Alternatively Spliced Tissue Factor Levels in the Plasma of Patients with Liver Fibrosis, Liver Cirrhosis, and Hepatocellular Carcinoma: A Single-Center Retrospective Study

Background:

Chronic liver diseases (CLD) such as chronic viral hepatitis, metabolic liver diseases, alcoholic liver disease, primary and secondary hemochromatosis, Wilson's disease, and cholestatic or autoimmune liver diseases are the major cause of morbidity and mortality worldwide ranking as the 12^th leading cause of overall mortality. CLD induce liver fibrosis characterized by the accumulation of extracellular matrix proteins, such as collagen, in the liver interstitial space. If untreated, liver fibrosis may evolve to end stage cirrhosis, which is associated with a high risk of developing hepatocellular carcinoma (HCC) and liver failure.

CLD is accompanied by changes in the coagulation system that embrace not only hypo-, but also hyper-coagulability. In this rebalanced situation, hypercoagulability can contribute, like genetic predisposition or environmental factors, to the progression of fibrosis and complications of cirrhosis.

Full-length tissue factor (TF), a glycosylated transmembrane protein, is the only initiator of the extrinsic coagulation cascade in vivo. It is elevated in patients with pancreatic cancer, blood disorders, diabetes, and cardiovascular diseases. During TF pre-mRNA processing, exon 5 can be spliced out, resulting in a frameshift that yields to a soluble TF isoform called alternatively spliced TF (asTF). While its involvement in thrombosis and/or hemostasis is still debated, asTF induces angiogenesis and was found highly expressed in pancreatic ductal adenocarcinoma and promoted cancer progression. More recently, it was reported that asTF mRNA expression levels may constitute a prognostic marker in human gastric cancer.

Methods

In a single-center study, we analyzed retrospectively asTF plasma levels in healthy subjects, patients with liver fibrosis, liver cirrhosis, and HCC. asTF plasma levels were measured using a sandwich enzyme-linked immunosorbent assay (ELISA). In addition, immunostaining for asTF was performed in HCC tissue biopsies using an anti-asTF specific antibody. Values were expressed as mean±SD.

Results

The patients with HCC displayed the highest mean asTF levels (n=128, 552.2±469.7pg/ml), followed by those with liver fibrosis (n=70, 524.4±453.9pg/ml) and liver cirrhosis (n=62, 357.2±389.8pg/ml). There was a significant difference in asTF levels between healthy subjects (n=60, 127.3±136.3pg/ml) and patients with liver fibrosis (p<0.0001), healthy subjects and cirrhotic patients (p<0.0001), healthy subjects and HCC patients (p<0.0001), cirrhotic patients and HCC patients (p=0.004) and between fibrotic and cirrhotic patients (p=0.03). Between fibrotic patients and HCC patients, no significant difference in asTF levels was detectable (p=0.70). There were no effects of gender, age and underlying liver disease (hepatitis B and C, alcohol abuse, nonalcoholic steatohepatitis) in each patient group on asTF levels. Furthermore, asTF levels did not correlate with Child-Pugh score, grade of liver fibrosis, and HCC tumor size. Preliminary results indicate that asTF is expressed in liver tissue by hepatocytes, cholangiocytes, and endothelial cells.

Conclusion

Our study revealed that asTF plasma levels were elevated not only in patients with HCC, but also in patients with cirrhosis and even in those with liver fibrosis. This suggests that asTF is produced during fibrotic remodeling of the liver. The lack of correlation between grade of liver fibrosis and asTF levels could be due to the fact that asTF levels did not correlate with the amount of fibrosis deposited in the parenchyma but with the process of producing the fibrosis. In summary, our observations suggest that asTF might play a role in hepatic fibrogenesis.