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J. Biol. Chem., Vol. 283, Issue 27, 18493-18504, July 4, 2008

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The ATP-gated P2X₁ Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine^*

Magnus Grenegård¹, Karin Vretenbrant-Öberg, Martina Nylander, Stéphanie Désilets, Eva G. Lindström², Anders Larsson^||, Ida Ramström, Sofia Ramström²³, and Tomas L. Lindahl

From the Department of Medicine and Health, Division of Drug Research and Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Cardiovascular Inflammation Research Center, Linköping University, Linköping SE-581 85 Sweden and the ^||Department of Medical Sciences, University Hospital, Uppsala SE-75105, Sweden

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via _2A-adrenergic receptors to provoke aggregation, secretion, and Ca²⁺ mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X₁, P2Y₁, and P2Y₁₂ (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with _2A-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X₁-receptor and the _2A-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

Received for publication, January 14, 2008 , and in revised form, May 14, 2008.

^* This study was supported by the strategic research area "Cardiovascular Inflammation Research Center," sponsored by the County Council of Östergötland and Linköping University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by Swedish Research Council Project K2007-64X-15060-04-3.

³ Supported by Forsknings-och forskarutbildningsnämnden at Linköping University.

¹ To whom correspondence should be addressed. Tel.: 46-13-221082; Fax: 46-13-149106; E-mail: Magnus.Grenegard{at}imv.liu.se.

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