Newly Formed Reticulated Platelets Undermine Pharmacokinetically Short-Lived Antiplatelet Therapies

Abstract

Objective: Aspirin together with thienopyridine P2Y₁₂ inhibitors, commonly clopidogrel, is a cornerstone of antiplatelet therapy. However, many patients receiving this therapy display high on-treatment platelet reactivity, which is a major therapeutic hurdle to the prevention of recurrent thrombotic events. The emergence of uninhibited platelets after thrombopoiesis has been proposed as a contributing factor to high on-treatment platelet reactivity. Here, we investigate the influences of platelet turnover on platelet aggregation in the face of different dual-antiplatelet therapy strategies.

Approach and results: Traditional light transmission aggregometry, cytometry, advanced flow cytometric imaging, and confocal microscopy were used to follow the interactions of populations of platelets from healthy volunteers and patients with stable cardiovascular disease. Newly formed, reticulated platelets overproportionately contributed to, and clustered at, the core of forming aggregates. This phenomenon was particularly observed in samples from patients treated with aspirin plus a thienopyridine, but was absent in samples taken from patients treated with aspirin plus ticagrelor.

Conclusions: Reticulated platelets are more reactive than older platelets and act as seeds for the formation of platelet aggregates even in the presence of antiplatelet therapy. This is coherent with the emergence of an uninhibited subpopulation of reticulated platelets during treatment with aspirin plus thienopyridine, explained by the short pharmacokinetic half-lives of these drugs. This phenomenon is absent during treatment with ticagrelor, because of its longer half-life and ability to act as a circulating inhibitor. These data highlight the important influences of pharmacokinetics on antiplatelet drug efficacies, especially in diseases associated with increased platelet turnover.

Keywords: aspirin; blood platelets; platelet aggregation; thienopyridines; thrombosis.

Figure 1.

Ticagrelor reduces aggregation and prevents formation of drug-free platelet cores during aggregate formation in vitro. Platelet-rich plasma (PRP) derived from blood preincubated with aspirin (30 μmol/L) and prasugrel active metabolite (PAM; 3 μmol/L) or ticagrelor (1.35 μmol/L) was mixed in a range of proportions with PRP from blood preincubated with respective vehicles or with ticagrelor (1.35 μmol/L) to reflect mid-dose t=6-h levels. Aggregation in response to (A) ADP 20 μmol/L or (B) arachidonic acid 1 mmol/L was determined by light transmission aggregometry. Data presented as mean±SEM and compared by 2-way ANOVA (n=4, **P<0.01, ***P<0.001). C, Multiple images captured by ImageStreamX of aggregates (mixtures of 85% aspirin+PAM-pretreated platelets or aspirin+ticagrelor pretreated platelets plus 15% uninhibited platelets). Each panel contains columns with following image sets: drug-free (green), inhibited platelets (red), merged image. D, Representative confocal images of aggregates (left) formed from mixtures comprising 85% aspirin+PAM-pretreated platelets or aspirin+ticagrelor-pretreated (green) and 15% uninhibited platelets (red). E, Images were analyzed for size of the uninhibited platelet particles. Data are presented as mean±SEM and compared by t test (n=4; **P<0.01).

Figure 2.

Drug-free platelets restore ex vivo aggregation responses differentially in the presence of prasugrel or ticagrelor. Platelet-rich plasma (PRP) isolated from individuals 6 h after receiving aspirin+prasugrel or aspirin+ticagrelor was mixed with increasing proportions of drug-free platelets and then stimulated. Final aggregation of samples stimulated with (A) ADP (20 μmol/L) or (C) arachidonic acid (1 mmol/L). B and D, Aggregates (%) where cores comprise naive platelets were blind scored and calculated from flow cytometric images of platelet aggregates from corresponding samples. One hundred four aggregates assessed per individual sample, with data presented as mean±SEM and compared by 2-way ANOVA (n=10 samples; *P<0.05, **P<0.01, ***P<0.001). E, Representative flow cytometric imaging (×60 objective) of aggregates formed in response to ADP (20 μmol/L) from 80%:20% mixtures of aspirin+prasugrel–inhibited platelet-rich plasma (PRP) or aspirin+ticagrelor-inhibited PRP obtained 6 h after the last drug dose was administered (red) and drug-free platelets (green). Scale bars, 7 μm.

Figure 3.

Drug-free platelets form cores within aggregates in the presence of prasugrel but not of ticagrelor. Representative confocal images of (A) ADP-stimulated or (B) arachidonic acid–stimulated aggregates formed from 80%:20% mixtures of aspirin+prasugrel–inhibited platelet-rich plasma (PRP) or aspirin+ticagrelor–inhibited PRP obtained 6 h after the last drug dose was administered (red) and drug-free platelets (green), conditions as in Figure 2. Images were analyzed for (C and E) volume of the drug-free platelet particles relative to the total aggregate volume and (D and F) average size of drug-free platelet clusters. Scale bars, 5 μm. Data are presented as mean±SEM and compared by t test (n=7–10; *P<0.05, ***P<0.001).

Figure 4.

Reticulated platelets display elevated reactivity in response to both arachidonic acid (AA) and ADP. The proportion of reticulated platelets among nonaggregated platelets was assessed by flow cytometry in platelet-rich plasma incubated with vehicle, (A) AA, or (B) ADP. C, Representative flow cytometric images of nonreticulated and reticulated (mRNA stain green) single platelets (red), as well as aggregates formed in response to ADP. Scale bars, 7 μm. Data presented as individual data points with overlaid mean±SEM and compared by paired t test (n=6; *P<0.05, ***P<0.001).

Figure 5.

In patients the response of reticulated platelets to ADP is inhibited to a greater extent by ticagrelor than by clopidogrel. The reticulated platelet subpopulation among nonaggregated single platelets was assessed by flow cytometry in platelet-rich plasma incubated with vehicle, ADP, or arachidonic acid (AA). Samples were obtained from patients taking (A) ticagrelor or (B) clopidogrel (in addition to aspirin). Representative flow cytometric images of ADP-stimulated platelet aggregates (platelets red; mRNA green) formed in samples from patients taking aspirin plus (C) ticagrelor or (D) clopidogrel. Scale bars, 7 μm. Data presented as individual data points with overlaid mean±SEM and compared by paired t test (n=9–10; ***P<0.001).