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Blood, 1 December 2002, Vol. 100, No. 12, pp. 4245-4246
CORRESPONDENCE
To the editor:
TRP channels and calcium entry in human platelets
An elevation in cytosolic calcium concentration
([Ca2+]i) plays a central role in the
physiologic activation of platelets. Although a number of
calcium entry pathways are believed to exist in human platelets,1 information on the identity of the channels
concerned is limited. The recent paper by Hassock and
colleagues,2 which attempts to characterize the expression
of homologues of the Drosophila transient receptor potential
channel (TRPC) mutant in platelets, is thus valuable in this respect.
However, the data reported by Hassock et al conflict with other
published data in several respects. Until these discrepancies are
resolved much uncertainty will remain. Hassock et al provide evidence for at least 2 Ca2+ entry
pathways in the platelet plasma membrane, one dependent and another independent of depletion of the intracellular Ca2+
stores.2 The store-independent entry is suggested to be
activated by diacylglycerol (DAG) and mediated by TRPC6. Activation of
this nonselective pathway by the DAG analog 1-oleoyl-2-sn-glycerol (OAG) and by thrombin was demonstrated, and was suggested to be independent of protein kinase C (PKC) because the PKC inhibitor bisindolylmaleimide was without effect. The divalent cation entry evoked by OAG was modest and slow. We have also reported the activation of Ca2+ entry in platelets independently of
Ca2+ store depletion.3 As in the experiments
of Hassock et al, this pathway was nonselective and could be activated
by OAG, phorbol-12-myristate-13-acetate, and thrombin. However, in
contrast to Hassock and colleagues, we found that the above agents
evoked rapid divalent cation entry that was blocked by the PKC
inhibitor Ro-31-8220. Although the presence of a PKC-stimulated
Ca2+ entry pathway seems at odds with the well-established
inhibitory (negative feedback) effects of PKC on platelet
Ca2+ signal generation,4 we have shown that
the effect of PKC stimulation on platelet
[Ca2+]i is time dependent. Ca2+
entry was observed on initial PKC stimulation, but inhibition was
observed after longer treatments.3 Hassock and colleagues conclude that the store-independent pathway
suggested to involve TRPC6 is distinct from the store-dependent pathway
on the basis of selectivity experiments. They suggest that the
store-dependent pathway is selective for Ca2+ and does not
admit Ba2+, in contrast to the store-independent route.
This conflicts with other reports that store depletion using low
concentrations of ionomycin5 or thapsigargin6
stimulated the entry of Ba2+, Sr2+, and
Ca2+ across the plasma membrane. The use of
Ba2+ alone in such studies is inadvisable since it has
relatively little effect on fura-2 fluorescence compared with
Ca2+.7 Also, Ba2+ blocks
many types of potassium channels,8 so membrane
depolarization may account for reduced cation influx. As well as TRPC6 expression, Hassock and colleagues demonstrate the
expression of TRPC1 in human platelets. However, they report a low
level of detection and suggest that TRPC1 is located in the
inner-membrane rather than plasma-membrane fraction after membrane separation by free-flow electrophoresis. Thus they suggest against a role for TRPC1 in store-mediated Ca2+ entry.
These results contrast with our own in at least 2 respects. First, we
can readily detect TRPC1 in human platelets using an anti-TRPC1
antibody from Alomone Laboratories (Jerusalem,
Israel)9,10 and have confirmed the detection of
the same protein of about 100 kDa using the T1E3 anti-TRPC1 antibody
characterized by Xu and Beech.11 Furthermore, we have
demonstrated de novo coupling of TRPC1 and the type II inositol
trisphosphate receptor (InsP3RII) when the intracellular
Ca2+ stores are depleted.9,10 This has led us
to propose that the activation of store-mediated Ca2+
entry (SMCE) in platelets occurs by a secretionlike coupling mechanism
involving TRPC1 and InsP3RII.9,10 The
anti-TRPC1 antibody from Alomone is raised against the extracellular
amino acid sequence 557 to 571, which is predicted to lie in the
pore-forming region of the protein. In accordance with this, we have
reported that this anti-TRPC1 antibody blocks both Ca2+ and
Mn2+ entry evoked following Ca2+ store
depletion using thapsigargin.12 This strongly supports a
role for TRPC1 in SMCE in human platelets and furthermore suggests that
TRPC1 must be located at least in part in the plasma membrane. It is difficult to explain the conflicts between our data and those of
Hassock et al. One factor may be the age of the cells. We used freshly
isolated platelets prepared with minimal handling and conducted all
experiments within a few hours of venipuncture. Hassock et al prepared
membranes by free-flow electrophoresis using older cells obtained via
blood banks, which necessarily have to be subjected to chemical
treatment. Another factor may be the specificity of the antibodies
concerned. In our hands, the anti-TRPC1 from Alomone and the T1E3
antibody (a gift from Prof D. J. Beech, University of Leeds,
United Kingdom) recognize the same single band of about 100 kDa. The
anti-Xenopus TRPC1 antibody used by Hassock et al to assess
TRPC1 distribution between the inner and plasma membranes detected
multiple protein bands, many more strongly than that of the
predicted size of TRPC1. Hence we believe much remains to be done to characterize
Ca2+ entry pathways in platelets.
Stewart O. Sage, Sharon L. Brownlow, and Juan A. Rosado
Correspondence: Stewart O. Sage, Department of Physiology,
Downing St, Cambridge, CB2 3EG, United Kingdom; e-mail:
sos10{at}cam.ac.uk
References
1.
Rosado JA, Sage SO.
Platelet Signalling: Calcium. In:
Gresele P,Page CP,Fuster V,Vermylen J, eds.
Platelets in Thrombotic and Non-Thrombotic Disorders. Cambridge United Kingdom: Cambridge University Press; 2002:260-271.
2.
Hassock SR, Zhu MX, Trost C, Flockerzi V, Authi KS.
Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel.
Blood.
2002;100:2801-2811[Abstract/Free Full Text].
3.
Rosado JA, Sage SO.
Protein kinase C activates non-capacitative calcium entry in human platelets.
J Physiol.
2000;529:159-169[Abstract/Free Full Text].
4.
Zavoico GB, Halenda SP, Sha'afi RI, Feinstein MB.
Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilisation and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.
Proc Natl Acad Sci U S A.
1985;82:3859-3862[Abstract/Free Full Text].
5.
Jenner S, Farndale RW, Sage SO.
Effects of Ca2+ store depletion and refilling with Ca2+, Sr2+ or Ba2+ on tyrosine phosphorylation and Mn2+ entry in human platelets.
Biochem J.
1994;303:337-339[Medline]
[Order article via Infotrieve].
6.
Dobrydneva Y, Williams RL, Blackmore PF.
Trans-resveratrol inhibits calcium influx in thrombin-stimulated human platelets.
Br J Pharmacol.
1999;128:149-157[CrossRef][Medline]
[Order article via Infotrieve].
7.
Schilling WP, Rajan L, Strobl-Jager E.
Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells.
J Biol Chem.
1989;264:12838-12848[Abstract/Free Full Text].
8.
Rudy B.
Diversity and ubiquity of K+ channels.
Neuroscience.
1988;25:729-749[CrossRef][Medline]
[Order article via Infotrieve].
9.
Rosado JA, Sage SO.
IP3 receptors couple with hTrp1 channels when Ca2+ stores are depleted in human platelets.
Biochem J.
2000;350:631-635[CrossRef][Medline]
[Order article via Infotrieve].
10.
Rosado JA, Sage SO.
Activation of store-mediated calcium entry by secretion-like coupling between the IP3 receptor type II and hTrp1 channels in human platelets.
Biochem J.
2001;356:191-198[CrossRef][Medline]
[Order article via Infotrieve].
11.
Xu S-Z, Beech DJ.
TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells.
Circ Res.
2001;88:84-87[Abstract/Free Full Text].
12.
Rosado JA, Sage SO.
Endogenous Trp1 conducts store-mediated Ca2+ entry in human platelets.
J Physiol.
2001;533:6P.
Response:
TRPC channels and Ca2+ entry in human platelets
Ca2+ entry is an important event in platelet
activation but little is established regarding the details of the
molecular components involved. Store-operated Ca2+ entry
(SOCE) represents a major pathway, in addition to SOCE-independent mechanisms, that may involve direct activation by surface receptors or
the involvement of second messengers.1 However, the
identities of the entry channels are not known, and from the known
properties of the SOCE pathway the molecular composition of the SOCE
channels may differ between cells. Members of the transient receptor
potential (TRP) family, of which there are 3 subfamilies (TRPC, TRPM,
and TRPV), have been suggested as candidates for SOCE and non-SOCE channels.2 In our recent study3 we examined
the expression and role of the TRPC family in human platelets. We
reported the strong expression of TRPC6 located in the plasma membrane
(PM) and suggested that it forms a SOCE-independent Ca2+
entry channel. We also reported the low expression of TRPC1 that was
found in intracellular membranes (IMs). Our results contrast with data
published by Rosado and Sage4,5 who, using an anti-TRPC1 antibody from Alomone Laboratories (Jerusalem, Israel),
reported strong expression of TRPC1 and its coupling to the
type II inositol 1,4,5-trisphosphate receptor (IP3R) upon
store depletion. In the preceding letter, Sage and colleagues
highlighted these discrepancies and suggested that age of cells and
specificity of antibodies used may explain the differences. We believe
that our data are totally in line with the known properties of TRPC
proteins. We reject the idea that age of cells could
contribute to differences in the results obtained. All of our studies
concerning Ca2+ measurements, phosphorylation,
immunoprecipitation, and overexpression are carried out with freshly
isolated cells. Only the data on purified PM and IM preparations are
obtained using one-day-old platelets from the blood bank (with the
delay arising because of the compulsory testing of pathogens), because
it is simply not ethical to take one liter of blood from laboratory
colleagues. On closer examination of the studies involved, we suggest
that the high doses and poorly established specificity of the reagents used by the Sage group, in addition to methodological differences, may
better explain the discrepancies involved. In our study we presented evidence that, in line with the expression of
TRPC6 in the PM, 60 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated
Ca2+/Ba2+ entry that was essentially
independent of protein kinase C (PKC), as the inhibitor
bisindolylmaleimide I (Bis I) had no effect on the OAG-induced entry.
Further, in line with the known negative-feedback properties of
PKC,6 Bis I enhanced 1 U/mL thrombin-stimulated Ba2+ entry. Bis I totally inhibited OAG-induced platelet
aggregation confirming this response to be PKC dependent. Under control
conditions, a faster entry of Ba2+ by thrombin compared
with OAG may be explained by the correct diacylglycerol (DAG) produced
by thrombin in a tightly coupled system. Activation of Ba2+
entry was independent of store depletion and thapsigargin (Tg) was a
poor stimulator for Ba2+ entry, under conditions where it
induced Ca2+ entry.3 Our results are
entirely in agreement with the published properties of
TRPC6,7 where TRPC6 is described as a
store-independent, nonselective channel, activated by DAG in a
membrane-delimiting manner. In contrast, Rosado and
Sage8 reported that high concentrations of OAG and of
phorbol-12-myristate-13-acetate (PMA) stimulated Ca2+ and
Sr2+ entry in a PKC-dependent manner as the entry was
blocked by the inhibitor Ro-31-8220. The PMA concentration used was 1 µM at a cell density of 1 × 108 platelets/mL. In our
hands 100 nM PMA is sufficient to maximally activate PKC even with
normal cell counts of 2 ×108/mL to
3 × 108/mL. It is possible that overstimulation
of PKC may induce cation entry. However, the possibility that the
higher concentrations of these agents used with low cell numbers may
alter the cell integrity and activate entry of cations needs to be
addressed. There is no doubt that PKC provides a negative-feedback
role for the control of Ca2+ elevation as
confirmed in our study. Sage and colleagues caution against the use of
Ba2+, which may possibly block certain potassium channels.
On the contrary, measurement of Ba2+ entry under defined
conditions has proved very useful in monitoring the gating properties
of many TRPC proteins, for example.9 While the
role of K+ channels in Ca2+ fluxes in platelets
needs to be better clarified, our experiments under identical
conditions indicate that thrombin is a powerful mediator of
Ba2+ entry but Tg is not. Sage and colleagues further
suggest that store depletion by low concentrations of ionomycin and Tg
stimulates the entry of Ca2+, Ba2+, and
Sr2+, citing their study by Jenner et
al.10 However, closer examination of this study reveals a
vastly different methodology from that which we used. Very high levels
(500 µM) of ionomycin were added to platelets for 20 minutes, followed by 2 centrifugations, incubation with cations, 2 further centrifugations, and analysis of cation release.10
Our studies were carried out in spectrofluorimeter cuvettes monitoring
fluorescence changes over 9 minutes3 and clearly showed a
marked selectivity for Ca2+ more than
Ba2+ upon store depletion. Perhaps the largest difference concerns the expression and role of
TRPC1. We have used 2 antibodies to TRPC1 (Ank and anti-XTRP1), which
we show recognize hTRPC1 overexpressed in QBI-293A cells. These antibodies show a low level of expression of TRPC1 in platelet IM. These data have received support from 2 important studies recently
published. Firstly, Mori and colleagues11 have shown that
the knock-out of TRP1 (avian TRPC1) in the hematopoietic DT40 cell line
resulted in a reduction of agonist-mediated release of Ca2+
from the stores and a reduction of IP3-induced
Ca2+ release from the endoplasmic reticulum. These results
would be incompatible if all of the TRP1 was located in the PM.
Further, Hofmann et al12 have defined the subunit
composition of TRPC channels. They report that TRPC1 can exist as a
heterotetramer with TRPC4 or TRPC5 but not with TRPC3, TRPC6, or TRPC7.
Additionally, they report that TRPC1 stayed in membrane compartments in
the cytoplasm unless it was coexpressed with either TRPC4 or TRPC5, in
which case it located to the PM. We have shown that platelets express
little or no TRPC4 or TRPC5, hence the location of TRPC1 in the IM
entirely supports the findings of Hofmann et al.12 Our
results do not rule out a more important role for TRPC1 in the SOCE
activity of other cells (Hassock et al3) where the subunit
composition of the TRP channels need to be determined. Rosado and
Sage4,5 have used the anti-TRPC1 antibody from Alomone.
The manufacturer's website
(http://www.alomone.com/Site/p_home/home.htm) states that in rat brain
it recognizes 2 products: a protein larger than 250 kDa and
more faintly a protein at approximately 120 kDa. The molecular size of
hTRPC1 is 80 kDa and the full length isoform is 34 amino acids
longer.3 Recently Ong et al13 showed that the
Alomone antibody did recognize a 120 kDa protein in mouse liver and
mouse brain. However, it did not recognize overexpressed hTRPC1 under conditions in which a number of other antibodies did,
including the anti-XTRP1 antibody used in our study. Therefore the
identity of the proteins recognized by the Alomone antibody needs to be
clarified. Using the Alomone anti-TRPC1 antibody, we have been unable
to reproduce the findings of Rosado and Sage,4,5 although
we cannot rule out variations of antibody specificity between
different batches supplied by the manufacturer. We therefore suggest that results obtained with the Alomone anti-TRPC1
antibody should be verified with other better-established antibodies. In conclusion, we feel that our work represents an important advance in
our knowledge of the expression and role of TRPC proteins in platelet
Ca2+ homeostasis. We have demonstrated the expression of
TRPC6 and its role as a SOCE-independent Ca2+ entry channel
in platelets. Clearly much remains to be determined regarding the
molecules and mechanisms involved with the SOCE pathway in platelets.
Kalwant S. Authi, Sheila Hassock, Michael X. Zhu, Veit Flockerzi, and Claudia Trost
Correspondence: Kalwant Authi, King's College London, Centre
for Cardiovascular Biology and Medicine, New Hunt's House, Guy's
Campus, London, SE1 1UL, United Kingdom; e-mail:
kalwant.authi{at}kcl.ac.uk
References
1.
Putney JW Jr, Broad LM, Braun FJ, Lievremont JP, Bird GS.
Mechanisms of capacitative calcium entry.
J Cell Sci.
2001;114:2223-2229[Medline]
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2.
Clapham DE, Runnels LW, Strubing C.
The TRP ion channel family.
Nat Rev Neurosci.
2001;2:387-396[Medline]
[Order article via Infotrieve].
3.
Hassock S, Zhu MX, Trost C, Flockerzi V, Authi KS.
Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel.
Blood.
2002;100:2801-2811[Abstract/Free Full Text].
4.
Rosado JA, Sage SO.
Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted.
Biochem J.
2000;350:631-635[CrossRef][Medline]
[Order article via Infotrieve].
5.
Rosado JA, Sage SO.
Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets.
Biochem J.
2001;356:191-198[CrossRef][Medline]
[Order article via Infotrieve].
6.
Zavoico GB, Halenda SP, Sha'afi RI, Feinstein MB.
Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.
Proc Natl Acad Sci U S A.
1985;82:3859-3862[Abstract/Free Full Text].
7.
Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G.
Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol.
Nature.
1999;397:259-263[CrossRef][Medline]
[Order article via Infotrieve].
8.
Rosado JA, Sage SO.
Protein kinase C activates non-capacitative calcium entry in human platelets.
J Physiol.
2000;529:159-169[Abstract/Free Full Text].
9.
Venkatachalam K, Ma HT, Ford DL, Gill DL.
Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells.
J Biol Chem.
2001;276:33980-33985[Abstract/Free Full Text].
10.
Jenner S, Farndale RW, Sage SO.
The effect of calcium-store depletion and refilling with various bivalent cations on tyrosine phosphorylation and Mn2+ entry in fura-2-loaded human platelets.
Biochem J.
1994;303:337-339[Medline]
[Order article via Infotrieve].
11.
Mori Y, Wakamori M, Miyakawa T, et al.
Transient receptor potential 1 regulates capacitative Ca2+ entry and Ca2+ release from endoplasmic reticulum in B lymphocytes.
J Exp Med.
2002;195:673-681[Abstract/Free Full Text].
12.
Hofmann T, Schaefer M, Schultz G, Gudermann T.
Subunit composition of mammalian transient receptor potential channels in living cells.
Proc Natl Acad Sci U S A.
2002;99:7461-7466[Abstract/Free Full Text].
13.
Ong HL, Chen J, Chataway T, et al.
Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis.
Biochem J.
2002;364:641-648[CrossRef][Medline]
[Order article via Infotrieve].
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