Secreted protein profile from HepG2 cells incubated by S(-) and R(+) enantiomers of chiral drug warfarin - An analysis in cell-based system and clinical samples

Purpose: Warfarin is a commonly prescribed oral anticoagulant with narrow therapeutic index. It interferes with the vitamin K cycle to achieve anti-coagulating effects. Warfarin has two enantiomers, S(-) and R(+) and undergoes stereoselective metabolism, with the S(-) enantiomer being more effective. Our target is to discover the biological differences of the two enantiomers for better warfarin therapy.Experimental design: We reported the extracellular protein profile in HepG2 cells incubated with S(-) and R(+) warfarin, using iTRAQ-coupled 2-D LC-MS/MS. In addition, clinical sera from 30 patients taken warfarin were also analyzed by the same method as a long-term batch.Results: In cell line batch in samples incubated with S(-) and R(+) warfarin alone, inter-α-trypsin inhibitor heavy chain H4, apolipoprotein A-I and α-2-HS-glycoprotein showed variations in cells incubated with S(-) warfarin and R(+) warfarin. For other proteins like α-2-macroglobulin and Fibrinogen γ chain, the expressions each were found to be the same in cells incubated with either S(-) or R(+) warfarin. Clinical results showed the same trends for protein ratio changes.Conclusion and clinical relevance: Our results indicated that those proteins may interfere with blood coagulation process, as well as contribute to the warfarin's side-effect response. Taken together, our findings provided molecular evidence on a comprehensive protein profile on warfarin-cell interaction which may shed new lights on future improvement of warfarin therapy.