Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats

Abstract

Background: Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function.

Objectives/hypothesis: Evaluate the safety of DAT in healthy cats and compare, ex vivo, platelet-dependent thrombin generation and agonist-induced platelet activation and aggregation in cats treated with clopidogrel, rivaroxaban, or DAT. We hypothesized that DAT would safely modulate agonist-induced platelet activation and aggregation more effectively than single agent treatment.

Animals: Nine apparently healthy 1-year-old cats selected from a research colony.

Methods: Unblinded, nonrandomized ex vivo cross-over study. All cats received 7 days of rivaroxaban (0.6 ± 0.1 mg/kg PO), clopidogrel (4.7 ± 0.8 mg/kg PO), or DAT with defined washout periods between treatments. Before and after each treatment, adenosine diphosphate (ADP)- and thrombin-induced platelet P-selectin expression was evaluated using flow cytometry to assess platelet activation. Platelet-dependent thrombin generation was measured by fluorescence assay. Platelet aggregation was assessed using whole blood impedance platelet aggregometry.

Results: No cats exhibited adverse effects. Of the 3 treatments, only DAT significantly decreased the number of activated platelets (P = .002), modulated platelet activation in response to thrombin (P = .01), dampened thrombin generation potential (P = .01), and delayed maximum reaction velocity (P = .004) in thrombin generation. Like clopidogrel, DAT inhibited ADP-mediated platelet aggregation. However, rivaroxaban alone resulted in increased aggregation and activation in response to ADP.

Conclusion and clinical importance: Treatment combining clopidogrel and rivaroxaban (DAT) safely decreases platelet activation, platelet response to agonists, and thrombin generation in feline platelets more effectively than monotherapy with either clopidogrel or rivaroxaban.

Keywords: cardiology; cardiovascular; clopidogrel resistance; factor Xa inhibitor; hypertrophic cardiomyopathy; saddle thrombus; thromboembolism.

FIGURE 1

Flow diagram illustrating drug administration and blood sampling timeline. Clop, Clopidogrel; DAT, dual antithrombotic treatment; Riva, Rivaroxaban.

FIGURE 2

Scatter dot plots demonstrating platelet inhibition, measured as percent (%) inhibition, in 9 cats after 7 days of rivaroxaban (RIVA), clopidogrel, or dual antithrombotic treatment (DAT) with rivaroxaban and clopidogrel. ADP‐induced whole blood impedance aggregometry was measured as maximum aggregation (Ag max), velocity and area under the cure (AUC). Percent inhibition >0% indicates platelet inhibition (up arrows) and inhibition (%) < 0% indicates platelet activation (down arrows) after 7 days of treatment. Clopidogrel alone and DAT resulted in significant inhibition compared to Riva alone. Riva alone resulted in platelet activation in 7 of the 9 cats. Line represents median and error bars represent interquartile range. *P < .05; **P < .005; ***P < .001.

FIGURE 3

Scatter dot pots demonstrating platelet activation measured as number of P‐selectin positive platelets (%) or response to agonists, ADP or thrombin, measured as fold change in median fluorescence intensity (MFI FC log ₁₀), by flow cytometry in 9 cats treated with 7 days of rivaroxaban (RIVA), clopidogrel (CLOP) or dual antithrombotic treatment (DAT). (A) Only cats treated with DAT had significantly lower number of P‐selectin‐positive (%) resting (unstimulated) platelets compared to single agent treatment with RIVA or CLOP. (B) When platelets were treated with 20 μM ADP, CLOP or DAT significantly modulated response to ADP, while 7 days of RIVA treatment resulted in an increase response to ADP, which were significantly higher on D7 compared to CLOP or DAT. (C) Only DAT modulated platelet response to 0.005 U/mL thrombin, as shown by decreased percentage of P‐selectin‐positive platelets, and MFI FC from D0 to D7. Response to thrombin was significantly higher on D7 in cats treated with RIVA alone. *P < .05; **P < .005; ***P < .001; ****P < .0005.

FIGURE 4

Representative thrombograms demonstrating continuous thrombin generation, measured as fluorescent signal in arbitrary unit (AU), in platelet rich plasma in a cat before and after 3 days of rivaroxaban treatment (RIVA) (A) and 7 days of dual antithrombotic treatment (DAT) with rivaroxaban and clopidogrel (B). Data points were derived from means of duplicate measurements at a single time point for 60 minutes. Note the flattening of thrombogram following DAT compared to rivaroxaban treatment alone.

FIGURE 5

Scatter dot plots demonstrating platelet dependent thrombin generation over time in 9 cats, measured as (A) time (seconds) to maximum thrombin generation (T _max), (B) peak thrombin generation (Relative Fluorescence Unit [RFU]), (C) thrombin generation potential (Area Under the Curve [AUC]) over 1 hour and (D) maximum velocity (V _max, unit/s). After 7 days of treatment with rivaroxaban (RIVA), T _max and peak thrombin generation were significantly modulated from D0 to D7. In addition to T _max and peak thrombin generation, dual agent treatment (DAT) modulated thrombin generation potential and maximum velocity of thrombin generation from D0 to D7. Compared to RIVA, DAT also significantly prolonged T _max compared to clopidogrel (CLOP) alone on D7. *P < .05; **P < .005; ***P < .001.