Synthesis of factor VIII in human hepatocytes in culture.
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/10(6) 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 judged 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 x 10(6) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ingerslev
J
J
Department of Clinical Immunology, University Hospital Aarhus, Denmark.
Christiansen
B S
BS
Heickendorff
L
L
Munck Petersen
C
C
D002478:Cells, Cultured; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000032:analysis
D010455:Peptides; D005169:Factor VIII; D003101:Collodion