Dichotomous Roles of Thrombin in Acetaminophen Hepatotoxicity

凝血酶在对乙酰氨基酚肝毒性中的二分作用

• 批准号: 8038828
• 负责人: Robert Andrew Roth
• 金额: $ 40.78万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038828
• 关键词: Dichotomous; Roles; Thrombin; Acetaminophen; Hepatotoxicity

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. PUBLIC HEALTH RELEVANCE: Coagulation cascade activation and associated thrombin generation occur in human patients who suffer from acetaminophen overdose, a major cause of acute liver failure in the US. Studies in mice revealed that thrombin has both harmful and beneficial roles in the progression of acetaminophen-induced liver injury. The proposed studies are designed to understand the mechanisms behind these dichotomous roles, and the results could lead to improved therapy and survival for patients with acetaminophen overdose.

描述(由申请人提供)：过量服用对乙酰氨基酚(APAP)是人类急性肝功能衰竭的最常见原因。APAP的代谢生物激活启动了一系列导致肝细胞坏死的事件，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生成伴随着APAP肝毒性的进展。在APAP治疗的小鼠中，组织因子(TF)激活凝血级联反应，导致凝血酶受体-1(PAR-1)的激活，并导致纤维蛋白在肝脏中沉积。初步结果提示，凝血系统的激活在APAP肝毒性中起着二元性作用。Tf依赖的凝血酶生成和PAR-1的刺激似乎参与了肝损伤的早期进展，因为缺乏Tf或PAR-1可以减轻APAP诱导的早期肝细胞损伤。一氧化氮和细胞因子如肿瘤坏死因子-α和白介素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激活已被证明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物-1(PAI-1)是一种血浆蛋白，其表达受转录因子缺氧诱导因子-1α(HIF-1a)的诱导，从而促进纤维蛋白的沉积。根据我们的初步数据，我们假设APAP过量导致依赖于TF的凝血酶的产生，凝血酶通过激活PAR-1而促进肝损伤的早期进展，但也通过产生纤维蛋白来限制出血和肝细胞坏死的进展。这一假说将在小鼠体内和体外进行一系列实验，使用实质和非实质肝细胞，并使用遗传方法，包括使用条件基因敲除、Cre-loxP技术产生的新小鼠，以及适当的药物干预。目的1探讨肝细胞和肝细胞源性促凝血微粒表达的转铁蛋白在凝血酶生成中的重要作用，以及谷胱甘肽降低在APAP中毒时转铁蛋白活化中的作用。目的探讨非实质细胞PAR-1活化在APAP肝损伤相关因子表达中的作用。最终目的将集中在纤维蛋白凝块对损伤的限制影响以及HIF-1a介导的PAI-1在肝脏纤维蛋白沉积中的作用。阐明APAP诱导的肝损伤的进展机制对于确定预防患者肝功能衰竭的新策略和全面了解药物诱导的肝损伤的发病机制是至关重要的。 公共卫生相关性：在美国，对乙酰氨基酚过量的患者会出现凝血级联激活和相关的凝血酶生成，这是导致急性肝功能衰竭的主要原因。对小鼠的研究表明，凝血酶在对乙酰氨基酚诱导的肝损伤的进展中既有有害的作用，也有有益的作用。拟议的研究旨在了解这些二分作用背后的机制，结果可能会改善对乙酰氨基酚过量患者的治疗和存活率。