Dichotomous Roles of Thrombin in Acetaminophen Hepatotoxicity

描述（由申请人提供）：过量使用对乙酰氨基酚（APAP）是人类急性肝衰竭的最常见原因。APAP的代谢生物激活引发一系列事件，导致肝细胞坏死，正是在这一进展阶段，解毒剂治疗最有可能成功。在人类患者中，凝血级联激活和凝血酶的产生伴随着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，导致凝血酶受体、蛋白酶激活受体-1 （PAR-1）的激活，并导致肝内纤维蛋白的沉积。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依赖性凝血酶的产生和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，PAR-1在其他组织中的非实质细胞的激活已被证明可以刺激这些因子的产生。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）的抗纤溶活性增强了纤维蛋白的沉积，PAI-1是一种由转录因子缺氧诱导因子-1 α (HIF-1a)诱导表达的血浆蛋白。根据我们的初步数据，我们假设APAP过量导致tf依赖性凝血酶的产生，凝血酶通过激活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的进展。这一假设将在小鼠身上进行一系列体内和体外实验，采用实质和非实质肝细胞，并使用遗传方法，包括使用条件敲除、Cre-LoxP技术以及适当的药物干预产生的新型小鼠。目的1将探讨肝细胞和肝细胞衍生的促凝微粒表达的TF在凝血酶生成中的重要性，以及APAP毒性过程中谷胱甘肽减少在TF激活中的作用。目的2将确定PAR-1对非实质细胞的激活在apap诱导的肝损伤发病机制相关因子表达中的作用。最终目的将集中在纤维蛋白凝块的损伤限制作用以及hif -1a介导的PAI-1表达在肝脏纤维蛋白沉积中的作用。阐明apap诱导的肝损伤进展机制对于确定预防患者肝功能衰竭的新策略以及对药物性肝损伤发病机制的总体理解至关重要。

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.