Does the P_2X1del 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 al1reported on the expression of a novel structurally altered P_2X1 receptor in platelets and in megakaryocytic cell lines. This P_2X1 variant lacks 17 amino acids in its extracellular domain due to a deletion within exon 6 of theP_2X1 gene (GenBank accession no. 17481172). The authors showed that, after heterologous expression in the 1321N1 astrocytoma cell line, P_2X1del 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 P_2X1mRNA of platelets, thus claiming that P_2X1del 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 P_2X1 receptor is an adenosine triphosphate (ATP)–gated ion channel that is unresponsive to high-performance liquid chromatography–purified ADP. Indeed, the activation of the P_2X1 receptor by ATP or by its stable analogs, α,β-methylene ATP and L-β,γ–methylene ATP, produces a rapid, quickly desensitized Ca²⁺ influx4 that is responsible for reversible platelet shape change,3,5and 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 P_2X1 receptors (P_2X1wt).3In addition, platelet receptors for ADP have been well characterized and are identified as 2 P_2Y receptors: P_2Y1 and P_2Y12 (reviewed in Gachet6). Both receptors are required for normal platelet responses to ADP, a conclusion recently corroborated in P_2Y1−/− and P_2Y12−/− knock-out mice.7,8 Thus, the existence in platelets of an ADP-activated variant of the P_2X1 ion channel as the major platelet P_2X 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 P_2X1del in platelet function. First, RT-PCR analyses of independent platelet RNA samples followed by sequencing of the PCR products showed major abundance of the P_2X1wt mRNA (Figure 1A, lane 2; Figure 1B), whereas the platelet P_2X1del mRNA appeared as a minor product. In contrast to the findings of Greco et al,1 we found comparable relative amounts of the P_2X1del 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 P_2X1del transcripts in platelets the authors designed 2 different sets of primers, S1/AS1 to amplify both P_2X1wt and P_2X1del cDNAs and S2/AS1 to amplify only P_2X1del cDNAs. However, this strategy does not enable a quantitative assessment of P_2X1del levels. Indeed, when the pcDNA3-P_2X1wtplasmid was used as a PCR template, we showed that the primer set S2/AS1 annealed to the P_2X1wt cDNA leading to artificial amplification of the 51 base pair deleted cDNA-encoding P_2X1del (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 P_2X1wtand P_2X1del 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 P_2X1del cDNA (del) or the P_2X1wt cDNA (wt) revealed only low amounts of P_2X1del proteins at the expected size in comparison to the P_2X1wt protein levels (Figure 1C). To ensure that the antibody used in this detection would recognize the 2 proteins with equal sensitivity the P_2X1del(del) and P_2X1wt (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 P_2X1del protein is not properly produced or is mainly unstable in the transfected 1321N1 cells.

Taken together, our data indicate that the P_2X1del 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.6The quantitative and functional relevance of the platelet P_2X1del variant should therefore be reconsidered.3 4