4 Results
4.1 Characterization of VKORC1 and VKORC1L1 in vitro
4.1.4 Inhibition profile of various oral anticoagulants in VKORC1 and
To test the susceptibility of either VKORC1 or VKORC1L1 to OACs the VKORC1L1 KO and VKORC1 KO cells were exposed to different drugs. The experiments were per-formed as described in 3.2.2 with K1 as substrate. The FIX activity assessed in cells incubated without drug was set to 100% and measurements from different drug concen-trations were related to the control. The inhibition curves and calculations were made with GraphPad Prism 5.
Coumarin (Figure 22, left graph) showed no ability to reduce FIX activity as normalized FIX activity did not fall below 80%, whereby concentrations up to 1 mM of coumarin were tested. In contrast, 4-hydroxycoumarin (Figure 22, right graph) was capable to in-hibit VKORC1 (measured in VKORC1L1 KO cells) and VKORC1L1 (measured in VKORC1 KO cells), although a diverse inhibition pattern was observed. VKORC1 showed higher susceptibility to 4-hydroxycoumarin compared to VKORC1L1. However, both enzymes were completely inhibited by 1 mM 4-hydroxycoumarin, i.e. no FIX activity was measured.
Coumarin 4-hydroxycoumarin
10-1 100 101 102 103
0 20 40 60 80 100 120
IC50
concentration in µM
Normalized FIX activity (%)
10-1 100 101 102 103
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 22: Inhibition curves of coumarin and 4-hydroxycoumarin tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells.
Measurements were performed in triplicates, values are shown as mean and error bars are rep-resented as SEM.
Next, therapeutically used coumarins were investigated (Figure 23), namely dicoumarol, warfarin, phenprocoumon, and acenocoumarol.
Dicoumarol Warfarin
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Phenprocoumon Acenocoumarol
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 23: Inhibition curves of OACs with 4-hydroxycoumarin backbone tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells.
Measurements were performed in triplicates, values are shown as mean and error bars are rep-resented as SEM.
This group of coumarins showed the same inhibition pattern as obtained for 4-hydroxycoumarin, with VKORC1 being more sensitive than VKORC1L1. However, the difference in sensitivity for dicoumarol and warfarin was greater than for phenprocoumon and acenocoumarol since the curves showed higher divergence for VKORC1 and its paralog. A remark concerns the inhibition pattern of dicoumarol. It was not possible to fit the 4-parameter logistic curve for dicoumarol data, because it appeared to be biphasic.
Therefore, direct measurements were used to estimate the half maximal inhibitory con-centration (VKORC1 49.1% at 5 nM, VKORC1L1 48.2% at 100 nM of mean normalized activities with respect to control).
The other group of OACs, the synthetic indandione derivatives, was investigated as well.
Phenindione and fluindione (Figure 24) both showed high enzyme specificity since VKORC1 was inhibited at markedly lower concentrations than VKORC1L1.
Phenindione Fluindione
10-4 10-3 10-2 10-1 100 101 0
20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100 101 0
20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 24: Inhibition curves of OACs with 1,3-indandione backbone (phenindione and fluindione) tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells.
Measurements were performed in triplicates, values are shown as mean and error bars are rep-resented as SEM.
Synthetic coumarins were originally intended for rodent control. Coumatetralyl and cou-machlor belong to the group of rodenticides of the first generation, whereby bromadio-lone and brodifacoum were developed later and are referred to second generation of rodenticides. Dose-response curves of the before mentioned rodenticides are shown in Figure 25.
Coumatetralyl Coumachlor
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Bromadiolone Brodifacoum
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 25: Inhibition curves of rodenticides belonging to the first (coumatetralyl and coumachlor) and the second generation (bromadiolone and brodifacoum).
Compounds were tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells. Measurements were performed in triplicates, values are shown as mean and error bars are represented as SEM.
Coumatetralyl and coumachlor showed less enzyme specificity compared to OACs since inhibition curves do not differ markedly. Indeed, those two rodenticides are slightly more effective to inhibit VKORC1. In contrast, bromadiolone and brodifacoum had a higher impact on VKORC1L1 compared to VKORC1, although the effect is not as prominent as seen for other anticoagulants (e.g. warfarin or fluindione).
Ferulenol, a natural occurring 4-hydroxycoumarin, was tested in VKORC1 KO and VKORC1L1 KO cells (Figure 26). The inhibition pattern of VKORC1 and VKORC1L1 differed as VKORC1 showed a higher susceptibility to ferulenol treatment than VKORC1L1.
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 26: Inhibition curves of ferulenol tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells.
Measurements were performed in triplicates, values are shown as mean and error bars are represented as SEM.
Half-maximum inhibitory concentration (IC50) was calculated as described by Fregin et al. by solving the 4-parameter logistic equation representing dose-response curve fits for the independent variable (drug concentration), as a function of the dependent variable (normalized activity), and substituting a dependent variable value of 50 since FIX mean activities were normalized to a maximum value of 100% in the absence of a drug (e.g.
warfarin) [30]. Calculations were performed using GraphPad Prism 5 and the results are summarized in Table 2.
Table 2: Overview of OACs and rodenticides with K1 supplementation.
The compound class as well as half maximum inhibitory concentrations (IC50) assessed in VKORC1L1 KO and VKORC1 KO HEK 293T cells are shown. Measurements were performed in triplicates. Enzyme activities assessed up to 1 mM coumarin;**Biphasic dose-response not fit-table with logistic curve; values from n=3 direct measurements (VKORC1 49.1%, VKORC1L1 48.2% mean normalized activities with respect to 0 nM dicoumarol); NA - not applicable, 4HC – 4-hydroxycoumarin, C1 – VKORC1, L1 – VKORC1L1.
Compound Compound class VKORC1
IC50 (nM)
VKORC1L1 IC50 (nM)
Ratio L1/C1
Coumarin Inactive backbone Not
inhibitory*
Not
inhibitory* NA 4-Hydroxycoumarin Active 4HC backbone 1978 25110 12.7 Dicoumarol Naturally occurring and
therapeutic 4HC 5.0** 100.0** 20.0
Warfarin Human therapeutic
4HC and rodenticide 1.9 25.2 13.4
Acenocoumarol Human therapeutic
4HC 1.5 5.8 3.9
Phenprocoumon Human therapeutic
4HC 3.6 10.5 2.9
Phenindione Human therapeutic
indandione 2.9 258.0 89.0
Fluindione Human therapeutic
indandione 4.8 268.5 55.9
Coumatetralyl 1st generation
rodenticide 1.9 2.8 1.5
Coumachlor 1st generation
rodenticide 0.7 1.0 1.4
Bromadiolone 2nd generation
rodenticide 1.6 0.8 0.5
Brodifacoum 2nd generation
rodenticide 0.5 0.4 0.8
Ferulenol Naturally occurring
4HC 0.8 3.0 3.8
In addition, VKOR activity was assessed in VKORC1L1 and VKORC1 KO cells. In Fig-ure 27 the inhibition curves are shown for warfarin, fluindione and coumachlor repre-senting the three most important categories of OACs tested. In comparison to K1, the inhibition curves do not differ markedly when K1>O was used. One exception was ob-served when VKORC1L1 KO cells were tested with fluindione as represented with high-er dose requirements for inhibition.
Warfarin Fluindione
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
10-4 10-3 10-2 10-1 100 101 0
20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Coumachlor
10-4 10-3 10-2 10-1 100
0 20 40 60 80 100
IC50
concentration in µM
Normalized FIX activity (%)
Figure 27: Inhibition curves of warfarin, fluindi-one and coumachlor tested in VKORC1 KO (blue curves) and VKORC1L1 KO (red curves) HEK 293T cells.
In contrast to previous experiments, K1>O (12 µM) was used. Measurements were performed in triplicates, values are shown as mean and error bars are represented as SEM.
IC50 values where calculated for above mentioned compounds using K1>O as a sub-strate and are summarized in Table 3.
Table 3: Overview of compounds tested with supplementation of K1>O.
Half maximum inhibitory concentrations (IC50) were assessed in VKORC1L1 KO and VKORC1 KO HEK 293T cells. C1 – VKORC1, L1 – VKORC1L1
Compound VKORC1
IC50 (nM)
VKORC1L1
IC50 (nM) Ratio L1/C1
Warfarin 2.4 27.0 11.3
Fluindione 203.8 419.6 2.1
Coumachlor 0.7 1.5 2.1
4.1.5 Investigation of antibiotics suspected to interfere with coagulation