Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures

We have reported a rapid method for the quantitation of proteins secreted in culture media (F.M. LaDuca, C.V. Dang, and W.R. Bell (1986) Anal. Biochem. 158, 262-267). Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture. The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin. Since heparin is known to affect certain cellular functions, the fates of [35S]methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments. Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin. At 8 h during the chase, there was a 40-50% reduction in fibrinogen-antigen in spent culture medium lacking heparin. The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis. In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen. The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation. Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.