A Novel Function of Thrombin-activatable Fibrinolysis Inhibitor during Rat Liver Regeneration and in Growth-promoted Hepatocytes in Primary Culture

Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl₄)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.Thrombin-activatable fibrinolysis inhibitor (TAFI)³ is a 60-kDa plasma glycoprotein secreted by hepatocytes as an inactive form. Activation of TAFI is known to be mediated by thrombin (1), thrombin-thrombomodulin complex (2), or plasmin (3). Activated TAFI down-regulates fibrinolysis by removing the plasminogen-anchoring structure from fibrin. This fibrin structure contains C-terminal lysine residues, to which plasminogen binds via its lysine-binding site (4). Thus, TAFI is thought to exhibit negative regulatory activity in the binding of plasminogen to fibrin or cell surfaces.It is well known that plasminogen on fibrin or a cell surface exerts its maximum activity there, because this binding is not only a prerequisite for plasminogen activators to convert plasminogen to plasmin efficiently, but also a guarantee for the plasmin to be protected from inactivation by specific inhibitors, such as α2-plasmin inhibitor (5). As such, plasminogen can fulfill its roles for both thrombolysis and pericellular proteolysis, when it is located on the surface (6).It has been reported that activation of plasminogen is observed at the early stage of liver regeneration in rats and that plasmin contributes to the priming of hepatocytes for proliferation through the reorganization of extracellular matrix (ECM) components (7). The impairment of liver regeneration and abnormalities in liver repair have been observed in plasminogen-deficient mice, when they have undergone partial hepatectomy (8, 9) or been treated with CCl₄ (10). We demonstrated that the plasminogen activator/plasmin system acts to enhance the formation of hepatocyte spheroids (11) and to promote the proliferation of hepatocytes in vitro (12). These results strongly suggest that plasmin(ogen) is a positive regulator for liver regeneration.Although the function of TAFI in fibrinolysis has been well investigated (13), its function, including the regulation of TAFI gene expression in pericellular fibrinolytic events, remains to be investigated. In studies using TAFI-deficient mice, it has been found that such mice develop normally, reach adulthood, and are fertile. No gross physical abnormalities are observed up to 24 months of age (14). On the other hand, abnormalities in the cutaneous wound healing with delayed skin closure due to the altered epithelial migration and colonic anastomosis with bleeding complications are observed in these TAFI-deficient mice (15). Regarding TAFI gene expression, there is a report by Boffa et al. (16) showing that the combination of interleukin-1 and interleukin-6, but neither alone, reduces the abundance of TAFI mRNA in cultured human hepatoma (HepG2) cells by decreasing the stability of the mRNA.In the present study, we investigated the roles of TAFI in liver regeneration. The gene expression of TAFI was found to be strictly controlled by factors responsible for hepatocyte growth such as growth factors or physical conditions. In other words, suppression of TAFI action enhanced the growth of hepatocytes by providing higher plasmin activity on their surfaces. We employed the RNA interference technique to clarify the substantial role of TAFI. In TAFI-silenced hepatocytes, plasmin activity on hepatocyte surface increased, and hepatocyte proliferation was enhanced proportionally. Thus we demonstrated for the first time that TAFI plays an important role in the proliferation of hepatocytes in vivo through its regulatory effect on the cell surface-bound plasmin.