Background/Aim: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. Methods: Fibrosis was induced in rats by administration of CCl 4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl 4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin (ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. Results: After three weeks of CCl 4 , treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p = 0.03) and the expression of TIMP-1 mRNA by 52% (p = 0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl 4 , treatment with SSR182289 resulted in a significant decrease in fibrosis (230%, p = 0.04) and ASMA positive areas (235%, p = 0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl 4 as shown by measuring levels of serum aminotransferases and the area of necrosis. Conclusions: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.
The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmouliè re, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.
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