Ex vivo synergistic effects of apixaban with dual anti-platelet therapy (DAPT) in lowering platelet reactivity and thrombin generation (SEARCH)

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Abstract Direct oral anticoagulants such as apixaban are increasingly being evaluated clinically for the secondary prevention of cardiovascular events; however, their effects on platelet function in combination with dual anti-platelet therapy (DAPT) have not been fully investigated. The purpose of this translational, in vitro study was to determine if apixaban via inhibition of thrombin generation exhibits synergistic activity with DAPT to reduce platelet reactivity. Consented subjects with a prior history (<12 mo) of ACS on DAPT regime with aspirin and clopidogrel (n=15; DAPT-C) or aspirin and ticagrelor (n=15; DAPT-T) were recruited, along with the age-matched healthy subjects as controls. Enrolled DAPT subjects had taken their prescribed regimen >7 days prior to blood collection. Platelet-rich plasma from TSC anticoagulated blood was prepared and treated in vitro with nothing, a carrier control or apixaban (40, 90 and 220 ng/mL). The range of 40 to 220 ng/mL brackets the expected apixaban exposure at steady state with all three approved regimens with the 40 ng/mL treatment corresponding to <5th percentile for the 2.5 mg bid dose, the 90 ng/mL corresponding to Cmax after the 2.5 mg bid or to Cmin after the 5 mg bid dose, and the 220 ng/mL corresponding to the Cmax after 10 mg bid dose. Platelet aggregation was measured by light transmission aggregometry (LTA) with tissue factor (TF) as agonist. Platelet p-selectin expression was measured by flow cytometry and thrombin generation was quantified. TF agonist was chosen to evaluate endogenous thrombin effects via Factor Xa activation (fXa). The CaCl2 concentration in the TF was titrated in the presence of peptide GPRP which minimized fibrin generation. The baseline maximal aggregation (MA) response was similar for both DAPT-T and DAPT-C (64%). Compared to DAPT alone, 90 and 220 ng/mL apixaban treatments decreased MA from 64% to 36% and 17% in the DAPT-T group and from 64% to 28% and 9% in the DAPT-C group (p<0.009), respectively. Platelet P-selectin expression decreased by 53% in the DAPT-T group with 220 ng/mL apixaban treatment (p<0.02) and in the DAPT-C group by 70% and 76% with 90 and 220 ng/mL apixaban treatment (p<0.004), respectively, compared to DAPT alone. Apixaban treatment (90 and 220 ng/mL) significantly increased thrombin generation lag time and time-to-peak results and significantly decreased peak thrombin in both DAPT groups (p<0.05). ACS patients on a DAPT regimen were susceptible to thrombin-mediated platelet activation via fXa. Apixaban treatment in vitro caused a larger reduction in thrombin-mediated platelet activation in the clopidogrel group compared to the ticagrelor group, consistent with ticagrelor having a more potent anti-platelet effect compared to clopidogrel. The in vitro addition of apixaban that corresponded to currently approved dosing regimens and at plasma drug levels routinely achieved demonstrated synergy with DAPT to reduce platelet reactivity and thrombin generation. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Bristol-Myers Squibb