛋白的沉积，PAI-1是一种血浆蛋白，其表达由转录因子低氧诱导因子-1 α（HIF-1a）诱导。基于我们的初步数据，我们假设APAP过量导致TF依赖性凝血酶产生，其通过激活PAR-1促进肝损伤的早期进展，但也通过产生纤维蛋白限制出血和肝细胞坏死进展。将采用实质和非实质肝细胞并使用遗传方法（包括使用条件性敲除、Cre-LoxP技术以及适当的药理学干预产生的新型小鼠），在一系列体内和体外实验中对该假设进行测试。目的1探讨肝细胞和肝细胞源性促凝微粒表达的TF在凝血酶生成中的重要性，以及还原型谷胱甘肽在APAP毒性过程中TF活化中的作用。目的2将确定PAR-1激活的非实质细胞的作用，在表达的相关因素的发病机制APAP诱导的肝损伤。最终的目标将集中在损伤限制的影响，纤维蛋白凝块和HIF-1 α介导的派-1的表达在纤维蛋白沉积在肝脏中的作用。阐明APAP诱导的肝损伤进展的机制对于确定预防患者肝衰竭的新策略以及对药物诱导的肝损伤的发病机制的一般理解至关重要。

DESCRIPTION (provided by applicant): Overdose with acetaminophen (APAP) is the most common cause of acute liver failure in humans. Metabolic bioactivation of APAP initiates a cascade of events that causes hepatocellular necrosis, and it is during this progression phase when antidotal therapy is most likely to be successful. In human patients, coagulation cascade activation and thrombin generation accompanies the progression of APAP hepatotoxicity. In APAP- treated mice, tissue factor (TF) activates the coagulation cascade, resulting in activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), and in the deposition of fibrin in liver. Preliminary results suggest that coagulation system activation has dichotomous roles in APAP hepatotoxicity. TF-dependent thrombin generation and stimulation of PAR-1 appear to contribute to the early progression of liver damage, since deficiency in TF or PAR-1 reduces early APAP-induced hepatocellular injury. Generation of nitric oxide and of cytokines such as tumor necrosis factor-alpha and interleukin 1-beta are associated with early progression of APAP hepatotoxicity, and PAR-1 activation of nonparenchymal cells in other tissues has been shown to stimulate production of each of these factors. Conversely, thrombin-mediated fibrin deposition limits later hepatocellular injury and hemorrhage. The deposition of fibrin is enhanced by the antifibrinolytic activity of plasminogen activator inhibitor-1 (PAI-1), a plasma protein the expression of which is induced by the transcription factor, hypoxia inducible factor-1alpha (HIF-1a). Based on our preliminary data, we hypothesize that APAP overdose results in TF-dependent production of thrombin, which contributes to the early progression of liver injury by activating PAR-1, but also limits hemorrhage and hepatocellular necrosis progression by generating fibrin. This hypothesis will be tested in mice in a series of experiments in vivo and in vitro employing parenchymal and nonparenchymal liver cells and using genetic approaches including novel mice generated using conditional knockout, Cre-LoxP technology, as well as appropriate pharmacological interventions. Aim 1 will explore the importance of TF expressed by hepatocytes and hepatocyte-derived procoagulant microparticles in thrombin generation, and the role of decreased glutathione in TF activation during APAP toxicity. Aim 2 will determine the role of PAR-1 activation on nonparenchymal cells in the expression of factors associated with the pathogenesis APAP-induced liver injury. The final aim will focus on the injury-limiting influence of fibrin clots and role of HIF-1a-mediated expression of PAI-1 in fibrin deposition in liver. Elucidating mechanisms by which APAP-induced liver injury progresses is essential for defining novel strategies to prevent liver failure in patients and for the general understanding of the pathogenesis of drug-induced liver injury.