The contribution of bile acid transport and endoplasmatic reticulum stress to tissue factor activation in hepatocytes

Objective Fibrin deposition in the liver parenchyma contributes to cholestatic liver injury, but the origin of tissue factor activity leading to thrombin generation has not been fully elucidated. We discovered that long-time exposure of viable HepG2 cells to unconjugated bile acids results in tissue factor decryption with a strong correlation to FXR-activation. HepG2 cells do not express sodium taurocholate cotransporting polypeptide (NTCP), which regulates hepatocellular uptake of bile acids. We hypothesized this to be the reason, why no effect was observed with conjugated bile acids. Endoplasmatic reticulum stress (ER-stress) might be the link between prolonged FXR overstimulation and tissue factor decryption. Hence, we wanted to test the effect of conjugated bile acids as well as ER-stress on tissue factor decryption in primary hepatocytes.

Material and Methods Primary hepatocytes from healthy donors exposed to chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA) in concentrations of 0-200 µM for 24 hours. Furthermore, cells were exposed to the ER-stressor tunicamycin in concentrations of 0-60 µM for 24 hours. Tissue factor activity was measured via conversion of Factor X to Factor Xa in the presence of Factor VIIa and calcium. Generated Factor Xa was determined by measuring the conversion rate of a chromogenic substrate. Cell viability was determined with the MTT assay.

Results Tissue factor activity showed a dose-dependent increase with a lower threshold in primary hepatocytes (>50 µM) than in HepG2 cells (>200 µM). In contrast to HepG2 cells, GCDCA in primary hepatocytes exhibited a higher potential to induce tissue factor activity than CDCA (200µM GCDCA: 31.7±6.9 pM FXa/min; 200 µM CDCA: 15.2±4.7 pM FXa/min; P<0.001). Tunicamycin dose-dependently induced tissue factor activity with a threshold of 20 µM (60 µM tunicamycin: 503 ± 62% of vehicle). Viability of cells was unimpaired with all employed concentrations.

Conclusion Expression of NTCP in primary hepatocytes facilitates FXR-overstimulation resulting in tissue factor decryption at lower bile acid concentrations, specifically with conjugated bile acids. ER-stress mediated tissue factor decryption hints to a possible link between prolonged FXR-stimulation and ER-stress. Further studies will have to investigate specifics of this signaling cascade as well as the exact mechanism of tissue factor decryption in hepatocytes.

Publication History

Article published online:
18 June 2021