Mechanism of the abnormal vitamin k‐dependent γ‐carboxylation process in human hepatocellular carcinomas

Background. An important marker for hepatocellular carcinoma is the presence of des‐γ‐carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown. Methods. Two groups of patients were defined according to the absence (Group I, n = 7) or presence (Group II, n = 8) of des‐γ‐carboxy prothrombin. The enzymatic activity of y‐carboxylase and the total microsomal prothrombin concentration were determined in all tumors. The kinetic parameters for the synthetic peptide Phe‐Leu‐Glu‐Glu‐Leu (FLEEI.) were measured in eight tumors. The γ‐carboxylase mRNA expression was evaluated by Northern blot analysis in 12 of 15 tumors. In addition, the total vitamin K content (K, K, epoxide, and menaquinones 4‐10) in 10 tumors was investigated by high performance liquid chromatography. Results. Concentrations of menaquinones 4‐10 were normal in the nontumorous part of the liver but significantly decreased (P = 0.02) in all the tumors (Groups I and II). This decrease was more severe in Group II (P = 0.02). The tumors in Group I had normal or increased γ‐carboxylase activity and increased mRNA expression (P < 0.02) as compared with their nontumorous counterparts. The tumors in Group II were heterogeneous. Five tumors displayed low y‐carboxylase activity, associated with low mRNA expression in two, whereas two others had high γ carboxylase activity and mRNA expression. The concentration of FLEEL at half‐maximal velocity was normal in all the tumors examined (Groups I and II), and a relation was found between the level of expression of γ‐carboxylase and the maximal velocity for FLEEL carboxylation in the tumors in Group II (r = 0.98; P < 0.01). The microsomal content of normal prothrombin was within normal limits in all tumors (Groups I and II). Conclusions. Tumor vitamin K content has a critical role in the synthesis of des‐γ‐carboxy prothrombin. Furthermore, the γ‐carboxylase defect, which is observed in some secreting tumors, is the result of the defective gene expression of a normal enzyme and not the consequence of the presence of a competitive inhibitor. It is possible that a 75% reduction in γ‐carboxylase gene expression could take a part in the secretion of des‐γ‐carboxy prothrombin, but this mechanism is not predominant.