Novel mechanisms linking blood coagulation to liver fibrosis

项目摘要/摘要 这项提议的首要目标是培养所需的技能，以实现我成为一名 独立研究人员，专注于凝血因子在细胞信号转导中的作用 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 在肝脏学、毒理学、血栓形成和止血方面进行跨学科研究。强壮 临床和实验证据表明，凝血级联在高血压病的发病机制中起着重要的作用。 肝纤维化的进展，即慢性损伤的肝脏形成“疤痕”。一种假说认为凝血 肝纤维化活性提示凝血酶驱动肝星状细胞(HSCs) 通过激活其主要受体--蛋白水解酶激活受体--表达胶原的肌成纤维细胞表型。 1(PAR-1)。然而，将PAR-1激活与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，PAR-1的凝血酶激活通过以下方式推动HSC向促纤维化表型转变 放大凝血因子VIIa跨膜受体组织因子的信号功能 (FVIIA)为了验证这一假设，我提出了三个具体目标。首先，我将确定 PAR-1的激活驱动肝星状细胞的促纤维化表型。我假设PAR-1的激活 通过诱导Tf：FVIIa信号放大HSC促纤维化表型。Tf：FVIIa信号转导途径的作用 在PAR-1介导的肝星状细胞激活将使用体外方法的组合，包括 外源性FVIIa治疗和缺乏Tf或PAR-2(即介导Tf：FVIIa信号的受体)的HSC。这个 HSC转铁蛋白在实验性肝纤维化中的作用将通过使用HSC特异性转铁蛋白的新小鼠来确定。 缺乏症。接下来，我将确定凝血酶：PAR-1信号在实验性肝纤维化中的作用。这个 凝血酶介导的PAR-1激活在肝纤维化中的确切作用在体内从未被研究过 因为PAR-1可以被多种酶切割。我假设凝血酶驱动PAR-1的激活 实验性肝纤维化。为了验证这一假设，我将使用包括老鼠在内的一系列策略 表达~10%的正常凝血酶原水平和选择性表达PAR-1的新突变小鼠 对凝血酶或其他激动型蛋白水解酶对特定残基的切割不敏感。最后，我将确定 在受损肝脏中驱动凝血激活的机制。我假设凝血系统的激活 实验性CCl4诱导的慢性肝损伤是由内源性凝血途径驱动的。我会用 互补的遗传学和药理学方法来确定外在和内在的确切作用 实验性慢性肝损伤所致肝纤维化中凝血级联激活的途径。这个 拟议中的研究将使我能够开辟一个独特的研究领域，研究细胞信号转导功能 凝血因子，并最终将推动针对局部的新疗法的开发 凝血介导的细胞信号转导事件在对正常止血影响最小的情况下减少肝纤维化。

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.