Synthesis of Factor VIII in Human Hepatocytes in Culture

Abstract

SummaryAlthough 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.

Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII

“…In contrast, others have identified FVIII mRNA or FVIII:Ag in hepatocytes but not in LSECs . FVIII:Ag has also been detected in the supernatant of in vitro cultures of purified hepatocytes . Owing to these contradictory reports, it is currently unclear which liver cells produce FVIII in humans.…”

Secretion of functional plasma haemostasis proteins in long‐term primary cultures of human hepatocytes

“…Data on the synthesis and secretion of the haemostasis proteins from human hepatocytes in primary cultures remain very limited (Ingerslev et al , 1988; Mazzorana et al , 1991; Ferrini et al , 1997). Ingerslev et al (1988) found that lysates of freshly isolated and purified human hepatocytes contained and secreted FVIII. Mazzorana et al (1991) tested the reliability of their hepatocyte culture model using FV detection.…”

Genetic Induction of a Releasable Pool of Factor VIII in Human Endothelial Cells

“…9 Even within the liver, the cell type or types in which the FVIII is synthesized are not clearly established. 8,10 The synthesis of vWF has been demonstrated in endothelial cells and megakaryocytes, where it is stored in specialized vesicles: Weibel-Palade bodies (WPBs) and ␣-granules, respectively. 11,12 In the late trans-Golgi network, vWF is sorted into releasable dense-core granules.…”

“…3 Organ transplantation studies in recent decades have identified that the liver, 4 as well as several extrahepatic sources of FVIII (lung, lymphatics, and spleen), [5][6][7] produces FVIII in humans and dogs with hemophilia A. Although FVIII synthesis by hepatocytes has been demonstrated, 8 end-stage liver disease is not associated with a decrease in plasma FVIII levels. 9 Even within the liver, the cell type or types in which the FVIII is synthesized are not clearly established.…”