Elevated Bile Acid Levels Induce Tissue Factor Activity in Hepatocytes

Scientific Research Question: Animal models of cholestatic liver injury show elevated levels of bile acids (BA) in hepatocytes and exhibit activated coagulation and platelets in correlation with disease severity. Under physiological conditions, tissue factor (TF) is present in hepatocytes in a non-activated encrypted form. Exposure of hepatocytes to very high levels of the BA taurocholic acid (TCA) has been shown to rapidly increase the procoagulant activity of TF, suggesting that BA could play a key role in triggering intrahepatic coagulation in cholestatic diseases.The mechanism of TF decryption in cholestasis is still unknown.

We hypothesized that highly concentrated BA in hepatocytes exhibit differing potentials to induce TF activation. We hence sought to investigate the effects of different BA on TF activity, postulating that therapeutic admission of BA might change the coagulation activation potential in chronic cholestasis.

Methodology: We incubated HepG2 cells with various concentrations of TCA, chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UCDA) for 15 minutes, 24 hours, 48 hours and 72 hours respectively, and evaluated cell viability with the MTT assay.

TF activity was tested via FXa generation by addition of factor X (150 nM), factor VIIa (150 nM), and calcium chloride (5 mM), as well as EDTA (5 mM) after 15 minutes incubation time. Generated factor Xa was detected using Perfachrome FXa 8595 (0.667 mM) on a plate reader identifying the absorbance increase at 405 nM for 60 minutes. Factor Xa concentrations were calculated via calibration with FXa.

Findings: TCA and UDCA did not induce TF activity up to concentrations of 1000 µM and 72 h incubation time. TF activity was increased with 100 µM und 200 µM CDCA after 72h (100 µM: 16.3±5.8 pM FXa/min; 200 µM: 9.7±4.6 pM FXa/min; Vehicle: 5,9±3,1 pM FXa/min; P < 0.001). With 400 mM and 600 µM CDCA TF activity was substantially higher after 24h (400 µM: 44.2±34.8 pM FXa/min; 600 µM: 820±337 pM FXa/min; Vehicle: 10,3±9,5 pM FXa/min; P < 0.001), and continually increased after 48h and 72h. At higher CDCA concentrations TF activity was already increased after 15 minutes. The highest TF-activity was detected under conditions where overall cell viability started to decrease, but the majority of cells was still alive.

Conclusions: We found that TF activity is enhanced after incubation with CDCA, but not with TCA and UDCA, arguing for a varying potential of these BA to trigger coagulation. Further studies will have to investigate the mechanism by which BA induce TF activity.

No conflict of interest has been declared by the author(s).