Platelet Activation and Clopidogrel Effects on ADP-Induced Platelet Activation in Cats with or without the A31P Mutation in MYBPC3

Abstract

Background: Clopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects.

Objective: To determine effects of clopidogrel on platelet responsiveness in cats with or without the A31P mutation in the MYBPC3 gene. A secondary aim was to characterize variability in feline platelet responses to clopidogrel.

Animals: Fourteen healthy cats from a Maine Coon/outbred mixed Domestic cat colony: 8 cats homozygous for A31P mutation in the MYPBC3 gene and 6 wild-type cats without the A31P mutation.

Methods: Ex vivo study. All cats received clopidogrel (18.75 mg PO q24h) for 14 days. Before and after clopidogrel treatment, adenosine diphosphate (ADP)-induced P-selectin expression was evaluated. ADP- and thrombin-induced platelet aggregation was measured by optical aggregometry (OA). Platelet pVASP and ADP receptor response index (ARRI) were measured by Western blot analysis.

Results: Platelet activation from cats with the A31P mutation was significantly (P = .0095) increased [35.55% (18.58-48.55) to 58.90% (24.85-69.90)], in response to ADP. Clopidogrel treatment attenuated ADP-induced P-selectin expression and platelet aggregation. ADP- and PGE₁ -treated platelets had a similar level of pVASP as PGE₁ -treated platelets after clopidogrel treatment. Clopidogrel administration resulted in significantly lower ARRI [24.13% (12.46-35.50) to 11.30% (-7.383 to 23.27)] (P = .017). Two of 13 cats were nonresponders based on OA and flow cytometry.

Conclusion and clinical importance: Clopidogrel is effective at attenuating platelet activation and aggregation in some cats. Cats with A31P mutation had increased platelet activation relative to the variable response seen in wild-type cats.

Keywords: Cat; Hypertrophic cardiomyopathy; Platelet hyper-reactivity; Thromboembolism.

Figure 1

Flow cytometric analysis of P‐selectin expression in 14 cats before clopidogrel treatment. (A) Representative histogram of resting platelets (blue) and ADP‐activated platelets (red) indicating the significant increase in platelet P‐selectin expression before clopidogrel treatment. ADP‐induced stimulation resulted in significant increase of percentage (%) in P‐selectin‐positive platelets (B) and P‐selectin MFI (C) as compared to resting platelets. The bolded line represents the mean, and the lower and upper lines represent the standard deviations.

Figure 2

Flow cytometric analysis of P‐selectin expression in 14 cats after clopidogrel treatment (day 15). (A) Representative histogram of resting platelets (blue) and ADP‐activated platelets (red) after clopidogrel treatment. After 14 days of clopidogrel treatment, no significant changes in P‐selectin expression were seen in resting (blue) and ADP‐activated platelets (red). ADP‐induced stimulation did not result in a significant increase of percentage (%) of P‐selectin‐positive platelets (B) and P‐selectin MFI (C) as compared to resting platelets. The bolded line represents the mean and the upper and lower lines represent the standard deviations.

Figure 3

Line plots illustrating the changes in platelet P‐selectin expression in resting and ADP‐stimulated platelets from HO (A, B) and WT (C, D) cats before 14 days of clopidogrel treatment. Medians and interquartile ranges are shown beside the line plots.

Figure 4

Flow cytometric analysis of platelet P‐selectin expression from HO and WT cats after 14 days of clopidogrel treatment. ADP‐induced stimulation did not result in significant increase in percentage (%) of P‐selectin‐positive platelets (A) and P‐selectin MFI (B).

Figure 5

Percentages (%) of maximum aggregation measured by optical aggregometry in 13 cats before (day 0) and after clopidogrel (day 15). The bolded line represents the mean, and lower and upper lines represent the standard deviations.

Figure 6

Representative Western blot analysis of platelet vasodilator‐stimulated phosphoprotein (VASP) phosphorylation in 3 different cats. (A) The molecular weight of total VASP is approximately at 46 kDa (Arrow head). Phosphorylation of serine 239 of VASP (pVASP) caused the protein to migrate to 50 kDa (black arrow). Immunoblots were shown to demonstrate the differences in molecular weight. (B) PGE ₁ serves as a positive control, as the rise in cAMP after PGE ₁ treatment results in strong pVASP expression. Platelets were either unstimulated/resting or treated with 20 μM ADP, 10 μM PGE ₁ (positive control) or a combination of 20 μM ADP and 10 μM PGE ₁. Before clopidogrel treatment, ADP inhibited pVASP and partially inhibited PGE ₁‐induced pVASP. (C) After clopidogrel treatment, irreversible antagonism of P2Y₁₂ abolished the effect of ADP on PGE ₁‐induced pVASP. The degree of VASP phosphorylation in resting and ADP‐treated platelets also was similar to positive control.

Figure 7

(A) Relative phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) quantified by Western blot analysis in platelets from 12 cats before (day 0) and after 14 days (day 15) of clopidogrel treatment. (B) ADP receptor response index (ARRI) in 12 cats before (day 0) and after 14 days (day 15) of clopidogrel treatment. The bolded line represents the mean, and the upper and lower lines represent the interquartile range.

Figure 8

Line plots illustrating the changes in P‐selectin expression before and after 14 days of clopidogrel treatment in 11 responders and 2 nonresponders. On day 15, the differences in P‐selectin mean MFI between resting and ADP‐stimulated platelets in nonresponders were higher than those on day 0. All responders but one had an attenuated response to ADP stimulation.