Legacy of blood: does prasugrel inhibit megakaryocytes and do juvenile platelets inherit this inhibition?

Acute coronary syndromes (ACS) are the first cause of death in the Western world. ACS are caused by a ruptured atherosclerotic plaque on the coronary arteries with the formation of a superimposed thrombus which occludes coronary circulation.1 Although novel therapies for atherosclerosis are under study,2 since the establishment of the role of platelets in ACS pathogenesis, platelet inhibition remains the cornerstone of medical therapy for ACS. The P2Y₁₂ receptor inhibitor prasugrel has been demonstrated to reduce recurrent ischemic events in ACS patients.3 Thienopyridines (clopidogrel and prasugrel) are prodrugs requiring biotransformation into an active metabolite. As thienopyridines irreversibly bind and antagonize the P2Y₁₂ receptor for the entire platelet lifespan, the formation of new platelets is required to recover platelet function. The clinical implications of this pharmacological effect are obvious; the slow offset of the antiplatelet effect due to this irreversible P2Y₁₂ receptor inhibition may be potentially problematic in the management of patients who are treated before coronary angiography and then require coronary artery bypass graft surgery or who need other unanticipated surgical procedures or who experiment severe bleeding. Given that we do not have a complete understanding of the offset period after discontinuation of thienopyridines, Baaten et al.4 aimed to investigate the dynamics of platelet functional recovery after prasugrel cessation.

Megakaryocytes in the vascular niche of the bone marrow generate platelets by extending long filaments or pseudopods termed proplatelets which protrude through the vascular endothelium into the sinusoid lumen, where platelets, stemming from the proplatelet tips, are released into the bloodstream. Juvenile or immature platelets (also termed reticulated platelets because the presence of mRNA produces a reticulated pattern after staining with thiazole orange very similar to erythroid reticulocytes) represent the youngest component of the circulating platelet pool in animals (less than 24 h old).5 Juvenile platelets exhibit larger volume, a greater number of dense granules, and more aggregation/reactivity than older circulating platelets. This increased thrombotic potential seems to be due to their content of cytosolic mRNA that is translationally active, which enables the expression of ADP receptors and prothrombotic factors. The number of juvenile platelets inversely correlates with responsiveness to clopidogrel6 and prasugrel,7 and also predicts adverse cardiovascular prognosis and future ACS.8 Whether levels of juvenile platelets are a marker of risk or a risk factor9 for ACS has still not been clarified.

In their present manuscript, Baaten et al.4 studied 16 STEMI patients on aspirin and prasugrel, who suspended prasugrel after one year. The authors first confirmed that P2Y₁₂-inhibited platelets participate less in thrombosis. Second, the authors showed that ADP-induced platelet aggregation (both using light-transmission aggregometry and the Multiplate assay) and thrombus formation in whole blood (using a flow chamber) exhibited gradual recovery during the washout period; this regained platelet reactivity was fully due to increased P2Y12 receptor function because it was completely abrogated with ex vivo spiking with clopidogrel active metabolite. Third, prasugrel discontinuation resulted in the formation of an emerging subpopulation of ADP-responsive platelets (with high expression of IIb/IIIa receptor). These ADP-responsive platelets were specifically juvenile platelets, as determined by both the established thiazole orange and the more sensitive 5′Cy5-oligo-dT probes. During the washout period, juvenile platelets were more reactive than older platelets, as previously discussed. In the most important part of the article, the authors discovered that, during the washout period, platelet reactivity of these juvenile platelets (and also of the older platelets) progressively increased along time: it was minimal at day 2 of offset and it progressively augmented on days 5, 12 and 30. This result strongly suggests partial inhibition of juvenile platelets even after five days of offset (when there is no active metabolite of prasugrel in the blood).

The key finding of the current manuscript4 is that juvenile platelets that are newly synthesized following prasugrel discontinuation paradoxically exhibit impaired platelet reactivity (even though the blood is devoid of prasugrel active metabolite), which strongly suggests a residual inhibitory prasugrel effect on the megakaryocyte level. Juvenile platelets (less than 24 h of life) synthesized during the washout period (when no prasugrel active metabolite is present in the body) should theoretically have full ADP-induced reactivity due to having 100% of uninhibited P2Y₁₂ receptors already at day 2 of offset. The authors, however, found that the functionality of juvenile platelets was impaired at day 2 of offset, with a gradual recovery of reactivity along time. Given that prasugrel does not affect proplatelet formation,10 and also that prasugrel is present in the bone marrow in animal models,1211 the most plausible hypothesis for this impairment in juvenile platelet function is that prasugrel also irreversibly inhibits the P2Y₁₂ receptors of the megakaryocytes13 (and not only of the circulating platelets); those inhibited P2Y12 receptors of the megakaryocyte are subsequently “inherited” by the nascent platelets leading to the production of juvenile platelets with partial pharmacological impairment for aggregation, thus creating a “blood legacy” which prolongs the antithrombotic effects of prasugrel. Over time, newly synthesized and uninhibited P2Y₁₂ receptors would progressively substitute the originally prasugrel-bound P2Y₁₂ receptors in the megakaryocytes, which explains the gradual recovery in the functionality of juvenile platelets at the end of the washout period.

Aggregation of the global platelet population, and not only specifically of the juvenile subpopulation, also supports this inhibitory effect of prasugrel on megakaryocytes. ADP-induced platelet aggregation was minimal at day 2 of offset, and showed a gradual increase at day 5 and a further improvement at day 30. Until now, this could be theoretically explained by the fact that at day 5 there has not been a complete replenishment of the original prasugrel-inhibited platelets by fresh uninhibited platelets. However, platelet reactivity at day 12, despite all the circulating platelets being prasugrel-naïve, was intermediate between day 5 and day 30, while it should have theoretically be completely recovered. This partial impairment of platelet reactivity at day 12 can only be explained if the initial nascent platelets released from megakaryocytes following prasugrel discontinuation exhibit a subpopulation of still inhibited P2Y₁₂ receptors (i.e. synthesized during prasugrel treatment and transferred to nascent platelets during the washout period) and another subpopulation of fully functional P2Y₁₂ receptors (i.e. synthesized after prasugrel interruption).

The main clinical conclusion of this paper relates to the duration of the washout period following prasugrel interruption in order to undergo surgery. Price14 demonstrated that, after prasugrel discontinuation, more than 75% of patients returned to base-line reactivity by washout day 7 compared with day 5 after clopidogrel. These findings are consistent with, and provide pharmacodynamic support for current guidelines regarding the recommended waiting time for surgery after prasugrel discontinuation.15 The impaired reactivity of juvenile platelets during the initial days of the washout period as discovered by Baaten4 may contribute to a risk for surgical bleeding and thus does not advocate a shortening of this period. Therefore, the shortening of this wash-out period is not recommended in elective procedures; in fact, a lengthening of this washout period could even be advocated, thus allowing for a more completely recovery of platelet function from this “legacy” of partial inhibition of P2Y12 receptors. Conversely, emergent procedures represent a completely different clinical situation; in the case of either urgent surgery being required or the need to control severe bleeding, platelet transfusions seem to be an attractive therapeutic strategy for restoring hemostasis. Specifically, Zafar et al.16 estimated that 6 h is the earliest time after a prasugrel loading-dose when added platelets are no longer inhibited by prasugrel’s active metabolite.

In addition, the current manuscript4 sheds light over a controversial phenomenon: the existence of “rebound effect” in platelet reactivity after the interruption of P2Y₁₂ inhibitors. Recurrence of ischemic effects following cessation of P2Y₁₂ inhibitors has been described1917 and “rebound” platelet hyperreactivity after discontinuation of thienopyridines has been proposed to explain it. While it is not surprising that platelet reactivity increases relative to reactivity while on treatment with thienopyridines (i.e. it would return to pre-treatment levels), there is no consistent evidence indicating that platelet reactivity rises beyond base-line levels. Moreover, recent studies designed specifically to address this question have not confirmed the existence of rebound platelet hyperreactivity.2220 The present study does not support the presence of this postulated “rebound” effect. Given that the juvenile platelets population after discontinuation of prasugrel will initially exhibit a combination of both inhibited and uninhibited P2Y₁₂ receptors according to Baaten,4 platelet reactivity during the first weeks of the wash-out period will be partially impaired, thus reducing the probability of the existence of a rebound effect.

In conclusion, this study clearly represents a significant advance in our understanding of platelet function. The authors demonstrate for the first time that prasugrel also inhibits the megakaryocytes and not only the circulating platelets, as had been believed until now. This prasugrel-induced irreversible inhibition of P2Y₁₂ inhibitors at the megakaryocyte level will prolong the impairment in platelet platelet reactivity when the juvenile platelets “inherit” these inhibited P2Y₁₂ receptors, thus prolonging the antiplatelet effect of prasgurel and hence creating a “blood legacy”. The clinical implications of this “legacy” are evident and warrant further investigation.

Footnotes

• Financial and other disclosures provided by the author using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are available with the full text of this paper at www.haematologica.org.