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

Department of Medicine and Health, Division of Drug Research, Division of Clinical Chemistry, Cardiovascular Inflammation Research Center, Linköping University, Linköping SE-581 85 Sweden.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2008; 283(27):18493-504. DOI: 10.1074/jbc.M800358200
Source: PubMed


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 alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) 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 P2X(1), P2Y(1), and P2Y(12) (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 alpha(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 P2X(1)-receptor and the alpha(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.

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Available from: Anders Larsson, Jul 16, 2015
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    • "Platelet agonists (e.g., ADP, thrombin, thromboxane A 2 (TXA 2 ), and epinephrine) activate platelets via G proteincoupled receptor (GPCR) pathways [1] [2], and the activations of distinct G proteins are integrated by interacting downstream molecules [3] [4]. Thus, once a specific GPCR is activated by an agonist, other GPCRs are eventually activated by recruited GPCR ligands and form a positive feedback loop which greatly amplifies activation signals [1] [5]. ADP and TXA 2 , two factors secreted during platelet activation, serve as both mediators and agonists. "
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    • "Similarly, we have described a mechanism independent of calcium influx, in which calcium increases induced by carbachol can be blunted by the activation of a P2X receptor with low affinity for ATP [46]. However, this inhibitory effect of ATP on intracellular calcium increases is controversial, it has also been reported that the ATP released from activated platelets induces intracellular calcium increases through P2X1 receptors [47]–[49]. These contrary results are probably due to differences in the experimental procedures. "
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    • "In platelets, thrombin cleaves protease-activated receptor 1 (PAR1) and PAR4, which induces signaling via Gα 12/13 and Gα q proteins [1]. Differences in downstream signaling and platelet responses following PAR1 and PAR4 activation have been reported [2] [3] [4] [5] [6] [7] [8] [9], but are still not fully understood [10]. ADP and ATP secretion from dense granule enhances platelet activation, and P-selectin from platelet α-granules becomes expressed on the platelet surface upon activation. "
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