Novel mechanisms linking blood coagulation to liver fibrosis

将凝血与肝纤维化联系起来的新机制

• 批准号: 10452687
• 负责人: Lauren G Poole
• 金额: $ 8.7万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452687
• 关键词: Agonist; Automobile Driving; Blood Coagulation Factor; Blood coagulation; Carbon Tetrachloride; Cell physiology; Cells; Cicatrix; Clinical; Coagulation Process; Collagen; Deposition; Development; Discipline; Disease Progression; Event; Factor VIIa; Factor XI; Factor XII; Foundations; Genetic; Goals; Hemostatic function; Hepatic Stellate Cell; Hepatology; In Vitro; Inflammation; Lead; Ligands; Link; Liver Fibrosis; Liver diseases; Mediating; Morbidity - disease rate; Mus; Mutant Strains Mice; Myofibroblast; Outcome; PAR-1 Receptor; Pathologic; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Pharmacology; Phenotype; Play; Prothrombin; Publishing; Research; Research Personnel; Research Proposals; Resolution; Role; Scheme; Signal Transduction; Testing; Thrombin; Thromboplastin; Thrombosis; Tissues; Toxicology; Up-Regulation; base; career; chronic liver disease; chronic liver injury; design; hepatotoxin; in vivo; liver injury; mortality; new therapeutic target; novel; novel therapeutics; programs; receptor; skills; tissue injury; translational study

Project Summary/Abstract The overarching goal of this proposal is to develop the skills required to achieve my career goal of becoming an independent investigator with a research focus on the role of blood coagulation factors in cell signaling during tissue injury and inflammation. Accordingly, I have developed a Research Plan that will build a strong foundation for conducting research across disciplines in hepatology, toxicology, and thrombosis and hemostasis. Strong clinical and experimental evidence suggests that the blood coagulation cascade plays a pathologic role in the progression of hepatic fibrosis, i.e., “scarring” of the chronically injured liver. One hypothesis linking coagulation activity to hepatic fibrosis proposes that the coagulation protease thrombin drives hepatic stellate cells (HSCs) to a collagen-expressing myofibroblast phenotype by activating its primary receptor, protease-activated receptor- 1 (PAR-1). However, the precise mechanisms linking PAR-1 activation to the HSC pro-fibrotic phenotype are unknown. My central hypothesis is that thrombin activation of PAR-1 drives HSCs to a pro-fibrotic phenotype by amplifying the signaling functions of tissue factor (TF), the transmembrane receptor for coagulation factor VIIa (FVIIa). To test this hypothesis, I propose three Specific Aims. First, I will determine the mechanisms whereby PAR-1 activation drives a pro-fibrotic phenotype in hepatic stellate cells. I hypothesize that PAR-1 activation amplifies the HSC pro-fibrotic phenotype through induction of TF:FVIIa signaling. The role of TF:FVIIa signaling in PAR-1-mediated HSC activation will be determined using a combination of in vitro approaches including exogenous FVIIa treatment and HSCs lacking TF or PAR-2 (i.e., the receptor mediating TF:FVIIa signaling). The role of HSC TF in experimental hepatic fibrosis will be determined using novel mice with HSC-specific TF deficiency. Next, I will determine the role of thrombin:PAR-1 signaling in experimental hepatic fibrosis. The precise role of thrombin-mediated PAR-1 activation in hepatic fibrosis has never been investigated in vivo because PAR-1 can be cleaved by multiple proteases. I hypothesize that activation of PAR-1 by thrombin drives experimental hepatic fibrosis. To test this hypothesis, I will use a combination of strategies including mice expressing ~10% of normal prothrombin levels and novel mutant mice expressing PAR-1 that is selectively insensitive to cleavage at specific residues by thrombin or by other agonist proteases. Finally, I will identify the mechanisms driving coagulation activation in the injured liver. I hypothesize that coagulation activation in experimental CCl4-induced chronic liver injury is driven by the intrinsic coagulation pathway. I will use complementary genetic and pharmacologic approaches to determine the precise role of the extrinsic and intrinsic pathways of coagulation cascade activation in hepatic fibrosis driven by experimental chronic liver injury. The proposed studies will allow me to carve out a unique research niche investigating the cell signaling functions of blood coagulation factors, and would ultimately drive development of novel therapeutics which target local coagulation-mediated cell signaling events to reduce hepatic fibrosis with minimal impact on normal hemostasis.

项目总结/摘要 这份建议书的总体目标是培养实现我的职业目标所需的技能， 独立研究员，研究重点是凝血因子在细胞信号传导中的作用， 组织损伤和炎症。因此，我制定了一个研究计划，将建立一个坚实的基础， 从事肝病学、毒理学、血栓形成和止血等学科的研究。强 临床和实验证据表明，血液凝固级联反应在血液凝固过程中起病理作用。 肝纤维化的进展，即，慢性损伤肝脏的“疤痕”。一种假设将凝血 活性肝纤维化提出凝血蛋白酶凝血酶驱动肝星状细胞（HSC） 通过激活其主要受体，蛋白酶激活受体- 1（PAR-1）。然而，将PAR-1活化与HSC促纤维化表型联系起来的确切机制是 未知我的中心假设是，凝血酶激活PAR-1驱动HSC的促纤维化表型， 增强组织因子（TF）的信号传导功能，TF是凝血因子VIIa的跨膜受体 （FVIIa）。为了验证这个假设，我提出了三个具体目标。首先，我将确定 PAR-1激活驱动肝星状细胞中的促纤维化表型。我假设PAR-1的激活 通过诱导TF：FVIIa信号传导放大HSC促纤维化表型。TF：FVIIa信号传导的作用 PAR-1介导的HSC活化将使用体外方法的组合来确定，包括 外源性FVIIa治疗和缺乏TF或PAR-2的HSC（即，介导TF：FVIIa信号传导的受体）。的 将使用具有HSC特异性TF的新小鼠来确定HSC TF在实验性肝纤维化中的作用 缺陷接下来，我将确定凝血酶的作用：PAR-1信号在实验性肝纤维化。的 凝血酶介导的PAR-1活化在肝纤维化中的确切作用尚未在体内研究 因为PAR-1可以被多种蛋白酶切割。我假设凝血酶激活PAR-1 实验性肝纤维化为了验证这一假设，我将使用包括小鼠在内的策略组合 表达~10%正常凝血酶原水平的小鼠和表达PAR-1的新型突变小鼠， 对凝血酶或其它激动剂蛋白酶在特定残基上的切割不敏感。最后，我将确定 驱动受损肝脏中凝血激活的机制。我推测凝血激活 实验性CCl 4诱导的慢性肝损伤由内源性凝血途径驱动。我会用 互补的遗传学和药理学方法，以确定外在和内在的确切作用， 实验性慢性肝损伤驱动的肝纤维化中凝血级联激活的途径。的 拟议的研究将使我能够开辟一个独特的研究利基调查细胞信号功能， 凝血因子，并最终推动新的治疗方法的发展，靶向局部 凝血介导的细胞信号传导事件，以减少肝纤维化，对正常止血的影响最小。