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Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2002-03-0812.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular and Vascular Biology,
University of Leuven, Belgium.
Adenosine triphosphate (ATP) and its stable analog,
Present in very high concentrations in the
platelet-dense granules, both adenosine diphosphate (ADP) and adenosine
triphosphate (ATP) are secreted during platelet
activation.1 ADP has long been recognized as an important
activator of platelets, playing an essential role in enhancing
secretion, and stabilizing platelet aggregates induced by other
agonists.2 ADP activates 2 receptors (for a review, see
Gachet3); P2Y1, coupled to a Gq protein, is
responsible for shape change and initiation of platelet aggregation, and P2Y12,4 a target for specific
antithrombotic drugs, leads to adenylate cyclase inhibition through a
Gi protein and promotes the completion and amplification of platelet
responses. Recently, ATP was found to be the agonist of the ionotropic
P2X1 receptor, causing a rapid influx of
Ca++.5,6 Because platelet studies have mainly
been performed at low extracellular Ca++ concentrations in
citrated plasma, but also due to the difficulties in preserving
P2X1 functionality during platelet handling because of its
fast desensitization by spontaneously released ATP,7 the
function of the platelet P2X1 ion channel only recently
started to be unraveled. Two studies described the ability of the
P2X1 selective agonists, P2X1 belongs to an emerging family of ionotropic receptors
that are involved in many physiologic processes.10 The
ionotropic P2X receptors regulate intracellular Ca++ levels
through the ligand-stimulated increase in calcium permeability. However, how these receptors are linked to intracellular signaling pathways subserving their biologic actions still is poorly understood. In PC12 cells, Swanson and coworkers11 have shown that
P2X2 mediates the activation of the extracellular
signal-regulated kinase (ERK1/2) members of the mitogen-activated
protein kinase (MAPK) family of serine/threonine kinases12
via a protein kinase C (PKC)- and proline-rich tyrosine kinase 2 (PYK2)-dependent pathway.
The prototype ERK pathway consists of a cascade of protein kinases,
Raf1, the MAPK kinase MEK1, and ERK1/2, which sequentially activate a
downstream kinase. In platelets, ERKs have been shown to be activated
after stimulation by thrombin,13 collagen,14 or phorbol esters15 as well as to play a role during
store-mediated Ca++ entry.16 ERK2 was also
shown to be down-regulated by In this study, we found that Materials
Preparation of hirudinized PRP and washed platelets
Platelet aggregation and ATP secretion analyses Light transmission during collagen-induced platelet aggregation was recorded in hirudinized PRP or apyrase-treated washed platelets on a Chrono-Log Aggregometer (Havertown, PA). ATP secretion was monitored in hirudinized PRP or in washed platelets in parallel with platelet aggregation by adding firefly luciferase and luciferin and comparing the luminescence generated by platelet ATP release or by an ATP standard (Kordia, Leiden, The Netherlands). P2X1 desensitization occurs within a few milliseconds; therefore desensitization could be accomplished by adding , -meATP
(0.5 µM) simultaneously with collagen to prevent released ATP from
activating the ion channel.7 P2X1 antagonism
was achieved by adding ADP (0.5 µM) 1 minute prior to
collagen.9 The MEK1/2 inhibitor U-0126, the PKC inhibitor
GF109203-X, or vehicle was added 2 minutes prior to collagen. EGTA
(ethylene glycol tetraacetic acid) was added 5 minutes prior to
collagen. At least 3 independent experiments were performed on
platelets from different individuals. The data are represented as the
mean ± SEM. Statistical analysis of the data were made using
nonpaired Student t test.
Phospho-ERK1/2 detection Serum-starved stable P2X1- and P2X1delL-expressing HEK293 cells (0.5 × 105 cells),20 or stirred washed platelets (0.3-mL aliquots) that had been treated with apyrase (1 U/mL) for 30 minutes, were stimulated with agonists at 37°C. The reaction was stopped by the addition of sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue). Then 40-µL sample aliquots were loaded on SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and subjected to Western blotting using the PhosphoPlus p44/42 MAP kinase antibody kit (New England Biolabs, Hitchin, United Kingdom) according to the instructions of the manufacturer. The data are representative of at least 3 independent experiments performed on different cell extracts or on platelets from different individuals.
P2X1 stimulation selectively causes Ca++- and PKC-dependent ERK2 activation We have investigated the intracellular signaling pathways selectively activated through P2X1 and that could subserve the role of this ion channel during platelet activation. We found that in apyrase-treated washed platelets,,-meATP (2 µM) caused
rapid and reversible ERK2 phosphorylation (about 5-fold stimulation), reaching a maximum at 2 minutes and disappearing after 10 minutes (Figure 1A). Figure 1B shows the dose
response to this agonist, which is in agreement with the previously
described concentrations for ,-meATP-induced platelet shape
change.9 The ,-meATP-elicited ERK2 phosphorylation
was fully inhibited by GF109203-X, an inhibitor of the , ,  ,
and  isoforms of PKC and by the highly specific MEK inhibitor,
U-0126 (Figure 2A). ERK2 phosphorylation
also required extracellular Ca++ because it no longer
occurred in the presence of EGTA (Figure 2B) and was almost absent when
no extracellular Ca++ was added (data not shown).
HPLC-purified ADP (from 0.5 up to 20 µM) was unable to induce ERK2
phosphorylation at any time point (Figure 2C). These data revealed
that, in platelets, the P2X1-mediated Ca++
influx activates a PKC-dependent ERK2 signaling pathway.
The dominant-negative P2X1delL mutant is unable to activate ERK2 We have previously identified and characterized a nonfunctional dominant-negative mutant of P2X1 (P2X1delL).20 To further demonstrate the ability of the P2X1-elicited Ca++ influx to selectively activate the ERK2 pathway, apyrase-treated HEK293 cells stably expressing either the normal P2X1 protein (P2X1wt) or P2X1delL were stimulated with,-meATP for 2 minutes before analyzing ERK phosphorylation.
,-meATP induced phosphorylation of ERK1/2 in
P2X1wt-expressing cells but not in cells expressing the
nonfunctional P2X1delL channels (Figure
3). The nontransfected cells did not
respond to ,-meATP (data not shown). These results thus
established a direct correlation between the presence of functional
P2X1 channels, triggering Ca++ influx, and the
activation of ERK.
Inhibition of P2X1 or ERK2 activation impairs dense granule release in platelets stimulated with low concentrations of collagen Collagen-induced platelet aggregation largely depends on secretion, and the importance of ADP, acting at P2Y1 and P2Y12, is well documented.3 We recently reported that P2X1, through secreted ATP, is also required for platelet aggregation elicited by low doses of collagen.9 We wanted to determine whether the P2X1-PKC-ERK2 signaling pathway plays a role in this process. Therefore, we have recorded ATP secretion in parallel with platelet aggregation and analyzed the effects of P2X1 desensitization and MEK inhibition on these platelet responses. These recordings were performed in hirudinized PRP to maintain physiologic Ca++ concentrations and preserve enough P2X1 functionality for the purpose of the experiment. Despite the fact that,-meATP could not induce ATP release by itself, these analyses
revealed that both P2X1 desensitization with ,-meATP
and MEK inhibition by U-0126 potently impaired ATP release triggered by
low doses of collagen (0.4-1 µg/mL). Collagen (0.5 µg/mL) induced
4.29 ± 0.22 µM ATP release (Figure
4), which was reduced to 0.14 ± 0.08
µM (n = 7, P < .0001) in the presence of
,-meATP (Figure 4), and to 0.35 ± 0.08 µM (n = 4,
P < .0001) by U-0126 (Figure 4). Because in the
Ca++-rich environment of hirudinized PRP, indomethacin only
slightly inhibited the collagen-induced platelet aggregation (data not shown), no major role in aggregation is attributed to thromboxane A2. Therefore, the inhibition of platelet aggregation by
U-0126 is not due to nonselective inhibition of
cyclooxygenase.21 Consistent with an event requiring
Ca++ influx through P2X1, ATP secretion was
similarly inhibited in the presence of the Ca++ chelator
EGTA (0.56 ± 0.11 µM, n = 3; Figure 4). In contrast, neither
,-meATP nor U-0126 affected platelet ATP secretion and aggregation evoked by higher concentrations of collagen (2 µg/mL; data not shown); likewise, EGTA only slightly impaired ATP secretion induced by 2 µg/mL collagen (data not shown). These data thus suggest
an essential role for the P2X1-PKC-ERK2 signaling pathway in enhancing dense granule release and completing platelet aggregation initiated by low doses of collagen.
The P2X1-PKC-ERK2 signaling pathway is not involved in the early collagen-induced secretion Platelets stimulated with collagen release ATP before the onset of shape change22 (Figure 4). This early release was totally blocked by the PKC inhibitor GF109203-X (not shown), which also abolished collagen-induced platelet shape change and aggregation (Figure 5A). The inhibition of collagen-induced platelet shape change by the P2Y1 receptor antagonist A2P5P was indicative of the involvement of secreted ADP (data not shown). We then wondered whether this early secretion responsible for platelet shape change precedes or depends on the activation of the P2X1-PKC-ERK2 signaling pathway. Figure 4 revealed that neither,-meATP nor U-0126 and EGTA were able to
fully inhibit ATP release induced by collagen (0.5 µg/mL), and
platelet shape change occurred normally. Accordingly, the ATP release
induced by 0.3 µg/mL collagen (0.33 ± 0.05 µM, n = 3), which
elicited platelet shape change without causing aggregation (data not
shown), was not affected by these treatments either (data not shown).
These results indicate that the PKC-ERK2 pathway is not involved in the
early secretion preceding platelet shape change, and further indicate
that activation of this pathway is secondary to the action of the
limited amount of ATP secreted during the initial response of platelets
to collagen.
At low-dose collagen, ERK2 phosphorylation is mediated via P2X1 In platelets, collagen activates the ERK signaling pathway.14 To further investigate the contribution of P2X1 to this pathway, we have analyzed the effect of P2X1 desensitization on the ability of collagen to cause ERK2 phosphorylation in apyrase-treated washed platelets. Platelet aggregation was recorded in parallel. In apyrase-treated washed platelets, similarly as in hirudinized PRP, P2X1 desensitization with,-meATP (0.5 µM) inhibited platelet
aggregation induced by low concentrations of collagen (0.75-1 µg/mL;
Figure 5A). Western blot analyses indicated that the collagen (0.75-1 µg/mL)-induced ERK2 phosphorylation reaches a maximum after 4 minutes following addition of the agonist (Figure 5B); the appearance
of ERK2 phosphorylation at minute 2 correlated with the onset of
platelet aggregation (Figure 5A,B). Interestingly, P2X1
desensitization with ,-meATP (0.5 µM), able to inhibit platelet
aggregation, also led to the loss of ERK2 phosphorylation at the time
where maximal activation occurred in the control (4 minutes; Figure
5A,B), even despite the fact that ,-meATP is capable of
triggering ERK2 phosphorylation itself (Figure 1). We then used a low
concentration of ADP (0.5 µM) that selectively antagonizes the
ATP-induced activation of P2X1 as well as the
,-meATP-evoked platelet shape change9 without causing
ERK2 phosphorylation (Figure 2C). Similarly as ,-meATP, ADP
pretreatment prevented the collagen-induced ERK2 phosphorylation
(Figure 5C) as well as platelet aggregation (Figure 5A and Oury et
al9). These results show that the observed
inhibition of ERK2 activation can be caused both by P2X1
antagonism (ADP) and desensitization (,-meATP), the 2 P2X1 neutralizing approaches leading to impaired platelet
aggregation. The collagen-induced ERK2 activation and aggregation were
also abolished in the presence of EGTA (Figure 5A,C), supportive of the
involvement of P2X1-mediated Ca++ influx
(Figure 2B). The PKC inhibitor GF109203-X that prevents platelet
secretion (data not shown), shape change, and aggregation evoked by
collagen (Figure 5A), and the ,-meATP-elicited ERK2 activation
(Figure 2A), inhibited ERK2 activation caused by collagen, as expected
(Figure 5C). The inhibition of collagen-induced ERK2 phosphorylation by
U-0126 (Figure 6B) equally blocked
platelet aggregation (Figure 5A), confirming the importance of the
P2X1-ERK2 pathway, also when studied in apyrase-treated
washed platelets. The further inhibition of U-0126-treated platelets
with GF109203-X abolished platelet shape change and residual ATP
secretion (data not shown), confirming that the minor early dense
granule release occurs independently of ERK2. Platelet aggregation was
prevented by the P2Y12 antagonist ARC-69931MX in agreement
with a role for secreted ADP (data not shown), and was also inhibited
by indomethacin (Figure 5A), indicative of a contribution of
synthesized thromboxane A2 during the process of
aggregation of washed platelets performed in the presence of apyrase.
The relative contribution of thromboxane A2 during
aggregation of apyrase-treated washed platelets thus differs from that
studied in hirudinized PRP. Nevertheless, indomethacin (Figure 5C) did
not affect ERK2 phosphorylation, showing that thromboxane
A2 is not involved in the activation of the ERK2 pathway. Our data thus indicate that ERK2 phosphorylation induced by low doses
of collagen depends on the action of secreted ATP at P2X1, contributing to the completion of platelet aggregation.
Platelet aggregation induced by high doses of collagen no longer depends on P2X1 and ERK2 activation Desensitization of P2X1 with,-meATP did not
inhibit ERK2 phosphorylation and aggregation of washed apyrase-treated
platelets in response to high concentrations of collagen (2 µg/mL;
Figure 6A,B). Similarly, collagen-induced ERK2 activation was no longer inhibited in the presence of EGTA (Figure 6B), showing that, in agreement with the absence of inhibition by ,-meATP, no role can
be attributed to P2X1 in these conditions of strong
platelet stimulation. Interestingly, total blockade of the ERK pathway by U-0126 (Figure 6B) had no effect on platelet aggregation induced by
the high concentration of collagen (Figure 6A). These data thus
indicate that, at high doses of collagen, platelet aggregation occurs
independently of the P2X1-ERK2 pathway. These results also show that, at high collagen concentrations, platelet aggregation and
ERK2 phosphorylation can be dissociated, ERK2 no longer being involved
in secretion reactions leading to platelet aggregation.
In the present study, we have identified the ERK2 signaling pathway as an intracellular mechanism subserving the function of the ATP-gated P2X1 ion channel during platelet aggregation induced by low concentrations of collagen. We have shown that the P2X1-mediated ERK2 activation is a Ca++- and PKC-dependent process needed to amplify dense granule release initiated by this agonist. In a previous study, we provided evidence that during
collagen-initiated platelet activation, the early secretion of ATP
results in the activation of P2X1 acting as a positive
regulator of subsequent platelet responses.9 To
investigate the role of the P2X1-PKC-ERK2 pathway in
collagen-induced platelet aggregation, we took advantage of the
possibility to selectively antagonize ATP-induced P2X1 activation with ADP9 and to prevent Ca++ influx
through the ion channel via chelation of extracellular Ca++
by EGTA. Collagen appeared to evoke the secretion of ATP (and ADP) rapidly, prior to the onset of shape change. The collagen-induced platelet shape change was found to depend on secretion, because it was inhibited by the PKC inhibitor, GF109203-X, which totally prevents secretion elicited by this agonist; shape change was also inhibited by the P2Y1 antagonist, A2P5P. In contrast, neither EGTA nor the MEK1/2 inhibitor U-0126 affected this platelet shape change, therefore excluding a role for the P2X1-PKC-ERK2 signaling pathway in the very early dense granule release triggered by collagen. This indicates that another PKC-dependent secretory pathway is responsible for the minor early collagen-induced secretion. Combining these findings, in the model depicted in Figure
7, we propose a
P2X1-dependent mechanism essential for the completion of
platelet aggregation induced by low doses of collagen. According to
this model, platelet stimulation with low doses of collagen rapidly
causes ERK2-independent minor dense granule release (step 1); ATP
secreted during this early event activates the
P2X1-PKC-ERK2 pathway (step 2), which is needed to complete
platelet aggregation by enhancing the release reaction from
collagen-primed dense granules (step 3). The notion of dense granule
priming refers to our finding that, on one hand, the minor
early release induced by collagen (activation of PKC1) is insufficient
to cause platelet aggregation unless the
P2X1-PKC2-ERK2 pathway is activated; on the other
hand, the sole activation of the latter pathway with
Our model describes the role of the P2X1-PKC-ERK2 pathway during platelet activation with low concentrations of collagen. The fact that desensitization of P2X1 can no longer inhibit the collagen-induced ERK2 phosphorylation, ATP secretion, and aggregation when platelets are activated with higher concentrations of collagen implies that ERK can be activated via alternative pathways; because the blockade of the ERK pathway by U-0126 no longer inhibited the collagen-induced platelet aggregation, it appears that ERK activation no longer plays an important role in aggregation under those conditions. Platelet aggregation progressively relies more on the engagement of other activation pathways, such as production of thromboxane and other secreted products. The inability of HPLC-purified ADP to activate ERK2 is in agreement
with a previous study in which ADP was rather linked to p38 MAPK
phosphorylation through P2Y1.23 Because p38
MAPK is not phosphorylated following P2X1 stimulation (data
not shown), it seems that the 2 MAPK pathways, ERK and p38 MAPK, are
differentially triggered by ATP and ADP, respectively. Here, the
selectivity of the P2X1-ERK2 cascade was further
demonstrated in HEK293 cells heterologously expressing P2X1
or its dominant-negative mutant P2X1delL.20
Indeed, only those cells expressing functional P2X1 channels were able to activate ERKs in response to Despite the biologic significance of secretion for platelet function,
the molecular mechanisms governing secretion are only partially
understood.24,25 Although the ERK pathway has been implicated in secretion processes in other cell types,26
how ERK activation could be linked to the cellular secretory machinery remains to be investigated. Interestingly, we found that, although In other tissues, P2X-type receptors have also been shown to play a
role in secretion. Thus, in cardiac sympathetic nerve endings, ATP
appeared to induce norepinephrine release by acting at P2X
receptors27; in the RBA-2 type-2 astrocyte cell line, ATP
stimulates Our data that the P2X1-activated secretion involves PKC is
in agreement with the study by Yoshioka et al31 who
identified PKC In conclusion, we provide evidence for a distinct role of the P2X1 ion channel in the amplification of platelet granule release initiated by low doses of collagen; this amplification occurs via selective activation of a PKC-dependent ERK2 signaling pathway. These results support the notion that platelet activation by this agonist leads to a concomitant signaling both through the ADP receptors P2Y1 and P2Y12, and the ATP-gated ion channel P2X1.
Submitted March 18, 2002; accepted May 15, 2002.
Prepublished online as Blood First Edition Paper, June 14, 2002; DOI 10.1182/blood-2002-03-0812.
Support was obtained from the bilateral scientific and technological cooperation between Flanders and Hungary (BIL00/12) and from the Fonds Wetenschappelijk Onderzoek Vlaanderen (FWO) project G 0376.01. E.T.Z. is recipient of a doctoral KU Leuven scholarship. C.O. is holder of a postdoctoral research mandate of the FWO.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marc F. Hoylaerts, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium; e-mail: marc.hoylaerts{at}med.kuleuven.ac.be.
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A. Kauskot, F. Adam, A. Mazharian, N. Ajzenberg, E. Berrou, A. Bonnefoy, J.-P. Rosa, M. F. Hoylaerts, and M. Bryckaert Involvement of the Mitogen-activated Protein Kinase c-Jun NH2-terminal Kinase 1 in Thrombus Formation J. Biol. Chem., November 2, 2007; 282(44): 31990 - 31999. [Abstract] [Full Text] [PDF]
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E. Toth-Zsamboki, C. Oury, H. Cornelissen, R. De Vos, J. Vermylen, and M. F Hoylaerts P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation J. Biol. Chem., November 21, 2003; 278(47): 46661 - 46667. [Abstract] [Full Text] [PDF]
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C. Oury, M. J. E. Kuijpers, E. Toth-Zsamboki, A. Bonnefoy, S. Danloy, I. Vreys, M. A. H. Feijge, R. De Vos, J. Vermylen, J. W. M. Heemskerk, et al. Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothrombotic phenotype Blood, May 15, 2003; 101(10): 3969 - 3976. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
1 µg/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 collagen-induced secretion
and ERK2 activation. In contrast, when elicited by high collagen
concentrations (2 µg/mL), platelet aggregation and secretion no
longer depended on P2X1 or ERK2 activation, as shown by the
lack of their inhibition by 
