Novel mechanisms linking blood coagulation to liver fibrosis

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.