(Hypertension. 1998;31:603-607.)
© 1998 American Heart Association, Inc.
Mechanism of Epinephrine-Induced Platelet Aggregation
Aaron Spalding;
Henrikas Vaitkevicius;
Scott Dill;
Steven MacKenzie;
Alvin Schmaier;
; Warren Lockette
From Wayne State University School of Medicine, Detroit, Veterans
Administration Medical Center, Detroit, and the University of Michigan Medical
School, Ann Arbor, Mich.
Correspondence to Warren Lockette, MD, Division of Endocrinology, Department of Medicine, Wayne State University School of Medicine, 4H University Health Center, 4201 St Antoine, Detroit, MI 48201. E-mail ricidulo{at}umich.edu
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Abstract
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Abstract—We report that a genetic
polymorphism of the 
2-adrenergic receptor (A2AR)
encoded by chromosome 10 is associated with hypertension and an
increase in epinephrine-mediated platelet aggregation in
humans. The mechanism responsible for this heritable contrast in
sensitivity to epinephrine is unknown. We tested our hypothesis
that epinephrine-induced platelet aggregation is mediated
by activation of chloride transport. We measured
epinephrine-mediated increases in optical density of
gel-filtered platelets suspended in a bicarbonate-buffered
physiological salt solution. Compared with
platelets incubated in the control buffer (130 mmol/L NaCl),
platelets incubated with either bumetanide, a Na/K/2Cl cotransport
inhibitor; anthracene-9-carboxylic acid, a chloride channel
blocker; or acetazolamide, an agent that blocks
ATP-dependent chloride transport had significantly decreased
aggregation responses to epinephrine. When measured
fluorometrically, epinephrine significantly increased
intraplatelet chloride concentrations. Chloride-dependent
modifications of epinephrine-induced platelet aggregation
were not attributable to changes in A2AR ligand binding characteristics
or to the concentration of platelet cAMP. Finally, subthreshold
concentrations of epinephrine also potentiated thrombin-induced
platelet aggregation, and blockade of chloride transport diminished
this synergistic action of epinephrine on thrombin-stimulated
platelet aggregation. Heritable differences in
epinephrine-mediated platelet aggregation may be
attributable to genetic differences in chloride transport in
platelets. Furthermore, because we observed a necessary role for
chloride transport in epinephrine-mediated platelet
aggregation, pharmacological agents that block chloride transport, such
as diuretics, may provide salutary protection against vascular
thrombosis in patients with hypertension independent of the effect of
these drugs on blood pressure.
Key Words: adrenergic receptor • diuretics • thrombosis • chloride • humans • polymorphism • blacks • genetics
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Introduction
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The mechanism by
which epinephrine induces platelet aggregation is unknown.
In some tissues, such as the respiratory epithelium, activation of the
A2AR increases transcellular sodium and chloride
cotransport.1 We reasoned that
epinephrine-induced platelet aggregation could also be
mediated by A2AR-dependent sodium and chloride cotransport.
Accordingly, we tested our hypothesis by measuring
epinephrine-mediated platelet aggregation under
experimental conditions that would inhibit A2AR-mediated sodium
chloride transport, and we measured epinephrine-mediated
changes in intracellular chloride concentrations fluorometrically in
platelets. Because platelet aggregation may also be dependent
on A2AR-mediated changes in the accumulation of intracellular cAMP, we
determined the effect of chloride transport inhibition on postreceptor
signal transduction by cAMP.
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Methods
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Subject Recruitment
We recruited healthy, fasting, male and female college-age
subjects who were taking no prescription medications or
over-the-counter drugs. These protocols were approved by our
institutional committee for the protection of humans in research, and
each volunteer gave informed consent.
Platelet Aggregation Measurements
All reagents were obtained from Sigma Chemical Co unless
otherwise indicated. Platelet aggregation was measured by standard
aggregometry techniques based on optical density. Briefly, 45 mL of
whole blood was collected from resting subjects in a citrate
anticoagulant (final ratio, 9:1 whole blood/citrate). PRP was obtained
after centrifugation of the whole blood at
200g for 15 minutes. PPP served as the appropriate blank,
and it was obtained by centrifugation of an aliquot of
the PRP at 17 000g for 3 minutes. The remainder of the PRP
was washed over a Sepharose 2B gel column with a PSS of the following
composition (in mmol/L): NaCl 130, KCl 4.7,
NaHCO3 14.9,
KH2PO4 1.18,
MgSO4 · 7H2O 1.17,
and CaCl2 1.6, pH=7.4. In some experiments, the
chloride concentration of the PSS was decreased by substitution of
chloride with an iso-osmotic concentration of sodium gluconate. To
study the role of chloride/bicarbonate countertransport, experiments
were also performed in which we replaced HEPES buffer (10 mmol/L,
pH 7.4) with sodium bicarbonate PSS. Chloride transport was also
blocked in some experiments by preincubation of the platelets for
at least 10 minutes in PSS to which either 0.5 mmol/L
acetazolamide, 5 µmol/L anthracene-9-carboxylic
acid, or 50 µmol/L bumetanide had been added. In every case,
each subject served as his or her own control. Platelet aggregation
induced by 3 µmol/L and 10 µmol/L epinephrine was
measured in 390 µL of PRP after 5 minutes in an aggregometer
(Chronolog Co) at 37°C. These concentrations of epinephrine
represented ED50 and
ED100 doses, respectively, for platelet
aggregation under these experimental conditions.2
Platelet aggregation was defined as the difference in light
transmission measured in PPP and PRP. Finally, because
epinephrine could potentiate aggregation induced by other
agonists, changes in optical density in response to gamma thrombin were
also measured in PRP that had been incubated with subthreshold
concentrations of epinephrine (0.3 µmol/L).
Determinations of Platelet [Cl]i
Venous blood from healthy volunteers was collected as described
above except that the EDTA was used as the anticoagulant. PRP was then
incubated with 2.5 mmol/L
N-ethoxycarbonylmethyl-6-methoxy-quinolium bromide at 37°C
for 2 hours. The PRP was centrifuged at 1000g, and
the resulting pellet was washed with 5 mL of calcium-free PSS and
resuspended in 6 mL of bicarbonate PSS. Fluorometric data were obtained
with a spectrofluorometer (Spex Fluorolog, Spex Industries) at 37°C.
An excitation wavelength of 359 nm and an emission wavelength of 464 nm
were used with monochromator slits set at 3 nm. The platelets were
diluted to 1:6 and incubated in PSS to which
CaCl2 (1.6 mmol/L), epinephrine
(10 µmol/L), and/or acetazolamide (0.5 mmol/L)
was added. The ratio of fluorescence at time 0 and over 5
minutes was measured and compared with the ratios obtained with a known
[Cl–]i determined from a
calibration curve with a double-ionophore
technique.3
Platelet A2AR Binding Studies
A2AR receptor binding and number were determined as described
previously4 with Scatchard analysis of
[3H]yohimbine binding to platelets
incubated in different buffers. Briefly, platelets were isolated
after centrifugation of 10 mL of PRP. Aliquots of
platelets were then washed and suspended in equal volumes of an
isotonic buffer containing 50 mmol/L Tris-HCl, 100 mmol/L
NaCl, and 5 mmol/L EDTA (pH 7.5) or in a Tris-HCl buffer in which
the NaCl was replaced with 100 mmol/L choline chloride or 100
mmol/L sodium gluconate. Platelets were then incubated with
increasing concentrations of [3H]yohimbine (1.0
to 20.0 nmol/L, Amersham) with a specific activity of 84.5 mCi/mol.
Phentolamine (2 mmol/L, Regitine, CIBA) was used to block
specific binding. The reaction was allowed to proceed at room
temperature for 45 minutes, and then the reaction was stopped by the
addition of ice-cold buffer and rapid filtration. The amounts of bound
and free ligand were calculated, and the saturation binding isotherms
were derived by Scatchard analysis as
described.5
Platelet cAMP
Whole blood (20 mL) was collected in EDTA, and PRP again was
obtained by low-speed centrifugation. Platelet
concentrations were adjusted to 105
platelets/µL with a calcium-free PSS.
Epinephrine-mediated inhibition of forskolin-stimulated
(10 µmol/L) adenylate cyclase activation was
measured in the presence of 1 mmol/L
isobutylmethylxanthine in 500-µL aliquots of
platelets incubated for 1 hour in the bicarbonate-buffered PSS,
HEPES/bicarbonate–free PSS, or bicarbonate-buffered PSS with 1
mmol/L acetazolamide at 37°C. The reaction was terminated
with the addition of ice-cold ethanol, and platelet cAMP was
extracted and measured with a commercially available radioimmunoassay
(Amersham) following the directions of the manufacturer.
Statistical Analysis
Statistical analysis was performed using Excel 5.0
(Microsoft). A Student's t test was used to compare mean
and median responses between treatment groups, and Bonferroni
adjustments were made for multiple comparisons. In cases in which
variances between comparison groups were unequal, the
nonparametric Wilcoxon rank sum test was used. All
values are expressed as mean±SEM, and P=.05 was considered
significant.
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Results
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Attenuation of chloride transport by relatively low concentrations
of various pharmacological agents significantly decreased
epinephrine-mediated platelet aggregation. As demonstrated
in Fig 1
, inhibition of chloride
transport by antagonists relatively specific for various
chloride transporters such as anthracene-9-carboxylic acid, bumetanide,
or acetazolamide, or inhibition of the chloride/bicarbonate
exchanger by substitution of a bicarbonate buffer with HEPES, all
decreased A2AR-mediated platelet aggregation. Furthermore,
epinephrine markedly increased
[Cl–]i, as shown in Fig 2; this A2AR-dependent change in
platelet [Cl–]i was
calcium-dependent. In the absence of physiological
concentrations of extracellular calcium, baseline
[Cl–]i was much higher
in the unstimulated platelets (110±6 mmol/L) than in
platelets incubated in 1.6 mmol/L calcium (53±9 mmol/L,
P<.05), and the addition of epinephrine caused no
increase in the intraplatelet chloride concentration over the
baseline value (data not shown).
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Figure 1. Effect of bicarbonate-free HEPES buffer,
anthracene 9-carboxylic acid (9AC), bumetanide, and
acetazolamide on platelet aggregation induced by 3
µmol/L epinephrine. Compared with responses in a control,
bicarbonate-buffered PSS, epinephrine-mediated platelet
aggregation was significantly reduced (values expressed as mean optical
density±SEM) in the HEPES buffer (from 15±10 to 10±6,
P=.006), PSS containing the 9AC (from 14±9 to 9±3,
P=.01), PSS with 50 µmol/L bumetanide (from 23±5
to 14±3, P=.05), or with the incubation of 0.5
mmol/L acetazolamide (from 28±6 to 15±6,
P=.0001). There were significant reductions in
A2AR-mediated platelet aggregation under these conditions when a
higher concentration (10 µmol/L) of epinephrine was used
(data not shown).
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Figure 2. Effect of epinephrine on
[Cl–]i in human platelets incubated in
bicarbonate PSS with 130 mmol/L NaCl and 1.6 mmol/L calcium.
[Cl–]i averaged 53±4 mmol/L.
Epinephrine more than doubled platelet
[Cl–]i to 113±2 mmol/L.
Acetazolamide alone decreased basal
[Cl–]i to 21±7 mmol/L. The
A2AR-mediated increase in chloride was completely inhibited by
acetazolamide.
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We next used [3H]yohimbine binding to quantify
A2AR and binding affinity in platelets. When platelets were
incubated in sodium-free PSS, A2AR binding was completely inhibited
(data not shown); however, incubation of platelets in a buffer in
which chloride was replaced iso-osmotically with gluconate modestly
increased the number of receptors available for ligand binding (Fig 3), and this manipulation tended to
increase the affinity of this receptor for its ligand. Replacement of
NaCl with sodium gluconate increased A2AR number (values expressed in
binding sites/platelet±SEM) from 66±10 to 88±12
(P<.05) without any significant change in the affinity
binding constant (values expressed in nmol/L±SEM): 1.27±0.20 versus
0.91±0.07 (P=NS). Neither bumetanide nor
acetazolamide had any effect on
[3H]yohimbine binding to platelets (data
not shown).
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Figure 3. Effect of chloride replacement on A2AR ligand
binding. Because A2AR-mediated binding of epinephrine can be
modulated by the concentration of sodium and chloride, we measured
receptor number and affinity binding constants for platelet A2ARs
in 100 mmol/L NaCl or 100 mmol/L sodium gluconate buffers.
Removal of sodium completely inhibited [3H]yohimbine
binding in platelets (data not shown). However, replacement of NaCl
with sodium gluconate increased A2AR number (values expressed in
binding sites/platelet±SEM) from 66±10 to 88±12
(P<.05) without any change in the affinity binding
constant (values expressed in nmol/L±SEM): 1.27±0.20 vs 0.91±0.07
(P=NS).
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A2AR-mediated platelet aggregation can be dependent on changes in
the intraplatelet concentration of cAMP. As demonstrated in Fig 4, inhibition of chloride/bicarbonate
exchange by incubation of the platelets in a bicarbonate-free HEPES
buffer not only significantly attenuated forskolin-stimulated increases
in cAMP, but this maneuver also decreased epinephrine-mediated
reductions in cAMP in forskolin-stimulated platelets. On the other
hand, inhibition of chloride transport by acetazolamide had
no discernible effect on epinephrine-induced changes in cAMP
concentrations in forskolin-stimulated platelets (Fig 4).
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Figure 4. Effect of HEPES buffer and
acetazolamide on epinephrine-induced decreases in
forskolin-stimulated [cAMP] in platelets. A2AR-mediated
platelet aggregation is associated with reductions in [cAMP]. In
bicarbonate PSS, 10 µmol/L forskolin increased cAMP (values
expressed in fmol cAMP/108 platelets per 30
minutes±SEM) from 502±35 to 2214±251, and this increase in cAMP was
completely inhibited by 30 µmol/L epinephrine.
Forskolin-induced augmentation of cAMP was dramatically reduced in
HEPES buffer (from 53±7 to 196±40). Although
acetazolamide caused the greatest decreases in
epinephrine-mediated platelet aggregation, this
inhibitor of ATP-dependent chloride transport had no
significant effect on platelet [cAMP]. IBMX indicates
isobutylmethylxanthine.
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Localized increases in endogenous epinephrine could
be directly responsible for the precipitation of thrombotic events in
humans. However, it is equally likely that epinephrine
potentiates the proaggregatory effect of other autocoids, such as
thrombin. Indeed, we found that subthreshold concentrations of
epinephrine dramatically augmented aggregation in washed
platelets that were stimulated with 
-thrombin; this potentiation
by epinephrine was dependent on chloride, and this synergistic
action of epinephrine was blocked by yohimbine, a specific A2AR
antagonist (Fig 5).
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Figure 5. Effect of epinephrine (Epi.) on
thrombin-mediated platelet aggregation. -Thrombin induced
aggregation in platelets incubated in bicarbonate PSS. Subthreshold
concentrations of epinephrine (0.3 µmol/L) more than
doubled thrombin-induced aggregation, as measured by changes in optical
density, from 18±3 to 37±4. This potentiating effect of thrombin was
inhibited by acetazolamide, and this effect of
epinephrine was blocked by yohimbine, an A2AR
antagonist.
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Discussion
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The gene for the A2AR encoded by chromosome 10 is polymorphic,
and Southern blotting with a cDNA probe after restriction enzyme
digestion of this gene results in fragments of either 6.7 or 6.3 kb in
size.2 6 In a population-based study of urban
Detroit blacks, we reported a strong association between homozygosity
for the 6.3-kb allele of the C10 A2AR gene and
hypertension.2 Subsequently, we noted that
normotensive individuals carrying at least one allele of the 6.3-kb
C10 A2AR had increased epinephrine-mediated platelet
aggregation compared with individuals homozygous for the 6.7-kb
allele, and transfection studies with these genotypes
demonstrated that this polymorphism was
functional.2 We theorized that the increased
frequency of this 6.3-kb allele in blacks compared with whites can
place blacks at risk for stroke or coronary thrombosis, and we
postulated that increased platelet aggregation may be the result of
genetic variation and heritability in epinephrine-mediated
platelet aggregation that occurs independently of pathological
elevations in blood pressure in this ethnic
group.2 Accordingly, we deemed it necessary to
determine the mechanism by which epinephrine mediates
platelet aggregation.
The mechanism by which A2AR ligands induce platelet aggregation was
unknown. We found that manipulations designed to inhibit chloride
transport in platelets were associated with attenuation of
epinephrine-mediated platelet aggregation. Specifically,
inhibition of Na/K/2Cl cotransport with bumetanide, blockade of anion
channels with anthracene-9-carboxylic acid, or inhibition of
Cl–/HCO3 exchange by
substituting bicarbonate buffer with HEPES all diminished A2AR-mediated
platelet aggregation. Acetazolamide most clearly
inhibited epinephrine-mediated platelet aggregation.
Acetazolamide is believed to block ATP-dependent chloride
transport.7 8 Alternatively, when carbonic
anhydrase is inhibited by acetazolamide, there is a
decrease in the intraplatelet bicarbonate concentration. This
decrease in
[HCO3–]i
can result in a loss of bicarbonate/chloride exchange and a subsequent
reduction in [Cl–]i.
Furthermore, inhibition of carbonic anhydrase with
acetazolamide results in the equimolar loss of a strong
acid (H+) and comparatively weak base
(HCO3), and a net intracellular alkalinization
ensues. It remains to be seen whether the
acetazolamide-induced increase in intraplatelet pH
diminishes platelet responsiveness to epinephrine and
thrombin. Indeed, when measured fluorometrically,
acetazolamide increased intraplatelet pH by 
0.2 pH
units (data not shown). It is likely that epinephrine-mediated
increases in intraplatelet chloride concentration contribute, in
part, to A2AR-dependent aggregation; our data support this
hypothesis.
The interrelationship among
[Cl–]i, chloride
movement through conductance channels, and ATP-dependent chloride
transport in post-A2AR receptor signal transduction in platelets is
uncertain. Although it has been reported that a reduction of
extracellular sodium decreases A2AR binding,9 we
also found that a reduction in intracellular chloride concentrations
did not decrease the availability of the A2AR for binding or change the
affinity binding constants of the A2AR. Stimulation of the A2AR results
in the activation of the heterotrimeric GTP-binding proteins, and a
pertussis toxin–sensitive Gi subunit decreases
adenylate cyclase activity and platelet [cAMP].
Chloride transport has been shown to be a necessary mediator of
platelet volume, and it is possible that
epinephrine-induced platelet aggregation results from a
chloride-dependent increase in platelet volume and surface area
that can increase aggregatability. It has been shown that
epinephrine can increase platelet
volume,11 and platelets isolated from
individuals with a history of stroke are indeed larger than those
platelets found in patients with no history of
stroke.12
In addition, the interrelationship of intraplatelet chloride and
calcium remains to be determined. We found that
epinephrine-dependent increases in
[Cl–]i relied on the
presence of calcium in the extracellular buffers.
Epinephrine-mediated platelet aggregation is also dependent
on calcium. Similar to our finding in platelets, it has been
demonstrated that chloride is necessary for agonist-induced increases
in the ionized intracellular calcium concentration of renal
mesangial cells.13 It is possible
that chloride fluxes in platelets contribute to the control of the
intracellular calcium milieu in thrombocytes, and changes in
intraplatelet calcium may ultimately control A2AR-dependent
platelet aggregation. Alternatively, it could also be argued that
the dependence of epinephrine-mediated increases in chloride on
extracellular calcium is attributable to an effect of
epinephrine on calcium-dependent chloride
transport.14 The exact nature of the interaction
between intracellular calcium and chloride in modulating platelet
responsiveness remains unclear. The argument for a primary role for
chloride in platelet aggregation, however, is supported by the
observation that U46619, a synthetic thromboxane agonist,
also induces platelet aggregation through a chloride-dependent
mechanism.15
It has been reported that epinephrine serves primarily to
increase platelet aggregation induced by other
autocoids.16 Our data on the effect of
epinephrine on potentiating thrombin-induced platelet
aggregation were striking. It remains to be determined whether
epinephrine acts synergistically with other autocoids in
addition to thrombin in facilitating thrombosis. Furthermore, it is
exciting to speculate that other vasoactive effects of thrombin, such
as the mitogenic property of this autocoid, could be
potentiated by the A2AR. Should this prove to be the case, it would be
of interest to determine whether increases in
[Cl–]i in
endothelial and vascular smooth muscle cells are also
dependent on the net flux of chloride into the cell. We have found that
A2AR-mediated vascular contraction is dependent on the presence of
extracellular chloride in preparations of isolated vascular smooth
muscle (unpublished observations), and others have reported that the
increased vascular reactivity found in deoxycorticosterone/salt
hypertension is attributable to abnormalities in ATP-dependent chloride
transport in blood vessels.17
Recently, debate has focused on the selection of first-line agents in
the treatment of hypertension. The goal in the treatment of these
patients should not be limited to the reduction in blood pressure but,
instead, to reduce the morbidity associated with hypertension, such as
the increased prevalence of strokes and coronary thrombosis.
Agents that specifically block chloride transport may decrease
thrombosis independent of their effect of blood pressure.
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Selected Abbreviations and Acronyms
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| A2AR |
= |
2-adrenergic receptor |
| [Cl–]i |
= |
intraplatelet chloride concentration |
| PPP |
= |
platelet-poor plasma |
| PRP |
= |
platelet-rich plasma |
| PSS |
= |
physiological salt solution |
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Acknowledgments
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These studies were supported by National Institutes of Health
grants HL-50849, HL-35553, HL-52772, and HL-56415. W.L. is an
Established Investigator of the American Heart Association. These
studies were conducted in partial fulfillment for the requirements
(A.S.) for graduation with Honors, College of Literature, Science, and
the Arts, from the University of Michigan.
Received July 29, 1997;
first decision August 12, 1997;
accepted October 3, 1997.
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References
|
|---|
1.
Liedtke CM, Furin J, Ernsberger P. Alpha-2
adrenergic, but not imidazole, agonists activate NaCl
cotransport in rabbit tracheal epithelial cells. Am J
Physiol. 1993;263:C568–C576.
2.
Freeman K, Farrow S, Schmaier A, Freedman R, Schork
NA, Lockette W. Genetic polymorphism of the A2AR is associated with
increased platelet aggregation, baroreceptor sensitivity, and salt
excretion in normotensive humans. Am J Hypertens. 1995;8:863–869.[Medline]
[Order article via Infotrieve]
3.
Koncz C, Daugirdas JT. Use of MQAE for measurement of
intracellular [Cl–] in cultured aortic smooth
muscle cells. Am J Physiol. 1994;267:2114–2123.
4.
Farrow S, Mers A, Banta G, Steigerwalt S, Lockette W.
Effect of the A2AR antagonist yohimbine on
orthostatic tolerance. Hypertension. 1990;15:877–880.[Abstract/Free Full Text]
5.
Lockette W, McCurdy R, Smith S. Endurance training and
A2AR on platelets. Med Sci Sports Exerc. 1987;19:7–10.[Medline]
[Order article via Infotrieve]
6.
Lockette W, Ghosh S, Farrow S, MacKenzie S, Baker S,
Miles P, Schork NA, Cadaret L. A2AR gene polymorphism and
hypertension in blacks. Am J Hypertens. 1995;8:390–394.[Medline]
[Order article via Infotrieve]
7.
Chipperfield AR, Davis JPL, Harper AA. An
acetazolamide-sensitive inward chloride pump in vascular
smooth muscle. Biochem Biophys Res Commun. 1993;194:407–412.[Medline]
[Order article via Infotrieve]
8.
Gerencser GA. The chloride pump: a chloride
translocating P-type ATPase. Crit Rev Biochem Mol Biol. 1996;31:303–337.[Medline]
[Order article via Infotrieve]
9.
Motulsky HJ, Insel PA. Influence of sodium on the A2AR
system of human platelets: role for intraplatelet sodium in
receptor binding. J Biol Chem. 1983;258:3913–3919.[Abstract/Free Full Text]
10.
Connoly TM, Limbird LE. Influences of sodium on the
A2AR system of human platelets: a method for removal of
extraplatelet Na+, effect of
Na+ removal on aggregation, secretion, and cAMP
accumulation. J Biol Chem. 1983;258:3907–3912.[Free Full Text]
11.
Kjeldsen SE, Weder AB, Egan B, Neubig R, Zweifler AJ,
Julius S. Effect of circulating epinephrine on platelet
function and hematocrit. Hypertension. 1955;25:1096–1105.[Abstract/Free Full Text]
12.
O'Malley T, Langhorne P, Elton RA, Stewart C.
Platelet size in stroke patients. Stroke. 1996;26:995–999.[Abstract/Free Full Text]
13.
Kremer SG, Zeng W, Hurst R, Ning T, Whiteside C,
Skorecki KL. Chloride is required for receptor-mediated divalent cation
entry in mesangial cells. J Cell Physiol. 1995;162:15–25.[Medline]
[Order article via Infotrieve]
14.
Mahaut-Smith MP. Chloride channels in human
platelets: evidence for activation in internal calcium. J
Membr Biol. 1990;118:69–75.[Medline]
[Order article via Infotrieve]
15.
Yokoyama K, Kudo I, Nakamura H, Inoue K. A possible
role for extracellular bicarbonate in U-46619-induced rat platelet
aggregation. Thromb Res. 1994;74:369–376.[Medline]
[Order article via Infotrieve]
16.
Steen VM, Holmsen H, Aarbakke G.
Platelet-stimulating effect of adrenaline through A2AR requires
simultaneous activation by a true stimulatory platelet
agonist. Thromb Haemost. 1993;70:506–513.[Medline]
[Order article via Infotrieve]
17.
Davis JPL, Chipperfield AR, Harper AA. Accumulation of
intracellular chloride by Na-K-Cl co-transport in rat
arterial smooth muscle is enhanced in deoxycorticosterone
acetate (DOCA)/salt hypertension. Mol Cell Cardiol. 1993;25:233–237.[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
