(Received for publication, May 15, 1995; and in revised form, July 18, 1995)
From the
ADP is an important platelet agonist which initiates platelet
shape change, aggregation, exposure of fibrinogen receptors, and
calcium mobilization. Because of the limitations of previously used
affinity analogs and photolabeling studies as well as controversies
surrounding the identity of an ADP receptor on platelets, we have used
an affinity label capable of alkylating a putative exofacial receptor
on platelets. We now report that
8-(4-bromo-2,3-dioxobutylthio)adenosine-5`-diphosphate (8-BDB-TADP),
which is an analog of the natural ligand ADP, blocked ADP-induced
platelet shape change, aggregation, exposure of fibrinogen-binding
sites, secretion, and calcium mobilization. Following modification by
8-BDB-TADP, the rates of aggregation of platelets induced by thrombin,
a calcium ionophore (A23187) or a stimulator of protein kinase C
(phorbol myristate acetate) were minimally affected. However, the
8-BDB-TADP-modified platelets exhibited decreased rates of aggregation
in response to ADP, as well as collagen and a thromboxane mimetic
(U46619), both of which partially require ADP. Autoradiograms of the
gels obtained by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of solubilized platelets modified by either
[-
P]8-BDB-TADP, or 8-BDB-TADP and
NaB[
H]
showed the presence of a
single radiolabeled protein band at 100 kDa. The intensity of this band
was reduced when platelets were preincubated with ADP, ATP, and
8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The
results show that 8-BDB-TADP selectively and covalently labeled
aggregin (100 kDa), a putative ADP receptor, resulting in a loss of
ADP-induced platelet responses.
Although ADP (Fig. 1A) is one of the earliest
known agonists for platelet activation(1, 2) , the
identity of the receptor is still uncertain. ADP receptors on platelets
and magakaryocytes are unique(3) ; they constitute a subtype
P of a class of P
purinergic receptors where
ADP is the strongest agonist and ATP is an antagonist(4) . The
presence of a P
receptor on human erythroleukemia cells
has recently been demonstrated(5) . Previous work from our
laboratory demonstrated that
5`-p-fluorosulfonylbenzoyladenosine (FSBA)
(Fig. 1B), an ADP affinity label, blocked
ADP-induced platelet shape change(6) , aggregation, and
exposure of fibrinogen-binding sites (7) with concomitant
covalent modification of a single surface protein, aggregin (100 kDa),
an ADP receptor on platelet surface(8, 9) . Covalent
modification of platelets by FSBA was shown to block platelet
aggregation induced by U46619 (a thromboxane mimetic) (10) and
collagen (11) , suggesting that aggregation induced by these
agonists, in part, depends on interaction of ADP with aggregin.
Figure 1: Chemical representations of ADP, 8-BDB-TADP, and FSBA. The representations of ADP (A), FSBA (B), and 8-BDB-TADP (C) were created by the CHEMDRAW computer program (Cambridge Scientific Computing, Cambridge, MA).
Other investigators have proposed different candidates for a
putative ADP rceceptor on platelet surface. Greco et al.(12) suggested that there is an ADP-binding site on platelet
glycoprotein IIb (GPIIb) based on results obtained by photolabeling of
platelets by
[S][adenosine-5`-(1-thiotriphosphate)],
ATP-
-S. The same group has recently proposed that an ADP-binding
site does not reside on GPIIb but is in close proximity to
it(13) . However, only ATP (but not ADP analogs) inhibits
binding of fibrinogen to its receptor, the GPIIb-IIIa
complex(12, 14) . Patients with thromboasthenia,
lacking this receptor, exhibit normal ADP-induced platelet shape change
and mobilization of intracellular Ca
(15) .
Although 2-azido-ADP (2-N
-ADP) was shown to block
ADP-induced aggregation of platelets(16) , no covalently
labeled ADP-binding protein was detected by gel electrophoresis.
Photolabeling by
2-(p-azidophenyl)-ethylthioadenosine-5`-diphosphate
(AzPET-ADP) was shown to label several proteins on the platelet surface (17) . The fact that labeling of one of these proteins (43 kDa)
was reduced in the presence of ADP led the authors to conclude that the
43-kDa protein might be an ADP receptor on platelet surface.
Although photoaffinity labeling has provided useful information concerning the structure-function relationship of both purified enzymes (18) and functional proteins in intact cellular systems(14, 19) , it suffers from a number of drawbacks. The nature of the chemical reaction and the product(s) formed during ``photoaffinity'' labeling(13, 17) remain uncertain. Photoaffinity labeling often yields multiple labeling patterns, thus complicating the process of identification of labeled proteins(13, 17) . UV radiation can mimic the action of small ligands in activating signal transduction pathways that activate and/regulate mammalian cell functions(20, 21, 22) .
FSBA previously
used to probe the ADP receptor also has certain limitations. Although
FSBA contains a carbonyl group in a position sterically equivalent to
the -phosphoryl group of ADP, it has a benzoyl group and thus
lacks the hydrophilicity of ADP. Since the reagent includes an ester
bond (Fig. 1B), hydrolysis of FSBA yields adenosine,
which is an inhibitor of platelet functions and thus adenosine
deaminase has to be included in the incubation mixtures(23) .
Furthermore, the available method of synthesis of
[
H]FSBA yields a product of relatively low
specific radioactivity. These properties have frustrated attempts to
purify the ADP receptor using [
H]FSBA as a
covalent label.
In light of the limitations on the use of FSBA and
the controversy surrounding the identity of an ADP receptor on the
platelet surface, we decided to investigate the effect of other
nucleotide-based affinity labels with the potential for alkylation of a
putative receptor. 8-(4-Bromo-2,3-dioxobutylthio)ADP (8-BDB-TADP) (Fig. 1C) has been previously used as an ADP affinity
label to probe the structure-function relationship of ADP-requiring
enzymes such as pyruvate kinase (24) and glutamate
dehydrogenase(25, 26) . 8-BDB-TADP has the
characteristic diphosphate of the natural ligand ADP, and is both
hydrophilic and negatively charged at neutral pH. Furthermore, it can
be synthesized as the P-labeled reagent with high specific
radioactivity. In order to further investigate and test our hypothesis
that aggregin is a putative ADP receptor, we chose 8-BDB-TADP as a true
ADP affinity ligand to label the ADP receptor and ascertain its effect
on ADP-induced platelet responses. In this case the nature of the
chemical reaction is well understood and chemically defined products
have been isolated(26, 27) . This report describes in
detail the effect of 8-BDB-TADP on ADP-induced platelet responses and
the protein(s) modified on the surface of human platelets. A
preliminary account of the work has previously appeared(28) .
The P-labeled compound,
[
-
P]8-BDB-TADP, was synthesized from
8-thioadenosine-5`-monophosphate, prepared, and purified as reported
previously(29) , by phosphorylation using
[
P]phosphoric acid (5 mCi added to 500 µmol
of the unlabeled phosphoric acid). Tributylammonium
[
P]phosphate was prepared after passage of the
radioactive sodium phosphate through a column of AG50-X4 (pyridinium
form) as described (30) and was dissolved in dimethylformamide.
Tributylammonium salt of 8-thioadenosine-5`-monophosphate was prepared
similarly (30) and was dissolved in dimethylformamide. The
phosphorylation was conducted by the method of Kozarich et
al.(31) . A solution of 1,1`-carbonyldiimidazole (200
µmol) in dimethylformamide (1.5 ml) was added to a solution of 100
µmol tributylammonium-8-(thio)adenosine-5`-monophosphate in
dimethylformamide (5 ml) and stirred for 45 min. Methanol (450
µmol) was added, and the reaction mixture was stirred for an
additional 30 min. Tributylammonium[
P]phosphate
(250 µmol in 2.5 ml of dimethylformamide) was added with stirring
and was allowed to stand at room temperature for 20 h. Methanol (9 ml)
was then added and the reaction mixture evaporated to dryness.
8-Thioadenosine-5`-diphosphate was purified by chromatography on
DEAE-cellulose column using a linear gradient from 10 to 500 mM NH
HCO
, essentially as described for
2-thioadenosine-5`-diphosphate(32) . Purified 8-TADP was
converted to the free acid form by application to a column of
AG-50W-X4(H
) and elution with distiilled water.
8-Thioadenosine-5`-[
-
P]diphosphate was
converted into [
-
P]8-BDB-TADP by reaction
with 1,4-dibromobutanedione, as described for the unlabeled 8-BDB-TADP (24) .
Figure 2:
Effect
of 8-BDB-TADP on ADP-induced platelet shape change. A,
concentration dependence: platelets were incubated in the dark with
various concentrations of 8-BDB-TADP for 20 min at 25 °C.
Incubation mixtures were challenged by 30 µM ADP,
monitored for shape change (), and data plotted as described under
``Experimental Procedures.'' B, time course:
platelets were incubated with 200 µM 8-BDB-TADP as
described above. Aliquots were withdrawn at various times and
challenged by 30 µM ADP to monitor residual shape change
(
). The data are expressed as percent of rate of maximum shape
change (LAU/min: LAU = light absorption units, arbitrary scale)
compared with an identical control (100%). The results are
representative of the data obtained with platelets from the blood of
three different donors.
Figure 3:
Effect of 8-BDB-TADP on ADP-induced
platelet aggregation. A, concentration dependence: platelets
were incubated in the dark with various concentrations of 8-BDB-TADP
for 30 min at 25 °C, challenged by 30 µM ADP, and
monitored for aggregation (), and data plotted as
described under ``Experimental Procedures.'' B, time
course: platelets were incubated with 150 µM 8-BDB-TADP.
Aliquots were withdrawn and challenged by 30 µM ADP and
monitored for aggregation (
). The data are expressed as percent
of rate of maximum aggregation (LTU/min: LTU = light
transmission units, arbitrary scale) compared with an identical control
(100%). The data are typical of those obtained with platelets from the
blood of three donors.
Figure 4:
Effect of 8-BDB-TADP on ADP-induced
exposure of fibrinogen binding sites in platelets. For total binding
washed platelets (1 10
/200 µl) in the presence
of 1 mM Ca
were incubated with increasing
concentration of
I-fibrinogen (0.2 mCi/mg protein) for 1
min followed by 30 µM ADP for 3 min at 25 °C. Three
aliquots (50 µl) were withdrawn from each incubation mixture and
layered over a mixture of silicon oils as described under
``Experimental Procedures'' and centrifuged in a microfuge
for 3 min. The pellet at the bottom of the centrifuge tube was excised
and assayed for radioactivity. Nonspecific binding of
I-fibrinogen to washed platelets was determined as
described above except that the incubation mixtures contained 2 mM EDTA and 10-fold molar excess of unlabeled fibrinogen. Binding to
platelets in the presence of 8-BDB-TADP was performed as described in
the case of total binding except that the washed platelets were
preincubated in the dark with 300 µM 8-BDB-TADP at 25
°C for 30 min. Specific binding of
I-fibrinogen to
platelets in the absence (
) and presence (
) of 8-BDB-TADP
were computed by subtracting nonspecific binding from the total binding
in each case. The data are expressed as mean of the molecules of
I-fibrinogen bound ± S.E./platelet versus concentration of
I-fibrinogen.
Figure 5:
Effect of 8-BDB-TADP on ADP-induced
platelet secretion. ATP release following exposure of platelets (5
10
/ml) to thrombin (2 nM) and ADP (30
µM) was measured by the commercial luciferase-luciferin
reagent as described under ``Experimental Procedures.''
Platelets (5
10
/ml) were preincubated in the dark
with 50 µM of 8-BDB-TADP for 15 min at 25 °C followed
by treatment with 30 µM ADP. 8-BDB-TADP had no effect on
the intensity of chemiluminiscence produced by solutions of known
concentration of ATP (used for calibration of the assay) treated with
luciferase-luciferin reagent under identical conditions. Thrombin- and
ADP-induced secretion were computed from two separate calibrations
employing standard solutions of ATP. Similar results were obtained with
platelets from the blood of two different
donors.
Figure 6:
Effect of 8-BDB-TADP on ADP-induced
increase in intracellular calcium levels in platelets. Platelets were
loaded with Fura 2/AM fluorophore as described under
``Experimental Procedures.'' The loaded platelets were then
stirred with various concentrations of ADP in the presence of 1
mM external Ca. Ca
release
from the platelets was monitored as described under ``Experimental
Procedures.'' To evaluate the effect of 8-BDB-TADP, washed
platelets were preincubated in the dark with 230 µM 8-BDB-TADP for 30 min at 25 °C followed by treatment with 1
mM Ca
and 30 µM ADP.
Figure 7:
SDS-PAGE of platelets labeled with
[-
P]-8-BDB-TADP. a, platelets (2
10
/0.2 ml) were labeled with
[
-
P]8-BDB-TADP in the absence (lanesA-C) and presence of 2 mM ADP (lane
D) and the labeled platelets subjected to SDS-PAGE as described
under ``Experimental Procedures.'' Bands of radioactivity
near the dye front correspond to unbound radioactivity. b,
platelets (5
10
/0.2 ml) were preincubated with
buffer (lane A), 10 mM ADP (lane
C), and 10 mM ATP (lane D) at 25 °C for 2
min before labeling by [
-
P]8-BDB-TADP as
described under ``Experimental Procedures.'' Platelets were
modified by 100 µM FSBA, and FSBA-modified platelets (5
10
/0.2 ml) were then labeled by
[
-
P]8-BDB-TADP (lanes E and F). Lane B corresponds to an aliquot of
[
-
P]8-BDB-TADP added to the sample buffer
used in the electrophoresis. The gel was calibrated with molecular
weight standards derivatized by the blue dye using the molecular
weights provided by the supplier (Life Technologies, Inc.). A band of
radioactivity at the top of the gel in lane A is due
to incomplete solubilization of much larger concentration of platelets
used in this experiment. c, platelets (2
10
/0.2 ml) were labeled by
[
-
P]8-BDB-TADP in the absence of any
modulator (lane A) and in the presence of 2 mM
concentration of ADP (lane B), 2-methylthio-ADP (laneC), 8-bromo-ADP (lane D), ATP
S (lane
E), adenosine (lane F), AMP (lane G), GDP (lane H), and p-chloromercuribenzenesulfonate (lane I). Experiments described in a-c were
performed with platelets obtained from the blood of three different
donors.
Figure 8:
SDS-PAGE of platelets labeled by
8-BDB-TADP and NaB[H]
. Platelets (2
10
/0.2 ml) were labeled by
8-BDB-TADP, and the modified platelets were then exposed to
NaB[
H]
. The reaction mixtures were
centrifuged, pellets solubilized, and subjected to SDS-PAGE as
described under ``Experimental Procedures.'' Lanes
A-C, correspond to platelets labeled in the absence of any other
reagent, 10 mM ATP, and 10 mM ADP, respectively.
NaB[
H]
alone did not label platelets.
The gel was calibrated with prestained molecular mass standards
(Bio-Rad) each of which was covalently attached to a dye of different
color and consisted of the following: myosin, 217 kDa;
-galactosidase, 135 kDa; bovine serum albumin, 72 kDa; carbonic
anhydrase, 42 kDa, and soybean trypsin inhibitor, 31 kDa. The values of
the molecular weight correspond to those supplied by
Bio-Rad.
8-BDB-TADP inhibited ADP-induced platelet shape change and
aggregation in a concentration- and time-dependent manner signifying
that the reagent covalently modifies ADP-binding sites on platelet
surface. Preincubation of the platelets with 8-BDB-TADP effectively
blocked ADP-induced binding of I-fibrinogen to platelets.
These results are consistent with the fact that the 8-BDB analog
inhibits ADP-induced platelet aggregation. When 8-BDB-TADP-modified
platelets were examined for their ability to aggregate by exposure to
various agonists, only ADP-induced aggregation was almost completely
blocked. The results that the rates of collagen- and U46619-induced
platelet aggregation were significantly reduced in 8-BDB-TADP-modified
platelets are in accord with the previous findings that platelet
aggregation induced by these two agonists proceeds, at least in part,
by ADP-dependent mechanisms(10, 11) . The rates of
aggregation induced by thrombin A23187, PMA, and A23187+PMA were
minimally affected by chemical modification of ADP-binding sites. These
results are in accord with the fact that aggregation of platelets
induced by the above agonist follow ADP-independent mechanisms (43, 44, 45) . 8-BDB-TADP also blocked
ADP-induced secretion of nucleotides by platelets.
ADP-induced
release of Ca from the dense tubular system plays an
important role in the platelet responses mediated by the binding of ADP
to its receptor(46) . Our results show that 8-BDB-TADP itself
was not an agonist of intracellular mobilization of
Ca
, but it effectively blocked similar mobilization
by ADP. ADP is a non-penetrating reagent, and platelet responses
elicited by this agonist are mediated through its interaction with
specific receptors on platelet surface(9, 47) . In
addition to inducing intracellular increase in Ca
,
ADP also has another important function: it antagonizes elevation of
intracellular levels of cAMP induced by prostaglandins(42) .
Our observation that covalent modification of platelets by 8-BDB-TADP
blocked the ability of ADP to inhibit stimulated adenylate cyclase
activity is consistent with the previous studies.
The ADP-binding
site mediating ADP-induced platelet shape change may be different from
the one mediating intracellular Ca mobilization(48) . Other investigators suggested that
that ADP-binding site for platelet shape change might be different from
those antagonizing stimulated adenylate cyclase activity(49) .
Savi et al.(50) recently presented experimental
evidence for two types of ADP-binding sites that differ in their
affinity for ADP. Unlike 8-BDB-TADP, FSBA was not able to antagonize
the stimulated adenylate cyclase activity, but this may be due to its
lower affinity for aggregin(23) .
Although the two-receptor
hypothesis for different ADP-induced platelet responses has been widely
discussed, the identity of two distinct ADP-binding proteins on
platelet surface has never been established. Results presented in this
investigation, for the first time, show that
[H]FSBA and
[
-
P]8-BDB-TADP label the same protein,
aggregin (100 kDa), the ADP receptor on the platelet surface. Covalent
modification of aggregin by 8-BDB-TADP not only inhibits ADP-induced
shape change, aggregation, and exposure of fibrinogen-binding sites but
also blocks the ability of ADP to antagonize stimulated adenylate
cyclase activity suggesting that all of these responses are mediated by
aggregin. Complete prevention of incorporation of
[
-
P]8-BDB-TADP into aggregin required
either prior covalent modification of aggregin by FSBA or preincubation
of platelets with a concentration of ADP or ATP as high as 10
mM. Our experience has shown that those concentrations of
2`,3`-dialdehyde derivative of ADP (10 mM) that far exceeded
those necessary to inhibit ADP-induced platelet shape change and
aggregation, could not completely block labeling of platelets by
[
H]FSBA. (
)We have recently shown that
7-chloro-4-nitro-2-oxa-1,3-diazole blocks ADP-induced platelet
responses by covalently modifying aggregin(51) . Preincubation
of platelets with 30 mM ADP, 30 mM ATP, or covalent
modification of platelets by FSBA was necessary to block completely
labeling of aggregin by
[
C]7-chloro-4-nitro-2-oxa-1,3-diazole. Other
investigators have shown that a concentration of ATP, as high as 30
mM, was needed to block ADP-induced mobilization of
Ca
in human erythroleukemia cells(5) .
Cristalli and Mills (17) found that ADP (0.8 mM)
reduced the intensity of the 43 kDa radiolabeled protein band and
claimed that it was the ADP receptor. Higher concentration of ADP
(>0.8 mM) or its analogs might have reduced the intensity
of other protein bands shown in the autoradiograph presented in that
report. It is noteworthy that the autoradiograph in the above report
contains a dense radiolabeled protein band around 100 kDa(17) .
Our results, however, strongly suggest that only one ADP-binding
protein on platelet surface, aggregin (100 kDa), mediates all of the
ADP-induced platelet responses. Our results do not rule out the
possibility that more than one class of ADP-binding site may exist on
this receptor which differ in their affinity for ADP. Another
explanation is that different signal transducing mechanisms exist for
ADP-induced platelet shape change and inhibition of stimulated
adenylate cyclase activity. Various macrophage cell lines have been
reported to express G protein-coupled
purinoreceptors(52, 53, 54) . We further
demonstrated that 8-BDB-TADP and NaB[
H]
also label aggregin, and their ability to modify aggregin was
blocked by the presence of ADP or ATP. These results are inconsistent
with the possibility that a 43-kDa protein on platelet surface is the
ADP receptor suggesting that labeling of this protein by AzPET-ADP
might be due to nonspecific interactions.
In summary, we have demonstrated that 8-BDB-TADP, an ADP-affinity analog, blocks ADP-induced shape change, aggregation, exposure of fibrinogen-binding sites, secretion, calcium mobilization, and inhibition of stimulated adenylate cyclase activity by ADP in washed platelets by covalently modifying a single surface protein, aggregin (100 kDa), a putative ADP receptor.