Mechanisms Involved in Adenosine Triphosphate–Induced
Platelet Aggregation in Whole Blood
Nicholas P. Stafford, Andrew E. Pink, Ann E. White, Jacqueline R. Glenn, Stan Heptinstall
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Objective—Effects on platelet aggregation of adenosine triphosphate (ATP) released from damaged cells and from platelets
undergoing exocytosis have not been clearly established. In this study we report on the effects of ATP on platelet
aggregation in whole blood.
Methods and Results—Aggregation, measured using a platelet-counting technique, occurred in response to ATP and was
maximal at 10 to 100 ␮mol/L. It was abolished by MRS2179, AR-C69931, and creatine phosphate/creatine
phosphokinase, implying that conversion to adenosine diphosphate (ADP) is required. ATP did not induce aggregation
in platelet-rich plasma, but aggregation did occur when apyrase or hexokinase was added. Aggregation also occurred
after addition of leukocytes to platelet-rich plasma (as a source of ecto-ATPase), and this was potentiated on removal
of adenosine by adenosine deaminase, indicating that adenosine production modulates the response. Dipyridamole,
which inhibits adenosine uptake into erythrocytes, inhibited aggregation induced by ATP in whole blood, and adenosine
deaminase reversed this. DN9693 and forskolin synergized with dipyridamole to inhibit ATP-induced aggregation.
Conclusions—ATP induces aggregation in whole blood via conversion of ATP to ADP by ecto-ATPases on leukocytes.
This is inhibited by agents that prevent adenosine removal. Reduced aggregation at high concentrations of ATP (⬎100
␮mol/L) may be a consequence of inhibition by ATP of ADP action at ADP receptors. (Arterioscler Thromb Vasc Biol.
2003;23:1928-1933.)
Key Words: ATP 䡲 ADP 䡲 platelet aggregation 䡲 leukocytes 䡲 ecto-ATPase
L
arge quantities of adenosine triphosphate (ATP) and
adenosine diphosphate (ADP) are present in erythrocytes, platelets, and other cells and tissues and can leave the
cells via physical damage or exocytosis.1,2 The amount of
ATP released from erythrocytes is approximately 10 times
the amount of ADP,3 and ATP and ADP in platelet secretory
granules are present in approximately equimolar concentrations.4 The released ADP can interact with P2Y1 and P2Y12
(formally known as P2T) receptors on platelets and induce
platelet aggregation,5–7 which contributes to normal hemostasis and to thrombus formation. However, the effect of the
released ATP is unclear. It is known that ATP can interact
with P2X1 receptors on platelets, causing a transient Ca2⫹
mobilization,8,9 but this does not result in platelet aggregation, and the importance of P2X1 receptors to overall platelet
function is unknown. It is also known that ATP acts as an
antagonist of the effects of ADP at P2Y1 and P2Y12 receptors10,11 and that high concentrations can inhibit ADP-induced
platelet aggregation.12
When considering the possible effects of ATP on platelets
in vitro and in vivo, the presence of enzymes present on blood
cells and endothelial cells and in plasma that metabolize
ATP must be taken into account. These include enzymes
that convert ATP to ADP and ADP to AMP (NTPDase-1,
also known as ATP diphosphohydrolase, CD 39 and EC
3.6.1.5), 13,14 ATP to AMP and ADP to AMP (5⬘monophosphate phosphoanhydrolase/phosphodiesterase,
NMPP),15 and AMP to adenosine (5⬘-nucleotidase),13 the
latter being an inhibitor of platelet aggregation.10,16 Also,
adenosine can be taken up and neutralized by erythrocytes
and other blood cells, thus limiting its potential inhibitory
action.17,18 Thus, the overall effect of ATP could depend on
several competing influences.
In this study, we have investigated the effects of ATP on
platelet aggregation in whole blood and in platelet-rich
plasma (PRP). We used hirudin as the anticoagulant to ensure
that the conditions used were as near physiological as
possible. We found that ATP induces platelet aggregation in
whole blood but not in PRP, and we have investigated the
mechanisms that are involved.
Methods
Materials
Hirudin (recombinant desulphato-hirudin, Revasc) was a gift from
Novartis. Mono-Poly resolving medium was supplied by ICN Biomedicals. AR-C69931, a purinergic P2Y12 receptor antagonist, was a
gift from Astra Charnwood. Dipyridamole, an adenosine uptake
inhibitor, was obtained from Boehringer Ingelheim. ATP, adenosine
Received May 20, 2003; revision accepted July 2, 2003.
Fom the Centre for Integrated Systems Biology and Medicine, University of Nottingham, UK.
Correspondence to Professor Stan Heptinstall, Cardiovascular Medicine, Queen’s Medical Centre, Nottingham NG7 2UH, UK. E-mail
[email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1928
DOI: 10.1161/01.ATV.0000089330.88461.D6
Stafford et al
ATP-Induced Platelet Aggregation in Whole Blood
1929
deaminase (ADA), creatine phosphate (CP), creatine phosphokinase
(CPK), hexokinase, apyrase, and MRS2179, a purinergic P2Y1
receptor antagonist were from Sigma Chemical Co. The plateletfixing solution contained 150 mmol/L NaCl, 4.6 mmol/L Na2EDTA,
4.5 mmol/L Na2HPO4, 1.6 mmol/L KH2PO4, and 0.16% wt/vol
formaldehyde, pH 7.4. PBS was from Unipath Ltd, and saline
(sodium chloride, 0.9% wt/vol) was from Baxter Healthcare Ltd.
Blood Collection
Venous blood was obtained from volunteers who denied taking any
medication in the 2 weeks before sampling. It was taken by forearm
venepuncture using a polypropylene syringe and 19G needle and
immediately dispensed into polystyrene tubes containing hirudin (fc
50 ␮g/mL) or EDTA (final concentration 4 mmol/L) as anticoagulant. The blood was then kept at 37°C for 30 minutes before
experimentation (whole blood investigations) or immediately processed to obtain PRP. The EDTA blood was used for leukocyte
preparation.
PRP Preparation
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The blood was centrifuged (180g, 10 minutes), and the resulting
supernatant (PRP) was removed. The remaining blood was centrifuged (1500g, 10 minutes) to obtain platelet-poor plasma. The
platelet count of the PRP was determined using a Sysmex KX-21
hematology analyser, and the PRP was diluted using autologous
platelet-poor plasma to a standard platelet count; the final platelet
count in each experiment was 300⫻109/L.
Leukocyte Preparation
Polymorphonuclear leukocytes (PMNLs) and mononuclear leukocytes (MNLs) were isolated from EDTA blood using Mono-Poly
resolving medium. Briefly, blood was centrifuged (180g, 10 minutes), and the resulting PRP was removed and discarded. The
remaining blood was reconstituted to the original volume with saline
and then carefully layered (3.5 mL) onto the Mono-Poly resolving
medium (3 mL) in polystyrene tubes, which were then centrifuged
(200g, 30 minutes). The resulting leukocyte layers (PMNLs and
MNLs) were removed and washed twice in PBS. Each cell population was finally resuspended in PBS at a concentration ⫻10 that was
obtained in whole blood, such that when added to PRP, the number
of leukocytes was equivalent to the white cell count in the original
whole-blood sample. (The mean leukocyte counts for MNLs and
PMNLs were 2900 and 4100/␮L, respectively.)
Platelet Aggregation
An aliquot of whole blood or PRP was dispensed into a polystyrene
LP3 tube (64⫻11 mm) containing a stirrer bar and any agent under
investigation (20 ␮L) or leukocyte preparation (50 ␮L). The final
volume of whole blood or PRP was always 500 ␮L. The tube was
placed in the stirring well of a Multi-Sample Agitator (University of
Nottingham) operating at 1000 rpm at 37°C. After 2 minutes, 20 ␮L
of a solution of ATP was added. Aliquots (15 ␮L) of the sample were
removed and mixed with fixative solution (30 ␮L) at various time
points after the addition of ATP. The platelet count of the fixed
samples was determined using an Ultra-Flo 100 whole-blood platelet
counter (whole blood) or a Sysmex KX-21 hematology analyser
(PRP).19,20 Platelet aggregation was calculated as the percentage loss
of single platelets compared with the platelet count of a fixed sample
of unstimulated whole blood or appropriately diluted PRP. Data were
analyzed using ANOVA (repeated measures) on SPSS 11.0 software.
Significance was assigned to P⬍0.05. Platelet aggregation was
confirmed by microscopy and flow cytometry.
Figure 1. Extent of platelet aggregation induced by addition of a
range of concentrations of ATP to whole blood (WB) and PRP.
Aggregation was measured 4 minutes after the addition of ATP
to the blood. Mean⫾SEM, n⫽11 (WB) and n⫽6 (PRP).
the ATP concentration beyond 100 ␮mol/L resulted in a
reduced aggregation response. In contrast, ATP was unable to
induce an aggregation response in PRP. The time course of
aggregation induced in whole blood by selected concentrations of ATP is shown in Figure 2.
To investigate the possibility that the aggregatory effect of
ATP in whole blood is mediated by conversion of ATP to
ADP, we determined the effects of the ADP antagonists
MRS2179 (a P2Y1 antagonist, 100 ␮mol/L)21,22 and ARC69931 (a P2Y12 antagonist, 1 ␮mol/L)23–25 (Figure 3). We
also studied the effect of removing ADP from the system
using CP/CPK (300 ␮mol/L CP and 22U/mL CPK).26 The
extensive aggregation response induced in whole blood by
ATP was virtually abolished in the presence of either ADP
antagonist or CP/CPK.
From the experiments described above, it would seem that
the aggregatory effects of ATP in whole blood are mediated
by conversion of ATP to ADP and that this does not occur in
PRP. Consequently, we carried out experiments in PRP in
which we sought to bring about the conversion of ATP to
ADP. We investigated the effects of ATP in PRP to which
either apyrase (an enzyme with adenosine 5⬘ triphosphatase
activity and adenosine 5⬘ diphosphatase activity) or hexokinase (an enzyme that converts ATP to ADP but has no
Results
The effects of adding various concentrations of ATP to whole
blood and to PRP are shown in Figure 1. Addition of ATP to
whole blood resulted in platelet aggregation, the extent of
which was dependent on the concentration used and was most
extensive at concentrations of 10 to 100 ␮mol/L. Increasing
Figure 2. Platelet aggregation induced by selected concentrations of ATP added to whole blood. Mean⫾SEM, n⫽11.
1930
Arterioscler Thromb Vasc Biol.
October 2003
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Figure 3. Inhibitory effects of 100 ␮mol/L MRS2179 (䡩), 1
␮mol/L AR-C69931 (▫), and 300 ␮mol/L CP with 22 U/mL CPK
(‚) on the platelet aggregation in whole blood induced by ATP
(30 ␮mol/L). The results are compared with the aggregation that
occurred in whole blood on addition of ATP alone (䢇).
Mean⫾SEM, n⫽4.
additional phosphatase activity) had been added. The results
are shown in Figure 4. As before, the addition of ATP to PRP
did not produce an aggregation response. However, in the
presence of apyrase or hexokinase, ATP brought about a
marked aggregation response.
Subsequent experiments were designed to investigate the
role of leukocytes and also the influence of any adenosine
that may be produced from the ATP. To examine the
contribution of leukocytes, we carried out experiments in
which PMNLs and MNLs were isolated from whole blood
and then added back to autologous PRP (at a concentration
equivalent to that in whole blood) before stimulation with
ATP. The results are shown in Figure 5. As previously, ATP
produced an aggregation response in whole blood but not in
PRP. However, on addition of leukocytes to PRP, a small
degree of aggregation was observed that was slightly more
extensive in the presence of PMNLs compared with MNLs.
Similar results were obtained in response to 10 ␮mol/L ATP
(results not shown). The results of control experiments
showed that neither leukocyte preparation caused platelet
aggregation in the absence of ATP (results not shown). To
Figure 5. Effects of MNLs (▫), PMNLs (‚), or PBS as control (䡩)
added to PRP on the platelet aggregation induced by ATP (100
␮mol/L). The experiments were repeated in the presence of 1.2
U/mL ADA (corresponding closed symbols). The results are
compared with the aggregation that occurred in whole blood in
response to ATP (dotted line). Mean, n⫽3 to 7. **P⬍0.01 PMNL
vs PBS; 䉫P⬍0.05 MNLs with ADA vs MNLs; *P⬍0.05 PMNLs
with ADA vs PMNLs.
examine the contribution of adenosine in this system, the
experiment was also performed in the presence of ADA,
which converts adenosine to inosine. When ADA was present, this aggregation response became much more extensive,
approaching that obtained in whole blood. Again similar
results were obtained in response to 10 ␮mol/L ATP (results
not shown).
To additionally investigate the contribution of adenosine,
we studied the effects of dipyridamole, an agent that inhibits
the uptake of adenosine into red cells,17,18 on ATP-induced
aggregation in whole blood. We also looked at the effects of
DN 9693 (a cAMP PDE inhibitor) and forskolin (a stimulator
of adenylate cyclase), low concentrations of which are known
to synergize with adenosine to raise platelet cAMP.27 The
results are shown in Figure 6. Dipyridamole (10 ␮mol/L)
limited the extent of aggregation observed in response to
ATP, and this inhibitory effect of dipyridamole was significantly enhanced by DN9693 and forskolin. This inhibition of
aggregation by dipyridamole used alone and in combination
with DN9693 or forskolin was completely prevented in the
presence of ADA.
Discussion
Figure 4. Potentiatory effects of 0.03 U/mL apyrase (䡩) and 0.05
U/mL hexokinase (▫) on the platelet aggregation induced by the
addition of ATP (30 ␮mol/L) to PRP. ATP alone produced virtually no aggregation when added to PRP (䢇). Mean⫾SEM, n⫽3.
In considering the factors that might determine the overall
effect of ATP on platelet aggregation, we thought that some
of the following might be relevant: possible promotion of
aggregation via conversion to ADP by ecto-ATPases on
blood cells (platelets or leukocytes), competitive inhibition by
ATP of the effects of ADP at ADP receptors on the platelet
surface, and inhibition of aggregation via conversion of ATP
to adenosine and consequent elevation of platelet cAMP.
Our first results confirmed earlier preliminary findings28
(that so far have only been reported in abstract form) and
demonstrated that ATP induces platelet aggregation in whole
blood but not in PRP. The aggregation was dependent on the
concentration of ATP that was used and was maximal at
concentrations between 10 and 100 ␮mol/L. Less aggregation
Stafford et al
ATP-Induced Platelet Aggregation in Whole Blood
1931
Figure 6. Effects of 10 ␮mol/L dipyridamole, both alone and in combination
with 0.3 ␮mol/L DN9693 or 1 ␮mol/L
forskolin, in the absence and presence of
1.2 U/mL ADA, on the platelet aggregation induced by the addition of ATP (100
␮mol/L) to whole blood. Aggregation was
measured 8 minutes after the addition of
ATP to the blood. Mean⫾SEM, n⫽4 to 8.
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was obtained when lower or higher concentrations of ATP
were used.
Although it is known that there are P2X1 receptors on the
platelet surface that interact with ATP,8,9 there is little
evidence that this interaction plays a significant role in
platelet aggregation.29 –31 The fact that ATP did not cause
aggregation in PRP seemed to confirm this. Also, the lack of
aggregation in PRP in response to ATP showed that the
aggregation was not merely a consequence of ADP present in
the ATP preparation. Consequently, we believed we needed
to look elsewhere for an explanation of the effects of ATP
that we had observed.
To test the possibility that the aggregation in whole blood
might be brought about by ADP generated from the added
ATP, we investigated the effects of 2 agents, MRS2179 and
AR-C69931, on ATP-induced platelet aggregation. ADPinduced aggregation is mediated by ADP interacting with
P2Y1 and P2Y12 receptors.5,6 MRS217921,22 and ARC6993123–25 act as antagonists at the P2Y1 receptors and the
P2Y12 receptors, respectively, and it is well-known that
blocking ADP interaction with either receptor inhibits ADPinduced platelet aggregation. We found that both these agents
markedly inhibited ATP-induced aggregation in whole blood.
Additional confirmation of a role for ADP in the response
came from an experiment in which we added CP/CPK to
remove any ADP that might be generated from the ATP.26
This also abolished the aggregation.
Because ATP was unable to induce aggregation in PRP, we
considered that platelets themselves may have insufficient
ATPase activity to generate enough ADP for aggregation to
occur. This is despite the fact that platelets themselves are
known to have an ecto-ATPase.32,33 Consequently, we looked
to see whether addition to PRP of an enzyme that converts
ATP to ADP would be sufficient for ATP to induce an
aggregation response and thus reproduce the results obtained
in whole blood. This proved to be the case with both apyrase
(which has combined ATPase and ADPase activity) and
hexokinase (an ATPase) able to bring about an aggregation
response to ATP.
Having identified an involvement of ADP in ATP-induced
aggregation in whole blood and a possible involvement of an
ATPase, we went on to try to identify the source of the
ATPase. Leukocytes were obvious candidates for which the
presence of ecto-nucleotidase activity has been de-
scribed.1,13,34,35 We performed experiments in which purified
PMNLs and MNLs were added to autologous PRP. The
number of leukocytes added was always equivalent to the
number in the whole blood from which they were derived.
Introduction of either type of leukocyte was sufficient for
ATP to induce a small, reversible aggregation response in the
PRP. This was slightly more extensive in the case of PMNLs
compared with MNLs, but the aggregation induced by the
ATP proved to be nothing like as extensive as that obtained
in whole blood itself. We thus looked for an explanation for
this.
In whole blood and PRP, ATP and ADP are metabolized to
adenosine,13,15 which is an inhibitor of platelet aggregation.10,16 It stimulates adenylate cyclase and increases the
intracellular level of cAMP, which in turn inhibits calcium
mobilization and other signal transduction pathways. We
thought it possible that differences in response to ATP in
whole blood and in PRP containing leukocytes might relate to
the relative availability of generated adenosine in the 2
systems. Erythrocytes rapidly take up and remove adenosine
and thereby might make it unavailable to act as an inhibitor of
platelet aggregation.17,18 To investigate the role of adenosine,
we used adenosine deaminase, an enzyme that rapidly deaminates any adenosine that is generated. Whereas ADA had no
effect on ATP-induced aggregation in whole blood, it was
found to markedly potentiate the aggregation induced by ATP
in PRP that contained leukocytes as a source of ATPase.
It would seem that ATP-induced aggregation in PRP
containing leukocytes and ADA is a consequence of ATP
conversion to ADP via ecto-ATPases on the leukocytes. The
presence of ADA removes any adenosine that is produced by
additional breakdown of ATP and ADP, thus making it
unavailable to reduce the degree of aggregation that occurs
via an effect on platelet cAMP. It follows that ATP-induced
platelet aggregation in whole blood could also be via ATP
conversion to ADP, with the erythrocytes providing the
means of removing any adenosine that is produced through
rapid uptake of the latter.
To explore this possibility further, we decided to study the
effects of dipyridamole, a drug that inhibits uptake of adenosine into erythrocytes.17,18 We found that dipyridamole
limited the extent of aggregation obtained after addition of
ATP to whole blood and that ADA reversed this inhibition,
1932
Arterioscler Thromb Vasc Biol.
October 2003
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results that are entirely in accord with the mechanism
proposed.
For additional confirmation of an inhibitory role for adenosine under circumstances where it is not removed from the
system (eg, in PRP or in whole blood containing dipyridamole), we carried out 2 additional experiments. It is known that
the effects of adenosine as an inhibitor of platelet aggregation
can be amplified by forskolin27 and also by cAMP phosphodiesterase inhibitors such as DN 9693.27 The former synergizes with adenosine in stimulating cAMP production and the
latter maintains cAMP by blocking its breakdown. These
agents were found to potentiate the inhibitory effects of
dipyridamole on ATP-induced aggregation in whole blood,
and, again, this was totally reversed by introducing ADA as
a means of removing adenosine from the system.
The results of these investigations suggest that ATPinduced platelet aggregation in whole blood occurs mainly
via conversion to ADP by ecto-ATPases on blood cells and
that the cells involved are both PMNLs and MNLs. The ADP
induces aggregation via interaction with P2Y1 and P2Y12
ADP receptors. Reduced aggregation at very high concentrations of ATP may be a consequence of antagonism by ATP of
ADP interaction with P2Y1 or P2Y12 receptors.10,11 It is
unlikely that this is an artifact of the high concentrations used,
because addition of high concentrations of ADP to whole
blood or PRP did not result in reduced platelet aggregation
(data not shown). There seems to be little inhibition of this
aggregation by adenosine generated from the ATP, probably
because the adenosine is rapidly removed by the erythrocytes
in the blood. Blockade of adenosine uptake by dipyridamole
reduces the extent of the aggregation response, and this effect
of dipyridamole can be enhanced by other agents that
promote the inhibitory effect of adenosine on platelet aggregation. In PRP, ATP does not induce aggregation, because in
the absence of blood leukocytes, insufficient ADP is generated to cause an aggregation response, and in the absence of
erythrocytes, adenosine generated from the ATP remains
available to inhibit platelet aggregation.
It should be noted that all of the experiments that were
performed here were conducted in whole blood or PRP that
contained hirudin as the anticoagulant. Use of hirudin (unlike
citrate, which is more commonly used in investigations of
platelet function) maintains plasma divalent cations at normal
physiological levels.36 In some experiments, we used citrate
in place of hirudin and found that the extent of aggregation
induced by ATP in whole blood was less extensive (results
not shown). It is therefore possible that normal physiological
levels of divalent cations are required for optimum conversion of ATP to ADP to occur.
In considering the relevance of these findings in whole
blood and in PRP in vitro to the situation in vivo, we must
note that in vivo platelets will also be subject to the influence
of endothelial cells on the luminal surface of vessel walls.
Endothelial cells are a major source of ecto-ATPase and
5⬘-nucleotidase, enzymes that collectively dephosphorylate
ATP, ADP, and AMP with production of additional adenosine.14,37 These activities will clearly add to the enzyme
activities present on blood leukocytes and in plasma. Endothelial cells are also able to synthesize and release prostacy-
clin, which, like adenosine, inhibits platelet aggregation via
stimulation of adenylate cyclase. Also, endothelial cells are
able to produce nitric oxide, another agent that inhibits
platelet aggregation, in this case through stimulation of
guanylate cyclase. Presumably, this powerful protective armoury makes it unlikely that platelets will be activated by
ATP/ADP under normal conditions, where blood flows over
intact endothelium. However, this may not be the case at sites
where the endothelium has been breached and where initiation of a hemostatic plug is required or where a platelet
thrombus develops. It is already known that ADP emerging
from damaged cells and tissues or from previously activated
platelets contributes to normal hemostasis and to thrombosis.
For example, patients with platelets with defective P2Y12
receptors exhibit bleeding problems,38 and agents that inhibit
ADP-induced platelet aggregation (agents such as clopidogrel
which blocks the effects of ADP at P2Y12 receptors on
platelets) have been shown to be effective antithrombotic
agents.39 The role of ATP released in parallel with the ADP
has been less certain, despite the fact that very large amounts
of ATP can be released. For example, in erythrocytes, the
concentration of ATP approaches 1 mmol/L and is 10 times
higher than the ADP that is present. The findings described
herein suggest that the overall effect of ATP released into
whole blood is to add to the proaggregatory effects of
released ADP and that ATP should be considered as an agent
making an additional contribution to normal hemostasis and
to thrombosis.
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Mechanisms Involved in Adenosine Triphosphate−Induced Platelet Aggregation in Whole
Blood
Nicholas P. Stafford, Andrew E. Pink, Ann E. White, Jacqueline R. Glenn and Stan Heptinstall
Arterioscler Thromb Vasc Biol. 2003;23:1928-1933; originally published online July 31, 2003;
doi: 10.1161/01.ATV.0000089330.88461.D6
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