P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation.
BACKGROUND
Thromboxane A2 (TXA2) 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 TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation.


RESULTS
PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 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 TXA2 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 cPLA2 phosphorylation and TXA2 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 TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.
Shankar
H
H
Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Garcia
A
A
Prabhakar
J
J
Kim
S
S
Kunapuli
S P
SP
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D002070:Butadienes / Q000494:pharmacology; D000077144:Clopidogrel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011743:Pyrimidines; D044463:Receptor, PAR-1 / Q000187:drug effects; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin / Q000187:drug effects; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000378:metabolism*; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D002070:Butadienes; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009570:Nitriles; C548576:P2RY12 protein, human; C548577:P2ry12 protein, mouse; D010975:Platelet Aggregation Inhibitors; D011743:Pyrimidines; D044463:Receptor, PAR-1; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; C113580:U 0126; D000249:Adenosine Monophosphate; D013928:Thromboxane A2; C117446:cangrelor; D000077144:Clopidogrel; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; C112908:protease-activated receptor 4; D013988:Ticlopidine