title: The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine.
abstract: 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.
authors.affiliation: Department of Medicine and Health, Division of Drug Research, Division of Clinical Chemistry, Cardiovascular Inflammation Research Center, Linköping University, Linköping SE-581 85 Sweden. Magnus.Grenegard@imv.liu.se
authors.forename: Magnus
authors.initials: M
authors.lastname: Grenegård
authors.forename: Karin
authors.initials: K
authors.lastname: Vretenbrant-Oberg
authors.forename: Martina
authors.initials: M
authors.lastname: Nylander
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Désilets
authors.forename: Eva G
authors.initials: EG
authors.lastname: Lindström
authors.forename: Anders
authors.initials: A
authors.lastname: Larsson
authors.forename: Ida
authors.initials: I
authors.lastname: Ramström
authors.forename: Sofia
authors.initials: S
authors.lastname: Ramström
authors.forename: Tomas L
authors.initials: TL
authors.lastname: Lindahl
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D001241:Aspirin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009842:Oligopeptides / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010446:Peptide Fragments / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058913:Purinergic Agonists; D058910:Purinergic P2 Receptor Agonists; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism; D013917:Thrombin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D000316:Adrenergic alpha-Agonists; D001241:Aspirin; D002118:Calcium; D002867:Chromones; D004791:Enzyme Inhibitors; D004837:Epinephrine; D006490:Hemostatics; D009025:Morpholines; D009842:Oligopeptides; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010975:Platelet Aggregation Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D058913:Purinergic Agonists; D058910:Purinergic P2 Receptor Agonists; D044463:Receptor, PAR-1; D018341:Receptors, Adrenergic, alpha-2; D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D018179:Receptors, Thrombin; D013917:Thrombin; C547768:ADRA2A protein, human; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; C082835:thrombin receptor peptide (42-47); C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C112908:protease-activated receptor 4