BACKGROUND: Although the liver is the major site of coagulation factor VIII (FVIII) synthesis, the type of cells producing FVIII within the liver is still unclear. OBJECTIVES: To measure FVIII in extracts of primary liver sinusoidal endothelial cells (LSECs) and hepatocytes, thereby preventing potential bias resulting from the modifications of the cell phenotype that can take place during in vitro culture. METHODS: LSECs were purified by flow cytometry cell sorting on the basis of their coexpression of Tie2 and CD32b. The purity of the cells was controlled by RNA sequencing. FVIII activity (FVIII:C) in extracts of purified cells was measured with a sensitive FVIII chromogenic assay, in which the specificity of the reaction is controlled by neutralization of FVIII activity with specific inhibitor antibodies. RESULTS: The FVIII:C concentration in purified LSECs ranged from 0.3 to 2.8 nU per cell. In contrast, FVIII:C was undetectable in hepatocytes. The intracellular FVIII:C concentrations are therefore at least 10-100-fold higher in LSECs than in hepatocytes. CONCLUSIONS: Our data demonstrate that LSECs, but not hepatocytes, contain measurable amounts of FVIII:C, and suggest that the former are the main cells producing FVIII in the human liver.
BACKGROUND: Although the liver is the major site of coagulation factor VIII (FVIII) synthesis, the type of cells producing FVIII within the liver is still unclear. OBJECTIVES: To measure FVIII in extracts of primary liver sinusoidal endothelial cells (LSECs) and hepatocytes, thereby preventing potential bias resulting from the modifications of the cell phenotype that can take place during in vitro culture. METHODS: LSECs were purified by flow cytometry cell sorting on the basis of their coexpression of Tie2 and CD32b. The purity of the cells was controlled by RNA sequencing. FVIII activity (FVIII:C) in extracts of purified cells was measured with a sensitive FVIII chromogenic assay, in which the specificity of the reaction is controlled by neutralization of FVIII activity with specific inhibitor antibodies. RESULTS: The FVIII:C concentration in purified LSECs ranged from 0.3 to 2.8 nU per cell. In contrast, FVIII:C was undetectable in hepatocytes. The intracellular FVIII:C concentrations are therefore at least 10-100-fold higher in LSECs than in hepatocytes. CONCLUSIONS: Our data demonstrate that LSECs, but not hepatocytes, contain measurable amounts of FVIII:C, and suggest that the former are the main cells producing FVIII in the human liver.
Authors: John M Stewart; Alice F Tarantal; Wayne J Hawthorne; Evelyn J Salvaris; Philip J O'Connell; Mark B Nottle; Anthony J F d'Apice; Peter J Cowan; Mary Kearns-Jonker Journal: Xenotransplantation Date: 2014-05-08 Impact factor: 3.907
Authors: Steffen Rosen; Stefan Tiefenbacher; Mary Robinson; Mei Huang; Jaydeep Srimani; Donnie Mackenzie; Terri Christianson; K John Pasi; Savita Rangarajan; Emily Symington; Adam Giermasz; Glenn F Pierce; Benjamin Kim; Stephen J Zoog; Christian Vettermann Journal: Blood Date: 2020-11-26 Impact factor: 22.113