Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets - PubMed (original) (raw)

Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

Analia Garcia et al. Biochem J. 2007.

Abstract

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the G(i)-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or G(i)+G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.

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Figures

Figure 1

Figure 1. Role of P2Y receptors in 2MeSADP-induced phosphorylation of ERK in platelets

Washed and aspirin-treated human platelets were stimulated with 100 nM 2MeSADP for different time points or 100 ng/ml convulxin (CVX) with stirring as indicated at 37 °C and ERK phosphorylation was measured using Western blot analysis using a phospho-ERK-specific antibody (A). Washed human platelets, without aspirin-treatment, were stimulated with 2MeSADP for 60 s in stirring conditions in the presence or absence of the GPIIb/IIIa antagonist, GR 144053 without added calcium. Platelet aggregation (B) and ERK phosphorylation (C) were measured by lumi-aggregometry and Western blot analysis respectively. Washed and aspirin-treated platelets were stimulated with 100 nM 2MeSADP in the presence of a P2Y1 receptor antagonist, MRS2179, or a P2Y12 receptor antagonist, AR-C69931MX, and ERK phosphorylation was measured by Western blot analysis (D). Total ERK antibodies were used in the Western blot analysis to ensure similar protein loading in all lanes. The data are representative of experiments performed using platelets from at least three different donors.

Figure 2

Figure 2. Effect of MEK inhibition on ADP-induced platelet functional responses

Washed human platelets were stimulated with 2MeSADP (100 nM) or ADP (10 μM), without added calcium, in the presence of the MEK kinase inhibitor, U0126 (10 μM) or its inactive analogue, U0124 (10 μM) as indicated. Pre-incubation times for U0126 and U0124 were 10 min at 37 °C. Platelet aggregation (A), platelet secretion (B) and thromboxane A2 generation (C) were measured as described in the Experimental section. Washed platelets treated with aspirin were stimulated with 2MeSADP and platelet aggregation was measured (D). All the above data are representative of experiments performed using platelets from at least three different donors. The maximum level of thromboxane seen upon stimulation with ADP was 4452 pg/ml. TXB2, thromboxane B2.

Figure 3

Figure 3. Role of Gq pathway signalling mediators on ADP-induced ERK phosphorylation and thromboxane A2 generation

Washed human platelets, treated with either aspirin or vehicle, were stimulated, without added calcium, with 2MeSADP (100 nM) or ADP (10 μM) in the presence or absence of a Gq blocker (YM-254890), a PLC inhibitor (U73122), a calcium chelator (BAPTA) or a PKC inhibitor (GFX) as indicated in the Figure. All of the inhibitors were incubated for 10 min at 37 °C prior to stimulation. ERK phosphorylation in aspirin-treated platelets (A) and thromboxane A2 generation in vehicle-treated platelets (B) were measured by Western blot analysis and ELISA respectively. All of the above data are representative of experiments performed using platelets from at least three different donors. TXB2, thromboxane B2.

Figure 4

Figure 4. Role of ERK in Gi+Gz-mediated activation of platelets

Washed and aspirin-treated human platelets were stimulated with 10 μM ADP alone or with 10 μM ADP+100 μM MRS2179+10 μM adrenaline (epinephrine) for 1 min, without added calcium, with stirring at 37 °C, and ERK phosphorylation was measured using Western blot analysis using a phospho-ERK-specific antibody (A). Total ERK antibodies were used in the Western blot analysis to ensure similar protein loading in all lanes. The data are representative of experiments performed using platelets from at least three different donors. (B) Washed human platelets were stimulated with 10 μM ADP+100 μM MRS-2179+10 μM adrenaline in the presence of the MEK kinase inhibitor, U0126 (10 μM) or its inactive analogue, U0124 (10 μM) as indicated. Pre-incubation times for U0126 and U0124 were 10 min at 37 °C. Thromboxane A2 generation was measured as described in the Experimental section and normalized to 100%. All of the above data are generated using platelets from at least three different donors. TXB2, thromboxane B2.

Figure 5

Figure 5. Effect of Src kinase inhibition on ADP-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation

Washed human platelets, without aspirin treatment, were stimulated with 2MeSADP (100 nM) or ADP (10 μM) for 60 s, without added calcium, in the presence or absence of the Src kinase inhibitor (PP2). The inactive analogue, PP3, was used as a negative control. Both PP3 and PP2 were pre-incubated at 37 °C for 10 min prior to stimulation. Platelet aggregation (A), ERK phosphorylation (B), and thromboxane A2 (C) were measured by lumiaggregometry, Western blot analysis and ELISA respectively. All of the above data are representative of experiments performed using platelets from at least three different donors. TXB2, thromboxane B2.

Figure 6

Figure 6. Effect of extracellular calcium on (A) 2MeSADP- or (B) Gi+Gz-induced ERK phosphorylation

Washed and aspirin-treated human platelets were resuspended in Tyrodes buffer with or without 2 mM extracellular calcium, and stimulated with 100 nM 2MeSADP (A) or 10 μM ADP+100 μM MRS-2179+10 μM adrenaline (epinephrine) (B) with stirring at 37 °C, and ERK phosphorylation was measured by Western blot analysis. The effect of various concentrations of extracellular calcium (C) and the time course of ERK2 phosphorylation in the presence or absence of extracellular calcium (D) were measured. Each Western blot is representative of experiments performed using platelets from at least three different donors.

Figure 7

Figure 7. Model depicting the mechanism of activation and functional role of ERK in ADP-induced thromboxane A2 generation

Signalling through both the P2Y1 and the P2Y12 receptors is essential for ADP-induced ERK2 activation in platelets. Activation of PLC and subsequent intracellular calcium increases are necessary for ADP-induced ERK2 activation. Src activation occurring downstream of the P2Y receptor activation is required for ADP-induced ERK activation. Extracellular calcium and specific PKC isoform(s) negatively regulate ADP-induced ERK2 phosphorylation and thromboxane generation through as yet undefined mechanisms. The identity of the specific PKC isoform(s) mediating this negative regulation is yet to be identified.

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