Summary
Although several investigators have attempted to identify the site of synthesis of
factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII
has not been clearly defined. Indications point at hepatocytes and certain endothelial
cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes
obtained by two-step collagenase digests of human liver pieces. Following Percoll
gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates
of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human
FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from
the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions.
A haemophilic antibody specific for epitopes of the light chain of FVIII, employed
in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide
pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to
that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose
and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance
of a doublet at 78–79 kDa in both these materials indicated the presence of the light
chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted
20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion
of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human
blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all
of these cell cultures, vWf : Ag was indetectable or present as trace. Our results
suggest that the human hepatocyte is a production site of FVIII.
Keywords
Factor VIII - Human hepatocytes - ELISA
