Antiplatelet effects of aspirin vary with level of P2Y₁₂ receptor blockade supplied by either ticagrelor or prasugrel

‘Dual antiplatelet therapy’, comprising aspirin and a P2Y₁₂ receptor inhibitor, is firmly established for the secondary prevention of thrombotic events with the rationale that they inhibit thromboxane A₂- (TxA₂) and ADP-P2Y₁₂-dependent pathways of platelet activation, respectively. We have recently reported that strong P2Y₁₂ receptor blockade alone, however, can provide inhibition of platelet aggregation to a broad range of agonists that is not further enhanced by aspirin [1]. While the clinical relevance of these observations is unclear, we have speculated that administration of aspirin to individuals achieving sufficiently strong P2Y₁₂ receptor blockade, has the potential to produce effects secondary to inhibition of cyclo-oxygenase at non-platelet sites, without providing additional antithrombotic activity [2]. The degree of P2Y₁₂ pathway blockade that is achieved in clinical practice, however, is quite variable [3], reflecting both the choice of drug and large inter-individual differences in drug metabolism [4]. Here, we have extended our previous observations of the interactions between aspirin and strong P2Y₁₂ blockade [1] by considering what additional anti-aggregatory effects aspirin provides when only partial P2Y₁₂ blockade is achieved, which may better reflect the clinical reality of these drugs.

We measured aggregation responses of platelet-rich plasma (PRP), using 96-well plate light transmission aggregometry, as previously described [1]. Blood was collected by venepuncture into tri-sodium citrate (0.32% final) from healthy volunteers who had abstained from non-steroid anti-inflammatory drug consumption for 14 days. To model the effects of P2Y₁₂ blockade and cyclo-oxygenase inhibition in vitro, PRP was incubated with the irreversible thienopyridine P2Y₁₂ inhibitor, prasgurel-active metabolite (PAM; 0.1–10 μmol L⁻¹), the reversible, cyclo-pentyl-triazolo-pyrimidine P2Y₁₂ antagonist, ticagrelor (0.1–10 μmol L⁻¹) and/or aspirin (1–100 μmol L⁻¹) for 30 min at 37 °C before addition of the agonist. Additional methodological details are provided as online supplementary information.

Using this approach we determined the inhibitory potencies of ticagrelor, PAM and aspirin against aggregations induced by ADP (0.625–20 μmol L⁻¹), the thromboxane-mimetic U46619 (0.1–30 μmol L⁻¹) and arachidonic acid (0.1–1 mmol L⁻¹). Both ticagrelor and PAM caused concentration-dependent inhibition of aggregations induced by ADP (Fig. S1), with ticagrelor displaying greater potency than PAM (log IC₅₀ values for inhibition of aggregation to 20 μmol L⁻¹ ADP: ticagrelor, −6.46; PAM, −5.64). Notably, the potency of ticagrelor, but not PAM, varied with the concentration of ADP (e.g. log IC₅₀ values for inhibition of aggregation to 2.5 μmol L⁻¹ ADP: ticagrelor, −7.05; PAM, −5.63). Aspirin, at concentrations up to 100 μmol L⁻¹, was without significant effect upon ADP-induced aggregations. Ticagrelor and PAM, but not aspirin, produced complete, concentration-dependent inhibition of platelet aggregations induced by U46619 (Fig. S2) with similar potency as for inhibition of ADP-induced aggregations (log IC₅₀ values for inhibition of aggregation to 30 μmol L⁻¹ U46619; ticagrelor, −6.24; PAM, −5.25). This is consistent with earlier reports that the second, irreversible wave of platelet aggregation that follows TP receptor activation is dependent upon platelet-derived ADP acting upon platelet P2Y₁₂ receptors [5]. Ticagrelor and PAM, as well as aspirin, also produced complete, concentration-dependent inhibitions of platelet aggregations induced by AA (Fig. S3; log IC₅₀ values for inhibition of aggregation to 1 mmol L⁻¹ AA: ticagrelor, −6.88; PAM, −6.00; aspirin, −5.20). When the production of TxA₂ accompanying platelet aggregation induced by AA was measured (by immunoassay for the levels of TxB₂), ticagrelor, PAM and aspirin were all found to cause concentration-dependent reductions in TxA₂ formation (Fig. S3; log IC₅₀ values for inhibition of aggregation to 1 mmol L⁻¹ AA: ticagrelor, −6.88; PAM, −5.985; aspirin, −5.51). This is in agreement with our early findings [1, 6], and indicates that P2Y₁₂ receptors are important in supporting both the activation mechanisms of platelets that drive TxA₂ formation and pathways downstream of the TP receptor.

To explore further the interactions between P2Y₁₂ receptors and the TxA₂ system in platelets, we examined the effect of aspirin on aggregation induced by a range of agonists in the presence of concentrations of ticagrelor or PAM producing different degrees of partial P2Y₁₂ blockade. From the inhibitor curves to ADP described above, concentrations of ticagrelor showing approximate IC₅ (0.03 μmol L⁻¹), IC₁₀ (0.1 μmol L⁻¹), IC₅₀ (0.3 μmol L⁻¹) and IC₉₀ effects (3 μmol L⁻¹) were combined with 30 μmol L⁻¹ aspirin, a concentration approximately equivalent to the peak plasma levels following ingestion of a 75–100 mg dose of aspirin. In these experiments, responses to AA (Fig. 1A) were found to be completely inhibited by ticagrelor at the higher two concentrations (representing ∼IC₅₀ and IC₉₀ for ADP-induced aggregation) without the need for aspirin. The lower two concentrations of ticagrelor (representing ∼IC₅ and IC₁₀ for ADP-induced aggregation) also produced substantial, but incomplete, inhibitions (Table S1). Ticagrelor also inhibited aggregations induced by ADP, collagen, epinephrine, the PAR-1 activating peptide, TRAP-6 (SFLLRN-amide) and U46619, in a concentration-dependent manner (Fig. 1).

Figure 1.

Concentration-response curves for the inhibition by combinations of ticagrelor (0.3 or 3 μmol L⁻¹) and aspirin (30 μmol L⁻¹) of platelet aggregations induced by (A) arachidonic acid (AA), (B) ADP, (C) collagen, (D) epinephrine, (E) TRAP-6 or (F) U46619, and of platelet TxB₂ formation induced by (G) AA and (H) collagen. n = 4.

When applied alone, aspirin inhibited aggregations induced by AA (Fig. 1A), collagen (Fig. 1C) and epinephrine (Fig. 1D), and showed a weak effect against ADP (Fig. 1B) but did not alter aggregations induced by TRAP-6 (Fig. 1E) or U46619 (Fig. 1F). In contrast, aspirin did augment the anti-aggregatory effects of the lower three concentrations of ticagrelor (achieving incomplete P2Y₁₂ inhibition) against both collagen (Fig. 1C) and epinephrine (Fig. 1D). In the presence of the highest tested concentration of ticagrelor (3 μmol L⁻¹; ∼IC₉₀ for ADP-induced aggregation), aspirin provided no additional anti-aggregatory effects to those of ticagrelor against aggregations to any agonist (Fig. 1). In agreement with earlier experiments, production of TxA₂ induced by either AA (Fig. 1G) or collagen (Fig. 1H) was partially inhibited by 0.3 μmol L⁻¹ ticagrelor (∼IC₅₀ for ADP-induced aggregation) and abolished by 3 μmol L⁻¹ ticagrelor (∼IC₉₀ for ADP-induced aggregation). We have previously reported that TxA₂ production in response to epinephrine is inhibited by P2Y₁₂ blockade in the same manner for AA and collagen [1]. Aspirin (30 μmol L⁻¹) either alone or in combination with ticagrelor also completely inhibited TxA₂ production to either agonist (Fig. 1G,H). A similar pattern of results was obtained using equivalent inhibitory concentrations of PAM in place of ticagrelor (Table S2), and when the concentration of aspirin was increased to 120 μmol L⁻¹ (Tables S1 and S2).

These studies show that ticagrelor and PAM inhibit platelet aggregation induced by a range of platelet agonists through a mechanism consistent with blockade of platelet P2Y₁₂ receptors and that ticagrelor is more potent than PAM in this regard. As well as inhibiting aggregation following from direct activation of P2Y₁₂ receptors by the addition of exogenous ADP, ticagrelor and PAM inhibited aggregations resulting from stimulation of platelets with AA, a response which is well characterized as being TxA₂ dependent. In addition to inhibiting platelet responses to endogenously produced TxA₂, ticagrelor and PAM also inhibited the production of TxA₂ by platelets [6]. Interestingly, ADP itself is a poor stimulus for TxA₂ production [1], suggesting that released ADP, acting on the P2Y₁₂ receptor, acts to potentiate the stimulation of TxA₂ synthesis by other signaling pathways activated in parallel. These results are consistent with the idea that whereas aspirin may inhibit just the TxA₂-dependent pathway of platelet activation, ticagrelor and PAM can inhibit both the ADP-P2Y₁₂-dependent and the TxA₂-dependent pathways of platelet aggregation. The observation that aspirin adds anti-aggregatory effects to partial, but not complete, P2Y₁₂ receptor blockade, further supports this idea.

Taken together these results demonstrate that rather than ADP-P2Y₁₂ and TxA₂ pathways acting independently, the TxA₂-dependent pathway is dependent upon the ADP-P2Y₁₂ pathway both for the production of TxA₂ and fundamentally for the irreversible aggregation that follows activation of TP receptors. If these data accurately model the situation in vivo, this may have important implications for the use of dual antiplatelet therapy using potent P2Y₁₂ antagonists in clinical practice [2]. For example, one could postulate that addition of aspirin could produce side-effects secondary to inhibition of cyclo-oxygenase at non-platelet sites, as has recently become apparent for non-steroid anti-inflammatory drugs, while providing little additional anti-aggregatory effect [7, 8]. Clearly, the validity of this hypothesis remains to be determined by clinical studies.

Disclosure of Conflict of Interests

T.D. Warner has received honoraria and research grants from AstraZeneca; S. Nylander is an employee of AstraZeneca. The other authors state that they have no conflict of interest.

Supporting Information

Figure S1. Concentration-response curves forthe inhibition by (A) ticagrelor(0.1–10 μmol L⁻¹), (B)prasugrel active metabolite (PAM;0.1–10 μmol L⁻¹) and (C)aspirin (1–100 μmol L⁻¹) ofplatelet aggregation induced by ADP(0.625–20 μmol L⁻¹).n = 4.

Figure S2. Concentration-response curves forthe inhibition by (A) ticagrelor(0.1–10 μmol L⁻¹), (B)prasugrel active metabolite (PAM;0.1–10 μmol L⁻¹) and (C)aspirin (1–100 μmol L⁻¹) ofplatelet aggregation induced by U46619(0.1–30 μmol L⁻¹).n = 4.

Figure S3. Concentration-response curves forthe inhibition by (A, B) ticagrelor(0.1–10 μmol L⁻¹), (C, D)prasugrel active metabolite (PAM;0.1–10 μmol L⁻¹) and (E, F)aspirin (1–100 μmol L⁻¹) ofplatelet aggregation (A, C, E) and TxA₂ release (B, D,F; measured as TxB₂) induced by arachidonic acid (AA;0.03–1 mmol L⁻¹).n = 4.

Table S1. Area under the concentration-responsecurve summary data for the effect of combinations of ticagrelor(0.03, 0.1, 0.3 and 3 μmol L⁻¹) andaspirin (30 and 120 μmol L⁻¹) onplatelet aggregation (A–C) and platelet TxA₂release (D; measured as TxB₂).

Table S2. Area under the concentration-responsecurve summary data for the effect of combinations ofprasugrel-active metabolite (PAM; 0.5, 1, 2 and10 μmol L⁻¹) and aspirin (30 and120 μmol L⁻¹) on platelet aggregation(A–C) and platelet TxA₂ release (D; measured asTxB₂).