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Blood, 15 March 2002, Vol. 99, No. 6, pp. 2275-2277
CORRESPONDENCE
To the editor:
Does the P2X1del variant lacking 17 amino
acids in its extracellular domain represent a relevant functional ion
channel in platelets?
In a recent issue of Blood, Greco et al1
reported on the expression of a novel structurally altered
P2X1 receptor in platelets and in megakaryocytic
cell lines. This P2X1 variant lacks 17 amino acids in its
extracellular domain due to a deletion within exon 6 of the
P2X1 gene (GenBank accession no.
17481172). The authors showed that, after heterologous expression in
the 1321N1 astrocytoma cell line, P2X1del subunits
constitute a channel preferentially activated by adenosine diphosphate
(ADP). In reverse transcriptase-polymerase chain reaction (RT-PCR)
analyses, they described this variant as the major P2X1
mRNA of platelets, thus claiming that P2X1del may play an
important role as an ADP-activated ion channel in these cells. These
conclusions are in contradiction with other studies2,3 that
show that the functional platelet P2X1 receptor is an
adenosine triphosphate (ATP)-gated ion channel that is unresponsive to
high-performance liquid chromatography-purified ADP. Indeed, the
activation of the P2X1 receptor by ATP or by its stable
analogs,  , -methylene ATP and L-, -methylene ATP, produces a
rapid, quickly desensitized Ca2+ influx4 that
is responsible for reversible platelet shape change,3,5
and that also plays a pivotal role during platelet aggregation
induced by collagen.3 These platelet responses to ATP were
found to be antagonized by ADP similarly to the inward current produced
by ATP in Xenopus oocytes expressing wild-type P2X1 receptors (P2X1wt).3
In addition, platelet receptors for ADP have been well
characterized and are identified as 2 P2Y receptors: P2Y1 and P2Y12 (reviewed in
Gachet6). Both receptors are required for normal platelet
responses to ADP, a conclusion recently corroborated in
P2Y1 / and P2Y12/ knock-out
mice.7,8 Thus, the existence in platelets of an
ADP-activated variant of the P2X1 ion channel as the major
platelet P2X representative, as hypothesized by Greco et
al,1 can be questioned. In this letter, we present data that argue against the possibility for
a role of P2X1del in platelet function. First,
RT-PCR analyses of independent platelet RNA samples followed by
sequencing of the PCR products showed major abundance of the
P2X1wt mRNA (Figure 1A, lane 2;
Figure 1B), whereas the platelet P2X1del mRNA appeared as a
minor product. In contrast to the findings of Greco et
al,1 we found comparable relative amounts of the
P2X1del mRNA in platelets and in the Dami megakaryocytic
cell line (Figure 1A, lane 3; Figure 1B). Second, in order to further
demonstrate the presence of P2X1del transcripts in
platelets the authors designed 2 different sets of primers, S1/AS1 to
amplify both P2X1wt and P2X1del cDNAs and
S2/AS1 to amplify only P2X1del cDNAs. However, this
strategy does not enable a quantitative assessment of
P2X1del levels. Indeed, when the pcDNA3-P2X1wt
plasmid was used as a PCR template, we showed that the primer set
S2/AS1 annealed to the P2X1wt cDNA leading to artificial
amplification of the 51 base pair deleted cDNA-encoding
P2X1del (Figure 1A, lane 4, as confirmed by sequencing).
This phenomenon evidently also can occur during RT-PCR analyses of
platelet and Dami cell RNA samples containing both P2X1wt
and P2X1del mRNA (Figure 1A, lane 5 and 6, respectively). Third, Western blotting experiments performed after transient transfection of 1321N1 cells with a pcDNA3 vector containing either the
P2X1del cDNA (del) or the P2X1wt cDNA (wt)
revealed only low amounts of P2X1del proteins at the
expected size in comparison to the P2X1wt protein levels
(Figure 1C). To ensure that the antibody used in this detection would
recognize the 2 proteins with equal sensitivity the P2X1del
(del) and P2X1wt (wt) proteins were synthesized in an in
vitro T7-coupled transcription/translation rabbit reticulocyte system.
Western blotting analyses revealed identical amounts of the 2 in
vitro-translated (nonglycosylated) proteins (Figure 1D). These data
thus suggest that the P2X1del protein is not properly produced or is mainly unstable in the transfected 1321N1 cells.
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| Figure 1.
Analyses of P2X1del mRNA and protein.
(A) RT-PCR of platelet (lanes 2 and 5) and Dami cell (lanes 3 and 6)
RNA; the primer sets S1/AS1 and S2/AS1 are described by Greco et
al.1 In lanes 1 and 4, the pcDNA3-P2X1wt
plasmid was used as a PCR template. (B) Enlarged view of lanes 2 and 3. For these experiments, platelets were isolated from freshly drawn blood
of at least 10 unrelated healthy volunteers. (C) Western blots of
P2X1del and P2X1wt in transfected 1321N1 total
cell extracts. The pcDNA3-P2X1del vector was transfected in
2 independent experiments (del1 and del2) in
parallel with the pcDNA3-P2X1wt vector (wt). A
nontransfected cell extract is also shown (NT). (D) Western blots of
P2X1del (del) and P2X1wt (wt) proteins
synthesized in a in vitro T7-coupled transcription/translation rabbit
reticulocyte system. The rabbit polyclonal anti-human P2X1
antibody used in these experiments was previously
described.9 Bands corresponding to P2X1del and
P2X1wt PCR products and proteins are indicated. Molecular
weight ladder is shown on the left.
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Taken together, our data indicate that the P2X1del variant
is unlikely to be a major protein in platelets. Moreover, the fact that
Greco et al present this variant as a potential ADP-activated channel is not consistent with all the previous molecular and functional studies of platelet P2 receptors.6
The quantitative and functional relevance of the platelet
P2X1del variant should therefore be
reconsidered.3,4
Cécile Oury, Emese Toth-Zsamboki, Jos Vermylen, and Marc F. Hoylaerts
Correspondence: Marc 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
References
1.
Greco NJ, Tonon G, Chen W, Luo X, Dalal R, Jamieson GA.
Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells.
Blood.
2001;98:100-107[Abstract/Free Full Text].
2.
Mahaut-Smith MP, Ennion SJ, Rolf MG, Evans RJ.
ADP is not an agonist at P2X1 receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets.
Br J Pharmacol.
2000;131:108-114[CrossRef][Medline]
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3.
Oury C, Toth-Zsamboki E, Thys C, Tytgat J, Vermylen J, Hoylaerts MF.
The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen.
Thromb Haemost.
2001;86:1264-1271[Medline]
[Order article via Infotrieve].
4.
Rolf MG, Brearley CA, Mahaut-Smith MP.
Platelet shape change evoked by selective activation of P2X1 purinoceptors with ,-me-ATP.
Thromb Haemost.
2001;85:303-308[Medline]
[Order article via Infotrieve].
5.
MacKenzie AB, Mahaut-Smith MP, Sage SO.
Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets.
J Biol Chem.
1996;271:2879-2881[Abstract/Free Full Text].
6.
Gachet C.
ADP receptors of platelets and their inhibition.
Thromb Haemost.
2001;86:222-232[Medline]
[Order article via Infotrieve].
7.
Léon C, Hechler B, Freund M, et al.
Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.
J Clin Invest.
1999;104:1731-1737[Medline]
[Order article via Infotrieve].
8.
Foster CJ, Prosser DM, Agans JM, et al.
Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.
J Clin Invest.
2001;107:1591-1598[Medline]
[Order article via Infotrieve].
9.
Oury C, Toth-Zsamboki E, Van Geet C, et al.
A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue.
J Biol Chem.
2000;275:22611-22614[Abstract/Free Full Text].
Response:
Functional adenosine diphosphate-activated P2X1del
receptor
Oury et al have confirmed our recent identification of a
P2X1del variant of the P2X1wt receptor RNA in
platelets and megakaryocytic DAMI cells.1 The complex
array of nucleotide receptors they describe in different cell types
suggests that questions of identity and function may not be fully
resolved. What we have done, no more and no less, is to show that
transfection of nonresponsive 1321 cells with the
P2X1del-variant cDNA results in the expression in these
cells of a selective homomeric receptor sensitive to adenosine
diphosphate (ADP).1 Unfortunately, Oury et al have not provided adequate information
for an evaluation of their polymerase chain reaction (PCR) results. We
have previously shown that stringent annealing temperatures (60°C)
are needed to minimize nonspecific binding due to the high degree
of homology of the S2 and AS1 PCR primers.1(Fig1 legend)
There are similar difficulties in evaluating their protein blots because their antibody appears to recognize multiple proteins in
nontransfected cells.1(Fig 1C, lane 1, "NT")
In addition, Oury et al do not take into account the
cross-reactivity of available antibodies for the P2X1wt and
P2X1del receptors. We do not feel that their conclusion
that "the P2X1del protein is not properly produced or is
mainly unstable in the transfected 1321N1 cells" can be deduced from
these results. Furthermore, contrary to the comments of Oury et al we have made no
claims that "the P2X1del variant is a major protein in platelets." In fact, data from the literature2 would
suggest that the P2X receptor would be of low abundance in
platelets. However, our Ca2+ influx studies clearly show
that the P2X1del receptor is an ADP-activated channel, not
a potential ADP-activated channel. Consideration of these
problems, taken together with the extensive data reported in our
original paper,1 shows that the question posed by Oury et
al as the title of their communication must be answered in the affirmative.
Nicholas J. Greco, Giovanni Tonon, Weidong Chen, Xunyi Luo, Rakhi Dalal, and Graham A. Jamieson
Correspondence: Nicholas J. Greco, American Red Cross, Blood & Cell Therapy Development Department, 15601 Crabbs Branch Way,
Rockville, MD 20855; e-mail: greco{at}usa.redcross.org
References
1.
Greco NJ, Tonon G, Chen W, Luo X, Dalal R, Jamieson GA.
Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells.
Blood.
2001;98:100-107.
2.
Mahaut-Smith MP, Sage SO, Rink TJ.
Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.
J Biol Chem.
1992;267:3060-3065[Abstract/Free Full Text]
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