P2X1-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen

Abstract

Adenosine triphosphate (ATP) and its stable analog, ␣,␤-methylene ATP, activate the platelet P2X 1 ion channel, causing a rapid Ca ؉؉ influx. Here, we show that, in washed apyrase-treated platelets, ␣,␤-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca ؉؉ -and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. ␣,␤-Methylene ATP also activated the ERK2 pathway in P2X 1 -transfected HEK293 cells but not in cells expressing mutated P2X 1 delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X 1 -mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X 1 with ADP or desensitization of this ion channel with ␣,␤-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< 1 g/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca ؉؉ chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collageninduced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 g/mL), platelet aggregation and secretion no longer depended on P2X 1 or ERK2 activation, as shown by the lack of their inhibition by ␣,␤-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X 1 -PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed. (Blood.

Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase

“…These data support the mediating role of secreted ADP and its P2Y 12 receptor in these thrombin effects. Consistent with this conclusion, as well as with previously published data, 15 a selective P2X 1 receptor agonist (␣, ␤-MeATP, 2.5 M) failed to cause any p38 phosphorylation (data not shown). Preincubation of platelets with indomethacin also inhibited thrombin-stimulated p38 activation, Hsp27 phosphorylation, and Pselectin expression to a similar extent as that observed with AR-C69931MX ( Figure 1A).…”

Historical perspective on ADP-induced platelet activation

“…Vial et al [109] have suggested that in vivo, these ATP-induced reactions could synergize with the platelet response to ADP through the P2Y 1 receptor and amplify the increase in intracellular calcium. Oury et al [110,111] obtained evidence for the importance in hemostasis of a synergy between ADP and ATP released from platelets stimulated by adherence to collagen. They used transgenic mice overexpressing the P2X 1 receptor in the megakaryocyte cell lineage and found that their platelets showed enhanced aggregation and secretion in response to collagen, compared with platelets from wild-type mice.…”

P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation

“…The ␣,␤-meATP was purified by high pressure liquid chromatography on a Adsorbosphere HS C18, 7-m, 250 ϫ 4.6-mm column (Alltech, Bad Segeberg, Germany) (15,16). Purified samples, lyophilized and adjusted to pH 7.0 with potassium phosphate buffer, were kept at Ϫ80°C and were stable over the time interval studied.…”

“…P2X 1 is highly expressed in human platelets and megakaryocytes (13,14); because of the role of Ca 2ϩ /CaM in platelet MLC kinase activation, we have presently investigated the P2X 1 -mediated Ca 2ϩ influx in relation to MLC phosphorylation and platelet shape change. We have previously shown that the P2X 1 -mediated Ca 2ϩ influx triggers phosphorylation of the extracellular signal-regulated kinase (ERK2), a reaction found to contribute to platelet activation and secretion induced by low concentrations of collagen (15,16). We therefore have also investigated the relation between MLC kinase and ERK2 activation during collagen-induced platelet activation, in relation to P2X 1 -mediated Ca 2ϩ influx.…”