title: Factor VIIa binding and internalization in hepatocytes.
abstract: The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization of FVIIa in liver cells using a human hepatoma cell line (HEPG2), and primary rat and human hepatocytes as cell models. 125I-FVIIa bound to HEPG2 cells in a time- and dose-dependent manner. Anti-tissue factor antibodies reduced the binding by about 25%, whereas 50-fold molar excess of unlabeled FVIIa had no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2 cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes. In contrast, annexin V, which binds to phosphatidylserine, blocked the binding and internalization. Consistent with this, binding of gla-domain-deleted FVIIa to hepatocytes was markedly diminished. In summary, the data presented herein reveal differences between HEPG2 cells and primary liver cells in FVIIa binding and internalization, and suggest that the rapid turnover of membrane and not a receptor-mediated endocytosis may be responsible for internalization of FVII/FVIIa in primary hepatocytes.
authors.affiliation: Health Care Discovery, Novo Nordisk, Maalov, Denmark.
authors.forename: G
authors.initials: G
authors.lastname: Hjortoe
authors.forename: B B
authors.initials: BB
authors.lastname: Sorensen
authors.forename: L C
authors.initials: LC
authors.lastname: Petersen
authors.forename: L V M
authors.initials: LV
authors.lastname: Rao
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D015942:Factor VIIa / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D010718:Phosphatidylserines; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D013925:Thromboplastin / Q000502:physiology
chemical_list: D017304:Annexin A5; D010718:Phosphatidylserines; D013925:Thromboplastin; D015942:Factor VIIa