Signalment and patient demographics
A total of 51 client-owned cats with HCM were enrolled and 49 cats completed the study. Of the 49 cats, 39 were Domestic Short Hair, 7 were Domestic Long Hair, 2 were Sphynx, 1 was Maine Coon, and 1 was Siamese. One cat was excluded from the study due to poor animal compliance resulting in the failure to administer clopidogrel at home. Another cat was excluded as there was no concentration of clopidogrel detected in plasma after completion of the study, suggesting either poor animal compliance or a failure to follow the drug administration protocol. The mean and standard deviation (SD) of age was 6.5 (+/- 4.5) years old with 35 castrated males and 14 spayed females. The mean body weight (+/- SD) was 5.6 (+/- 1.4) kg. No adverse effects of clopidogrel administration were reported by the owners throughout the study period.
Missense SNPs in P2RY1, P2RY12, and CYP2C41 genes
In the present study, the frequencies of the missense SNPs in the P2RY1, P2RY12, and CYP2C41 genes were successfully determined in all cats and the results are listed in Table 1.
Table 1: Frequencies of genetic polymorphisms in three targeted genes (P2RY1, P2RY12, and CYP2C41).
Genes
|
Mutations
|
Polymorphisms
|
Distributions n/49 (%)
|
P2RY1
|
p.A236G
|
C/C (wildtype)
C/G
G/G
|
16/49 (32.7)
25/49 (51.0)
8/49 (16.3)
|
P2RY12
|
p.V34I
|
G/G (wildtype)
G/A
A/A
|
23/49 (46.9)
22/49 (44.9)
4/49 (8.2)
|
CYP2C41
|
p.H231R
|
G/G (wildtype)
G/A
A/A
|
20/49 (40.9)
23/49 (46.9)
4/49 (8.2)
|
Adenosine diphosphate-induced platelet aggregation is inhibited by clopidogrel therapy
The mean area under the curve (AUC), maximum aggregation (AU), and aggregation velocity (AU/min) before clopidogrel therapy were 140 +/- 40.2 (AU*min), 225.3 +/- 63.3 (AU), and 42.9 +/- 14.2 (AU/min), respectively. After clopidogrel therapy, mean AUC, aggregation, and velocity were significantly reduced at 35.9 +/- 15.2 (AU*min), 73.3 +/- 28.9 (AU), 7.4 +/- 2.3 (AU/min), respectively (Figure 1). The percent inhibition of AUC, aggregation, and aggregation velocity were 73.6 (+/- 11.3) %, 66.6 (+/- 12.0) %, and 81.5 (+/- 7.8) %, respectively. The Multiplate® parameters were all significantly decreased after 10 – 14 days of clopidogrel therapy compared to pre-treatment values (P < 0.0001) (Figure 1).
Clopidogrel inhibits platelet P2Y12 by activating cyclic adenosine monophosphate and subsequent phosphorylation of vasodilator-stimulated phosphoprotein in cats with HCM
Irreversible inhibition of platelet ADP receptor, P2Y12, was assessed by measuring the degree of phosphorylation of vasodilator-stimulated phosphoprotein (P-VASP) within platelets in the presence of prostaglandin E1 (PGE1), adenosine diphosphate (ADP) or both. PGE1 treated platelets served as positive control. P-VASP expression was reported with mean fluorescence intensity (MFI) measured by flow cytometry. Prior to starting clopidogrel administration, significantly lower levels of P-VASP expression was detected in ADP-activated platelets (MFI 1238, interquartile rage [IQR] 963.8 – 1632) compared to PGE1-treated platelets (MFI 3906, IQR 3201 – 5422) (P < 0.0001) (Figure 2a). P-VASP expression was also significantly lower in PGE1 and ADP-treated platelets (MFI 2924, IQR 2165 – 3631) than PGE1-treated platelets (P < 0.0001) but higher than ADP-treated platelets (P < 0.0001) (Figure 2a). These results indicate that activation of the P2Y12 receptor by ADP inhibits the adenyl cyclase-cyclic AMP pathway leading to lower P-VASP in platelets. After clopidogrel therapy, the P-VASP in platelets activated by ADP in the presence of PGE1(MFI 3925 IQR 2785 – 4680) was similar to that in PGE1-treated platelets (MFI 4344, IQR 3054-5164) (P = 0.16) (Figure 2b) indicating adequate inhibition of P2Y12 receptor. Platelets activated by ADP maintained the significantly lower level of P-VASP (MFI 1229, IQR 2785 – 4680) than platelets activated by PGE1 and both PGE1 and ADP (P < 0.0001) (Figure 2b). The mean platelet reactivity index (PRI) calculated from P-VASP expression level was compared before and after clopidogrel therapy. The median (IQR) PRI of P-VASP before and after clopidogrel therapy were 27.0% (16.9 – 38.2) and 6.1% (-0.85 – 12.9), respectively. The PRI was significantly lower in platelets measured after clopidogrel therapy than those measured before the treatment (P < 0.0001) (Figure 2c).
Clopidogrel inhibits ADP-mediated alpha-granule secretion in cats with HCM
Platelet surface P-selectin expression was measured by flow cytometry to determine if clopidogrel modulates alpha granule secretion. The percentage of platelets positive with P-selectin expression and the MFI of P-selectin expression were compared between resting (unstimulated) and ADP-stimulated platelets. Prior to clopidogrel therapy, the median percentage of platelets with P-selectin expression were 42.1% (25.3 – 61.2) in resting platelets and 61.7% (44.6 – 72.0) in ADP-treated platelets. The median MFI of P-selectin expression was 1364 (1017 – 2003) in resting platelets and 2378 (1830 – 3151) in ADP-stimulated platelets. The ADP stimulation resulted in a significantly higher percentage of P-selectin positive platelets (P < 0.0001) and P-selectin MFI (P < 0.0001) (Figure 3a).
After clopidogrel therapy, the median percentage of resting platelets with P-selectin expression [30.7% (15 – 48.6)] was not significantly different from that of ADP-activated platelets [30% (14.6 – 46.1)] (P=0.4). However, P-selectin MFI in ADP-activated platelets [967 (853 – 1242)] were significantly higher than in resting platelets [960.5 (814 – 1126)] (P = 0.0084) (Figure 3b). To determine platelet reactivity to ADP, the percent change in P-selectin MFI with or without ADP stimulation was calculated by dividing the difference between MFI with ADP stimulation and resting MFI (MFI without stimulation) by the resting MFI. The MFI percent change was then compared before and after clopidogrel therapy, and it was found to be significantly lower after clopidogrel therapy than before clopidogrel therapy (P < 0.0001) (Figure 3c).
Clopidogrel and clopidogrel metabolite concentrations do not correlate with clopidogrel-mediated platelet inhibition
Concentrations of clopidogrel, clopidogrel carboxylic acid, and derivatized clopidogrel active metabolite (CAM-D) were measured in plasma obtained from cats approximately two hours after the last dose of clopidogrel. CAM-D metabolic ratio was calculated based on the molarity of clopidogrel, clopidogrel carboxylic acid, and CAM to minimize the impact of variable absorption of parent drugs.25 The mean (+/- SD) concentrations of clopidogrel, clopidogrel carboxylic, and CAM-D were 33.8 +/- 33.9 ng/mL, 612.8 +/- 1303.3 ng/mL, and 15.2 +/- 13.9 ng/mL, respectively. The mean (+/- SD) of CAM-D metabolic ratio were 0.8 (+/- 0.96) %. The correlation analyses between the concentrations of clopidogrel and clopidogrel metabolites and platelet function were performed, and weakly or moderately significant correlations were observed between CAM-D metabolic ratio and platelet function assessed by Multiplate and P-selectin expression (Supplement Table 1).
Genetic polymorphisms alter inhibition of platelet aggregation by clopidogrel in cats with HCM
Statistical analyses were performed to determine the associations between genetic polymorphisms in P2RY1, P2RY12, CYP2C41 genes, and clopidogrel-mediated platelet inhibition. Platelet inhibition by clopidogrel was measured as percent inhibition of AUC, maximum aggregation, and aggregation velocity based on the results of whole blood platelet aggregometry. The percent inhibitions of AUC (P = 0.012), maximum aggregation (P = 0.0084), and aggregation velocity (P = 0.037) were all significantly lower in cats with P2RY1:A236G variants (combined homozygous and heterozygous variants) compared to cats with P2RY1:A236G wildtype. When the percent inhibition of AUC was compared among P2RY1:A236G wildtype and heterozygous and homozygous genotypes, the percent inhibition was significantly different overall (P = 0.034), and the percent inhibition between the wildtype and heterozygous remained significantly different as well (P = 0.025). However, no significant difference of the percent inhibition was noted between the wildtype and homozygous genotypes (P = 0.85) and between heterozygous and homozygous genotypes (P > 0.99) (Table 2, Figure 4a and d). The same comparisons were made for percent inhibition of maximum aggregation and aggregation velocity. When the percent inhibition of aggregation was compared among P2RY1:A236G wildtype and heterozygous and homozygous genotypes, they remained significantly different overall (P = 0.019), and the percent inhibition between the wildtype and heterozygous remained significantly different (P = 0.015). However, no significant difference was noted between the percent inhibition of wildtype and homozygous genotypes (P = 0.74) as well as between heterozygous and homozygous genotypes (P > 0.99) (Table 2, Figure 4b and e). When the percent inhibition of aggregation velocity was compared among P2RY1:A236G wildtype and heterozygous and homozygous genotypes, they were not significantly different (P = 0.093) (Table 2, Figure 4c and f).
Table 2. The percent inhibition (interquartile range) of area under the curve (AU*min), aggregation unit (AU), and aggregation velocity (AUC/min), measured by whole blood aggregometry, in cats with the wildtype (WT) and the mutant genotype (both homozygous and heterozygous) in various genotypes (P2RY12:V34I, P2RY1:A236G, and CYP2C41:H231R).
Genotype
|
Nucleotide
|
AUC
(AU*min) (IQR)
|
P-value (overall)
|
P-value
|
Aggregation unit (AU) (IQR)
|
P-value (overall)
|
P-value
|
Aggregation velocity (AU/min) (IQR)
|
P-value (overall)
|
P2RY1:A236G
|
c/c
|
81 (74.8 - 85.7)
|
0.03
|
c/c vs c/g: 0.025
|
76 (71.6 - 81.6)
|
0.019
|
c/c vs c/g: 0.015
|
86 (82.8 - 88.1)
|
0.093
|
|
c/g
|
77.7 (71.4 - 80.8)
|
|
c/c vs g/g: 0.85
|
66.2 (52.3 - 74.2)
|
|
c/c vs g/g: 0.74
|
83.1 (71.7 - 86.5)
|
|
|
g/g
|
85.2 (82.5 - 89.6)
|
|
c/g vs g/g: >0.99
|
71.1 (54.7 - 80.8)
|
|
c/g vs g/g: >0.99
|
84.8 (77.1 - 87.3)
|
|
P2RY12:V34I
|
g/g
|
72.9 (63.1 - 80.3)
|
0.034
|
g/g vs g/a: 0.96
|
66 (53.2. - 74.1)
|
0.019
|
g/g vs g/a: 0.36
|
82.5 (75.4 - 87.4)
|
0.35
|
|
g/a
|
77.7 (71.4 - 80.8)
|
|
g/g vs a/a: 0.031
|
72 (64.8 - 76.3)
|
|
g/g vs a/a: 0.024
|
83.8 (78.5 - 86.4)
|
|
|
a/a
|
85.2 (82.5 - 89.6)
|
|
g/a vs a/a: 0.13
|
81 (79 - 86.5)
|
|
g/a vs a/a: 0.21
|
85.6 (85.6 - 92.3)
|
|
CYP2C41:H231R
|
a/a
|
80.34 (65.1 - 83.0)
|
0.28
|
|
74.2 (60.1 - 80.4)
|
0.49
|
|
84.4 (82.4 - 87.7)
|
0.38
|
|
a/g
|
73.8 (61.7 - 80.9)
|
|
|
70 (55 - 75.8)
|
|
|
81.2 (71.8 - 86.5)
|
|
|
g/g
|
74.8 (69.5 - 78.3)
|
|
|
68.3 (60.2 71.6)
|
|
|
84.6 (82.1 - 85.6)
|
|
AUC; area under the curve, IQR; interquartile range
The percent inhibition of maximum aggregation was significantly lower in cats with the P2RY12:V34I variants (combined homozygous and heterozygous) than cats with P2RY12:V34I wildtype (P = 0.019), but this significant difference was not found in AUC (P = 0.068) and aggregation velocity (P = 0.57) (Figure 5a-c). When the percent inhibition of AUC was compared among P2RY12:V34I wildtype and heterozygous and homozygous genotypes, it remained significantly different among the three groups (P = 0.034). The percent inhibition between the wildtype and homozygous also remained significantly different (P = 0.031), whereas no significant difference was noted between the percent inhibition of wildtype and heterozygous genotypes (P = 0.96) as well as between heterozygous and homozygous genotypes (P = 0.13) (Table 2, Figure 5d). The same comparisons were made for percent inhibition on maximum aggregation and aggregation velocity among P2RY12:V34I genotypes. When the percent inhibition of maximum aggregation was compared among P2RY12:V34I wildtype and heterozygous and homozygous genotypes, it remained significantly different (P = 0.019), and the percent inhibition between the wildtype and homozygous remained significantly different (P = 0.024). However, no significant difference was noted between the percent inhibition of wildtype and heterozygous genotypes (P = 0.36) as well as between heterozygous and homozygous genotypes (P = 0.21) (Table 2, Figure 5e). When the percent inhibition of aggregation velocity was compared among P2RY12:V34I wildtype and heterozygous and homozygous genotypes, the percent inhibition among the three groups was not significantly different (P = 0.35) (Table 2, Figure 5f).
No other missense SNPs were significantly associated with diminished percent inhibition determined based on platelet aggregometry (Table 2). False discovery rate (FDR) analysis was performed to adjust the multiple comparisons with three genetic polymorphisms. The percent inhibition of AUC (P =0.035) and maximum aggregation (P = 0.025) remained significantly lower in cats with P2RY1:A236G variants than the wildtype. On multivariable regression analysis (comparison between wildtype and mutants), only P2RY1:A236G remained significantly associated with AUC (P = 0.012), maximum aggregation (P = 0.015), and aggregation velocity (P = 0.032).
Repeated measure analysis with the linear mixed model was conducted to assess the fixed effect of P2RY1:A236G and P2RY12:V34I variants, time, and the mixed effects of these genetic variant status and time on platelet activation before and after clopidogrel therapy assessed by P-selectin and P-VASP expression. Statistically significant effect with regards to P2RY1:A236G status on P-selectin expression (Figure 6a), but not on the PRI derived from P-VASP expression (Figure 6b), was detected. The same analysis was performed for P2RY12:V34I (Supplement Figure 1) and CYP2C41 variants but no significant effects with regards to these SNPs on P-selectin and P-VASP expressions were identified.
Genotypes did not alter clopidogrel metabolite concentrations
Association analysis between the genetic polymorphisms and clopidogrel, clopidogrel metabolites, and CAM-D metabolic ratio were performed. There were no significant differences in clopidogrel, clopidogrel metabolites (carboxylic acid, and CAM-D), and CAM-D metabolic ratio between the wildtype and heterozygous and homozygous variants of CYP2C:H231R (Supplement Figure 2). No other missense SNPs in P2RY12 and CYP2C genes were significantly associated with the concentrations of clopidogrel, clopidogrel metabolites, and CAM-D metabolic ratio.