Determination of Pleiotropic Effect of Warfarin in VKORC1 and CYP2C9 Genotypes in Patients With Heart Valve Replacement

Figures

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  FIGURE 1. Screenshot of allelic discrimination plot. The three colored dots represent an individual genotype: blue and red dots show homozygotes, whereas green dots show heterozygotes. The encircled dots show not tightly clustered.

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  FIGURE 2. Allelogram showing VKORC1 SNPs. 1639G > A (rs9923231) and C1173T (rs9934438) represented as heterozygous GA and CT phenotypes are in green dots obtained by TaqMan SNP genotyping assay. The genotype of samples of rs9923231 was further confirmed by direct sequencing.

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  FIGURE 3. Allelogram showing 3730G > A (rs7294) SNP. The SNP represented in red dotted area as homozygous GG phenotype obtained by TaqMan SNP genotyping assay. The genotype of samples of rs7294 was further confirmed by direct sequencing.

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  FIGURE 4. Allelogram showing CYP2C9 2* and 3* SNP genotyping in the blue dotted area.

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  FIGURE 5. (A) IL-6 mRNA expression on low warfarin dose. Samples are plotted on the x-axis and RQ on the y-axis. Each bar shows the set of sample values of a detector. The detector for the IL-6 gene is shown in purple. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value). (B) IL-6 mRNA expression on medium warfarin dose. The detector for IL-6 gene is shown in blue. The error bars show the RQ min/max expression levels that measure the standard error of the mean expression level (RQ value). (C) IL-6 mRNA expression on high warfarin dose. Samples are plotted on the x-axis and RQ on the y-axis. Each bar shows the set of sample values of a detector. The detector for the IL-6 gene is shown in blue. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value). (D) TNF-α mRNA expression on high warfarin dose. The detector for the TNF-α gene is shown in purple. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value).

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  FIGURE 6. (A) TNF-α mRNA expression on low warfarin dose. Samples are plotted on the x-axis and RQ on the y-axis. Each bar shows the set of sample values of a detector. The detector for the TNF-α gene is shown in blue. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value). (B) COX-2 mRNA expression on low warfarin dose. The detector for COX-2 gene is shown in green. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value). (C) Association of IL-6, COX-2, and TNF-α serum levels with warfarin dose: Serum IL-6, COX-2, and TNF-α levels were detected by ELISA with different warfarin doses (2.5, 5, 7.5, 10, 12.5, and 15) mg/day and compared with Control. (D) COX-2 mRNA Expression on high warfarin dose. Samples are plotted on the x-axis and RQ on the y-axis. Each bar shows the set of sample values of a detector. The detector for the COX-2 gene is shown in blue. The error bars show the RQ min/max expression levels that measured the standard error of the mean expression level (RQ value).

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  FIGURE 7. (A) Proposed model of warfarin’s pleiotropic effect: Illustrates that warfarin either in a low dose or a high dose inhibits the effect of the vitamin K cycle and thus gamma carboxylation in a controlled manner. Therefore, prolonging the coagulation pathway. The process of gamma carboxylation and inflammatory pathway goes side by side; thus, warfarin if affecting the coagulation pathway also influences the inflammatory pathway by directly inhibiting or stimulating the interacting cytokine levels. (B) Western blot protein expression analysis: IL-6 (23 kDa), TNF-α (17 kDa), and COX-2 (27 kDa) protein expression levels with a higher dose (>10 mg/day) of warfarin drug along with endogenous control GAPDH (37 kDa).

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  FIGURE 8. (A) 3D image of warfarin (green) docked with CYP2C9 (a) wild type, (b) p.Arg144Cys, and (c) p.Ile359Leu. (B) 2D image of warfarin (purple) docked with CYP2C9 (a) wild type, (b) p.Arg144Cys, and (c) p.Ile359Leu.

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  FIGURE 9. 2D image of warfarin (blue) docked with CYP2C9 (A) wild type, (B) p.Arg144Cys, and (C) p.Ile359Leu. Hydrogen bonds are shown in orange, whereas eye-lashes are indicating hydrophobic interactions.

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  FIGURE 10. Binding energies of warfarin with the wild type (A), p.Arg144Cys (B), and p.Ile359Leu (C) derived from 10 ns MD trajectory analysis. The blue line represents Coul–SR interactions, whereas the orange line depicts LJ–SR interactions.