The TRPV1 ion channel is expressed in human but not mouse platelets

To the Editor

We have previously reported the expression of the TRPV1 ion channel in human platelets and have demonstrated a role for this channel in mediating agonist-evoked Ca²⁺ signalling in these cells [1]. Our Western blot studies have been independently reproduced by Savini et al. using a different antibody [2]. Furthermore, TRPV1 has also been identified in a human platelet proteomic study [3], [4]. These earlier studies are, however, yet to address the role that this non-selective cation channel might play in regulating platelet activity in vivo. A common strategy to assess the functions of signalling molecules in platelets has been to knockout the gene for the protein of interest in mice and then compare the responses of platelets from the knockout and wild-type animals. As a TRPV1-knockout mouse has been generated [3], we conducted pilot experiments to assess whether this channel is expressed in the platelets of wild-type mice and thus whether the knockout mouse would provide a useful model for studying the function of TRPV1 in platelets in vivo.

Blood was taken by venepuncture from healthy human volunteers or by venepuncture of the abdominal vein in wild-type C57/Bl6 or Swiss mice terminally anaesthetised with ketamine (75 mg/kg) and medetomidine (1 mg/kg). After blood collection, mice were euthanized by cervical dislocation. Washed platelet suspensions from both species were prepared and the platelets were then loaded with Fura-2 by incubation with 5 µM Fura-2/AM for 30 minutes at 37°C. Platelets were then collected and resuspended in Hepes-buffered saline supplemented with glucose (10 mM), bovine serum albumin (1 mg/ml) and apyrase (0.1 U/ml) [1].

In Fura-2-loaded platelets from wild-type C57/Bl6 or Swiss mice, capsaicin (100 µM), in the presence of 1 mM external Ca²⁺, evoked no rise in [Ca²⁺]_cyt over that observed in vehicle-treated controls (Figure 1A; n = 6). This lack of response was not due to a failure of Fura-2 loading or an ability to detect changes in [Ca²⁺]_cyt as samples from all mouse platelet suspensions demonstrated changes in [Ca²⁺]_cyt upon treatment with thapsigargin in the absence of extracellular Ca²⁺ followed by the addition of Ca²⁺ to the medium to elicit store-operated calcium entry (data not shown). In parallel experiments, human platelets, at the same cell density, responded to capsaicin from the same stock solution with a rise in [Ca²⁺]_cyt as previously reported (Figure 1B; n = 6) [1]. We also examined whole cell lysates of murine (Swiss) and human platelets, prepared from the samples used for fluorescence experiments, for the presence of TRPV1 by Western blotting. We detected a band at around 100 kDa, consistent with the molecular weight of hTRPV1, in the human whole platelet lysates as previously reported [1], [2], but could not detect a band at around 95 kDa, as would be expected for mTRPV1 [5], in the mouse platelet lysates (Figure 1C; n = 3). The antibody was, however, able to detect a protein of around 95 kDa in mouse brain lysate (Figure 1D; n = 3). We also observed a secondary band in the mouse brain lysate, which we believe is likely to represent a truncated variant of mTRPV1 expressed in hypothalamic osmoreceptors [6]. Another N-terminal variant of TRPV1, TRPV1b, has also been reported to be present in mice [7]. The anti-TRPV1 antibody used in this study was directed to the C-terminal of TRPV1, thus our data suggest the absence of all known murine TRPV1 isoforms from mouse platelets. Recent transcriptome profiles of murine and human platelets have demonstrated low levels of TRPV1 mRNA in human platelets, whilst it appeared to be absent from murine platelets [8], supporting our observations.

Figure 1. TRPV1 is expressed in human but not mouse platelets. (A,B) Fura-2-loaded platelets from mice (A) or humans (B) were stimulated in the presence of 1 mM Ca²⁺, with either 100 µM capsaicin (Caps) or an equal volume of its vehicle DMSO. (C,D) Western blot analysis of whole cell lysates of mouse or human platelets (C) or whole cell lysates of mouse brain and platelets (D). Whole cell lysates were analyzed following sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) by Western blotting using an anti-TRPV1 polyclonal antibody. In panels marked “No Primary”, the primary antibody was omitted to rule out non-specific binding of the secondary antibody. Positions of molecular mass markers (kDa) are shown on the left. These results are representative of 6 (C) or 3 (D) independent experiments.

These results demonstrate that murine platelets lack TRPV1, and thus cannot provide a model system in which to study the physiological function of TRPV1 in platelets in vivo. However, the demonstration that these cells lack both the TRPV1 protein and responsiveness to capsaicin, even at high concentrations, does provide a cell that can be used as a negative control for screening for non-specific effects of TRPV1 agonists and antagonists in platelets. The negative results from the murine platelets also support our previous conclusion that capsaicin activates calcium signalling and serotonin secretion in human platelets through a specific effect on TRPV1 [1].

There is growing recognition of platelet involvement in a range of inflammatory conditions such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis [9]. We have previously suggested that the presence of TRPV1 in human platelets may be involved in a signalling pathway that could elicit platelet activation under these conditions [1]. The results presented here, however, suggest that the molecular identity of signalling pathways eliciting platelet activity under inflammatory conditions is likely to be different in mice.

The absence of TRPV1 from murine platelets adds to a growing body of evidence of differences in both the proteomes and transcriptomes of mouse and human platelets [8]. Thus, while platelets from genetically-modified mice provide a useful tool for an initial evaluation of the role of a variety of proteins in platelet function, caution must be exercised when extrapolating from mouse studies to consider human platelet function.

Acknowledgements

We thank Dr Pedro Redondo and Professor Gines Salido for assistance with some experiments.

Declaration of interest: The authors report no declaration of interest. This work was supported by The Spanish Ministry of Innovation (BFU2010-21043-C02-01).