Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 199-207
Marked Temperature Dependence of the Platelet Calcium Signal
Induced by Human von Willebrand Factor
By
John C. Kermode,
Qi Zheng, and
Elizabeth P. Milner
From the Department of Pharmacology and Toxicology, University of
Mississippi Medical Center, Jackson, MS.
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ABSTRACT |
Interaction of von Willebrand factor (vWF) with the platelet is
essential to hemostasis when vascular injury occurs. This interaction
elevates the intracellular free calcium concentration ([Ca2+]i) and promotes platelet activation.
The present study investigated the temperature dependence of
vWF-induced [Ca2+]i signaling in human
platelets. The influence of temperature can provide invaluable insight
into the underlying mechanism. Platelet
[Ca2+]i was monitored with Fura-PE3.
Ristocetin-mediated binding of vWF induced a transient platelet
[Ca2+]i increase at 37°C, but no
response at lower temperatures (20°C to 25°C). This temperature
dependence could not be attributed to a reduction in vWF binding, as
ristocetin-mediated platelet aggregation and agglutination were
essentially unaffected by temperature. Most other platelet agonists
(U-46619, 
-thrombin, and adenosine 5'-diphosphate
[ADP]) induced a [Ca2+]i
signal whose amplitude did not diminish at lower temperatures. The
[Ca2+]i signal in response to arachidonic
acid, however, showed similar temperature dependence to that seen with
vWF. Assessment of thromboxane A2 production showed a
strong temperature dependence for metabolism of arachidonic acid by the
cyclo-oxygenase pathway. vWF induced thromboxane A2
production in the platelet. Aspirin treatment abolished the vWF-induced
[Ca2+]i signal. These observations suggest
that release of arachidonic acid and its conversion to thromboxane
A2 play a central role in vWF-mediated
[Ca2+]i signaling in the platelet at
physiological temperatures.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a
multimeric, plasma glycoprotein that plays a major role in hemostasis
and thrombosis.1,2 It can interact with a receptor, the
glycoprotein Ib-IX-V complex (GP Ib-IX-V), on the platelet membrane and
thereby promote platelet adhesion. Such interaction is not spontaneous,
but may be triggered through surface immobilization of vWF3
or by an exogenous modulator, such as the antibiotic
ristocetin.4 Binding of vWF to exposed subendothelial
elements may play a similar role in eliciting its interaction with GP
Ib-IX-V in vivo.5 Recent data indicate that exposure to
high shear stress, such as encountered in a stenosed artery, also
promotes binding of vWF to GP Ib-IX-V.6
Considerable evidence has accrued to suggest that interaction of vWF
with GP Ib-IX-V causes platelet activation.1,3,7-9 The
signaling mechanism that leads to such activation is now under investigation. Several investigators have reported that binding of vWF
to GP Ib-IX-V evokes an increase in intracellular free calcium
concentration ([Ca2+]i) in the
platelet.10-17 By analogy with other platelet
agonists,18 this calcium signal is presumed to play an
essential role in platelet activation.
Studies of the vWF-induced [Ca2+]i signal
have used several approaches to elicit the interaction of vWF with
platelet GP Ib-IX-V. Some investigators have adopted a static system,
in which either an exogenous modulator (ristocetin or botrocetin)
mediates the binding of native human vWF11,15,17 or
spontaneous binding results from the use of a mutant human
vWF14 or of porcine vWF16; others have chosen a
dynamic approach, where shear stress acts as the trigger for vWF
binding.10,12,13 There have been several significant
discrepancies among the results obtained with these diverse approaches.
In particular, the intracellular signaling mechanism that is
responsible for the [Ca2+]i increase appears
to differ depending on the means for triggering vWF interaction.
Observations with the static approaches have suggested that activation
of the phospholipase A2 pathway and subsequent production
of thromboxane A2 may play a major role in vWF-induced
[Ca2+]i signaling.11,19 In
contrast, studies using shear stress to trigger vWF binding have
implicated calcium influx through channels in the platelet plasma
membrane as the primary signaling mechanism, with negligible
contribution from thromboxane A2.1,12,13
Such a divergence in the signaling mechanism for vWF would present an
attractive target for selective therapeutic intervention to prevent
thrombosis in a region of stenosis without impairing hemostasis in the
event of a vascular injury. It is premature, however, to conclude at
this time that the signaling mechanism differs in this way. Before
reaching such a conclusion, it is necessary to rule out any more
mundane, technical explanation for the divergent findings. For example,
the investigations of vWF-mediated platelet
[Ca2+]i signaling under shear stress appear,
for technical reasons, to have been conducted at room
temperature,10,12,13 whereas those under static conditions
were generally performed at 37°C.11,14,16,17,19 This
difference in temperature might conceivably explain the apparent difference in the signaling mechanism.
To address this concern, this study examines the influence of
temperature on the [Ca2+]i signal that arises
when ristocetin triggers interaction of vWF with platelet GP Ib-IX-V.
The pattern of temperature dependence is compared with that observed
for other platelet agonists. The influence of temperature on a
biological process may also provide insight into the nature of the
mechanism that underlies it, as a diffusion-limited process is usually
less affected by temperature than an enzyme-mediated
process.20 Not only do our findings stress the importance
of temperature as a factor that has a profound influence on vWF-induced
signaling in the platelet, they also highlight a major difference in
the signaling mechanism between vWF and most other platelet agonists.
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MATERIALS AND METHODS |
Evaluation of kd for binding of calcium to Fura-PE3.
The dissociation constants for binding of calcium to Fura-PE3 at
20°C and 37°C were assessed using a calcium calibration buffer kit (Molecular Probes, Eugene, OR) in accordance with the
manufacturer's instructions. In brief, for measurements at each
temperature, two solutions containing 2 µmol/L Fura-PE3
(K+ salt; Texas Fluorescence Laboratory, Austin, TX) were
prepared, one with 10 mmol/L EGTA and the other with 10 mmol/L CaEGTA.
Both solutions also contained 100 mmol/L KCl, 1 mmol/L free
Mg2+, and 10 mmol/L 3-(N-morpholino)
propanesulfonic acid (MOPS), and they were adjusted to pH
7.0 at the desired temperature. A series of 11 solutions of known free
Ca2+ concentration was prepared by appropriate mixing.
Fluorescence excitation spectra were collected for each solution at the
appropriate temperature with an emission wavelength of 510 nm. The kd
value was calculated from a Hill plot of the fluorescence intensity with excitation at 340 nm.17
Isolation of human platelets and loading with Fura-PE3.
Venous blood was drawn from healthy adult volunteers who had not taken
aspirin or a nonsteroidal antiinflammatory drug for at least 10 days
before phlebotomy. Female donors were not taking an oral contraceptive.
The protocol was approved by the local Institutional Review Board, and
informed consent was obtained from each donor. Platelets were isolated
using the method described previously.17 In brief, blood
was collected into 1/6 volume of acid-citrate-dextrose anticoagulant
(65 mmol/L citric acid, 85 mmol/L sodium citrate, 110 mmol/L
D-glucose). Platelet-rich plasma was treated with 5 mmol/L creatine
phosphate and 25 U/mL creatine phosphokinase (Sigma Chemicals, St
Louis, MO), and the platelets were isolated by centrifugation at
1,000g for 15 minutes at 23°C. The platelets were
resuspended in calcium-free, magnesium-free, Tyrode's solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 0.4 mmol/L
NaH2PO4, 5.5 mmol/L D-glucose),
supplemented with 10 mmol/L HEPES, pH 7.35, and 0.35% (wt/vol) bovine
serum albumin (BSA). This solution was also supplemented with 5 mmol/L creatine phosphate, 25 U/mL creatine phosphokinase, and 1 mmol/L EGTA
during indicator loading.
Platelets were loaded with fluorescent calcium indicator by a 1-hour
incubation at 23°C in the dark with 5 µmol/L Fura-PE3 acetoxymethyl ester (AM) (Texas Fluorescence Laboratory) in the presence of 0.004% (wt/vol) Pluronic F-127; before addition to the
platelets, the Fura-PE3/AM stock (3.3 mmol/L in dimethylsulfoxide) was
mixed with Tyrode's solution containing 4% BSA to facilitate its
dispersal. A separate portion of the platelet suspension was retained
as an autofluorescence control. After loading, the platelets were
washed twice by centrifugation and resuspension.17,21 The
final resuspension (in buffered Tyrode's solution with 0.35% BSA and
1 mmol/L MgCl2) was adjusted to a platelet count of 50,000 cells/µL.
Measurement of platelet [Ca2+]i signals.
Platelet [Ca2+]i was assayed on a Photon
Technology International (South Brunswick, NJ) RF-M2004
spectrofluorometer. All studies were conducted in a cylindrical glass
cuvette (8-mm diameter) designed for aggregometry (Payton Scientific,
Buffalo, NY). Reflections from its curved surface were eliminated by
using a vertical polarizer in the excitation beam and a horizontal one
in the emission beam. A specially designed stirrer was used; this
comprised a Teflon cylinder with a bar magnet embedded at its base and
an ultraviolet (UV)-grade methacrylate stirring vane
attached at the top.17 This stirrer ensures efficient
stirring of a platelet suspension without significant interference with
the optical signal, and its use has been validated extensively in
studies with both classical platelet agonists and multimeric human
vWF.17
Platelets (50,000 cells/µL) that had been loaded with fluorescent
calcium indicator were stirred continuously at the selected temperature. They were equilibrated briefly (1 to 2 minutes) with extracellular calcium (1 mmol/L CaCl2), unless otherwise
indicated. Stimulation was usually effected by sequential addition of 1 mg/mL ristocetin (Bio/Data Corp, Hatboro, PA) and 5 µg/mL purified
human vWF. Human vWF was either obtained from a commercial source
(Calbiochem, San Diego, CA) or purified from plasma by cryoethanol
precipitation, differential polyethylene glycol fractionation, and gel
filtration on Sepharose CL-4B.22,23 Similar results were
obtained with these two vWF preparations; each was essentially pure by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and included the full range of multimers present in plasma vWF. In other studies, platelets were stimulated by addition of 5 µmol/L adenosine
5'-diphosphate (ADP), 0.02 U/mL human 
-thrombin, 0.1 µmol/L
U-46619, or 20 µmol/L arachidonic acid (all from Sigma Chemicals).
Fluorescence intensity was monitored at 510 nm emission with excitation
alternating between 340 and 380 nm wavelengths. Autofluorescence was
determined in platelets that had not been loaded with Fura-PE3 and was
subtracted from each fluorescence measurement (before ratio calculation).
Calibration data were obtained in each experiment by the procedure of
Merritt et al24 (on a separate aliquot of platelets). The
maximal fluorescence ratio (Rmax) was assessed by adding 60 µmol/L digitonin (Calbiochem) in the presence of 1 mmol/L
CaCl2, and the minimal fluorescence ratio
(Rmin) determined by adding excess (20 mmol/L) EGTA,
buffered to pH 8.5. Experimental measurements of the fluorescence ratio
(R) were converted to [Ca2+]i values by the
equation25:
where Ff and Fb are the fluorescence
intensities with 380 nm excitation for free and calcium-bound
indicator, respectively. Calculations at 20°C and 37°C are
based on measured kd values of 251 and 146 nmol/L, respectively, for
binding of calcium to Fura-PE3 (see Results); calculations at
intermediate temperatures used interpolated kd estimates of 220 nmol/L
at 25°C and 189 nmol/L at 30°C.
Platelet aggregometry.
Platelet aggregation was assessed simultaneously with the
[Ca2+]i measurements in some studies. The
Photon Technology RF-M2004 spectrofluorometer was used in an unorthodox
configuration for these studies (Fig 1);
one emission monochromator (380 nm wavelength) was repositioned to
monitor transmitted light, and thereby assess aggregation, while
platelet [Ca2+]i was evaluated from Fura-PE3
fluorescence emission measurements (510 nm) perpendicular to the
excitation beam. The lower part of the transmitted light beam was
blocked with an opaque panel in a specially designed cuvette holder so
that the novel stirrer (which protrudes into the excitation beam) did
not interfere with the aggregation measurements. The excitation
wavelength alternated between 340 and 380 nm for the
[Ca2+]i measurements; transmitted light was
monitored during the 380-nm excitation phase only. Aggregation is
expressed as the change in light transmittance relative to the
transmittance in the absence of platelets. This experimental approach
yields aggregation data that are indistinguishable from data obtained
with a traditional aggregometer.
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| Fig 1.
Optical configuration and stirring arrangement for
simultaneous assessment of platelet aggregation and
[Ca2+]i signaling. Measurements are
conducted in a cylindrical glass aggregometer cuvette (8-mm diameter)
in a reconfigured dual-emission spectrofluorometer. The platelet
suspension is stirred with a novel stirrer, comprising an opaque Teflon
cylinder with a bar magnet at its base and a UV-grade methacrylate
stirring vane at its top. This stirrer protrudes into the excitation
beam of the spectrofluorometer to ensure that platelet aggregates
cannot settle below its detection zone.17 Platelet
aggregation is monitored through measurement of transmitted light
intensity, with the lower part of the transmitted light beam blocked so
that the stirrer does not interfere with these measurements. Platelet
[Ca2+]i is monitored through fluorescence
measurements perpendicular to the excitation beam. Reflections from the
curved surfaces of the cuvette are eliminated by use of a vertical
polarizer in the excitation beam and a horizontal one in the emission
beam. This arrangement provides efficient stirring of the platelet
suspension and ensures that the fluorescence signal is representative
of the entire population of platelets regardless of the extent of
aggregation.
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Thromboxane A2 production.
Production of thromboxane A2 was assessed in the samples
used to monitor the platelet [Ca2+]i signal.
Platelets were incubated with 1 mg/mL ristocetin and 5 µg/mL human
vWF for 10 minutes, a time sufficient for the
[Ca2+]i transient to be complete at 37°C.
Incubations of the same duration were performed at the lower
temperatures, and equivalent control incubations were conducted at each
temperature with 1 mg/mL ristocetin in the absence of vWF. Analogous
studies were conducted with 20 µmol/L arachidonic acid, except that
the incubation time was 3 minutes. Each reaction was stopped by adding
1 mmol/L aspirin and 10 mmol/L EDTA (in buffered Tyrode's solution);
the supernatant fraction was collected after centrifugation for 1 minute at 16,000g. Thromboxane A2 production during
the incubation was assessed by competitive enzyme immunoassay of
thromboxane B2 (its stable breakdown product) in the
supernatant fraction, using a commercial assay kit (Cayman Chemicals,
Ann Arbor, MI). All thromboxane B2 samples from studies on
platelets from a particular donor were included in a single assay; the
within-assay coefficient of variation for these assays averaged 4%.
Agglutination of paraformaldehyde-fixed platelets.
Human platelets were fixed with 0.6% (wt/vol) paraformaldehyde by the
method of Brinkhous and Read26 and washed extensively. Ristocetin-mediated agglutination of the fixed platelets by vWF was
assessed by monitoring light transmittance at 380 nm (as for the
aggregation measurements on live platelets).
Statistical analysis.
Most data on platelet [Ca2+]i and thromboxane
A2 production in this study were not normally distributed.
Such data are thus presented as a median value with the interquartile
range, and nonparametric tests were used to determine statistical
significance (specific tests indicated in Results). Data on kd values
for binding of calcium to Fura-PE3 were consistent with a normal
distribution and are presented as a mean value ± standard error of
mean (SEM).
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RESULTS |
Temperature dependence of the calcium-binding characteristics of
Fura-PE3.
The calcium-binding characteristics of Fura-PE3 were evaluated from
excitation spectra measured in the presence of a series of free calcium
concentrations. Three studies at 20°C yielded an average kd value
of 251 ± 9 nmol/L (mean ± SEM) for Fura-PE3 at pH 7.0. A
significantly lower kd value (146 ± 9 nmol/L,17 based
on four studies; P < .001, Student's t-test) was
found at 37°C and pH 7.0.
Temperature dependence of the vWF-induced platelet
[Ca2+]i signal.
Ristocetin-mediated binding of multimeric human vWF in the presence of
extracellular calcium induced a gradual and transient platelet
[Ca2+]i increase at 37°C
(Fig 2C). This
[Ca2+]i signal arose after an appreciable lag
phase. At 20°C or 25°C, in contrast, vWF did not induce a
perceptible increase in [Ca2+]i (Fig 2A and
Table 1). Studies at 30°C showed either
a small increase in platelet [Ca2+]i (Fig 2B)
or no discernible increase (four of the eight studies). Analogous
studies performed in the absence of extracellular calcium (with 1 mmol/L EGTA) showed the same pattern of temperature dependence (data
not shown).
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| Fig 2.
Temperature dependence of the
[Ca2+]i signal induced by
ristocetin-mediated binding of vWF. Human platelets were loaded with 5 µmol/L Fura-PE3/AM. Measurements were performed in an aggregometer
cuvette, with the platelet suspension (50,000 cells/µL) stirred by
the novel stirrer. The platelets were stimulated at 20°C (A),
30°C (B), or 37°C (C) by adding 1 mg/mL ristocetin and 5 µg/mL multimeric human vWF (solid traces) in the presence of
extracellular calcium (1 mmol/L CaCl2). Data obtained in
parallel control studies with 1 mg/mL ristocetin alone (dotted traces)
are also displayed. The fluorescence intensity at 510-nm emission
wavelength was measured with excitation alternating between 340 and 380 nm. Platelet [Ca2+]i was calculated from
the fluorescence ratio (340:380 nm), using the kd value for Fura-PE3 at
the corresponding temperature. Observations at 25°C (not shown)
were indistinguishable from those at 20°C. The results illustrated
are from a typical one of eight such studies.
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Stirring of the platelet suspension was required for vWF to induce a
[Ca2+]i increase at 37°C, suggesting that
platelet aggregation might be necessary. The role of vWF binding to the
glycoprotein IIb-IIIa complex (GP IIb-IIIa) in the signaling mechanism
was evaluated using a monoclonal antibody against GP IIb-IIIa (10E5;
kindly provided by Dr Barry Coller, Mount Sinai Medical Center, New
York, NY).27 An antibody concentration of 10 µg/mL proved sufficient to prevent binding of vWF to GP IIb-IIIa; it
abolished the aggregation by vWF of platelets that had been activated
by U-46619 (Fig 3A). In contrast,
preincubation with this concentration of antibody did not significantly
affect the platelet [Ca2+]i signal evoked by
ristocetin-mediated binding of vWF at 37°C (Fig 3B and C). Median
values (from four studies) for the peak [Ca2+]i increase were 98 nmol/L in untreated
platelets, 86 nmol/L in platelets pretreated with an isotype-matched
control Ig (IgG2a), and 89 nmol/L after treatment with 10E5 antibody
(P > .2, Friedman's test). Blockade of GP IIb-IIIa with the
tetrapeptide Arg-Gly-Asp-Ser (1 mmol/L) also had negligible effect on
the vWF-induced [Ca2+]i signal.17
These data imply that binding of vWF to GP Ib-IX-V triggers the
platelet [Ca2+]i increase.
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| Fig 3.
Effect of GP IIb-IIIa blockade on the vWF-induced
[Ca2+]i signal. (A) The ability of the 10E5
antibody to block GP IIb-IIIa was evaluated by aggregometry. Washed
human platelets (50,000 cells/µL, suspended in Tyrode's solution
with 1 mmol/L CaCl2) were preincubated (5 minutes at
37°C) with isotype-matched control IgG (solid trace) or with 10E5
IgG (dotted trace). Each IgG was essentially azide-free and was used at
a final concentration of 10 µg/mL. Platelets were stimulated with 0.1 µmol/L U-46619 and aggregation was assessed at 37°C in the
presence of 5 µg/mL multimeric human vWF. (B and C) The effect of the
10E5 antibody on the platelet [Ca2+]i
signal was evaluated. Human platelets (50,000 cells/µL, loaded with
Fura-PE3) were preincubated with 10 µg/mL control IgG (B) or 10E5 IgG
(C). The platelets were then stimulated at 37°C by 1 mg/mL
ristocetin and 5 µg/mL vWF (solid traces) in the presence of 1 mmol/L
CaCl2. Data obtained in parallel control studies with 1 mg/mL ristocetin alone (dotted traces) are also displayed. Fluorescence
measurements were performed in a cylindrical aggregometer cuvette, with
the platelet suspension stirred by the novel stirrer. Platelet
[Ca2+]i was calculated from the
fluorescence excitation ratio (340:380 nm). Results of a typical one of
four such studies are illustrated. Aggregation and platelet
[Ca2+]i were also assessed in the absence
of either IgG (not shown); results were indistinguishable from those in
the presence of the control IgG.
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The absence of a vWF-induced platelet [Ca2+]i
signal at the lower temperatures could reflect a strong temperature
dependence for either binding of vWF to the GP Ib-IX-V complex or a
subsequent step in the signal transduction process triggered by vWF.
These two possibilities were discriminated primarily by evaluating the temperature dependence of ristocetin-mediated platelet aggregation by
vWF. ("Aggregation" is used here in an inclusive sense to
encompass both physical agglutination through binding of vWF to
different platelets and true aggregation involving platelet activation. Both processes occur when live platelets are incubated with vWF in the
presence of ristocetin, and the measurement of light transmittance cannot distinguish between them.) Aggregation was assessed
simultaneously with the platelet [Ca2+]i
measurements by using a novel configuration of the spectrofluorometer (Fig 1). The aggregation of live platelets by vWF was essentially unaffected by temperature over the range 20°C to 37°C
(Fig 4A and B). Temperature also had
negligible effect when aggregation was assessed in the presence of
Arg-Gly-Asp-Ser (data not shown). Ristocetin-mediated agglutination of
fixed platelets by vWF declined marginally when the temperature was
lowered from 37°C to 20°C (Fig 4C and D), but there was no
significant reduction at 30°C (data not shown). These data together
imply that the marked temperature dependence of the vWF-induced
platelet [Ca2+]i signal cannot be attributed
to differences in vWF binding to GP Ib-IX-V.
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| Fig 4.
Temperature dependence of platelet aggregation and
agglutination by ristocetin-mediated binding of vWF. (A and B)
Aggregation of live platelets was monitored simultaneously with
platelet [Ca2+]i. The platelet suspension
(50,000 cells/µL) was incubated at 20°C (A) or 37°C (B) in
the presence of 1 mmol/L CaCl2. Aggregation was deduced
from the transmitted light intensity (during the 380 nm fluorescence
excitation phase). Aggregation patterns at 25°C and 30°C (not
shown) were indistinguishable from those at 20°C and 37°C. The
results illustrated were acquired in parallel with the
[Ca2+]i measurements shown in Fig 2; these
aggregation data are typical of four such studies. (C and D)
Agglutination of paraformaldehyde-fixed platelets (50,000 cells/µL)
was evaluated at 20°C (C) and 37°C (D) in an analogous manner.
Agglutination patterns at 30°C (not shown) were comparable to those
at 37°C. These data are typical of three such studies. Panels A
through D present the responses on incubation either with 1 mg/mL
ristocetin and 5 µg/mL multimeric human vWF (solid traces) or with 1 mg/mL ristocetin alone (dotted traces).
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Temperature dependence of platelet [Ca2+]i
signals for other agonists.
The influence of temperature on the [Ca2+]i
signal was also evaluated for other platelet agonists. For the stable
thromboxane A2 analog U-46619, the amplitude of the
agonist-induced [Ca2+]i transient was not
significantly affected by temperature (Fig 5 and Table 1; P > .2, Friedman's test). For -thrombin
and ADP, the amplitude of the [Ca2+]i signal
tended to diminish as the temperature was increased from 20°C to
37°C (Table 1); this diminution was marked for -thrombin, but
modest for ADP. The time course of the
[Ca2+]i transient for each of these three
agonists tended to be prolonged at the lower temperatures (Fig 5).
Studies with different concentrations of U-46619 (0.05 to 1 µmol/L)
and -thrombin (0.01 to 0.1 U/mL) yielded similar observations (data
not shown). Resting platelet [Ca2+]i rose
gradually with increasing temperature (Table 1). Analogous findings
were made in studies conducted with these agonists in the absence of
extracellular calcium (data not shown).
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| Fig 5.
Temperature dependence of the
[Ca2+]i signals induced by U-46619,
-thrombin, and ADP. The [Ca2+]i
transient in a stirred suspension of human platelets (50,000 cells/µL) was monitored with Fura-PE3. The platelets were stimulated
at 20°C (dotted traces) or 37°C (solid traces) by 0.1 µmol/L
U-46619 (A), 0.02 U/mL human -thrombin (B), or 5 µmol/L ADP (C) in
the presence of extracellular calcium (1 mmol/L CaCl2).
Platelet [Ca2+]i was calculated from the
fluorescence excitation ratio (340:380 nm) using the kd value for
Fura-PE3 at the corresponding temperature. Observations at 25°C and
30°C (not shown) were intermediate between those at 20°C and
37°C. The results shown are from a typical one of six such
studies.
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The platelet [Ca2+]i signal induced by
arachidonic acid showed a markedly different pattern of temperature
dependence than these direct platelet agonists. A robust
[Ca2+]i increase was observed at 37°C in
the presence of extracellular calcium (Fig
6C), a very modest increase generally occurred at 30°C (Fig 6B),
and no [Ca2+]i signal was seen at 20°C
and 25°C (Fig 6A). The same pattern was observed in the absence of
extracellular calcium (data not shown). This pattern of temperature
dependence was closely analogous to that observed with vWF (Table
1).
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| Fig 6.
Temperature dependence of the
[Ca2+]i signal induced by exogenous
arachidonic acid. The [Ca2+]i transient in
a stirred suspension of human platelets (50,000 cells/µL) was
monitored with Fura-PE3. The platelets were stimulated at 20°C (A),
30°C (B), or 37°C (C) by 20 µmol/L arachidonic acid (solid
traces) in the presence of extracellular calcium (1 mmol/L
CaCl2). Data obtained in parallel control studies with
0.2% (wt/vol) dimethylsulfoxide (vehicle for arachidonic acid; dotted
traces) are also displayed. Observations at 25°C (not shown) were
indistinguishable from those at 20°C. The results illustrated are
from a typical one of seven such studies.
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Temperature dependence of arachidonic acid metabolism by the
cyclo-oxygenase pathway.
The strong temperature dependence of the platelet
[Ca2+]i signal induced by arachidonic acid,
but lack of such dependence when U-46619 was the agonist, suggested
that metabolism of arachidonic acid by the cyclo-oxygenase pathway
might be severely curtailed at the lower temperatures. This possibility
was assessed directly by assay of thromboxane A2 production
in platelets incubated with exogenous arachidonic acid at different
temperatures. Metabolism of arachidonic acid to thromboxane
A2 was substantially abrogated when the incubation
temperature was reduced from 37°C to 30°C (Fig 7A; P < .01, Friedman's
test with a nonparametric Student-Newman-Keuls posthoc test) and was
virtually abolished at the lower temperatures (20°C and 25°C).
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| Fig 7.
Temperature dependence of thromboxane A2
production induced by exogenous arachidonic acid and vWF. A stirred
suspension of human platelets (500 µL at 50,000 cells/µL) was
stimulated with either 20 µmol/L arachidonic acid (A) or a
combination of 1 mg/mL ristocetin and 5 µg/mL multimeric human vWF
(B) at 20°C, 25°C, 30°C, or 37°C (as indicated).
Platelet thromboxane A2 production was assessed by enzyme
immunoassay of its stable breakdown product, thromboxane
B2. Control samples were incubated with dimethylsulfoxide
(vehicle for arachidonic acid) or 1 mg/mL ristocetin alone; thromboxane
A2 production in the control (typically, 0.4 ng in both
cases) was subtracted from that in the corresponding experimental
sample. Data are presented as a Tukey box plot: the central line in the
box shows the median value for the agonist-induced increment in
thromboxane A2 production from seven studies, the lower and
upper limits of the box designate the quartiles, and the error bars
extending below and above the box represent the 10th and 90th
percentiles, respectively. Statistical analysis was based on
Friedman's test with a nonparametric Student-Newman-Keuls posthoc
test; significant differences (P < .01) from measurements at
the higher temperatures are designated:  compared with 37°C,
and  compared with 30°C.
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vWF induces thromboxane A2 production in the platelet.
The similar patterns of temperature dependence for platelet
[Ca2+]i signaling with vWF and arachidonic
acid suggested that these signals might arise through similar
mechanisms. Assay of thromboxane A2 production in response
to the interaction of vWF with the platelet provided an independent
test of this hypothesis. Ristocetin-mediated binding of vWF promoted a
substantial increase in platelet thromboxane A2 production
at 37°C (Fig 7B). vWF-induced thromboxane A2 production was substantially diminished at 30°C (P < .01) and
abolished at 20°C and 25°C (Fig 7B). This pattern of
temperature dependence closely matches the patterns seen for
thromboxane A2 production with arachidonic acid and for
platelet [Ca2+]i signaling with both vWF and
arachidonic acid.
Effect of aspirin on the vWF-induced platelet
[Ca2+]i signal.
If thromboxane A2 plays an essential role in the mechanism
by which vWF elevates platelet [Ca2+]i,
treatment with a cyclo-oxygenase inhibitor should abolish the
[Ca2+]i signal. Studies were conducted in
aspirin-treated platelets to evaluate this possibility. Pretreatment
with 0.2 mmol/L aspirin consistently abrogated the platelet
[Ca2+]i increase induced by
ristocetin-mediated binding of vWF at 37°C (Fig 8A and B; P < .005, Wilcoxon
signed-rank test). Such treatment also abolished vWF-induced
thromboxane A2 production (Fig 8C; P < .005).
Platelet [Ca2+]i signaling and thromboxane
A2 production in response to 20 µmol/L arachidonic acid
were also eliminated (data not shown), confirming the effectiveness of
cyclo-oxygenase inhibition.
View larger version (16K):
[in this window]
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| Fig 8.
Effect of cyclo-oxygenase inhibition on the vWF-induced
[Ca2+]i signal and thromboxane
A2 production. Human platelets (50,000 cells/µL, loaded
with Fura-PE3) were preincubated (5 minutes at 37°C) with 0.1%
dimethylsulfoxide (vehicle) or 0.2 mmol/L aspirin. The platelets were
then stimulated at 37°C by 1 mg/mL ristocetin and 5 µg/mL
multimeric human vWF in the presence of 1 mmol/L CaCl2.
Parallel control studies were undertaken with 1 mg/mL ristocetin alone.
(A) The results from a typical one of nine such studies are shown. The
solid trace denotes the vWF-induced [Ca2+]i
signal in vehicle-treated platelets, and the dotted trace that after
aspirin treatment. (B) The amplitude of the vWF-induced
[Ca2+]i increment, relative to ristocetin
alone, in the whole set of studies is presented as a Tukey box plot.
(C) Thromboxane A2 production was assessed in parallel. The
vWF-induced increment in thromboxane A2 production,
relative to ristocetin alone (typically, 0.3 ng), is displayed as an
analogous Tukey box plot. The hatched bars in panels B and C summarize
data for platelets preincubated with vehicle and the open bars data for
aspirin-treated platelets.
|
|
| |
DISCUSSION |
Temperature has only a modest influence on the amplitude and kinetics
of [Ca2+]i signals in response to most
agonists in many types of cell.20,28,29 The limited nature
of this influence has served as a rationale for performing some
[Ca2+]i signaling studies at ambient
temperature when the demands of a particular experimental approach make
it technically difficult to perform the study at a physiological
temperature. For this reason, confocal microscopic evaluation of
[Ca2+]i signals in individual cells is often
performed at ambient temperature.30-32 This rationale also
explains the choice of an ambient temperature for studies of platelet
[Ca2+]i signaling under shear
stress.10,12,13
The limited influence of temperature on
[Ca2+]i signals induced by the classical
platelet agonists U-46619, -thrombin, and ADP in the present study
(Fig 5 and Table 1) is consistent with the patterns reported previously
for -thrombin and ADP in the platelet33,34 and for many
agonists in various cells.20,28,29 The platelet
[Ca2+]i transients with these classical
agonists tended to be somewhat prolonged at the lower temperatures.
This pattern parallels previous reports of a slower removal of excess
calcium from the cytosol at lower temperatures.35,36
U-46619 triggers a platelet [Ca2+]i increase
primarily through stimulation of phospholipase C and inositol
1,4,5-trisphosphate-mediated calcium release from the intracellular
stores,37 whereas influx of calcium through
receptor-operated channels in the plasma membrane is thought to play
the main role in ADP-induced [Ca2+]i
signaling.38,39 The amplitude of an agonist-induced
[Ca2+]i transient reflects a balance between
calcium entry into the cytosol and its extrusion. That the
[Ca2+]i signal with U-46619 is temperature
invariant might be related to the enzyme-mediated nature of both
calcium release from the intracellular stores and its extrusion from
the cytosol. A lesser effect of temperature on calcium influx with ADP
(a diffusion-limited process) than on extrusion might also explain the
modest increase in amplitude of its [Ca2+]i
signal at lower temperatures. The pattern of inverse temperature dependence with -thrombin, however, cannot be rationalized so readily.
In striking contrast to our findings with U-46619, ADP, and
-thrombin, the platelet [Ca2+]i signal
induced by ristocetin-mediated binding of vWF was inhibited substantially by a reduction in temperature to 30°C and abrogated completely by a further reduction to 20°C or 25°C (Fig 2 and
Table 1). This marked difference in temperature dependence strongly suggests a substantive difference in the signaling mechanism for vWF
compared with the other agonists. The protracted time course of the
[Ca2+]i signal with vWF (at 37°C) also
supports such a difference. In particular, the pronounced lag phase
between addition of vWF and the platelet
[Ca2+]i increase argues in favor of an
indirect signaling mechanism for vWF. Furthermore, the similarity in
temperature dependence of the platelet
[Ca2+]i signals with vWF (Fig 2) and
arachidonic acid (Fig 6) suggests that release of arachidonic acid may
be a proximal event in [Ca2+]i signaling with
vWF. The finding that vWF promotes thromboxane A2
production in the platelet (Fig 7B) lends further support to this
possibility. Moreover, the abolition of the
[Ca2+]i signal by the cyclo-oxygenase
inhibitor aspirin (Fig 8A and B) provides direct evidence that
thromboxane A2 plays an essential role in the signaling
mechanism for vWF. Metabolism of arachidonic acid to thromboxane
A2 by the cyclo-oxygenase pathway may exhibit such a strong
temperature dependence (Fig 7A) because it involves three catalytic
steps, cyclization and peroxidation by cyclo-oxygenase (prostaglandin H
synthase) and isomerization by thromboxane A synthase. The temperature
dependence of these steps, in combination, can explain the marked
temperature dependence for platelet [Ca2+]i
signaling with vWF.
Prior studies of vWF-induced [Ca2+]i
signaling in the platelet have yielded inconsistent findings. One of
the earliest studies by Kroll et al,11 using ristocetin to
trigger vWF binding, led its authors to postulate that activation of
the phospholipase A2 pathway and subsequent production of
thromboxane A2 play a major role in vWF-induced
[Ca2+]i signaling. There have been no further
detailed studies of the signaling mechanism using this experimental
approach. However, one subsequent study, using a mutant type 2B vWF
that binds spontaneously to platelet GP Ib-IX-V, yielded data that
broadly support this mechanism.19 In contrast, other
investigators have suggested that the proximal event in vWF signaling
leading to a [Ca2+]i increase is an influx of
calcium through channels in the plasma membrane1,10,12,13,16,40; these investigators contend that
thromboxane A2 does not contribute significantly to the
process. Support for the latter mechanism is derived largely from
dynamic studies where shear stress acted as the trigger for vWF binding to platelet GP Ib-IX-V.10,12,13 One study with a static
system, measuring the response to spontaneous binding of porcine vWF, has also lent support to this mechanism.16 The putative
ability of vWF to signal by two distinct mechanisms when its
interaction with platelet GP Ib-IX-V is instigated by different means
has not yet been explained. It does, nonetheless, present an attractive therapeutic target for prevention of pathological thrombosis without impairment of normal hemostasis.
It is not uncommon for a signal transduction pathway to bifurcate
downstream of a receptor.41,42 Such bifurcation could enable platelet GP Ib-IX-V, when occupied by vWF, to signal through both phospholipase A2 and a plasma membrane calcium
channel. This would provide a partial explanation for the
putative ability of vWF to stimulate a different primary
signaling pathway when its interaction with GP Ib-IX-V is initiated by
different means. This hypothesis, however, does not explain why one
signaling pathway should predominate (in general) under static
conditions, but a different pathway under shear stress.
There is not yet sufficient evidence to conclude that the signaling
mechanism for vWF really differs in this manner. Instead, a more
mundane explanation for the divergent findings among prior studies
should, perhaps, be sought. Could the discrepancies simply reflect
technical differences between the various experimental approaches? The
present study does provide a simple technical explanation for the
apparent lack of involvement of the phospholipase A2
pathway in signaling for vWF under shear stress. Prior investigations into platelet [Ca2+]i signaling under shear
stress appear to have been conducted at room
temperature.10,12,13 Our study shows that arachidonic acid
effectively cannot be metabolized to thromboxane A2 (a
necessary step in the phospholipase A2 pathway) in the
platelet at this temperature (Figs 6 and 7). Although the isolated
cyclo-oxygenase enzyme has demonstrable activity at
25°C,43 this need not pertain in the cellular milieu
where other competing reactions and regulatory mechanisms
exist.44 Clearly, further studies of vWF-induced [Ca2+]i signaling under shear stress must be
undertaken at 37°C to establish unequivocally whether the
phospholipase A2 pathway plays a significant role under
these circumstances. It is worth noting, in this context, that the
classical studies of vWF-mediated platelet adhesion and aggregation on
exposed vascular subendothelium in a Baumgartner chamber showed a
strong temperature dependence for platelet activation under shear
stress.45 Our earlier studies, moreover, have raised doubts
about the validity of most [Ca2+]i
measurements in aggregating platelets.17,46,47 As these doubts apply particularly to studies where a
[Ca2+]i signal is observed only in the
presence of extracellular calcium (Kermode et al, manuscript
submitted), the apparent ability of vWF to induce calcium
influx into the platelet (under any circumstances) should be
questioned. It is possible that the prior studies of vWF-induced
[Ca2+]i signaling under shear stress failed
to uncover the real signaling pathway because they were performed at
ambient temperature, but, instead, reported an artifactual
[Ca2+]i increase due to platelet aggregation.
In conclusion, platelet [Ca2+]i signaling
induced by ristocetin-mediated interaction of vWF with GP Ib-IX-V is
markedly influenced by temperature. A similar pattern of temperature
dependence is seen for [Ca2+]i signaling with
arachidonic acid, but not with other platelet agonists. Treatment of
platelets with the cyclo-oxygenase inhibitor aspirin abolishes the
vWF-induced [Ca2+]i signal. These findings
are consistent with the concept that stimulation of phospholipase
A2 is a necessary and proximal event in platelet
[Ca2+]i signaling with vWF.
| |
ACKNOWLEDGMENT |
We are greatly indebted to Joe Ed Smith for his expertise in
constructing the cuvette holder and novel stirrer (Fig 1). The generous
gift of the 10E5 antibody by Dr Barry Coller is most appreciated.
Thanks are due to Dr Rodney Baker for his critical review of the
manuscript. We are also grateful to the volunteer donors who provided
blood samples for these studies.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted March 4, 1999.
Supported by Grant-in-Aid MS-G-970048 from the American Heart
Association, Mississippi Affiliate, Inc, by National Heart Foundation Starter Grants No. NHF-97302 and NHF-98301 from the American Health Assistance Foundation, and by a Biomedical Research Support Grant from
the University of Mississippi Medical Center (all to J.C.K.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John C. Kermode, PhD, Department of
Pharmacology and Toxicology, University of Mississippi Medical Center,
2500 N State St, Jackson, MS 39216-4505.
| |
REFERENCES |
1.
Kroll MH, Hellums JD, McIntire LV, Schafer AI, Moake JL:
Platelets and shear stress.
Blood
88:1525, 1996[Free Full Text]
2.
Andrews RK, López JA, Berndt MC:
Molecular mechanisms of platelet adhesion and activation.
Int J Biochem Cell Biol
29:91, 1997[Medline]
[Order article via Infotrieve]
3.
Savage B, Shattil SJ, Ruggeri ZM:
Modulation of platelet function through adhesion receptors: A dual role for glycoprotein IIb-IIIa (integrin IIb
3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.
J Biol Chem
267:11300, 1992[Abstract/Free Full Text]
4.
Howard MA, Firkin BG:
Ristocetin: A new tool in the investigation of platelet aggregation.
Thromb Diath Haemorrh
26:362, 1971[Medline]
[Order article via Infotrieve]
5.
Dardik R, Ruggeri ZM, Savion N, Gitel S, Martinowitz U, Chu V, Varon D:
Platelet aggregation on extracellular matrix: Effect of a recombinant GPIb-binding fragment of von Willebrand factor.
Thromb Haemost
70:522, 1993[Medline]
[Order article via Infotrieve]
6.
Konstantopoulos K, Chow TW, Turner NA, Hellums JD, Moake JL:
Shear stress-induced binding of von Willebrand factor to platelets.
Biorheology
34:57, 1997[Medline]
[Order article via Infotrieve]
7.
De Marco L, Girolami A, Russell S, Ruggeri ZM:
Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation.
J Clin Invest
75:1198, 1985
8.
Francesconi MA, Casonato A, Pagan S, Donella-Deana A, Pontara E, Girolami A, Deana R:
Inhibitory effect of prostacyclin and nitroprusside on type IIB von Willebrand factor-promoted platelet activation.
Thromb Haemost
76:469, 1996[Medline]
[Order article via Infotrieve]
9.
Minamoto Y, Hato T, Nakatani S, Fujita S:
Detection of platelet adhesion/aggregation to immobilized ligands on microbeads by an aggregometer.
Thromb Haemost
76:1072, 1996[Medline]
[Order article via Infotrieve]
10.
Kawakami K, Fukuyama M, Sakai K, Itagaki I, Kawano K, Handa M, Ikeda Y:
Change in intracellular calcium ions during shear induced platelet aggregation.
ASAIO Trans
36:M696, 1990[Medline]
[Order article via Infotrieve]
11.
Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI:
von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.
J Clin Invest
88:1568, 1991
12.
Chow TW, Hellums JD, Moake JL, Kroll MH:
Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.
Blood
80:113, 1992[Abstract/Free Full Text]
13.
Ikeda Y, Handa M, Kamata T, Kawano K, Kawai Y, Watanabe K, Kawakami K, Sakai K, Fukuyama M, Itagaki I, Yoshioka A, Ruggeri ZM:
Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation.
Thromb Haemost
69:496, 1993[Medline]
[Order article via Infotrieve]
14.
Francesconi MA, Deana R, Girolami A, Pontara E, Casonato A:
Platelet aggregation induced by plasma from type IIB von Willebrand's disease patients is associated with an increase in cytosolic Ca2+ concentration.
Thromb Haemost
70:697, 1993[Medline]
[Order article via Infotrieve]
15.
Ozaki Y, Satoh K, Yatomi Y, Miura S, Fujimura Y, Kume S:
Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor.
Biochim Biophys Acta
1243:482, 1995[Medline]
[Order article via Infotrieve]
16.
Mazzucato M, De Marco L, Pradella P, Masotti A, Pareti FI:
Porcine von Willebrand factor binding to human platelet GPIb induces transmembrane calcium influx.
Thromb Haemost
75:655, 1996[Medline]
[Order article via Infotrieve]
17.
Milner EP, Zheng Q, Kermode JC:
Ristocetin-mediated interaction of human von Willebrand factor with platelet glycoprotein Ib evokes a transient calcium signal: Observations with Fura-PE3.
J Lab Clin Med
131:49, 1998[Medline]
[Order article via Infotrieve]
18.
Scrutton MC:
The platelet as a Ca2+-driven cell: Mechanisms which may modulate Ca2+-driven responses.
Adv Exp Med Biol
344:1, 1993[Medline]
[Order article via Infotrieve]
19.
Francesconi MA, Casonato A, Pontara E, Dalla Via L, Girolami A, Deana R:
Type IIB von Willebrand factor induces phospholipase A2 activation and cytosolic Ca2+ increase in platelets.
Biochem Biophys Res Commun
214:102, 1995[Medline]
[Order article via Infotrieve]
20.
Michel MC, van Tits LJ, Trenn G, Sykora J, Brodde OE:
Dissociation between phytohaemagglutinin-stimulated generation of inositol phosphates and Ca2+ increase in human mononuclear leucocytes.
Biochem J
285:137, 1992
21.
Mustard JF, Kinlough-Rathbone RL, Packham MA:
Isolation of human platelets from plasma by centrifugation and washing.
Methods Enzymol
169:3, 1989[Medline]
[Order article via Infotrieve]
22.
Sobel M, McNeill PM, Carlson PL, Kermode JC, Adelman B, Conroy R, Marques D:
Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.
J Clin Invest
87:1787, 1991
23.
Newman J, Johnson AJ, Karpatkin MH, Puszkin S:
Methods for the production of clinically effective intermediate- and high-purity factor-VIII concentrates.
Br J Haematol
21:1, 1971[Medline]
[Order article via Infotrieve]
24.
Merritt JE, McCarthy SA, Davies MPA, Moores KE:
Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils: Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.
Biochem J
269:513, 1990[Medline]
[Order article via Infotrieve]
25.
Grynkiewicz G, Poenie M, Tsien RY:
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:3440, 1985[Abstract/Free Full Text]
26.
Brinkhous KM, Read MS:
Fixation platelets and platelet agglutination/aggregation tests.
Methods Enzymol
169:149, 1989[Medline]
[Order article via Infotrieve]
27.
Coller BS, Peerschke EI, Scudder LE, Sullivan CA:
A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.
J Clin Invest
72:325, 1983
28.
Oosawa Y, Imada C, Furuya K:
Temperature dependency of calcium responses in mammary tumor cells.
Cell Biochem Funct
15:113, 1997[Medline]
[Order article via Infotrieve]
29.
Paltauf-Doburzynska J, Graier WF:
Temperature dependence of agonist-stimulated Ca2+ signaling in cultured endothelial cells.
Cell Calcium
21:43, 1997[Medline]
[Order article via Infotrieve]
30.
Blatter LA, Niggli E:
Confocal near-membrane detection of calcium in cardiac myocytes.
Cell Calcium
23:269, 1998[Medline]
[Order article via Infotrieve]
31.
Bootman MD, Berridge MJ, Lipp P:
Cooking with calcium: The recipes for composing global signals from elementary events.
Cell
91:367, 1997[Medline]
[Order article via Infotrieve]
32.
Faury G, Usson Y, Robert-Nicoud M, Robert L, Verdetti J:
Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells.
Proc Natl Acad Sci USA
95:2967, 1998[Abstract/Free Full Text]
33.
Sage SO, Reast R, Rink TJ:
ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets: Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.
Biochem J
265:675, 1990[Medline]
[Order article via Infotrieve]
34.
Alonso MT, Alvarez J, Montero M, Sanchez A, García-Sancho J:
Agonist-induced Ca2+ influx into human platelets is secondary to the emptying of intracellular Ca2+ stores.
Biochem J
280:783, 1991
35.
Shuttleworth TJ, Thompson JL:
Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes.
J Biol Chem
266:1410, 1991[Abstract/Free Full Text]
36.
Balasubramanyam M, Kimura M, Aviv A, Gardner JP:
Kinetics of calcium transport across the lymphocyte plasma membrane.
Am J Physiol
265:C321, 1993[Abstract/Free Full Text]
37.
Siess W, Boehlig B, Weber PC, Lapetina EG:
Prostaglandin endoperoxide analogues stimulate phospholipase C and protein phosphorylation during platelet shape change.
Blood
65:1141, 1985[Abstract/Free Full Text]
38.
Sage SO, Sargeant P, Heemskerk JWM, Mahaut-Smith MP:
Calcium influx mechanisms and signal organization in human platelets.
Adv Exp Med Biol
344:69, 1993[Medline]
[Order article via Infotrieve]
39.
Gachet C, Cazenave J-P:
Platelet ADP/purinergic receptors.
Handbook Exp Pharmacol
126:117, 1997
40.
Ruggeri ZM:
Mechanisms of shear-induced platelet adhesion and aggregation.
Thromb Haemost
70:119, 1993[Medline]
[Order article via Infotrieve]
41.
Neer EJ:
Heterotrimeric G proteins: Organizers of transmembrane signals.
Cell
80:249, 1995[Medline]
[Order article via Infotrieve]
42.
Clark EA, Brugge JS:
Integrins and signal transduction pathways: The road taken.
Science
268:233, 1995[Abstract/Free Full Text]
43.
van der Ouderaa FJG, Buytenhek M:
Purification of PGH synthase from sheep vesicular glands.
Methods Enzymol
86:60, 1982[Medline]
[Order article via Infotrieve]
44.
Kulmacz RJ:
Cellular regulation of prostaglandin H synthase catalysis.
FEBS Lett
430:154, 1998[Medline]
[Order article via Infotrieve]
45.
Turitto VT, Baumgartner HR:
Effect of temperature on platelet interaction with subendothelium exposed to flowing blood.
Haemostasis
3:224, 1974[Medline]
[Order article via Infotrieve]
46.
Kermode JC, Zheng Q, Cook EP:
Fluorescent indicators give biased estimates of intracellular free calcium change in aggregating platelets: Implication for studies with human von Willebrand factor.
Blood Cells Mol Dis
22:238, 1996[Medline]
[Order article via Infotrieve]
47.
Kermode JC, Zheng Q, Milner EP:
Ratiometric measurements with fluorescent indicators should be interpreted cautiously in studies of platelet calcium signaling for von Willebrand factor.
Thromb Haemost
78:1417, 1997[Medline]
[Order article via Infotrieve] (letter)
TOP
ABSTRACT
INTRODUCTION![[Ca<SUP>2+</SUP>]<SUB>i</SUB> = kd(F<SUB>f</SUB>/F<SUB>b</SUB>)(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R),](/content/vol94/issue1/fulltext/199/img001.gif)
