Báo cáo Y học: Irregular spiking in free calcium concentration in single, human platelets Regulation by modulation of the inositol trisphosphate receptors ppt

Irregular spiking in free calcium concentration
in single, human platelets
Regulation by modulation of the inositol trisphosphate receptors
Roosje M. A. van Gorp
1
, Marion A. H. Feijge
1
, Wim M. J. Vuist
1
, Martin B. Rook
2
and Johan W. M. Heemskerk
1
1
Departments of Biochemistry and Human Biology, University of Maastricht, the Netherlands;
2
Department of Medical Physiology,
University Medical Centre Utrecht, the Netherlands
Fluorescence ratio imaging indicates that immobilized,
aspirin-treated platelets, loaded with Fura-2, respond to
inositol 1,4,5-trisphosphate- (InsP
3
)-generating agonists
such as thrombin by high-frequency, irregular rises in
cytosolic [Ca
2+
]
i
with spikes that vary in peak level and
peak-to-peak interval. This differs from the regular [ Ca
2+

]
i
oscillations observed in other, larger cells. We found that the
thiol-reactive compounds thimerosal (10 l
M
) and U73122
(10 l
M
) evoked similar irregular Ca
2+
responses in platelets,
but in this case in the absence of InsP
3
generation. Throm-
bin-induced spiking was acutely abolished by inhibiting
phospholipase C or elevating intracellular cAMP levels,
while spiking with sulfhydryl reagents was only partially
blocked by cAMP elevation. Confocal laser scanning
microscopy using fluo-3-loaded platelets indicated that, with
all agonists or conditions, t he irregular spikes were almost
instantaneously raised in various regions within a single
platelet. When using saponin-permeabilized platelets, we
found that InsP
3
-induced Ca
2+
release from stores was
stimulated by modest Ca
2+
concentrations, pointing to a

mechanism of InsP
3
-dependent Ca
2+
-induced Ca
2+
release
(CICR). This process was completely inhibitable by heparin.
The Ca
2+
release b y InsP
3
, but not the C ICR sensor, was
negatively regulated by cAMP elevation. Thimerosal treat-
ment did n ot release Ca
2+
from intracellular stores, but
markedly potentiated the stimulatory effect of InsP
3
.In
contrast, U73122 caused a heparin/cAMP-insensitive Ca
2+
leak from stores that differed from those used by InsP
3
.
Taken together, these results demonstrate t hat Ins P
3
recep-
tor channels play a crucial role in the irregular, spiking Ca
2+

signal of intact platelets, even when induced by agents such
as thimerosal or U73122 which do not stimulate InsP
3
for-
mation. The irregular Ca
2+
release events appear to be
subjected to extensive regulation by: ( a) InsP
3
level, (b) the
potentiating effect of elevated Ca
2+
on InsP
3
action via
CICR, ( c) Ins P
3
channel sensitization by sulfhydryl (thim-
erosal) modification, (d) InsP
3
channel-independent Ca
2+
leak with U73122, and (e) down-regulation via cAMP
elevation. The observation that individual Ca
2+
peaks were
generated in various parts of a platelet at similar i ntervals
and amplitudes points t o effective c ooperation of the various
stores in the Ca
2+

-release process.
Keywords:Ca
2+
-induced Ca
2+
release; cyclic AMP ;
cytosolic Ca
2+
; inositol trisphosphate; platelets.
Most vertebrate cells respond to specific agonists by
repetitive spiking or oscillation in cytosolic [Ca
2+
]
i
as a
consequence of regenerative release of Ca
2+
from stores
into the c ytosol through inositol 1,4,5-trisphosphate (InsP
3
)
or ryanodine receptor channels, located in the membrane of
the endoplasmic o r sarcoplasmic reticulum, respectively [1].
For large cells such as oocytes and HeLa cells, evidence has
been collected that local clusters of InsP
3
receptors in the
reticular membrane function as discrete Ca
2+
release s ites.

Such local spots, being spaced at intervals of tens of
micrometers apart, are taken responsible for so-called
elementary Ca
2+
release events [2–4]. At low concentra-
tions, InsP
3
may trigger individual release sites, which
results i n t he appearance of local Ca
2+
ÔpuffsÕ, i.e. of brief
Ca
2+
release events of usually low amplitude. Higher I nsP
3
concentrations cause a summation in amplitude or fre-
quency mode of these release events, and lead to recruitment
of neighbouring r elease sites. As a consequence, global
increases in [Ca
2+
]
i
can develop that prop agate through the
entire cell as Ca
2+
oscillations or waves. These whole-cell
Ca
2+
responses are u sually regular in shape, such in
contrast to the local Ca

2+
puffs which are heterogeneous in
both amplitude and t ime of appearance.
In a variety of cells, the InsP
3
receptor channels play
crucial roles in eliciting [Ca
2+
]
i
oscillations and puffs [1–4].
Three different InsP
3
receptor isoforms are presently
recognized with subtle differences in the regulation of
Ca
2+
channel opening. Characteristic for the type 1 InsP
3
receptors is a biphasic effect of cytosolic Ca
2+
on the
channel activity, with Ca
2+
stimulating the Ca
2+
release
Correspondence to J. W. M. Heemskerk, Departments of Biochemis-
try/Human Biology, University of Maastricht, PO Box 616,
6200 mD Maastricht, the Netherlands.

Fax: + 31 43 3884160, Tel.: + 31 43 3881671,
E-mail:
Abbreviations:CICR,Ca
2+
-induced Ca
2+
release; InsP
3
, inositol
1,4,5-trisphosphate; PGE
1
, prostaglandin E
1
.
Note: Part of this paper appears in the PhD Thesis of
R. M. A. Beisser-van Gorp (University of Maastricht,
the Netherlands).
(Received 21 September 2001, revised 21 December 2 001, accepted
22 January 2002)
Eur. J. Biochem. 269, 1543–1552 (2002) Ó FEBS 2002
from stores up to 300 n
M
and inhibiting this activity at
higher levels [5–9]. This biphasic effect may control the
rising and falling phases of individual Ca
2+
spikes.Thus,at
a relatively low [Ca
2+
]

i
,InsP
3
-mediated Ca
2+
release is
facilitated by the sensitizing mechanism of Ca
2+
-induced
Ca
2+
release ( CICR), whereas at higher Ca
2+
levels the
InsP
3
receptors become desensitized. Other factors deter-
mining the open probability of the receptor channels are the
luminal Ca
2+
concentration in the endoplasmic reticulum
[9,10], modulation or oxidation of the receptor sulfhydryl
groups [11–14], and phosphorylation b y cAMP-dependen t
protein kinase [ 15,16].
Platelets are among the smallest cellular entities in the
mammalian body (diameter o f about 2 lm w ith e stimated
volume of 6 fL). They acutely respond to Ins P
3
-forming
agonists by regenerative Ca

2+
release [ 17–20]. T he [Ca
2+
]
i
spiking pattern of platelets is remarkably irregular in shape
in comparison to that of larger cells, e.g. of the smoothly
oscillating megakaryocytes [21,22]. All thre e InsP
3
receptor
isoforms have been identified in platelets, i.e. mostly type 1
and type 2 receptors in addition to some type 3 receptors
[23–26]. In p latelet membrane preparations it is shown that
the InsP
3
receptors are susceptible to sulfhydryl modifica-
tion and c AMP-dependent phosphorylation [ 24,25]. T here
is, however, little evidence that s uch modulation influences
InsP
3
receptor f unctioning also in intact platelets [27,28]. In
particular, it is controversial whether cAMP-dependen t
protein kinase may stimulate InsP
3
-induced Ca
2+
release
[29], cause modest inhibition [30,31], or is without effect [32]
on the release process.
In this report we consider the nature and subcellular

organization of the regenerative Ca
2+
release in platelets
triggered by I nsP
3
-mobilizing receptor agonists and non-
InsP
3
-mobilizing sulfhydryl reagents. We investigated the
importance of InsP
3
receptor-dependent CICR in the
irregular Ca
2+
signal generation by these agents, and the
sensitivity of this signal toward cAMP elevation. We found
that th e i rregular s piking Ca
2+
signal of platelets contains
several but not all characteristics of local, InsP
3
receptor-
dependent Ca
2+
puffs described f or other, larger cells.
EXPERIMENTAL PROCEDURES
Materials
H-Arg-Gly-Asp-Ser-OH (RGDS) was purchased from
Bachem (Bubendorf, Switzerland), a nd ultra-pure calcium-
free water from Baker (Phillipsburg, NJ, USA). Fura-2,

Fluo-3 and Indo-1 acetoxymethyl esters as well as noneste-
rified Fluo-3 were bought from Molecular P robes (Leiden,
the Netherlands). Manoalide, U73122, U73343 and InsP
3
came from Biomol (Plymouth Meeting, PA, USA), and
thimerosal (sodium ethylmercuri-thiosalicylate) was from
Janssen (Beerse, Belgium). Other chemicals were obtained
from Sigma (St Louis, MO, USA) or Merck (Darmstadt,
Germany).
Platelet preparation and loading with Ca
2+
probes
Blood was collected from healthy volunteers, w ho had n ot
taken medication for at least two weeks. Platelet-ric h plasma
was p repared by centrifugation [18]. It was incubated with
acetoxymethyl ester of Fura-2 (3 l
M
) or Fluo-3 (7 l
M
)in
thepresenceoflysineacetylsalicylate(aspirin,100l
M
)at
37 °C for 45 min. After this loading procedure, the platelets
were spun down, washed twice i n the presence of apyrase
(0.1 U ADPaseÆmL
)1
), and resuspended in buffer A
(pH 7 .45), which was composed of 136 m
M

NaCl, 10 m
M
glucose, 5 m
M
Hepes, 5 m
M
KCl, 2 m
M
MgCl
2
,0.1%(v/v)
bovine serum albumin and apyrase (0.2 U ADPaseÆmL
)1
).
The suspension was adjusted to 1 · 10
8
plateletsÆmL
)1
.
Measurement of [Ca
2+
]
i
in single, immobilized platelets
Aspirin-treated, Fura-2-loaded platelets were immobilized
on fibrinogen-coated glass coverslips, as described previ-
ously [18]. Briefly, the platelets were allowe d to bind to the
surface, and bathed in 0.5 mL buffer A supplemented with
10 l
M

RGDS, apyrase (0.2 U ADPaseÆmL
)1
) and CaCl
2
(2 m
M
)at23°C. Agonists and antagonists were given as
freshly prepared solutions in bathing medium (0.1 m L).
Changes i n Fura-2 fluorescence were recorded in in dividual
cells using an inverted N ikon microscope (Tokyo, Japan),
equipped with a dichroic mirror, computer-driven excitation
and emission filter wheels, and an intensified charge-coupled
device camera working at standard video rate (Photonic
Sciences, Robertsbridge, UK). A 100-W Xenon lamp was
used for illumination. The excitation wavelength was
alternated between 340 and 380 nm, and fluorescent light
was detected at 505 nm. The light was collected with a 40 ·
oil objective (Fluor Nikon, numerical aperture 1.3). Final
image resolution was 1.0 pixels Ælm
)1
, while confocality
giving half-maximal intensity in the x–y plane was deter-
mined at 2 .3 lm.
QUANTICELL
700 software (Visitech, Sun-
derland, UK) was used to control the filter wheels and
capture the images [33]. Four-times averaged, backgroun d-
subtracted fluorescence ratio images were obtained e very
second. Calibration of 340/380 nm fluorescence ratio to
[Ca

2+
]
i
, using lysed platelets, w as as described elsewhere [20].
Fluorescence measurements with suspensions of Fura-2- or
Fluo-3-loaded platelets were c arried out as described [20].
High-resolution, confocal images were collected with a
Nikon RCM 8000 real-time c onfocal laser scanning system,
equipped with an Argon laser. Light was collected with a
60 · oil objective (Apo Nikon, numerical aperture 1.4).
Fluo-3-loaded platelets were visualized at a laser power of
87–91 lW, and excitation and emission wavelengths of
488 nm and 500–550 nm, respectively. Using a small
pinhole, confocality in the x–y plane was experimentally
determined at 0.2 lm ( matching the fi nal image re solution
of 6.0 pixelsÆlm
)1
), while confocality along the z axis was
0.5 lm. Because of the limited fluorescence levels in the
platelets, image frames were eightfold averaged to give a
final temporal resolution of 10 Hz. The Fluo-3 fluorescence
level was expressed as a pseudo-ratio value (F/F
o
)ofthe
actual fluorescence intensity ( F) relative to the basal i ntensity
of the platelet at r est (F
o
), as described elsewhere [3,4].
Calibration was performed as described by Yao et al.[2].
The s ame confocal system was a lso used t o monitor Ind o-

1-labelled platelets, at settings described else where [34].
Measurement of [Ca
2+
] in suspensions
of saponin-permeabilized and intact platelets
Aspirin-treated platelets were suspended at a concentration
of 1–1.5 · 10
9
mL
)1
in buffer B (pH 7.45), composed of
1544 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002
136 m
M
NaCl, 20 m
M
glucose, 5 m
M
Hepes, 5 m
M
KCl,
2m
M
MgCl
2
and 0 .1 m
M
EGTA prepared in calcium-free
water. The platelets were permeabilized with saponin at
23 °C, basically as described elsewhere [31]. Immediately

before start of a measurement, a sample of 0.4 mL was
added to 1.6 mL of Hepes/KCl buffer pH 7.4 (buffer C),
composed of 100 m
M
KCl, 100 m
M
sucrose, 20 m
M
Hepes ,
1.4 m
M
MgCl
2
and 1.25 m
M
NaN
3
(preparedincalcium-
free water). The mixture, in a fluorescence cuvette, was
supplemented with 7.5 m
M
phosphocreatine, 1 m
M
ATP,
1m
M
KH
2
PO
4

,30lgÆmL
)1
creatine kinase, 0.6 lgÆmL
)1
oligomycin and 1 l
M
Fluo-3. Permeabilization of the
platelets was achieved by addition of 15–20 lgÆmL
)1
saponin. After 10 min of stirring, fluorescence was mea-
sured and the free Ca
2+
level was titrated to 110 n
M
by
stepwise additions from a 0.05-m
M
CaCl
2
solution. InsP
3
and other agents were given during the fluorescence
recording. Part of the experiments were carrie d out with
0.75 m
M
phosphocreatine and 0.1 m
M
ATP. In that case,
apyrase (2 UÆmL
)1

) was added after 6 m in of permeabili-
zation to degrade A TP. Free Ca
2+
was then adjusted to the
desired level, after which InsP
3
was added. Ultra-pure,
calcium-free water was used for preparation of all buffers,
supplements and agonists.
Fluo-3 fluorescence intensities (F) were continuously
recorded at 488 nm excitation and 526 nm emission wave-
lengths (slits of 4 nm), using an SLM-Aminco DMX-1100
spectrofluorometer (Rochester, NY, USA). Calibrations
were performed by adding excess amounts of CaCl
2
and
EGTA/Tris (1 : 1, mol/mol) to obtain F
max
and F
min
values,
respectively. Level of [Ca
2+
] in the suspe nsion was calcu-
lated from the binding equation [Ca
2+
] ¼ K
d
· b (F–F
min

)/
(F
max
–F). Th e s ame fluorometer was also u sed to measure
changes in [Ca
2+
]
i
in intact platelets loaded with Fura-2 o r
Fluo-3 [34].
Measurement of Ins
P
3
InsP
3
levels were determined in samples of resting and
activated platelets (180 lL, 3.5 · 10
8
cells). Cellular a ctivity
was stopped by addition of 75 lL ice-cold 10% (w/v)
HClO
4
. After standing on ice for 30 min and centrifuging at
2000 g for 10 min (strictly at 4 °C), supernatants were
collected and neutralized to pH 7 with a solution of 1.7
M
KOH a nd 75 m
M
Hepes. After 30 m in on ice, the precipi-
tated KClO

4
was removed by another centrifugation step
(4 °C). The supernatants were used to measure mass
amounts of InsP
3
with a Biotrak radioreceptor assay
system (Amersham-Pharmacia, UK). Freshly dissolved
InsP
3
was taken as a standard.
Statistics
Paired data were compared for significance of difference
using a Student t-test. Unpaired data were compared by
ANOVA
.
RESULTS
Irregular spiking in [Ca
2+
]
i
in single platelets
independently of Ins
P
3
formation
Fura-2-loaded p latelets immobilized on fibrinogen often
exhibit ÔspontaneousÕ, spiking increases in [Ca
2+
]
i

,which
can partially be prevented by treatment of the platelets with
aspirin and apyrase (blocking the effects of released
thromboxane A
2
and ADP, r espectively) [35]. U sing plate-
lets treated with a spirin and apyrase, we compared the
effects of v arious G
q
/phospholipase C-b stimulating recep-
tor agonists on Ca
2+
signal generation. Extracellular CaCl
2
was p resent to allow physiological, store-regulated influx of
Ca
2+
. Both platelet-activating factor (400 n
M
)andthe
thromboxane A
2
analogue, U46619 (1 l
M
), caused repetit-
ive increases in [Ca
2+
]
i
in single platelets for up to 3 min. In

these traces, individual C a
2+
spikesvariedinpeaklevels
and occurred after short but variable time intervals
(Fig. 1 A,B). The strong agonist thrombin (4 n
M
)also
elicited irregular, spiking rises in [Ca
2+
]
i
, but the signal
now persisted for more than 5 min (Fig. 1C). These
responses differ markedly from the quite regular and
symmetric oscillations in [Ca
2+
]
i
, which have been reported
for larger cells such as rat megakaryocytes [9,10]. To
determine the involvement of cytosolic InsP
3
in the irregular
spiking process in p latelets, we used t he phospholipase
C-inhibiting agents manoalide [36,37] a nd U73122 [38,39].
Addition of manoalide (10 l
M
) or a low dose of U73122
Fig. 1. Irregular spiking in [Ca
2+

]
i
induced b y
phospholipase C-activating agonists. Aspirin-
treated, Fura-2-loaded p latelets on a fib rin-
ogen surface were stimulated with 0 .4 l
M
platelet-activating factor ( PAF) (A), 1 l
M
U46619 (B) or 4 n
M
thrombin (Thr) (C–F) in
thepresenceof1m
M
CaCl
2
and apyrase (0.1
UADPaseÆmL
)1
). Where i ndicated, 10 l
M
manoalide (D), 2 l
M
U73122 (E), or 10 l
M
PGE
1
(F) was added after stimulation.
Fluorescence ratio images were collected from
microscopic fields using a came ra-based sys-

tem. Traces are Ca
2+
responses of single
platelets, representative for 50–100 cells from
at least four independent experiments.
Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1545
(2 l
M
) shortly after t hrombin completely cancelled t he
generation of new [Ca
2+
]
i
spikes (Fig. 1 D,E), whereas the
U73122 control substance U73343 (2 l
M
) was without
effect (data not shown). T hrombin-induced [Ca
2+
]
i
spiking
was also annulled by addition of the cAMP-elevating agent,
prostaglandin E
1
(PGE
1
, Fig. 1F). Thus, the irregular
spiking process with thrombin apparently depends on
continuous generation of InsP

3
and is down-regulated
by elevation of the cAMP concentration (see also below).
Note that similar, irregular Ca
2+
responses were also
obtained when using platelets loaded with Fluo-3 instead of
Fura- 2.
Membrane-permeable sulfhydryl reagents provide an
alternative way of evoking Ca
2+
responses, although
occurring in the apparent absence of phospholipase C
activation [40,41]. W e used thimerosal, a compound that
sensitizes the platelet InsP
3
receptor channels [28,42], and a
high dose of U73122 which acts as an N-ethylmaleimide
derivative thus affecting o ther en zymes than only phospho -
lipase C [38,39,43]. When aspirin-treated platelets on fibri-
nogen were treated with thimerosal ( 10 l
M
) or U73122
(10 l
M
), this resulted in prolonged, irregular spiking in
[Ca
2+
]
i

after a lag time of one or more minutes (Fig. 2A,B).
As U73122 inhibits phospholipase C activity already at
2 l
M
(see below), the spiking with U73122 is unlikely to
result from phospholipase C activation and InsP
3
genera-
tion. This conclusion was also drawn for thimerosal, as
neither pretreatment with manoalide (Fig. 3A) nor postad-
dition of manoalide (Fig. 3B) or a low dose of U73122 (not
shown) influenced the spiking induced by thimerosal. In
quantitative trms, after 5 min o f stimulation with thimero-
sal, peak amplitudes were 667 ± 80 n
M
[Ca
2+
]
i
in the
absence of manoalide pretreatment and 586 ± 48 n
M
after
manoalide pretreatment (mean ± SEM, n ¼ 22 cells,
P ¼ 0.38). In contrast, p reincubation of the p latelets with
10 l
M
PGE
1
lowered the amplitude of the thimerosal-

induced peaks to 390 ± 70 (n ¼ 24 platelets, P ¼ 0.009)
(Fig. 3 C). PGE
1
, when added after thimerosal, gradually
inhibited the appearance of new [Ca
2+
]
i
spikes, although it
did not restore [ Ca
2+
]
i
to the basal level (compare Fig. 3A
and D). When added after U73122, PGE
1
had a similar
effect on the spiking process ( Fig. 2C).
In experiments with aspirin-treated platelets in suspen-
sion, we verified the effects of these platelet-activating agents
on phospholipase C stimulation. Levels of InsP
3
levels were
measured at time points where the Ca
2+
signal was still
maximal. Thrombin, but not thimerosal, had a potent
InsP
3
-elevating effect that was largely abolished by a

preincubation with PGE
1
(Table 1). This is in agreement
with earlier data [44]. U73122 blocked the thrombin-in duced
increase in InsP
3
level at concentrations that also suppressed
the thrombin-induced Ca
2+
response. Together these results
indicate that both InsP
3
-generating (thrombin) and non-
InsP
3
-generating (sulfhydryl reagents) agents cause irregular
[Ca
2+
]
i
spiking in platelets. The thrombin-induced spiking
and to a lesser extent the thimerosal/U73122-induced
spiking appears to be sensitive to cAMP modulation.
Regulation of InsP
3
receptor function and store
depletion by Ca
2+
, cAMP and sulfhydryl reagents
To better understand the effects of these agents on the

spiking p rocess, we directly measured the Ca
2+
release
through the InsP
3
receptor channels. Therefore, platelets in
suspension were permeabilized with saponin under low
Ca
2+
-buffering and ATP-regenerating conditions using
Fluo-3 as a Ca
2+
probe [31]. In t his experimental system,
InsP
3
caused a (nonlinear) dose-dependent increase in
[Ca
2+
] from stores, which was completely suppressed by the
InsP
3
receptor antagonist heparin (Fig. 4A). A low con-
centration of InsP
3
(50 n
M
)causedaCa
2+
release of
27 ± 6 pmol per 10

8
platelets (mean ± SEM, n ¼ 7) at a
medium free Ca
2+
concentration of 110 n
M
.
The Ca
2+
-dependency of the InsP
3
-evoked Ca
2+
release
was evaluated by permeabilization experiments designed as
to prevent c hanges in the Ca
2+
store content. Platelets
were thus permeabilized at 110 n
M
[Ca
2+
], after which
apyrase was added (to block Ca
2+
re-uptake), followed by
different amounts of Ca
2+
and 50 n
M

InsP
3
(see Materials
and methods). Under these conditions, the Ca
2+
release
Fig. 2. Irregular spiking in [Ca
2+
]
i
induced by sulfhydry l-reac tive
agents. Aspirin-treated, Fura-2-loaded platelets on a surface were sti-
mulated with 10 l
M
thimerosal (TMS) (A) or 10 l
M
U73122 (B, C).
CaCl
2
and apyrase were present (see Fig. 1); PGE
1
(10 l
M
) was given
as indicated. Calcium r esponses are s hown of single platelets, and are
representative for > 50 cells.
1546 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002
increased about tenfold when the [Ca
2+
] was raised from 50

to 200 n
M
, whereas it declined at [Ca
2+
] above 400 n
M
(Fig. 4 B). This result thus resembles the biphasic effect of
Ca
2+
on InsP
3
-dependent CICR, p reviously observed in
preparations from cerebellum, synaptosomes and A7r5
smooth muscle cells [5–7,45], although in the latter sys-
tems higher levels of InsP
3
were needed to achieve Ca
2+
release. Preincubation of platelets with 10 l
M
PGE
1
before
permeabilization resulted in a 50% suppression of the
Ca
2+
-mobilizing e ffect of InsP
3
but w as without influence
on the biphasic effect of Ca

2+
(Fig. 4B and Table 2).
Control experiments indicated t hat PGE
1
treatment did not
influence the slow Ca
2+
release evoked by the endomem-
brane Ca
2+
-ATPase inhibitor thapsigargin (data not
shown). Thus, cAMP elevation seems to partially block
the InsP
3
receptor channel opening, but not to affect the
sensitization mechanism by Ca
2+
.
Further experiments with permeabilized platelets were
performed under conditions where the Ca
2+
release process
was most sensitive to modulation, i.e. at InsP
3
and Ca
2+
concentrations of 50 and 110 n
M
, respectively. Thrombin
activation of the platelets prior to p ermeabilization signifi-

cantly increased the amount of Ca
2+
released by InsP
3
(Table 2). This i s possibly due to a decrease in the platelet
cAMP level caused by this G
i
-stimulating agonist [46].
Thimerosal and U73122 had very different effects. Thim-
erosal (10 l
M
) did not elicit Ca
2+
release, but strongly
stimulated InsP
3
-induced Ca
2+
release (Fig. 5A), as repor-
ted for hepatocytes a nd other cells [13,40,41]. On the other
hand, a h igh dose o f U73122 (10 l
M
) caused stro ng release
of Ca
2+
by itself (Fig. 5A), which process was insensitive to
pretreatment with hep arin or PGE
1
(Table 2). This U73122
reaction was of little effect on subsequent InsP

3
-induced
Ca
2+
release. Control experiments showed that the inhib-
itory effects of heparin and PGE
1
on InsP
3
-evoked Ca
2+
release were not influenced by U73122 (Table 2). These data
thus suggest that InsP
3
and U73122 have additive effects on
Ca
2+
release from intracellular stores.
To confirm this, InsP
3
was applied at a higher, saturating
concentration. With 1 l
M
InsP
3
,increasing[Ca
2+
]from
100 to 200 n
M

resulted in a 1.7–fold (± 0.2, n ¼ 3) increase
in Ca
2+
release; PGE
1
pretreatment reduced the Ca
2+
release by 45%. When given after high InsP
3
(1 l
M
),
U73122 (10 l
M
) s till caused a rapid phase of Ca
2+
release
(Fig. 5 B). This suggested that its e ffect was mediated b y
Ca
2+
-leak channels different from the InsP
3
receptors.
Thapsigargin was used to determine the possible effect of
U73122 on (thapsigargin-releasable) Ca
2+
stores [18,27].
In permeabilized platelets, thapsigargin (1 l
M
)causeda

slow but progressive Ca
2+
release, when applied either
before or after InsP
3
. However, the release by U73122 was
not reduced, but even proceeded faste r, after InsP
3
/thaps-
igargin application (Fig. 5B). When applied to suspensions
of intact platelets in EGTA-containing medium, thapsigar-
gin caused slow and partial Ca
2+
release. In this system,
preincubation with U73122 accelerated and potentiated the
Ca
2+
release in a similar way as did the InsP
3
-generating
agonist thrombin (Fig. 5C). A synergism of thapsigargin-
and thrombin-evoked Ca
2+
mobilization in platelets has
Fig. 3. Regulation of thimerosal-induced
spiking in [Ca
2+
]
i
. Immobilized platelets were

stimulated with 10 l
M
thimerosal (TMS)
under conditions, as de scribed for Fig. 2.
Manoalide (10 l
M
) was added at either 5 min
before (A) or 1.5 min after (B) thimerosal. In
other e xperiments, PGE
1
(10 l
M
)wasadded
at 5 min before (C) or 1.5 min after (D)
thimerosal. Traces are typical responses from
a single platelet, representative for 50–75
analysed cells.
Table 1. Levels of InsP
3
activated platelets. Aspirin -treated platelets
(1 · 10
9
mL
)1
)in1m
M
CaCl
2
and apyrase remained unstimulated or
were activated with thrombin (10 n

M
)orthimerosal(10l
M
). The
platelets were preincubated with U73122 (2 l
M
)and/orPGE
1
(10 l
M
)
for 5 min, where indicated. Mass amounts of InsP
3
were determined
after 5 s (thrombin) or 60 s (thimerosal) of activation, i.e. when
maximal rises in [Ca
2+
]
i
were reached, as measured in parallel incu-
bations. Data are mean values ± SEM (n ¼ 4–6). ND, not deter-
mined.
Agonist
InsP
3
(pmol/10
8
platelets)
No pretreatment PGE
1

pretreatment
None 0.48 ± 0.06 ND
Thrombin 1.40 ± 0.15
a
0.75 ± 0.04
b
U73122 + thrombin 0.51 ± 0.08
c
ND
Thimerosal 0.58 ± 0.14 0.56 ± 0.10
a
P < 0.005;
b
P < 0.05 compared to the control condition, i.e. no
agonist (t-test, two-sided);
c
n ¼ 3.
Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1547
been described earlier [18], but this can now be extended to
thapsigargin- and U73122-evoked responses. From these
experiments we concluded that InsP
3
,Ca
2+
(via CICR) and
U73122 c ause additional a mounts of Ca
2+
release both in
intact and permeabilized platelets. The sulfhydryl reagent
U73122 seems to release Ca

2+
from stores that differ from
those used by InsP
3
, in a way insensitive to heparin and
cAMP.
Puff-like characteristics of [Ca
2+
]
i
spiking in single
platelets
To determine the involvement of different Ca
2+
stores in
the [Ca
2+
]
i
spiking process in single platelets, we monitored
this at higher spatial and temporal resolution. A fast
confocal fluorescence laser system was used to produce
fluorescent images from immobilized Fluo-3-loaded plate-
lets at an image resolution of 6.0 pixels per micrometer and
a scanning rate of 10 Hz. Because platelets spread on
fibrinogen increase in surface area from about 2–4 lmin
diameter (thickness of  0.5 lm), this set-up gave image
series of 250–450 pixels per platelet. We fi rst monitored the
characteristics of the Ca
2+

release events at low agonist
conditions, i.e. the ÔspontaneousÕ [Ca
2+
]
i
spikes that are due
to autocrine p roduced ADP [ 35]. Quite similar fluctuating
patterns in fluorescence were detected in different sub-
cellular regions (80–100 pixels) within a single platelet
(Fig. 6 A-B). The fluorescence pattern was completely
different in the adjacent region of a nearby platelet, proving
that the optical resolution was sufficiently high to detect
differences between the selected regions. The high temporal
resolution allowed precise analysis of the [Ca
2+
]
i
spikes. The
ÔspontaneousÕ peaks arose after long but variable intervals of
15.1 ± 1.6 s (mean ± SEM, n ¼ 63) (Fig. 6C). The ampli-
tudes of the individual peaks were highly variable, but rela-
ted to the total peak duration (Fig. 6D). When compared to
the usual criteria for low-amplitude Ca
2+
puffs (maximal
Fig. 4. InsP
3
-induced CICR in permeabilized platelets. (A) Traces of
InsP
3

-induced mobilization of Ca
2+
from stores. Asp irin-treated
platelets (3 · 10
8
ÆmL
)1
) permeabilized with saponin in the presence of
Fluo-3, as described in Materials and methods. The Ca
2+
level of the
medium was a djusted to 110 n
M
,InsP
3
was a dded at 50 or 200 n
M
concentrations, heparin (20 lgÆmL
)1
) was given at 2 min before InsP
3
where indicated. (B) InsP
3
–induced Ca
2+
release as a function of
[Ca
2+
] of the medium. Plate lets were pe rmeabilized with saponin
at 110 n

M
Ca
2+
, after which ATP generation was abolished with
apyrase, and Ca
2+
in the medium was changed to the indicated level
(x axis). The release o f C a
2+
by 50 n
M
InsP
3
was measured (y axis).
Before permeabilization, the platelets were treated with 10 l
M
PGE
1
(open circles) or r emained untreated (closed circles). Vertical line is at
standard [Ca
2+
]of110n
M
. Data are from three or more experiments
(mean ± SE M).
Table 2. Modulatio n of Ins P
3
-induced Ca
2+
release in permeabilized

platelets. Aspirin-treated platelets in KCl/ATP medium containing
Fluo-3 were left untreated or were treated with PGE
1
(10 l
M
,5min),
where indicated. Part of the platelets was activated with thrombin
(40 n
M
, 2 min). The pl atelets were permeabilized with saponin, and
[Ca
2+
] in t he medium wa s adjusted to 110 n
M
. Thimerosal ( 10 l
M
),
U73122 (10 l
M
) and/or h eparin (2 min, 20 lgÆmL
)1
) w er e added a t
8 min after saponin, as indicated. InsP
3
(50 n
M
) was given at 10 min
after saponin. Increases in [Ca
2+
] were measured i n response to the

agonist (thrombin, thimerosal or U73122) and I nsP
3
.TheCa
2+
release
by 50 n
M
InsP
3
under control conditions (no pretreatment) was taken
as 100% (82 ± 17 n
M
,equivalentto27±6pmolper10
8
platelets).
Data are mean values ± SEM (n ¼ 3–5). N D, not determined.
Agonist
Ca
2+
release (% of control)
Agonist,
no heparin
InsP
3
,
no heparin
InsP
3
with heparin
None – 100 (control) 10 ± 3

a
+ PGE
1
–46±8
a
ND
Thrombin ND 139 ± 13
a
5±2
a
Thimerosal 8 ± 4 217 ± 30
a,b
3±2
a
+ PGE
1
15 ± 5 92 ± 16 ND
U73122 472 ± 24 78 ± 12
c
3±1
a,c
+ PGE
1
495 ± 35 46 ± 9
c,d
ND
a
P < 0.001 compared to the release by InsP
3
under control con-

ditions, i.e. no pretreatment/no other agonist (t-test, two-sided).
b
Effect of thimerosal was 145 ± 17% of control with 500 instead
of 50 n
M
InsP
3
.
c
Relative to corresponding control value at
550 n
M
[Ca
2+
].
d
P < 0.01 compared to control conditions.
1548 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002
amplitude of < 200 n
M
and t otal durat ion o f 1–2 s) [2–4],
many of the low-amplitude Ca
2+
release events in platelets
(< 200 n
M
) appear to be of longer dur ation.
The high-resolution confocal scanning revealed irregular
trains of [Ca
2+

]
i
spikes when the Fluo-3-loaded platelets
were stimulated with thrombin (Fig. 7A). A gain, no more
than minor differences in peak generation were f ound
between different subcellular regions. The average peak-to-
peak interval was n ow decrea sed to 4 .8 ± 0.3 s (mean ±
SEM, n ¼ 67 peaks of 20 cells). This is similar to the highest
oscillation frequency reported for ATP-stimulated rat
megakaryocytes (peak-to-peak interval per cell varying
from 5 to 30 s) [22]. After platelet stimulation with
thimerosal, again trains of [Ca
2+
]
i
peaks started almost
simultaneously in vari ous subcellular parts (Fig. 7B). W ith
thimerosal, the average peak-to-peak interval was
8.9 ± 0.8 s (mean ± SEM, n ¼ 50; P < 0.001 compared
to thrombin). Thus, regardless of the peak generation
frequency, individual Ca
2+
-release events seemed to be
generated in various parts of a platelet at quite similar
intervals and amplitudes.
DISCUSSION
Here we describe that InsP
3
-mobilizing agonists (thrombin,
U46619 and p latelet-activating factor) as well as agents

acting independently of InsP
3
formation (thimerosal and
U73122 at 10 l
M
) evoke irregular [Ca
2+
]
i
spiking in aspirin-
treated platelets. T he thrombin-induced spiking app ears to
be strictly dependent on InsP
3
formation, because it is
abolished by manoalide or low U73122. It is also inhibited
by cAMP elevation with PGE
1
, in part due to reduced InsP
3
formation ( probably by phospholipase C inhibition) and in
part due to decreased InsP
3
-mediated Ca
2+
release from
intracellular stores. On the other hand, the sulfhydryl
reagent thimerosal elicits [ Ca
2+
]
i

spiking not by increasing
the InsP
3
level but by potentiating InsP
3
receptor-mediated
Ca
2+
release. This may explain why the Ca
2+
response with
thimerosal is only partially inhibitable by P GE
1
.The
N-ethyl maleimide derivative U73122, at a high dose of
10 l
M
, yet acts in a still different manner. In permeabilized
Fig. 6. Confocal monitoring of ÔspontaneousÕ spiking in [Ca
2+
]
i
in spread
platelets. Fluorescence changes were monitored by confocal laser
scanning microscopy in aspirin-treated, Fluo-3-loaded platelets spread
on fibrinogen. Apyrase was omitted from the incubation medium
(nominally Ca
2+
-free). High-resolution images of 250–450 pixels/
platelet were collected at 10 Hz. (A) Fluorescence recordings from

three selected regions of one spread platelet (a-c); and from a region of
interest of an adjace nt platelet ( r) (initial value of each trace, F/F
o
¼ 1).
Insert shows expanded part of curves a–c. (B) Selection of regions of
interest of the platelets ( areas  0.8 · 2.5 lm). (C) Histogram of
variation in peak-to-peak interval of 15 responsive platelets. (D) Plot
of total duration of individual peaks (90% decay) vs. peak amplitude.
Data are mean values plus SEM of analysis results from the three
regions per platelet. Regression analysis of all data: y ¼ 0.83 + 0.98 x
(R
2
¼ 0.68, P < 0.001).
Fig. 5. Calcium mobilization from stores in permeabilized and intact platelets. (A,B) Aspirin-treated platelets were permeabilized with saponin in
Fluo-3-containing me dium. A fter [ Ca
2+
] adjustment to 110 n
M
,thimerosal(TMS,10l
M
), U73122 (10 l
M
) and thapsigargin (TG, 1 l
M
)were
given, as indicated. (A) InsP
3
was added at a low concentration of 50 n
M
with 3 · 10

8
plateletÆmL
)1
(B) InsP
3
was given at a higher concentration
(1 l
M
), while the platelet concentration was 2 · 10
8
plateletsÆmL
)1
. Note that U73122-evoked Ca
2+
release leads to a h igher m edium [Ca
2+
], which
potentiates t he InsP
3
-evoked r elease. (C) Intact, a spirin-treated platelets in suspension (1 · 10
8
plateletsÆmL
)1
), loaded with Fura-2, were stimulated
with thrombin (T hr, 4 n
M
), thapsigargin (1 l
M
) and/or U73122 (10 l
M

) in the presence of 1 m
M
EGTA.
Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1549
platelets, it causes a cAMP/heparin-insensitive Ca
2+
leak
that seems to be independent of the InsP
3
receptor-mediated
Ca
2+
release. It can thus be envisioned that, in intact
platelets, the Ca
2+
release evoked by U73122 s timulates the
process of InsP
3
receptor-mediated CICR, and thereby the
generation of [Ca
2+
]
i
spikes.
In a variety of cells, thimerosal is known to react with
critical thiol groups controlling InsP
3
-receptor channel
opening, which results in repetitive Ca
2+

release at basal
levels of InsP
3
[13,14,40,41]. In platelets sulfhydryl groups
may s imilarly control I nsP
3
receptor functioning [42]. This
agrees with our finding that, i n permeabilized platelets,
heparin completely inhibits the t himerosal-enhanced Ca
2+
release by InsP
3
. Taken together, the present work thus
indicated t hat the platelet InsP
3
receptors p lay a key role in
the regenerative, spiking Ca
2+
release evoked by phospho-
lipase C -stimulating and InsP
3
receptor-modulating agents,
similarly as established f or other cell types.
Using saponin-permeabilized platelets, we foun d that the
InsP
3
-evoked Ca
2+
-mobilizing pote ncy changed with the
cytosolic Ca

2+
concentration in a biphasic way (Fig. 6 ),
similarly as firstly described for neuronal cells [5–7] and later
for pancreatic acinar c ells, hepatocytes and smooth muscle
cells [45,47,48]. Whereas in many cell t ypes micromolar
concentrations of InsP
3
were needed to detect a stimulating
effect of Ca
2+
on InsP
3
receptor-mediated Ca
2+
release
[5–8,45,47], this could be demonstrated in platelets already
low levels of 50–200 n
M
InsP
3
. It is noted that platelets are
relatively rich in type 1 InsP
3
receptors [26], which are quite
sensitive to Ca
2+
modulation.
For rabbit a nd mouse pancreatic acinar cells, it has been
shown that U73122 evokes [Ca
2+

]
i
oscillations by potenti-
ating the release of Ca
2+
from a InsP
3
-sensitive store
compartment [39,43]. This release may lead to increased
Ca
2+
influx from the e xternal medium and to subsequen t
overloading of InsP
3
-insensitive stores, which in turn can
trigger regenerative C a
2+
release [1,43]. A similar mechan-
ism, i.e. cooperation of store compartments in [Ca
2+
]
i
spiking, may also apply to platelets.
Typical for platelets is that the amount of Ca
2+
released
by a suboptimal InsP
3
concentration, but not the Ca
2+

sensitivity of the release, is suppressed upon cAMP eleva-
tion. There i s little doub t that most o r all cAMP-m ediated
effects in platelets are due to cAMP-dependent protein
phosphorylation, and that the platelet InsP
3
receptors are
targets of cAMP-dependent protein kinase [27]. Earlier, we
have reported that thrombin- and thapsigargin-induced
Ca
2+
responses in platelets are down-regulated by cAMP
analogues and inhibitors of cAMP phosphodiesterase, a nd
that cAMP-dependent protein kinase was important in this
effect. These cAMP-elevating interven tions also suppressed
the InsP
3
-induced Ca
2+
mobilization in saponin-permeabi-
lized platelets [ 46]. Together w ith the new evidence it thus
becomes clear that cAMP-dependent phosphorylation ren-
ders the InsP
3
receptor less active as a Ca
2+
channel
[30,31,49], and also that the phosphorylated receptor
remains sensitive to change s in [ Ca
2+
]

i
(this paper). In this
respect, platelets differ from other cells such as hepatocytes,
where a ctivation of c AMP-dependent kinase was found to
increase the amount of Ca
2+
released by InsP
3
[41].
The confocal laser scanning experiments with Fluo-
3-loaded platelets, permitting a simultaneously high tem-
poral and spatial resolution of the Ca
2+
signal, clearly
indicated that the [Ca
2+
]
i
release events in platelets are
highly irregular in s hape, amplitude and frequency, regard-
less of whether they are raised by InsP
3
-generating receptor
agonists or sulfhydryl-reactive compounds. The experi-
ments show that the irregular traces detected in Fura-
2-loaded platelets by camera-based microfluorometry are
most probably not artefacts of the ratio imaging procedure.
In addition, they detect similar Ca
2+
release events at

distant sites within a platelet: this holds not only f or single
[Ca
2+
]
i
spikes, but also for complex series of consecutive
spikes (Figs 6 ,7). Calcium puffs as recorded in large r cells
are commonly d efined as single Ca
2+
release events that
arise due to the action of multiple InsP
3
receptor c hannels
clustered in f unctional units [2–4]. The operating definitions
of a Ca
2+
puff vary somewhat, but congregate as a local
Ca
2+
release event (diameter about 1 lm) with a maximal
amplitude of < 200 n
M
, a rising time of < 0 .35 s and t otal
duration of 1–2 s. The Ca
2+
spikes of platelets resemble the
puffs seen in larger cells in local appearance, but differ from
these in at least two a spects. First, the platelet spikes appear
at a v ariable frequency (0.02–0.3 Hz), regardless o f whether
CaCl

2
or EGTA is externally present (see [35]). Second, they
are rather broad and do not sum up, i.e. the individual
Fig. 7. Uniform [Ca
2+
]
i
transients within act i-
vated, spread platelets. Fluo-3-loaded platelets
were stimulated with (A) thrombin (4 n
M
,
given a t t ¼ 8 s) o r (B) thimerosal (10 l
M
,
given a t t ¼ 0 s) in the presence of 1 m
M
CaCl
2
and apyrase. High-resolution images
were collected by co nfocal laser s canning
microscopy, as described for Fig. 6. Fluores-
cence recordings are show n from three non-
overlapping regions of one platelet (initial
value of each trace, F/F
o
¼ 1). Inserts give
extended parts. Data are representative for 3
or more experiments.
1550 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002

events do not seem to be subjected t o frequency or
amplitude recruitment, such as described for HeLa cells [4].
Because of the small size of platelets with nearby Ca
2+
-
ATPases throughout the cell, it is likely that the rate of
Ca
2+
pumping rather than the diffusion of released Ca
2+
into the c ytosol (as in bigger cells) d etermines the duration
of the platelet spikes.
In many cell types, the global release of Ca
2+
is
controlled by an intimate interplay between thapsigargin-
and InsP
3
-sensitive Ca
2+
store compartments. For instance,
in rabbit pancreatic a cinar cells the (thapsigargin-inhibited)
compensatory Ca
2+
pumping by endomembrane Ca
2+
-
ATPases restricts the Ca
2+
-store depletion by InsP

3
[47].
In mouse lacrimal cells, the thapsigargin-induced Ca
2+
mobilization is dependent on the basal level of InsP
3
and the
InsP
3
-receptor func tion [50]. Such a situation m ay also
exists in platelets, where both the InsP
3
- a nd thapsigargin-
sensitive Ca
2+
store compartments are likely to contribute
to the [Ca
2+
]
i
spiking [18,27]. In the present paper, we
describe that regardless of the type of a gonist, stimulating
(thrombin) or sensitizing (thimerosal) InsP
3
receptors or
acting primarily independently of InsP
3
receptors (U73122),
and regardless o f the type of stores u sed by these agonists,
the spiking process w as always irregular in amplitude and

frequency and occurred with no more than little subcellular
heterogeneity. This situation however, differs from that of
pancreatic acinar cells, where even within the voxel of a
Ca
2+
Ôhot spotÕ quite different patterns of spike-like events
can be observed [51]. This apparently points to a high
cooperation of Ca
2+
mobilization from the various stores in
platelets to g enerate s maller as well as l arger C a
2+
-release
events.
In summary, the small platelets forms an attractive model
to study the function of InsP
3
receptors, even when induced
by agents such as U73122 and thimerosal that do not cause
InsP
3
formation. The platelet InsP
3
receptors are subjected
to extensive regulation by at least four factors: (a) local
InsP
3
levels; (b) the potentiating effect of moderate increases
in [Ca
2+

]
i
on InsP
3
action via CICR; (c) InsP
3
receptor
channel sensitization (thimerosal) and desensitization
(mediated by cAMP); and (d) InsP
3
channel-independent
Ca
2+
leak with U73122. Given the importance of the Ca
2+
signal for the process of platelet activation, it is likely t hat
the highly regulated nature of the Ca
2+
signal plays a n
important role in ensuring rapid p latelet deposition a t the
right physiological sites during hemostasis and at athero-
sclerotic sites during thrombosis.
ACKNOWLEDGEMENTS
We ack nowledge grants from the Netherlands Heart Foundation and
the Netherlands Organization for Scientific Research.
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