ABSTRACT
We have investigated the purinoceptor subtypes responsible for
calcium signaling in human platelets, which previous studies have shown
to involve both Ca
influx via receptor-operated
cation channels and release of Ca
from intracellular
stores. Fura-2 measurements of
[Ca
]
in stirred
platelet suspensions showed that both ADP (40 µM) and the
non-hydrolyzable ATP analogue 
-meATP
(
,
-methyleneadenosine 5`-triphosphate, 10 µM)
activated a rapid Ca
influx whereas only ADP
mobilized Ca
from internal stores. In
``nystatin'' whole-cell patch clamp recordings, ATP, ADP, and
the non-hydrolyzable ATP analogues,
,
-meATP and ATP
S
(adenosine 5`-O-(3-thiotriphosphate), all activated a cation
channel permeable to both monovalent and divalent cations with a
single-channel conductance of 11 picosiemens in NaCl saline. The
current response to ATP (40 µM) was activated within 20 ms
and desensitized with a time constant of 47-107 ms in the
continued presence of agonist, which are characteristics of P
receptors in other tissues. We conclude that human platelets
possess a P
purinoceptor, which mediates a rapid phase of
ADP- or ATP-evoked Ca
entry via a cation channel,
whereas one or more separate ADP-selective P
purinoceptors
evoke release of calcium from intracellular stores.
In many cell types, extracellular ATP and ADP interact with a
family of P
purinoceptors(1) . Two subgroups have
been recently cloned: ionotrophic (P
, P
,
P
, P
) and G-protein-coupled
(P
, P
)
purinoceptors(2, 3, 4, 5, 6, 7, 8) .
Data suggest putative P
and P
purinoceptors (9, 10) also exist. Human platelets are reported to
possess a unique ADP-selective purinoceptor, termed P
,
which mediates shape change and aggregation(11) . Major actions
of ADP in platelets include mobilization of intracellular calcium
stores (13) and activation of a non-selective cation
channel(12) . In cell-attached patch clamp
recordings(14) , ADP evoked single channel activity if included
in the pipette but not when added to the bath saline, demonstrating
that this channel is activated by a direct receptor-operated or
G-protein-linked mechanism rather than via a diffusible second
messenger. Whether the actions of ADP in Ca
signal
generation in human platelets are mediated by one or multiple
purinoceptors is, however, uncertain. In the present study we have used
both whole-cell patch clamp recordings and fura-2 intracellular calcium
measurements to investigate the purinoceptor subtypes involved in human
platelet calcium signaling.
MATERIALS AND METHODS
Solutions and Reagents
Unless otherwise stated,
standard platelet saline contained (mM) 150 NaCl, 10 Hepes, 1
MgCl
, 1 EGTA at pH 7.35 (with NaOH). BaCl
saline contained (mM) 110 BaCl
, 10 Hepes, 1
MgCl
at pH 7.35 (with N-methyl D-glucamine base). The pipettes were filled with a solution
containing (mM) 50 KCl, 70 K
SO
, 10
Hepes, 5 MgCl
, 0.1 EGTA, pH 7.2 (KOH). 50-100
µM nystatin was added to the internal pipette saline from
a 50 mM stock, made in dimethyl sulfoxide, immediately before
an experiment. ATP was obtained from Boehringer Mannheim, ATP
S (
)from Calbiochem Novabiochem, and ADP, AMP, UTP,

-meATP, apyrase (Grade V), and nystatin from Sigma.
Isolation of Platelets
Venous blood was donated by
healthy volunteers with their informed consent and platelet-rich plasma
(PRP) prepared as described previously(13) . Apyrase (20
µg/ml) and aspirin (100 µM) were added to minimize
platelet activation by spontaneously released adenosine nucleotides and
thromboxane, respectively. For electrophysiological recordings, 1-ml
aliquots of PRP were spun for 1 min at low speed in a Microcenteur
microcentrifuge (MSE Scientific Instruments, UK), and the pellet was
resuspended in nominally calcium-free standard platelet saline
containing 20 µg/ml apyrase and 0.1% bovine serum albumin. The
addition of bovine serum albumin further reduced spontaneous platelet
activation. In the case of fluorescence measurements, platelets were
loaded while in PRP with the fluorescent indicator, fura-2, as
described previously(13) .
Fluorescence Recordings
Fura-2 fluorescence
measurements were made from stirred platelet suspensions using a Cairn
spectrophotometer system (Cairn Research Ltd., UK).
[Ca
]
was calculated from the
340/380 ratio based on calibration in the presence of 50 µM digitonin as described by Grynkiewicz et
al.(15) . Dose-response curves were fitted by a
four-parameter logistic function using Sigmaplot (Jandel Scientific),
from which pEC
values were derived.
Electrophysiology
Whole-cell patch clamp
recordings, formed by nystatin permeabilization of cell-attached
patches, were made with a List EPC7 amplifier, as described
previously(13) . Patch pipettes (filled resistances of
5-10 megaohms) were pulled from borosilicate-filamented glass
tubing (Clark Electromedical Instruments, UK). Membrane currents were
filtered at 3 kHz and sampled at 100 µs using a Digidata 1200
interface and pClamp6 software (Axon Instruments, CA). Agonists were
applied from a closely apposed pressure injection pipette, and the bath
was continuously counterperfused with saline containing 20 µg
ml
apyrase to minimize purinoceptor desensitization.
All experiments were performed at the ambient temperature (23-25
°C). Data were expressed as the mean ± S.E. with number of
observations (n) in parentheses.
RESULTS AND DISCUSSION
Intracellular Calcium Responses Evoked by ATP,
,
-meATP, and ADP
Fura-2-loaded human platelets in
stirred suspension were used to assess Ca
influx and
mobilization from intracellular calcium stores evoked by adenosine
nucleotides and a range of related analogues as well as by other
nucleotides. As previously reported(13) , in the presence of 1
mM external calcium, 40 µM ADP evoked a peak
[Ca
]
rise of 313 ± 12
nM (n = 9), which in Ca
-free
saline was reduced to 151 ± 50 nM (n =
10) (Fig. 1a). Our results now demonstrate for the
first time that ATP (40 µM) also evokes an elevation in
[Ca
]
, which peaked 138 ±
45 nM (n = 8) above basal levels in the
presence of external Ca
(Fig. 1b).
This response was reduced to a gradual increase in cytosolic
Ca
of 57 ± 37 nM (n =
6) in Ca
-free saline (Fig. 1b). Since
the adenosine nucleotidase, apyrase, was necessary in these experiments
to minimize desensitization of platelet purinoceptors by spontaneous
release of endogenous ATP and ADP, the ATP-evoked signal could arise
from ADP generated by this enzyme. Hence we investigated the effect of
a non-hydrolyzable analogue of ATP. As shown in Fig. 1c,
,
-meATP (10 µM)
elevated [Ca
]
by 90 ± 9
nM (n = 4) from resting levels in the presence
of 1 mM external calcium but had no effect in
Ca
-free saline. This is to be expected for an agonist
considered selective for P
purinoceptors, which do not
activate phospholipase C(2) .
Figure 1:
Effect of ADP, ATP, and
,
-meATP on [Ca
]
of human platelets. a-c,
[Ca
]
responses
recorded in stirred suspensions of fura-2-loaded human platelets
following addition (at arrow) of 40 µM ADP (a), 40 µM ATP (b), or 10 µM
,
-meATP (c) in the presence of 1 mM external Ca
or in Ca
-free
saline (2 mM EGTA). Each trace is representative of
6 experiments. d, dose-response curves for ADP and
,
-meATP for the peak
[Ca
]
increase in the
presence of 1 mM external
Ca
.
Dose-response curves (Fig. 1d) were constructed for ADP (10 nM to
30 µM) and
,
-meATP (10 nM to 10
µM) in the presence of 1 mM external calcium. In
the case of ADP, a calcium response was detectable at 10 nM,
was maximal at around 100 µM, and had a pEC
of -6.7 ± 0.2. The dose-response curve could also be
fitted by a Hill plot with a Hill coefficient of 0.76 (not shown),
which could be explained by the presence of two purinoceptors with
differing affinities for ADP. In contrast,
,
-meATP increased
[Ca
]
with a threshold
concentration of 0.3 µM, a maximal response at 3
µM, and a pEC
of -6.1 ± 0.13.
The
,
-meATP response curve was also fitted by a Hill plot
with a Hill coefficient of 1.5 (not shown). The pharmacological
properties of this response are similar to those for
,
-meATP-activating P
purinoceptors in smooth
muscle (16) and provide further evidence for the presence of
this class of purinoceptor in human platelets. Neither the purine
nucleotide, AMP (100 µM, n = 3), nor the
pyrimidine nucleotide, UTP (100 µM, n =
3), had an effect on [Ca
]
(data
not shown).
ATP Rapidly Activates a Non-selective Cation Current in
Whole-cell Recordings
Single human platelet responses to
adenosine nucleotides were studied using the ``nystatin''
whole-cell patch clamp technique. Application of ATP, at a holding
potential of -70 mV, evoked a transient inward current with a
delay of <20 ms and a peak in the range of 25.5-106 pA (n = 6) (Fig. 2a). The decay time of the
current was fitted by a single exponential with a time constant in the
range of 47-107 ms (n = 6). The single channel
current, when resolved from whole-cell current records (12) at
-70 mV, was 0.79 ± 0.04 pA (mean ± S.D., n = 6) in standard NaCl platelet saline and 0.77 ±
0.05 pA (mean ± S.D., n = 3) in 110 mM BaCl
saline. Permeability to Ca
is
predicted from this result since Ba
has proved a good
surrogate for Ca
in studies of
Ca
-permeable channels including the platelet ADP
receptor-operated channel(12, 14) . Following a 5-s
application of ATP, reapplication of agonist within 1-2 min
resulted in a much reduced inward current (Fig. 2b),
and recovery of the response required several minutes washing in normal
saline (not shown). The phenomenon of tachyphylaxis following
application of agonist is characteristic of P
purinoceptors(2) . As previously reported(12) ,
ADP activates a non-selective cation current in ``nystatin''
whole-cell recordings from human platelets. This was shown to be via a
direct receptor-operated (rather than second messenger-operated)
channel in cell-attached recordings(14) . In this study, ADP
(40 µM) activated a peak inward current in the range of
5-60 pA (n = 4) at -70 mV (not shown). The
single channel current was 0.71 ± 0.07 pA (mean ± S.D., n = 3), a value comparable with that determined in
earlier studies(12) . From the maximal peak currents recorded,
we estimate that ADP and ATP are capable of activating 80-130
channels in a single platelet.
Figure 2:
Membrane currents evoked by adenosine
nucleotides in whole-cell patch clamp recordings from human platelets.
Bath contained 150 mM NaCl saline. a-c, whole-cell currents evoked by ATP or ATP
S (40
µM) at a holding potential of -70 mV. The bar indicates superfusion of agonist. a and c show
the response to a single application of agonist, and b shows
superimposed responses to two successive applications of ATP, each of
total duration 5 s, separated by 1.5 min. d, single channel
current-voltage relationships for ADP (triangle), ATP
S (square), and 
-meATP (circle).
Unitary events were measured from the inactivation phase of the
whole-cell currents(12) .
Response to ATP
S and
,
-meATP in Whole-cell
Recordings
To ensure that the observed ATP-evoked currents were
not due to breakdown products of the adenosine nucleotide, the effects
of non-hydrolyzable analogues were investigated. A large transient
inward current was activated by 40 µM ATP
S in
standard platelet saline (Fig. 1c). This current had a
similar delay time (<20 ms) to the ATP-evoked current, but the decay
time was greater, being fitted by a single exponential with a time
constant of 236-372 ms in standard platelet saline (Fig. 2, a and c). The prolonged time course
of the ATP
S-induced membrane current is an intrinsic property of
the receptor as opposed to the inability of apyrase to catabolize
ATP
S, as all agonists were superfused in apyrase-free saline.
Furthermore, activation of P
purinoceptors by ATP
S
in smooth muscle also evoked a prolonged inward current(17) .
The unitary current-voltage relationships for both ATP
S and
,
-meATP showed inward rectification between the measured
range of -34 mV and -104 mV (Fig. 2d).
These relationships were comparable with that for the current activated
by ADP in standard platelet saline (Fig. 2d).
Therefore, ADP and ATP activated the same cationic current in
whole-platelet patch recordings.From these observations we conclude
that at least two different purinoceptor subclasses mediate calcium
responses in human platelets. One or more purinoceptors are responsible
for release of calcium from intracellular stores. This may include the
novel platelet purinoceptor that has been designated
P
(18) . Our results demonstrate for the first time
a P
purinoceptor activated by ATP and ADP, which is
coupled to a non-selective cation channel, with pharmacological and
kinetic properties similar to the P
purinoceptor recently
cloned from vas deferens(2) . Since damaged endothelial cells
will release both ADP and ATP, rapid Ca
influx via
P
-coupled cation channels is likely to be one of the
earliest events by which platelet activation occurs at sites of
vascular injury. Furthermore, platelets secrete ADP and ATP from their
dense storage granules(19) , which will activate yet more
platelets to increase the size of a developing thrombus. Therefore,
Ca
influx via the P
purinoceptor may
represent a more important pathway in platelet activation than has
previously been recognized.