Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Bauer, T.
The Influence of Bile Acids on Thrombin Generation in Cholestatic Liver Injury -
In vitro Studies and Translational Aspects
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2023. pp. 73
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Jahnel Jörg
- Schlagenhauf Axel
- Wagner Martin
- Altmetrics:
- Abstract:
- Background and aims: Besides their traditional metabolic functions, bile acids (BA) have been shown to affect intrahepatic coagulation processes. Elevated BA concentrations present in cholestatic liver disease seem to activate tissue factor (TF) decryption, resulting in intrahepatic thrombin generation. However, the precise process of TF decryption within the liver tissue and the contribution of farnesoid X receptor (FXR), a nuclear bile acid receptor, are still not understood. In this work, the impact of distinct BA on TF action and thrombin generation in hepatocytes were explored and linked to initiation of FXR-mediated signaling and apoptosis. Methods: HepG2 cells and primary human hepatocytes were incubated with various concentrations of chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), the synthetic FXR agonist GW4064 or the synthetic FXR antagonist DY268 for 12 and 24 hours respectively. MTT tests were used to determine cell viability. The activity of TF was evaluated by generating factor Xa and assessing thrombin generation through calibrated automated thrombography. Quantitative polymerase chain reaction (qPCR) was used to determine FXR activation. Western blotting was used to determine TF protein levels and fluorescence microscopy of stained HepG2 cells to denote overexpression of TF. Cleaved caspase-3 and increased Annexin V binding were tested as apoptotic markers. Results: Enhanced thrombin production in tandem with increased TF activity was seen with CDCA and GW4064 and in primary hepatocytes also with GCDCA. UDCA did not show any effect. TF activity significantly decreased when FXR activation was inhibited with the antagonist DY268. qPCR revealed that only CDCA and GW4064 induced the upregulation of FXR targets in HepG2 cells. Western blotting and fluorescence microscopy revealed no upregulation of TF, implying that TF decryption rather than upregulation occurred. No evidence of apoptosis was detected. Conclusion: Long-term contact of liver cells with elevated levels of endogenous BA could cause a disproportionate stimulation of FXR, resulting in the decryption of TF regardless of the amphiphilic nature of BA. The influence of BA on TF initiation is related to its capacity to enter the cells and activate FXR. TF decryption occurs in the absence of apoptotic mechanisms.