The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

C Scott Jamison; Bryan F Burkey; Sandra J Friezner Degen

doi:10.1055/s-0038-1656315

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1992; 68(01): 040-047
DOI: 10.1055/s-0038-1656315

Original Article

Schattauer GmbH Stuttgart

The Effects of Vitamin K₁ and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Authors

C Scott Jamison

The Children’s Hospital Research Foundation and the Developmental Biology Graduate Program, University of Cincinnati Medical Center, Cincinnati, Ohio, USA
Bryan F Burkey

The Children’s Hospital Research Foundation and the Developmental Biology Graduate Program, University of Cincinnati Medical Center, Cincinnati, Ohio, USA
Sandra J Friezner Degen

The Children’s Hospital Research Foundation and the Developmental Biology Graduate Program, University of Cincinnati Medical Center, Cincinnati, Ohio, USA

Abstract

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Summary

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K₁ or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K₁ treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K₁ treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with ³⁵S-methionine and immunoprecipitation of ³⁵S-labelled prothrombin demonstrated that, with vitamin K₁ treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K₁ nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K₁ and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

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References

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