VKORC1 Pharmacogenetics and Pharmacoproteomics in Patients on Warfarin Anticoagulant Therapy: Transthyretin Precursor as a Potential Biomarker

Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke.We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation.This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism

Prevention of Liver Fibrosis by Triple Helix-Forming Oligodeoxyribonucleotides Targeted to the Promoter Region of Type I Collagen Gene

Hepatic fibrosis leading to cirrhosis remains a global health problem. The most common etiologies are alcoholism and viral infections. Liver fibrosis is associated with major changes in both quantity and composition of extracellular matix and leads to disorganization of the liver architecture and irreversible damage to the liver function. As of now there is no effective therapy to control fibrosis. The end product of fibrosis is abnormal synthesis and accumulation of type I collagen in the extracellular matrix, which is produced by activated stellate or Ito cells in the damaged liver. Therefore, inhibition of transcription of type I collagen should in principle inhibit its production and accumulation in liver. Normally, DNA exists in a duplex form. However, under some circumstances, DNA can assume triple helical (triplex) structures. Intermolecular triplexes, formed by the addition of a sequence-specific third strand to the major groove of the duplex DNA, have the potential to serve as selective gene regulators. Earlier, we demonstrated efficient triplex formation between the exogenously added triplex-forming oligodeoxyribonucleotides (TFOs) and a specific sequence in the promoter region of the COL1A1 gene. In this study we used a rat model of liver fibrosis, induced by dimethylnitrosamine, to test whether these TFOs prevent liver fibrosis. Our results indicate that both the 25-mer and 18-mer TFOs, specific for the upstream nucleotide sequence from −141 to −165 (relative to the transcription start site) in the 5′ end of collagen gene promoter, effectively prevented accumulation of liver collagen and fibrosis. We also observed improvement in liver function tests. However, mutations in the TFO that eliminated formation of triplexes are ineffective in preventing fibrosis. We believe that these TFOs can be used as potential antifibrotic therapeutic molecules

Effect of <i>VKORC1</i> diplotypes [H1H1 (n = 24), H1H7/H1H9 (n = 18) and H7H7/H7H8H9 (n = 9)] on (A) free T₃ (FT₃), (B) total T₃ (TT₃), (C) free T₄ (FT₄), and (D) total T₄ (TT₄) levels in patients receiving warfarin.

<p>Patients harboring the H1H1 diplotype group had significantly lower FT₃ and TT₃ levels compared with patients carrying the high dose (H1H7/H1H9) associated diplotype groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015064#pone-0015064-g003" target="_blank">Figures 3A and B</a>; P<0.05 in each case). These findings probably reflect the higher levels of TTR expressed in patients carrying the low dose H1H1 diplotype, which probably leads to lower levels of FT₃ as well as TT₃.</p

Immunoblot expressions of TTR levels in HepG2 cells exposed to low (1.0 µg/mL) and high (10 ug/mL) concentrations of warfarin for 24 hrs.

<p>TTR expression levels were determined in (A) serum-rich, and (B) serum-free conditions to exclude the possibility of false positive results. The data (mean ± S.E.) represents expression ratios of biological replicates normalized to GAPDH. The decrease in expression of TTR was significantly greater in HepG2 cells exposed to higher concentration (10 ug/mL) of warfarin under serum-rich conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015064#pone-0015064-g004" target="_blank">Fig. 4A</a>; P = 0.01).</p

OptEIA™ quantification of IL-6 from low- and high-dose warfarin treated patients.

<p>Box plot shows the median IL-6 concentrations of low-dose (3.3 pg/mL; range: 0.14 to 19.05 pg/mL) and high-dose (4.4 pg/mL; range: 1.4 to 117.16 pg/mL) warfarin treated groups. Patients on high-dose warfarin (N = 37) showed significantly increased levels of IL-6 in comparison with those on low-dose (N = 39) warfarin [<i>P</i> = 0.018].</p

Influence of <i>VKORC1</i> diplotypes on TTR precursor level in warfarin treated Asian patients (N = 51).

<p>Patients carrying the H1H1 diplotype showed significantly higher levels of TTR precursor compared to patients harboring the H1H7/H1H9 (P = 0.001) and H7H7/H7H8H9 diplotypes (P<0.0001).</p

The distribution of <i>VKORC1</i> diplotypes in patients receiving low-dose (N = 25) and high-dose (N = 28) warfarin.

<p>The distribution of <i>VKORC1</i> diplotypes in patients receiving low-dose (N = 25) and high-dose (N = 28) warfarin.</p

Differential expression of plasma protein levels in patients treated at low- and high-dose warfarin identified by iTRAQ-coupled LC-MS/MS analysis.

<p>A total of 163 proteins were identified and 11 proteins with significant expression levels were identified based on ProtScore with a cut-off value of 2.0 at 99% confidence value. Of the eleven proteins, the median expression level of transthyretin precursor was highly significant between patients receiving low- and high-dose warfarin group (low-dose: 1.53, range: 0.828 to 3.83; and high-dose: 0.818, range: 0.534 to 1.483; P<0.0001).</p