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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Lack of evidence for functional ADP-activated human P2X1 receptors supports
a role for ATP during hemostasis and thrombosis
Catherine Vial, Samantha J. Pitt, Jon Roberts, Michael G. Rolf, Martyn P. Mahaut-Smith, and Richard J. Evans
Purine nucleotides acting through P2 receptors play key roles in platelet signaling. The
P2X1 receptor is an adenosine triphosphate
(ATP)–gated ion channel that mediates a
rapid calcium influx signal, but can also
synergize with subsequent adenosine
diphosphate (ADP)–evoked P2Y1 receptormediated responses and thus may contribute to platelet activation during hemostasis.
Recent studies have shown that P2X1 recep-
tors contribute to the formation of platelet
thrombi, particularly under conditions of
high shear stress. Based on intracellular
Ca2ⴙ measurements a previous report has
suggested that a splice variant of the P2X1
receptor, P2X1del, is expressed in platelets
and, in contrast to the full-length P2X1WT
receptor, is activated by ADP. In the present
study we show that the P2X1del receptor fails
to form functional ion channels and is below
the limit of detection in human platelets.
Furthermore, ADP does not contribute to
the rapid ionotropic P2X receptor-mediated
response in platelets. These results support
the notion that ATP is the principal physiologic agonist at P2X1 receptors and that it
plays a role in the activation of platelets.
(Blood. 2003;102:3646-3651)
© 2003 by The American Society of Hematology
Introduction
Purine nucleotides acting through P2 receptors play key roles in
platelet signaling and hemostasis. P2 receptors can be subdivided
into P2X ionotropic and P2Y metabotropic receptors, and transgenic mice models have now firmly established roles of P2X1,
P2Y1, and P2Y12 receptors in platelet function.1-6 P2X1 receptors
for adenosine triphosphate (ATP) mediate rapid transient increases
in calcium concentration that can synergize with the larger, more
sustained response through the adenosine diphosphate (ADP)–
dependent P2Y1 receptor, leading to speeding and amplification of
the calcium increase.4 In addition, P2X1 receptor-deficient mice
have recently been shown to have a decreased aggregation response
and to exhibit protection against thromboembolism.7 A major
problem that has complicated the understanding and characterization of P2 receptors is the use of agonists to define P2 receptors
because impurity or interconversion of the agonist has led to the
pharmacologic misclassification of receptors. For example uridine
diphosphate (UDP) appears as a weak agonist at the uridine
triphosphate (UTP)–sensitive P2Y4 receptor; however, when contaminating UTP is removed with hexokinase, agonist activity is
essentially abolished.8 The same problem has also influenced the
study of P2 receptors in platelets and characterization of the
specificity of action of different purines at individual receptors. For
the P2X1 receptor we have shown that the apparent activity of ADP
at the receptor can be accounted for by ATP contamination of
commercially available ADP.9,10 This has important implications
for the physiologic activation of platelet purinoceptors because
ATP acts as a competitive antagonist rather than an agonist at
platelet P2Y1 receptors as a result of their low level of expression.11,12 Thus, in platelets ATP and ADP are selective stimuli at
P2X1 and P2Y1 receptors, respectively, and synergy between these
2 receptors may be important during hemostasis. However, the
inability of ADP to activate platelet P2X receptors has been
questioned by a study of a deletion variant, P2X1del. This variant,
cloned from platelets and megakaryocytic cell lines, is deficient in
17 amino acids in the extracellular ligand-binding loop and has
been reported to act as an ADP receptor in calcium measurement
studies.13 Significant controversy exists as to whether the P2X1del
receptor is expressed at high levels in platelets and exerts a
functional role.4,14,15
In this study we have (1) characterized the expression and
properties of recombinantly expressed P2X1del receptors, both as
homomeric P2X1del receptors and in heteromeric assemblies with
full-length P2X1 receptors; (2) determined in human platelets the
relative concentrations of expression of P2X1 and P2X1del receptors; and (3) investigated whether P2X1del receptors contribute to
generating P2X receptor-mediated signals. We provide conclusive
evidence that platelet P2X1del receptors are not functional in cell
lines or platelets and that ATP and ADP separately stimulate the
native platelet P2X and P2Y receptors during hemostasis.
From the Department of Cell Physiology and Pharmacology, University of
Leicester, United Kingdom; and the Department of Physiology, University of
Cambridge, United Kingdom.
Reprints: Richard J. Evans, Department of Cell Physiology and
Pharmacology, Medical Sciences Bldg, University of Leicester, University Rd,
Leicester LE1 9HN, United Kingdom; e-mail: [email protected].
Submitted June 18, 2003; accepted July 22, 2003. Prepublished online as
Blood First Edition Paper, August 7, 2003; DOI 10.1182/blood-2003-06-1963.
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.
Supported by a Wellcome Trust Programme Grant (R.J.E.) and the British
Heart Foundation (BS/10, FS/02/033, and FS/97052) (M.P.M.S.).
3646
Materials and methods
Cell culture and transfections
Human embryonic kidney 293 (HEK293) cells were maintained in minimal
essential medium with Earle salts (with GlutaMAX I) supplemented with
10% fetal bovine serum, 1% nonessential amino acid (Invitrogen, Paisley,
United Kingdom) at 37°C in a humidified atmosphere of 5% CO2 and 95%
air. Native 1321-N1 astrocytoma cells (a gift from Dr T.E. Webb, De
Montfort University, Leicester, United Kingdom) and 1321-N1 cells
© 2003 by The American Society of Hematology
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
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BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
subcloned after transfection of either wild-type (WT) P2X1 or P2X1del
(clone 4) (1321-N1 G P2X1WT or 1321-N1 G4 P2X1del, respectively;
kindly provided by Dr N.J. Greco, Red Cross Research Laboratories,
Rockville, MD) were maintained in Dulbecco modified Eagle medium
(with GlutaMAX I, 4500 mg/L D-glucose) supplemented with 10% fetal
bovine serum (Invitrogen). Stably transfected 1321-N1 cells were maintained under permanent selection in 240 ␮g/mL G418 (Invitrogen).
HEK293 and 1321-N1 cells were transiently transfected with P2X1WT,
P2X1del, or both receptors using LipofectAMINE 2000 Reagent/Opti-MEM
(both from Invitrogen). For patch-clamp studies, as a control to identify
cells that were efficiently transfected, the pEGFP-N1 (Clontech, Palo Alto,
CA) vector (10% of total plasmid transfected) was cotransfected with P2X1
plasmids, and recordings were made only from fluorescent cells expressing
the pEGFP. After 24 hours, the cells were either lysed, for Western blotting
or were attached to glass coverslips and kept in culture for a maximum of 3
days for use in confocal microscopy and patch-clamp experiments.
Protein expression analysis
Western blots. Human platelets from 13 healthy donors were prepared as
described16 and then pelleted at 10 000 rpm for 10 minutes. Platelets,
human embryonic kidney 293 (HEK293), and 1321-N1 cells were homogenized in 20 mM Tris (tris[hydroxymethyl]aminomethane)–HCl (pH 8), 250
mM NaCl, 3 mM EDTA (ethylenediaminetetraacetic acid), 3 mM EGTA
(ethylene glycol-bis (b-aminoethylether)-N,N,N⬘,N⬘-tetraacetic acid), and
0.5% Triton X-100 complemented with protease inhibitor cocktail (1:100)
(Sigma-Aldrich, Poole, United Kingdom). Samples were separated under
nonreducing conditions on 10% sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS-PAGE) gels. After electrophoretic transfer to
nitrocellulose (100 V, 1 hour), the membrane was blocked overnight in
TTBS (20 mM Tris-HCl [pH 7.6], 145 mM NaCl, 0.05% Tween 20) and
10% dry skim milk. The membrane was then incubated with anti-P2X1
antibody (1:1000) (Alomone, Jerusalem, Israel) in TTBS and 10% dry skim
milk for 2 hours, washed in TTBS, and incubated for 2 hours in antirabbit
horseradish peroxidase secondary antibody (1:1500 dilution) (Sigma). After
washing, the visualization of the protein bands was achieved with an ECL (Plus)
kit (Amersham Biosciences, Little Chalfont, United Kingdom) according to the
manufacturer’s instructions.
Surface cell expression and immunoprecipitations. All cell surface
proteins were biotinylated with Sulfo-NHS-LC-Biotin (0.5 mg/mL in
phosphate-buffered saline (PBS) (Pierce, Perbio Science, Tattenhall, United
Kingdom) for 30 minutes. After washing with PBS, cells were homogenized in RIPA buffer (10 mM Tris-HCl [pH 7.4], 2 mM EDTA, 160 mM
NaCl, 1% Nonidet P-40, and 0.5% deoxycholic acid) for 10 minutes on ice
and centrifuged at 13 000 rpm for 5 minutes. The supernatant was incubated
in the presence of 300 ␮L anti-P2X1 antibody (Alomone) diluted at 1: 200
in TE (10 mM Tris-HCl, 2 mM EDTA [pH 7.4]) for 2 hours on ice. Protein
A Sepharose CL-4B (180 ␮L of 3% gel, reconstituted in 10 mM Tris-HCl,
2 mM EDTA [pH 7.4]) (Amersham Biosciences) was added, and the
samples were homogenized for 15 minutes at 4°C, washed successively in
RIPA buffer and TE, and resuspended in the gel sample buffer (180 mM
Tris-HCl [pH 6.8], 5.7% SDS, 29% glycerol) and heated at 60°C for 3
minutes. The samples were separated on SDS-PAGE gel and were
transferred onto nitrocellulose membrane as described, and the membrane
was blocked overnight in TTBS and 3% bovine serum albumin (BSA).
The membrane was then incubated with Immunopure streptavidin/
horseradish peroxidase conjugated (0.5 ␮g/mL) (Pierce) in TTBS ⫹ 3%
BSA for 30 minutes and washed; the visualization of the protein bands
was achieved as described above.
Patch-clamp recordings
Conventional whole-cell, patch-clamp experiments were performed at a
holding potential of ⫺60 mV at room temperature, as described previously.4
Agonists were rapidly applied through a U-tube.
Intracellular calcium measurements
From cell lines. 1321-N1 cells (1321-N1 G P2X1WT or 1321-N1 G4
P2X1del), attached to glass coverslips, were incubated with 1 to 3 ␮M
LACK OF ADP-DEPENDENT P2X1 RECEPTORS IN PLATELETS
3647
fluo-3-AM for 30 to 45 minutes, then washed gently and added to the
imaging chamber. The standard bath saline contained (in mM) 150 NaCl,
2.5 KCl, 1 MgCl2, 2.5 CaCl2, 10 HEPES (N-2-hydroxethylpiperazine-N’-2ethanesulfonic acid), pH 7.3 (NaOH); CaCl2 was omitted for experiments in
nominally Ca2⫹-free saline. Fluorescence recordings (488 nm excitation,
greater than 505-nm emission) were made from fields of approximately 20
to 30 cells using a Zeiss LSM 510 confocal microscope or an Olympus
Fluoview FV 300 (Solent Scientific, Portsmouth, United Kingdom).
Fluorescent signals were background-subtracted, and F/F0 ratios were used
to normalize fluorescence levels (F) against starting fluorescence (F0).
Figures show the Ca2⫹ responses from individual cells representative of 15
to 30 cells within the field of view. ATP and ADP were applied either
through a U-tube system or by perfusion of the entire chamber. Thapsigargin (Calbiochem, Meik Biosciences, Nottingham, United Kingdom) was
added by dilution directly to the chamber.
Human platelets. Blood was taken with informed consent from 13
donors, and fura-2 ratiometric fluorescence measurements of [Ca2⫹]i in
stirred, washed platelet suspensions were performed as described previously.17 Platelet saline contained (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1
MgCl2, 10 HEPES, 10 D-glucose, pH 7.35 (NaOH). The cuvette temperature was lowered to 13°C to slow the start of the P2Y receptor-dependent
Ca2⫹ response and to more clearly distinguish P2X1-evoked Ca2⫹ increases.9 The background-corrected F340/F380 fluorescence ratio was used as
a direct indication of [Ca2⫹]i to avoid errors in application of the standard
calibration methods in platelets at this temperature (for a discussion, see
Mahaut-Smith et al9). Agonists were added after insertion of Hamilton
syringes into a custom-built holder to avoid artefacts introduced by opening
the cuvette lid, and additions were marked electronically to allow comparison of timing between different experiments.
Data analysis
Data are presented throughout as mean ⫾ SEM; n represents the number of
observations. Differences between means were determined by the appropriate Student t test and were considered significant when P ⬍ .05.
Reagents
␣␤-Methylene ATP (␣␤-meATP), ATP, and ADP were all obtained from
Sigma. ADP was treated with hexokinase in a high-glucose–containing
saline at pH 8, as described previously,9 to remove contaminating levels of
ATP. Fluo-3-AM and fura-2-AM were from Molecular Probes (Leiden, the
Netherlands) and made as stocks of 1 mM in 20% (wt/vol) Pluronic in
dimethyl sulfoxide (DMSO).
Results
Electrophysiologic properties of P2X1 WT and P2X1del receptors
transiently expressed in HEK cells
ATP (100 ␮M) is a full agonist at P2X1 WT receptors and evokes rapidly
desensitizing inward currents through these receptors transiently expressed in HEK293 cells (mean amplitude, 4629 ⫾ 700 pA; n ⫽ 18)
(Figure 1A). Commercially available ADP (100 ␮M) evoked an inward
current (mean amplitude, 1514 ⫾ 502 pA; n ⫽ 7; data not shown);
however, after removing contaminating ATP with hexokinase, the
response to ADP was reduced by more than 95%, to ⫺71 ⫾ 44 pA
(n ⫽ 10; Figure 1A). We failed to detect any ATP or ADP (both 100
␮M) receptor-activated currents in HEK293 cells transfected with the
P2X1del receptor (n ⫽ 14, 18) (Figure 1B). Total expression levels of
P2X1WT and P2X1del were determined using Western blot analysis with
an anti-P2X1 receptor antibody. The anti-P2X1 receptor antibody
identified an approximately 50-kDa band in cells transfected with the
P2X1WT plasmid and an approximately 46-kDa band in cells transfected with the P2X1del plasmid (Figure 1D) (in addition, a nonspecific
band of approximately 60 kDa was present in nontransfected HEK293
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3648
VIAL et al
Figure 1. Expression and properties of recombinant P2X1WT and P2X1del
receptors in HEK293 cells. (A) ATP (100 ␮M; drug application is indicated by bar)
induced a transient inward current in HEK293 cells transiently transfected with P2X1
WT receptors, whereas ADP (100 ␮M) was ineffective. (B) ATP or ADP (both 100 ␮M)
failed to evoke a change in holding current from HEK293 cells transiently transfected
with P2X1 del receptors. (C) When P2X1 WT and P2X1 del receptors were transiently
coexpressed, ATP (100 ␮M) induced inward currents; however, ADP (100 ␮M) was
ineffective. (D) Western blot analysis on nontransfected HEK293 cells (NT) and cells
transiently transfected with either P2X1 WT (WT), P2X1 del (del), or both receptors
(WT ⫹ del). Total cell protein analysis (left panel) showed that P2X1WT and P2X1del
receptors were expressed. However, only P2X1 WT receptor was detected at the cell
surface (right panel). A weak nonspecific band was observed around 60 kDa in
nontransfected HEK293 cells.
cells, but not in 1321-N1 cells; see Figure 2B). The total level of
expression of the P2X1del was approximately 20% compared with the
P2X1WT. Cell surface biotinylation studies demonstrated that the
P2X1WT receptor was trafficked to the cell surface; however, surface
expression of P2X1del receptors was below the limit of detection (Figure
1D). These results indicate that the lack of agonist-evoked membrane
current in cells transfected with P2X1del receptors is at least in part
caused by poor surface expression of the receptor.
Heteromeric assembly of different P2X receptor subunits (eg,
P2X2 and P2X318) or WT and mutant P2X1 receptor subunits can
give rise to channels with altered properties.19 Both P2X1 WT and
P2X1del receptor RNAs are made by platelets and a range of
megakaryocytic cells,13 which raises the possibility that heteromeric channels may be formed with novel properties. We therefore
cotransfected P2X1WT and P2X1del receptors in HEK293 cells with
a DNA ratio of 1:9 to determine whether we could detect any
regulation of the properties of P2X1WT receptors. Agonist-evoked
currents from cells cotransfected with P2X1WT and P2X1del were
indistinguishable from those of WT P2X1 receptors—that is, there
was a rapidly desensitizing response to ATP, whereas ADP was
ineffective as an agonist (Figure 1C; n ⫽ 15, 10) (similar results
were found when the receptors were transiently cotransfected in
1321-N1 cells, ratio P2X1WT/P2X1del 1:9; n ⫽ 10; for Western blot
see Figure 2C). Western blot analysis indicated that the total level
of P2X1WT and P2X1del receptors was similar in HEK293 cells
(Figure 1D). However, cell surface biotinylation again showed that
only P2X1WT was detected at the cell surface (Figure 1D).
BLOOD, 15 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 10
was consistent with Western blotting results in which both P2X1
WT and P2X1del receptors were below the limit of detection in these
cell lines (Figure 2B).
When P2X1WT receptors were transiently transfected into a
1321-N1 cell background, ATP (100 ␮M) evoked inward currents
(in either the 1321-N1 G P2X1WT or the 1321-N1 G4 P2X1del mean
amplitude, ⫺1674 ⫾ 470 pA; n ⫽ 19) (Figure 2A), demonstrating
that P2X1WT receptors can form functional channels in this cell
line as described previously.20 As shown for the recombinant P2X1
receptors expressed in HEK293 cells, purified ADP was ineffective
as an agonist at activating P2X1WT receptor-mediated inward
currents in 1321-N1 cells (Figure 2Aiii, ⫺32 ⫾ 12 pA; n ⫽ 9). No
inward current was evoked by either ATP or ADP (100 ␮M) when
applied to native 1321-N1 cells transiently expressing P2X1del
receptors (data not shown; n ⫽ 8, 9). Western blotting was used to
estimate the total levels of expression of transiently transfected
P2X1WT and P2X1del receptors in 1321-N1 cells (Figure 2C). The
anti-P2X1 receptor antibody detected bands in total cellular lysates
corresponding to the P2X1WT and P2X1del receptors. These results
confirm that the P2X1del receptor can be expressed but that it fails to
form functional ion channels in 1321-N1 cells.
Characteristics of the P2 receptor-evoked [Ca2ⴙ]i responses in
cell lines used for previous P2X1del studies
Even though we could not detect P2X1 receptor current or protein
in the cell lines provided by Greco et al,13 we were able to detect
rapid increases in intracellular calcium in response to ATP and ADP
(Figure 3A) in 50% and 67% of cells tested (70 cells from 3
different studies) (Figure 3A). These results can be explained by
the presence of native P2Y receptors in the subclonal 1321-N1 G
P2X1WT and 1321-N1 G4 P2X1del cell lines. The standard approach to assess the relative contribution of P2Y versus P2X
receptors is to examine the Ca2⫹ response in the absence of external
Ca2⫹. P2X receptors rely entirely on Ca2⫹ influx, whereas the
response to P2Y receptors was at least partly caused by inositol 1,
4,5-triphosphate (IP3)–dependent Ca2⫹ release. However, this
proved difficult in 1321-N1 cells because of the rapid depletion of
the intracellular Ca2⫹ stores in Ca2⫹-free medium. Thus, after only
Functional properties of P2X1 receptors expressed
in 1321-N1 cells
In the study by Greco et al,13 P2X1del receptors were stably
expressed in the 1321-N1 cell line, and changes in calcium levels
were monitored from populations of cells. Because Ca2⫹ increases
evoked by ATP and ADP can result from both P2X and P2Y
receptors, we used patch-clamp recordings to detect P2X receptor
currents in individual cells from the lines used in the original study
by Greco et al.13 Applying ATP or ADP to 1321-N1 G P2X1 WT or
1321-N1 G4 P2X1del cell lines gave no change in holding current,
demonstrating that these cells do not express functional P2X
receptors (n ⫽ 19, 8, and 15, 15 respectively) (Figure 2A). This
Figure 2. Expression and properties of recombinant P2X1WT and P2X1del
receptors in 1321-N1 cells. (A) ATP and ADP (both 100 ␮M, application indicated by
bar) had no effect on the holding current in 1321-N1 cells stably transfected with
either (i) P2X1WT (1321-N1 G P2X1 WT) or (ii) P2X1del (1321-N1 G4 P2X1del)
receptors.13 When transiently transfected with P2X1WT receptors (iii), these cells
gave a transient inward current in response to ATP (100 ␮M) but not to ADP (100 ␮M).
(B) Western blot showing that no P2X1 receptor signal was detected in 1321-N1 G5
P2X1del, 1321-N1 G4 P2X1del, or 1321-N1 G P2X1WT cell clones13 and in nontransfected native 1321-N1 or HEK293 cells (NT). We used HEK293 cells transiently
transfected with P2X1 WT as a positive control of expression of the P2X1 receptor.
(C) However, bands corresponding to P2X1 receptors were detected when P2X1WT
(WT), P2X1del (del), or P2X1 WT ⫹ P2X1del (WT ⫹ del) receptors (ratio 1:9) were
transiently expressed in 1321-N1 G P2X1WT cells.
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3649
2 to 3 minutes in nominally Ca2⫹-free saline, ADP failed to evoke a
response, though this resulted from the depletion of the internal
stores because the subsequent addition of the endomembrane
Ca-ATPase inhibitor Thapsigargin had no effect on the cytosolic
Ca2⫹ level (Figure 3B). The extremely rapid depletion of Ca2⫹
stores in Ca2⫹-free medium in this cell line highlights the difficulty
of using this condition as a definitive test for P2X or P2Y receptors. In
Ca2⫹-containing medium, when the stores are replenished after agonist
exposure, Thapsigargin causes its expected effect of a substantial
increase in cytosolic Ca2⫹ (Figure 3C). However, ADP did evoke a
small Ca2⫹ response after shorter exposures to Ca2⫹-free medium (for
example, 60 seconds; see Figure 3D) in both 1321-N1 G P2X1WT and
1321-N1 G4 P2X1 del, confirming the presence of P2Y responses in these
cell lines.
Lack of evidence for ADP-evoked Ca2ⴙ responses through
native P2X receptors in human platelets
The lack of functional expression of recombinant P2X1del receptors
may reflect the absence of some accessory factor necessary for
efficient trafficking/processing of the receptor in HEK293 and
1321-N1 cell lines. Given that the P2X1del receptor was originally
isolated from platelets, we determined the levels of P2X1del
receptor expression in human platelets and the functional role of
P2X and P2Y receptor-mediated calcium signaling. In Western blot
analysis of platelet lysates (from 13 donors) the anti-P2X1 receptor
antibody detected a single band at approximately 50 kDa, corresponding to the P2X1WT receptor, but no lower molecular weight
band corresponding to the P2X1del receptor was detected (Figure
4A). To estimate the detection threshold for the anti-P2X1 antibody,
we diluted the amount of platelet sample run on the gel. P2X1WT
receptors could still be detected at a 1:24 dilution (Figure 4B).
Assuming that the antibody has the same sensitivity for P2X1WT
and P2X1del (which is valid given that the deletion is in the
extracellular portion of the receptor and the 2 variants share the
identical epitope at the C-terminal), this indicates that the P2X1del
receptor accounts for less than 4% of the total protein. Given the
additional reduction in efficiency of transfer to the cell membrane,
this indicates that the P2X1del accounts for a small proportion, if
any, of the cell surface P2X1 receptors. Studies of P2 receptor Ca2⫹
Figure 4. Lack of detection of P2X1del protein or ADP evoked P2X responses in
human platelets. (A) Ten ␮g platelet total protein from 13 healthy human donors was
analyzed by Western blotting (i) and (ii) as a control. Samples of recombinant
P2X1WT and P2X1del receptors in HEK293 cells are shown. P2X1WT receptors were
detected in the platelets from each donor; the P2X1 del receptor, however, was below
the limit of detection. (B) To overcome the possibility that P2X1 del receptor was not
detected because of the weak expression level in platelets, dilution of one of the platelet
protein samples (donor 4) was analyzed by Western blotting. The band corresponding to
the P2X1 WT receptor was still detectable when only 0.625 ␮g platelet total proteins were
separated (the small box represents the 3 last lanes for which the autoradiography has
been submitted to a longer exposure). Therefore, if P2X1 del receptor is also expressed in
human platelets, the ratio P2X1del/P2X1WT is less than 1:24. (C) [Ca2⫹]i measurements in
suspensions of fura-2–loaded human platelets, as indicated by the 340/380 fluorescence
excitation ratio, in response to either 10 ␮M ␣␤meATP or 10 ␮M hexokinase-treated ADP
(arrow). Traces from 2 donors (10 and 11 in panel Aii) are shown at 2 different time scales
and are representative of the results from 13 donors. The cuvette temperature was set at
13°C to allow clear separation of P2X and P2Y receptor-evoked responses, as described in
Mahaut-Smith et al.9
responses were also conducted in platelets from 13 donors,
including 6 of those used for the Western blot analyses of Figure
4A. The temperature was lowered to 13°C to clearly distinguish
rapid P2X1 increases from the slower P2Y1 response.9 In all donors
tested (n ⫽ 13), the P2X1 receptor agonist ␣␤-meATP evoked a
rapid transient calcium increase (Figure 4C), whereas the P2Y1
receptor agonist, ADP (40 ␮M, hexokinase purified) evoked Ca2⫹
increases with a prolonged delay of 2 to 3 seconds. The lack of
evidence for an ADP-induced P2X receptor-mediated rise in
calcium demonstrates that ADP-dependent P2X1del receptors do not
contribute to calcium signaling in human platelets.
Discussion
Figure 3. Intracellular Ca2ⴙ measurements in subclones of 1321-N1 cells
indicate the presence of P2Y receptor responses. Intracellular Ca2⫹ responses in
single 1321-N1 G P2X1 WT (A) and 1321-N1 G4 P2X1del cells (B-D)13 loaded with the
fluorescent calcium indicator fluo-3. Similar results were obtained in the 2 cell lines.
(A) In the presence of extracellular Ca2⫹, ATP (30 ␮M) and ADP (30 ␮M) evoked a
transient Ca2⫹ increase. (B) Loss of the response to ADP (100 ␮M) after only a short
period (120 seconds in this experiment) in nominally Ca2⫹-free medium. This lack of
response was caused by the rapid depletion of Ca2⫹ from intracellular stores because
Thapsigargin (TG) had no effect in Ca2⫹-free medium. (C) Demonstration that TG
evoked an expected large increase in Ca2⫹-containing medium after ADP exposure.
(D) Adding ADP earlier (60 seconds) after exposure to Ca2⫹-free medium evoked a
small, delayed Ca2⫹ increase.
In the present study we have investigated the expression and
function of the P2X1del receptor because it has been suggested that
this receptor may be sensitive to ADP and may play a functional
role in the activation of platelets. We have demonstrated that the
P2X1del receptor fails to form functional channels when expressed
in HEK293 and 1321-N1 cell lines and that this most likely results
from poor trafficking of the receptor to the cell surface. In addition,
in human platelets, P2X1del protein was below the limit of
detection. These results demonstrate that the P2X1del receptor does
not play a significant role in platelet function and that the P2X1
receptor in human platelets is activated by ATP not ADP.
P2X1WT receptors were transiently expressed in HEK293 and
1321-N1 cells as a positive control. Activation of this channel by
ATP evoked rapidly inactivating responses, and purified ADP was
ineffective as an agonist, confirming results described by several
independent laboratories.9,10,18,20-24 It is anomalous that in the study
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VIAL et al
of Greco et al,13 purified ADP was found to be an equi-effective agonist
at the P2X1WT receptor. Even when commercially available ADP
(impure) has been used, it only acts as a weak partial agonist with more
than 100-fold reduced potency compared with ATP in electrophysiologic9,21 or calcium-imaging studies.20 In the study by Greco et al,13
ADP evoked an intracellular calcium increase of amplitude comparable
with that of ATP when these agents were added sequentially, with an
interval of only 10 to 20 seconds in the P2X1WT stable cell line
(designated 1321-N1 G P2X1 WT in this study).13 This is unusual
because native and recombinant P2X1 receptors show profound desensitization to agonist application, such that long wash/recovery periods
between applications are required to generate reproducible responses.
Furthermore, agonist desensitization is used routinely to remove “contaminating” P2X1 receptor-mediated responses in pharmacologic studies.18,25 For example, the role of native P2X1 receptors in HL60 cells and
platelets had been overlooked because of problems associated with
receptor desensitization during cell preparation.17,26 For these reasons
the pharmacology of the P2X1WT responses reported by Greco et al13
appear inconsistent with the consensus of those reported for wild-type
P2X1 receptors (for recent review, see North18).
The patch-clamp technique provides a highly sensitive measure
of changes in membrane current and detection of functional ion
channels. The single-channel amplitude of the P2X1WT receptor
under the recording conditions used is approximately 0.6 pA
(R.J.E., unpublished observations, 2001), and signals of approximately 2 to 3 pA can be readily discriminated; therefore, theoretically the opening of as few as 5 channels can be detected. The
P2X1del receptor failed to produce functional ion channel currents
when transiently expressed in either HEK293 or 1321-N1 cells. Using
Western blot analysis (Figures 1, 2), we show that the P2X1del receptor is
produced in these cells and associates to form trimeric channels (data not
shown), as for the wild type. However, the total P2X1del receptor level
was reduced compared with equivalent transfection of P2X1WT (for a
discussion, see also Oury et al14), indicating that the P2X1del receptor is
either inefficiently made or broken down. In addition, we showed the
P2X1del receptor is below the limit of detection at the cell surface, and
this lack of receptor trafficking most likely accounts for the lack of
functional P2X1del receptor-mediated currents.
There was an approximately 3- to 4-kDa decrease in the
molecular mass of the P2X1del receptor monomer compared with
the full-length P2X1WT receptor, as described previously after in
vitro translation.13,14 This is to be expected because the deletion
removes 17 amino acids that include a glycosylation site (N184,
which contributes 2-3 kDa).27 The poor surface expression of the
P2X1del receptor in the present study is unlikely to result solely
from the removal of glycosylation at N184.27,22 Interestingly, for
the rP2X1a deletion mutant, which has a larger deletion (amino
acids 175-201 compared with 176-192 for P2X1del), the coexpression of P2X1WT can in part rescue cell surface channel expression
in HEK293 cells.22 This was not the case for the P2X1del receptor.
The reason the extra 10-amino acid deletion of rP2X1a encourages
cotrafficking to the cell surface remains to be determined.
We were unable to detect P2X1 or P2X1del receptor expression
either electrophysiologically or by Western blotting using the stable
cell lines expressing P2X1 and P2X1del receptors provided by Greco
et al.13 When the P2X1WT receptor was transiently transfected into
these 1321-N1 stable cell lines, ATP evoked robust P2X receptor
currents, and receptor protein of the appropriate molecular weight
was detected, confirming that the cells can produce functional
P2X1WT receptors.20 The fact that calcium responses to ADP and
ATP can be recorded from these cells in the absence of detectable
P2X1 receptor currents or protein indicates that endogenous P2
receptors other than P2X1 are expressed in these cells.13
Results following the expression of recombinant receptors
indicate that P2X1del receptors are not functional, though it is
possible that HEK293 and 1321-N1 cell lines lack some accessory
factor necessary for efficient trafficking/processing of these receptors. Previous estimates of relative levels of P2X1WT and P2X1del
in platelets have been made based on reverse transcription–
polymerase chain reaction (RT-PCR). In this study we measured
protein levels directly with Western analysis and investigated the
role of ADP-activated P2X1 receptors in functional assays. Western
blotting failed to detect the P2X1del receptor protein in total platelet
lysates from 13 donors (in assays that could detect a 24-fold
dilution of the WT receptor). Given the likely additional inefficient
trafficking to the cell surface, P2X1del accounts for a small, if any,
proportion of the P2X1 receptors at the cell surface. Because the
P2X1del receptor failed to be expressed on the cell surface of HEK
or 1321-N1 cells, it is still uncertain whether ADP is an agonist, as
shown for the WT receptor. Nevertheless, functional studies failed
to detect a rapid ADP-mediated P2X receptor Ca2⫹ response; thus,
ADP-activated receptors, whether P2X1del or other naturally occurring mutants, do not contribute to the P2X1 receptor phenotype, as
also shown in kinase assays.24 Previous work has shown that the
P2X1 receptor in human platelets synergizes with functional responses
through P2Y1 and collagen receptors.4,5 Thus, our present study supports
the concept that ATP, released from damaged vascular cells or cosecreted from platelets with ADP and other agonists, is an agonist through
P2X1 receptors during hemostasis. A recent study using P2X1⫺/⫺ mice
has suggested that the main role of this receptor is to enhance platelet
activation under conditions of high shear stress.7 This functional
importance of P2X1 receptors may be a consequence of the rapid nature
of the ATP-activated Ca2⫹ influx or its ability to potentiate P2Y1 and
collagen-receptor signaling.4,5
In summary, we can find no evidence to support a functional
role for an ADP-activated P2X1del receptor in human platelets and
conclude that ATP is the principal natural agonist at P2X receptors
in platelets mediating a rapid increase in intracellular calcium. This
calcium increase also synergizes with subsequent ADP-mediated
P2Y receptor signaling events associated with hemostasis, and
blockade of the ATP-sensitive P2X1 receptor may provide a novel
drug target for protection against thromboembolism.7
Acknowledgments
We thank Dr N. Greco for providing the P2X1del plasmid and transfected
1321-N1 cell lines, Dr T. Webb for providing native 1321-N1 cells,
Professor A. Goodall and Dr J. Appleby for providing samples of human
platelets, and J. Holdich for technical assistance.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2003 102: 3646-3651
doi:10.1182/blood-2003-06-1963 originally published online
August 7, 2003
Lack of evidence for functional ADP-activated human P2X1 receptors
supports a role for ATP during hemostasis and thrombosis
Catherine Vial, Samantha J. Pitt, Jon Roberts, Michael G. Rolf, Martyn P. Mahaut-Smith and Richard
J. Evans
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