Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide

Abstract

Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.

Keywords: anti-platelet therapy; atherothrombosis; prostacyclin.

Conflict of interest statement

Conflict of interest statement: T.D.W. has received consultancy fees from AstraZeneca and Daichii Sankyo/Eli Lilly relating to clinical development of P2Y12 inhibitors.

Figures

Fig. 1.

Blockade of the P2Y12 receptor sensitizes platelets to the inhibitory effect of NO against thrombin-induced aggregation, secretion, and Rap1 activation. Thrombin (1 U/mL)-induced aggregation of washed platelets, determined by changes in light transmission, was only weakly inhibited by the NO donor, DEA/NONOate (A) (n = 8), but this effect was greatly increased in platelets pretreated with the P2Y12 receptor blocker PAM (3 μM). Traces show the effects of 1 μM DEA/NONOate in the absence and presence of PAM. Similarly, inhibition by DEA/NONOate of platelet aggregation in hirudin-anticoagulated whole blood stimulated with SFLLRN-amide (10 μM) + AYPGKF-amide (30 μM) and measured by impedance aggregometry (B), thrombin (1 U/mL)-induced GPIIb/IIIa activation by flow cytometry (C) and ADP+ATP secretion, measured by a modified luminometric method (D), were also strongly potentiated by P2Y12 receptor blockade (n = 4 for each). Thrombin (1 U/mL) also stimulated rapid activation of Rap1, as determined by affinity purification of Rap1-GTP and Western blotting of lysates of platelets stimulated under stirring conditions for 15 s (E). Rap1 activation was little altered by DEA/NONOate or P2Y12 receptor blockade alone but was strongly inhibited by the combination. Platelet lysates incubated with GTPγS were included on blots as a positive control for affinity isolation of Rap1-GTP (n = 4 for each). *P < 0.05 by two-way ANOVA vs. vehicle.

Fig. 2.

P2Y12 receptor blockade does not alter the ability of NO to stimulate cAMP or cGMP formation or VASP phosphorylation. cGMP content, determined by immunoassay in lysates of washed platelets stimulated with thrombin (1 U/mL) under stirring conditions, was increased by DEA/NONOate (A). This response was not different in platelets pretreated with the P2Y12 receptor blocker PAM (3 μM; n = 9). cAMP content, measured in the same way, was not altered by either DEA/NONOate or P2Y12 receptor blockade (B) (n = 9). The level of phospho(Ser239)-VASP, measured by flow cytometry in washed platelets stimulated with thrombin under nonstirring conditions, was also increased by DEA/NONOate, and this response was not altered by P2Y12 receptor blockade (C). Representative histograms show the effect of 100 μM DEA/NONOate in the presence and absence of PAM (n = 4).

Fig. 3.

P2Y12 receptor blockade potentiates inhibition of platelet aggregation to exogenous cGMP but does not potentiate phosphorylation of VASP. Thrombin (1 U/mL)-induced aggregation of washed platelets, determined by changes in light transmission, was inhibited by the cell-permeable cGMP analog, 8-pCPT-cGMP (A), and this action was enhanced in platelets pretreated with the P2Y12 receptor blocker PAM (3 μM). An increase in platelet phospho(Ser239)-VASP levels also was produced by 8-pCPT-cGMP, as determined by flow cytometry, but this was not altered by P2Y12 receptor blockade (B). *P < 0.05 by two-way ANOVA vs. vehicle (n = 3–4).

Fig. 4.

P2Y12 receptor blockade does not alter the ability of NO to inhibit intracellular Ca2+ mobilization but facilitates its ability to inhibit PI3K signaling. Thrombin (1 U/mL)-induced mobilization of intracellular calcium in Fura2-loaded washed platelets was inhibited by DEA/NONOate, and this inhibition was not altered by the P2Y12 receptor blocker PAM (3 μM) (A). Thrombin (1 U/mL) also stimulated phosphorylation of Akt(Ser473) under stirring conditions measured in washed platelets lysed 2 min after agonist addition (B). Akt(Ser473) phosphorylation was strongly inhibited by P2Y12 receptor blockade (PAM; 3 μM) or the PI3K inhibitor, wortmannin (Wort; 200 nM) (B). DEA/NONOate only weakly inhibited thrombin-induced Akt(Ser473) phosphorylation, but this effect was greatly potentiated in the presence of PAM (C). In parallel studies, specific inhibition of PI3K by wortmannin enhanced the ability of DEA/NONOate to inhibit thrombin (1 U/mL)-induced aggregation of washed platelets in the absence, but not the presence, of P2Y12 blockade (D). (A, C, and D) *P < 0.05 by two-way ANOVA vs. vehicle. (B) *P < 0.05 by one-way ANOVA vs. thrombin. (D) #P < 0.05 by two-way ANOVA vs. the same treatment in the absence of wortmannin (n = 4).

Fig. 5.

Schematic and summary for interaction between P2Y12 receptors and NO signaling in platelets. Schematic showing proposed mechanism by which P2Y12 receptor activation by secreted ADP limits the antiplatelet effect of NO by providing a cGMP-insensitive pathway for platelet aggregation, which depends partly on PI3K signaling (A). Relative potencies for DEA/NONOate on platelet responses (B) highlight that although DEA/NONOate is active on platelets at nanomolar concentrations (cGMP, VASP, Ca2+), inhibition of strong activation (aggregation, secretion, etc.) requires millimolar concentrations of DEA/NONOate. However, when P2Y12 receptors are blocked, the inhibitory potency of NO against activation markers closely correlates with its potency on cGMP production, VASP, and calcium.