P2Y12 receptor‐mediated potentiation of thrombin‐induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

Abstract

Summary. Background: Thromboxane A 2 (TXA 2 ) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA 2 . The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA 2 generation. Results: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A 2 (cPLA 2 ), release of arachidonic acid (AA) from membranebound phospholipids, and subsequent TXA 2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y 12 receptor antagonist, AR-C69931MX, but not the P2Y 1 receptor antagonist, MRS2179. In addition, AYPGKF-and thrombin-induced TXA 2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA 2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA 2 phosphorylation and TXA 2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. Conclusions: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA 2 generation, which is potentiated by the P2Y 12 receptor-mediated signaling pathway but not the P2Y 1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.

Platelets, inflammation and anti-inflammatory effects of antiplatelet drugs in ACS and CAD

“…Recent studies showed an additional antiplatelet activity via the AA-TBXA2-COX pathway potentiating the effects of ASA, which implies that cessation of clopidogrel affects both, the ADP-and AA-induced platelet aggregation (55,(150)(151)(152)(153) contributing to the rebound effect.…”

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

“…[18][19][20] It is well-established that P2Y 12 blockade has significant effects upon platelet responsiveness to collagen, U46619, and PAR4 agonists consistent with its antithrombotic effect in man. [21][22][23] We found that the P2Y 12 receptor blocker PAM (3 mM) markedly prevented agonist-induced single platelet loss as compared with vehicle (0.5% DMSO). This inhibition persisted even at the highest concentrations tested of collagen (72 6 10% vs 58 6 11%), PAR4-amide (80 6 4% vs 11 6 6%), and U46619 (50 6 15% vs 1 6 1%) ( Figure 3A-C).…”

A single-centre, randomised study on platelet reactivity after abrupt or gradual discontinuation of long-term clopidogrel therapy in patients after percutaneous coronary intervention

“…As we also examined platelet reactivity using ASPI test and platelet count and did not find significant differences between the examined groups, we assumed that platelet aggregation differences are specifically due to the strategy of clopidogrel cessation. However, previous data suggested that there is an interaction between clopidogrel and the arachidonic acid-induced pathway of platelet reactivity [11][12][13][14]. Sambu et al [14] reported finding that when clopidogrel is withdrawn one year after DES implantation, there is a significant increase in AA-induced platelet reactivity in addition to an increase in ADP-induced platelet reactivity.…”