Mechanism of Lipopolysaccharide-induced Decrease of Plasma Protein S: Its Expression in Hepatocytes and Sinusoidal Endothelial Cells Is Mediated by NFkB Activation

Abstract number: P0133

Hayashi T, Kishiwada M, Fujii K, Yuasa H, Nishioka J, Gabazza EC, Suzuki K

Anticoagulant protein S (PS) is mainly synthesized in hepatocytes and endothelial cells. It functions as a cofactor of activated protein C (APC) that inactivates the blood coagulation factors Va and VIIIa. PS circulates in a free form and in complex with C4b-binding protein (C4BP), and only the free PS acts as a cofactor of APC. Patients with sepsis have decreased plasma level of PS, and this decrease is probably induced by lipopolysaccharide (LPS). Decreased circulating PS is associated with low anticoagulant activity in plasma and with thrombotic tendency. The mechanism of PS decrease in plasma of patients with sepsis is unclear. In this study, we examined the effect of LPS on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) of rats. Plasma PS antigen level was time-dependently decreased in LPS-treated rats (2 mg/kg). Real-time PCR analysis revealed that PS mRNA was transiently decreased in rat liver at 2 to 8 h and then recovered at 24 h after LPS treatment. LPS dose-dependently and significantly decreased plasma PS level and mRNA expression in both hepatocytes and SECs. After LPS injection, the plasma levels of tumor necrosis factor (TNF)-a, interleukin (IL)-6 and interferon (IFN)-g transiently increased. In vitro assays showed that IL-6 increased PS expression in hepatocytes, while TNF-a decreased it in SECs. Reverse transcriptase-PCR showed that both hepatocytes and SECs express CD14 and toll-like receptor (TLR)-4, and LPS-induced NFkB activation was inhibited by anti-rat CD14 and anti-rat TLR-4 antibodies in both hepatocytes and SECs. Furthermore, inhibitor of NFkB inhibited LPS-induced decreased expression of PS in both cells. These findings indicate that LPS decreases PS expression in hepatocytes and SECs, and this LPS-induced decreased PS expression in hepatocytes and SECs is caused by NFkB activation via membrane-bound CD14 and TLR-4.