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(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
[
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
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
[ 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 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
The
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 (
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 (
Figure 4:
Effect of 8-BDB-TADP on ADP-induced
exposure of fibrinogen binding sites in platelets. For total binding
washed platelets (1
Figure 5:
Effect of 8-BDB-TADP on ADP-induced
platelet secretion. ATP release following exposure of platelets (5
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
Figure 7:
SDS-PAGE of platelets labeled with
[
Figure 8:
SDS-PAGE of platelets labeled by
8-BDB-TADP and NaB[
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 ADP-induced
release of Ca The ADP-binding
site mediating ADP-induced platelet shape change may be different from
the one mediating intracellular Ca 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
[ 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.
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24482-24488
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-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.
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.
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. -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.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) .
Materials
AMP, ADP, ATP, 8-bromo-ADP, p-chloromercurisulfonate, benzamidine hydrochloride, FSBA,
leupeptin, PMA (phorbol-12-myristate-13-acetate), apyrase (from
potato), adenosine deaminase (from calf intestinal mucosa, Type VII),
bovine serum albumin, and luciferase-luciferin reagent (in glycine
buffer) were obtained from Sigma. U46619
(9,11-dideoxy-9a,11a-methanoepoxy prostaglandin
F) was from Cayman Chemicals (Ann Arbor, MI). A23187
(a calcium ionophore), collagen and p-APMSF (p-aminophenylmethylsulfonylfluoride) were from Calbiochem
(San Diego, CA). Adenosine, GDP, and ATP-
-S were obtained from
Boehringer Mannheim. 2-Methylthio-ADP was supplied by Research
Biochemicals, Inc. (Natick, MA). Prestained molecular weight standards
used as markers to estimate molecular weight of proteins by gel
electrophoresis were from Life Technologies, Inc., and Bio-Rad.
Quin-2/AM (AM = acetoxymethyl ester) and Fura-2/AM were obtained
from Molecular Probes (Eugene. OR). cAMP-binding protein assay kit was
purchased from Diagnostic Corp. (Los Angeles, CA).
NaB[H] was supplied by DuPont NEN.
[H]FSBA was prepared as described
previously(6, 7) . Human -thrombin (3203 NIH
units/mg protein) was a gift from Dr. John W. Fenton II (Division of
Laboratories and Research, New York State Department of Health, Albany,
NY). Fibrinogen used in labeling of platelets by I-fibrinogen was obtained from Sigma. In all other
experiments fibrinogen used was purchased from KabiVitrum (Franklin,
OH).
Synthesis and Characterization of
8-BDB-TADP
8-BDB-TADP was synthesized and characterized by
chemical and physical methods as described previously(24) .
Briefly, ADP was treated with Br to yield
8-bromoadenosine-5`-diphosphate (8-Br-ADP). 8-Bromo-ADP was converted
to 8-thioadenosine-5`-diphosphate (8-TADP) by reaction with LiHS. After
purification, 8-TADP was coupled with 1,4-dibromobutanedione to yield
8-[4(bromo-2,3-dioxobutyl)thio]adenosine-5`-diphosphate
(8-BDB-TADP). 8-BDB-TADP was stored dried at -80 °C.
Solutions of 8-BDB-TADP were prepared in 50 mM MES, pH 6.0 and
the concentration determined spectrophotometrically by using a molar
absorption of 19 mM cm
.
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) .
Platelet Isolation and Washing
Fresh human
blood was obtained from healthy volunteers with informed consent.
Platelets were isolated by differential centrifugation and washed as
described previously(33) . Washed platelets were suspended in
Tyrode's buffer (18.4 mM HEPES, 0.42 mM
NaHPO, 136 mM NaCl, 2.7 mM KCl, 11.9 mMD-glucose; 12 mM
NaHCO, and 3.5 mg/ml bovine serum albumin, pH 7.35). Shape Change and Aggregation of
Platelets
Shape change and aggregation of washed platelets were
performed in a Lumi-Aggregometer (Chronolog, Havertown, PA) under
constant stirring conditions at a concentration of 1 
10
/ml and 5 10/ml, respectively. Washed
platelets (1 10/ml) and suspension buffer were used
in reference cuvettes in shape change and aggregation experiments,
respectively. ADP-induced platelet shape change was performed in the
presence of 2 mM EGTA while platelet aggregation was performed
in the presence of 1 mM Ca and 1 mg/ml
fibrinogen.
Fibrinogen Binding
Human I-fibrinogen (specific radioactivity 2.12 mCi/mg protein)
was prepared by the iodogen method as described previously by Puri et al.(34) except that the equilibration and elution
buffer contained 184 mM HEPES, 136 mM NaCl, 2.7
mM KCl, and 0.35 mg/ml bovine serum albumin, pH 7.35. The
preparation of radioiodinated fibrinogen was found to be 93 ± 5% (n = 4) clottable. The radioiodinated fibrinogen was
diluted with unlabeled fibrinogen (20 mg/ml) in a 1:1 ratio before
using it in binding experiments. Binding of
I-fibrinogen
to washed platelets was measured as reported previously(34) .
For total binding of
I-fibrinogen, platelets (1
10/200 µl) were placed in an Eppendorf tube at 25
°C and incubated with increasing amount of I-fibrinogen in the presence of 1 mM Ca
for 1 min. The reaction mixture was then
incubated with 30 µM ADP for another 5 min. The reaction
was terminated by removing a 50-µl aliquot from each sample and
layering over 200 µl of silicon oil mixture in a microtube
(Sarsdet, Princeton, NJ) (34) and centrifuging in a Beckman
model E microcentrifuge for 3 min. The pellet at the bottom of the
microtube was excised and radioactivity in the pellet measured by
counting in an automatic gamma counter (model 1470 WIZARD, Wallac Oy,
Turku, Finland) equipped with RIaCalc WIZ software 2.2. Nonspecific
binding of
I-fibrinogen to platelets was determined
identically in the presence of 2 mM EDTA and 10-fold molar
excess of unlabeled fibrinogen. Binding in the presence of 8-BDB-TADP
was determined similarly by preincubating the platelets in the dark
with 8-BDB-TADP at 25 °C for 30 min.
Platelet Secretion
Secretion of ADP by
platelets (5 10/ml) following exposure to ADP in
the absence and presence of 8-BDB-TADP, was estimated, simultaneously
with aggregation, by measuring the intensity of chemiluminescence of
commercial luciferase-luciferin reagent (30 µl/ml of platelet
suspension; freshly constituted as described in the protocol provided
by the suppliers) in a dual-channel Lumi Aggregometer (Havertown, PA).
The ATP release in the incubation mixtures was computed from a standard
curve prepared by estimating chemiluminescence from solutions of known
concentration of ATP in the absence and presence of 8-BDB-TADP as
described above. 8-BDB-TADP, as high as 0.6 mM, had no effect
on the assay. Measurement of Intracellular Calcium
Levels
Platelets were loaded with fluorescent indicators by
incubating platelet-rich plasma with Fura-2/AM (5 µM) or
Quin-2/AM (20 µM) at 37 °C for 20 min. Stock solutions
of the indicators were prepared in MeSO. Maximum
fluorescence (F
), reflecting total content of
Ca in storage granules, was estimated by treating the
Fura-2- (
and
, 340 and 510 nm,
respectively) loaded platelets (2
10/ml) with 2
µM ionomycin (in MeSO), and autofluorescence
(AF) was determined by quenching the above incubation mixture with 10
mM MnCl. Platelets loaded with the fluorophores
were then incubated with 1 mM CaCl at 37 °C
for 1 min and then stirred with an increasing concentration of ADP to
monitor the fluorescence emission intensity (F).
The intracellular level of Ca
,
[Ca
]
, was calculated (35, 36) by using the equation
[F-F
/F-F]
K
(where F =
AF + (F-AF)/6, and K = 228 for Fura-2/AM). The loaded platelets were then
incubated with 8-BDB-TADP at 25 °C in the dark and ADP-induced
release of Ca determined as described above. When
Quin-2/AM was used as the fluorophore, the protocol used for
determining [Ca
]
was similar to
the one described above except that the and
used were 339 and 492 nm, respectively, and a value
of 114 was used for K
(37) . Measurement of Intracellular cAMP
Levels
Intracellular levels of cAMP in washed and
8-BDB-TADP-modified platelets (1 10/ml) were
determined by the commercial cAMP-binding assay as outlined by the
manufacturers and described previously by Puri et
al.(34) . Labeling of Platelets by
[
Washed platelets
(1-2 -P]8-BDB-TADP
10/0.25 ml) were incubated in the dark
with 200 µM [-P]8-BDB-TADP
(5.91 mCi/mol) in the absence or presence of various reagents for 30
min at 25 °C followed by addition of an ice-cold solution (1 ml)
containing Tris-buffered saline (TBS; 15 mM Tris-HCl and 139
mM NaCl, pH 7.4) and EDTA (10 mM). The reaction
mixture was centrifuged for 30 s in a Beckman model E microcentrifuge.
The pellet was suspended in a solution (35 µl) containing TBS and 1
mM EDTA and solubilized by addition of an equal volume of a
solution containing SDS (4%, w/v),
-mercaptoethanol (12%, v/v),
leupeptin (20 µM), p-APMSF (60 µM), and
benzamidine (2 mM). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed on 10% gels according to the
method of Laemmli (38) with the modification that the gels were
prepared from a solution of acrylamide-bisacrylamide mixture containing
leupeptin (10 µM), p-APMSF (30 µM), and
benzamidine (1 mM). Gels were calibrated with prestained
molecular weight standards. The dried gels were exposed to CRONEX x-ray
film (NEN-DuPont) at -70 °C for 7-14 days before
developing. Labeling of Platelets by 8-BDB-TADP and
NaB[
Washed platelets were
modified by unlabeled 8-BDB-TADP as described above, washed once with
ice-cold TBS buffer containing 10 mM EDTA (1 ml), and
centrifuged in a microfuge for 30 s. 8-BDB-TADP-modified platelets were
suspended in Tyrode's buffer, pH 7.35 (0.2 ml). The suspension
was treated with a NaB[H]H] solution
(25 µl; 50-70 Ci/mol) for 30 min at 25 °C with occasional
but gentle shaking. The NaB[H] solution used was not diluted with unlabeled reagent. Labeled
platelets were centrifuged for 30 s in a microfuge. The pellet was
suspended in the TBS buffer and solubilized and subjected to
electrophoresis as described in the previous section. Gels were
carefully shaken with ENLIGHTNER (NEN-DuPONT) for 20 min at 25 °C
and dried immediately. Autoradiography required exposure of the gels to
x-ray film for 2-4 weeks. Labeling of Platelets by
[
Washed platelets (5 H]FSBA
10/0.25 ml) were incubated with 100 µM [H]FSBA and adenosine deaminase at 25 °C
for 40 min. An ice-cold solution containing TBS and 1 mM EDTA
(1 ml) was added to the incubation mixture and the mixture centrifuged
for 30 s in a microfuge. The labeled platelets were resuspended,
dissolved, and electrophoresed as described above. Gels were carefully
shaken with ENLIGHTNER for 20 min at 25 °C and dried immediately.
Gels were exposed to the x-ray film at -70 °C for 1-2
months.
Shape Change
8-BDB-TADP inhibited ADP-induced
shape change in a concentration-dependent manner with an IC of 75 µM (Fig. 2A). Complete
inhibition of ADP-induced shape change required 400 µM of
the 8-BDB analog. The inhibition of shape change by the 8-BDB analog
followed biphasic kinetics. The time course of inhibition of
ADP-induced platelet shape change by 200 µM 8-BDB-TADP
showed complete inhibition of shape change in 70 min (Fig. 2B). However, loss of 50% of the rate of shape
change occurred in a relatively short time, 10 min. 8-BDB-TADP, at
concentrations as high as 400 µM, did not induce platelet
shape change. The results show that 8-BDB-TADP was a potent inhibitor
of ADP-induced 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.
Aggregation
8-BDB-TADP blocked ADP-induced
platelet aggregation in a concentration-dependent manner with an
IC of about 75 µM (Fig. 3A).
Inhibition of ADP-induced aggregation was also time-dependent, and the
8-BDB analog (150 µM) inhibited the rate of ADP-induced
aggregation by 50% in 10 min and 90% in 40 min (Fig. 3B). 8-BDB-TADP, as high as 400 µM,
did not act as an agonist of platelet aggregation. The results
demonstrate that 8-BDB-TADP is an effective inhibitor of ADP-induced
platelet 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.
Fibrinogen Binding
When platelets were exposed to
30 µM ADP, total binding of I-fibrinogen was
concentration dependent (Fig. 4). Nonspecific binding of
I-fibrinogen to platelets remained at about 10% level
over the 16-fold concentration range of the radiolabeled fibrinogen.
When 8-BDB-TADP-modified platelets were incubated with an increasing
concentration of the radiolabeled fibrinogen followed by exposure to 30
µM ADP, the total binding of fibrinogen was diminished to
a mean (±S.E.) value of 21 ± 5% compared with that of
unmodified platelets. The results show that specific binding of
I-fibrinogen to platelets (or exposure of GPIIb-IIIa
complex) induced by ADP was blocked by 80% when platelets were exposed
to the 8-BDB-TADP.
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.
Aggregation Induced by Various Agonists
When
platelets were modified by 8-BDB-TADP, centrifuged, washed, and
resuspended in Tyrode's buffer, pH 7.35, only 9% of the
ADP-induced aggregation was detectable (Table 1). Rates of
collagen- and U46619-induced aggregation of platelets modified by
8-BDB-TADP were significantly lower compared with the corresponding
controls (Table 1). In contrast, thrombin-induced aggregation of
8-BDB-TADP-modified platelets was comparable to that of the unmodified
platelets. Rates of aggregation of 8-BDB-TADP-modified platelets by
A23187, PMA, and A23187+PMA were minimally affected. The results
show that only rates of aggregation of platelets by ADP, U46619, and
collagen agonists that completely or partially utilize ADP-dependent
mechanism (7, 10, 11) were almost completely
or significantly affected as a result of covalent modification of the
ADP-binding sites on the platelet surface.
Secretion
Thrombin and ADP both caused secretion
of 5 and 2 µM ATP, respectively, from the storage granules
of platelets (Fig. 5). Whereas thrombin-induced secretion of
nucleotides and aggregation are synchronous events, both reaching
maximum within a minute, ADP-induced secretion only starts after
maximum aggregation has occurred. This lag may be due to the presence
of a higher concentration of ADP (some released from the platelets) in
the localized environment of platelets. The concentration of 8-BDB-TADP
(50 µM) which minimally affected ADP-induced platelet
aggregation completely blocked ADP-induced secretion. The results show
that (a) thrombin- and ADP-induced secretion of nucleotides
from dense granules proceed by two different mechanisms, and (b) ADP-induced secretion does require binding of the agonist
to the receptor, and chemical modification of this receptor by
8-BDB-TADP blocks this response.
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.
Intracellular Calcium Mobilization
Intracellular
levels of calcium in platelets exposed to ADP rose from a basal value
of 100 to 703 nM and 683 nM as measured by using
Fura-2/AM and Quin-2/AM, respectively (Fig. 6, data for
measurements by using Quin-2/AM not shown). Preincubation of platelets
with 230 µM 8-BDB-TADP for 30 min at 25 °C completely
blocked ADP-induced elevation of
[Ca]
. 8-BDB-TADP itself did not
cause any change in the basal levels of Ca in
platelets.
. 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.
Intracellular Levels of cAMP
There is little or no
intracellular cAMP present in resting platelets (Table 2,
1)(39, 40) . Iloprost (a stable carbocyclic derivative
of prostaglandin I) and papaverine (a phosphodiesterase
inhibitor) in combination raise and sustain intracellular levels of
cAMP by stimulating membranous adenylate cyclase which converts ATP to
cAMP (Table 2, 2). Thrombin (40, 41) to a larger
extent and ADP (Table 2, 5 and 6) (42) to a lesser extent
antagonize the increase in intracellular levels of cAMP in platelets
induced by prostaglandins. 8-BDB-TADP neither raised (Table 2, 3)
nor affected the ability of iloprost to raise intracellular levels of
cAMP (Table 2, 4). However, the 8-BDB analog impaired the ability
of ADP to antagonize the increase in cAMP levels in platelets exposed
to iloprost (Table 2, 8). These results show that covalent
modification of ADP-binding sites on platelet surface results in the
loss of the ability of ADP to modulate stimulated adenylate cyclase
activity.
Identification of 8-BDB-TADP-labeled Protein on Platelet
Surface
We have previously demonstrated that a single peak of
radioactivity at 100 kDa in the radioactivity distribution profile of
slices of gels obtained by SDS-PAGE of solubilized
[H]FSBA-labeled membranes obtained from
[H]FSBA-labeled platelets corresponds to an ADP
receptor, aggregin, on the platelet surface ((9) , and
references cited therein). However, because of the relatively low
specific radioactivity of the available [H]FSBA
and the low emitting threshhold of tritium, it is cumbersome and time
consuming to obtain good radiograms by autoradiography of gels obtained
by SDS-PAGE of solubilized [H]FSBA-labeled
platelets. Nevertheless, an autoradiograph of the gel obtained by
SDS-PAGE of solubilized [H]FSBA-labeled platelets
shows the presence of a single protein band corresponding to
radiolabeled aggregin (100 kDa) (data not shown).
[-P]8-BDB-TADP also labeled a single
protein (100 kDa) on the platelet surface; the intensity of this band
was reduced when labeling was carried out with platelets preincubated
with 2 mM ADP (Fig. 7a). When FSBA-modified
platelets or platelets exposed to 10 mM ADP or 10 mM ATP were incubated with
[
-P]8-BDB-TADP, no label was incorporated
into the protein corresponding to aggregin (Fig. 7b);
whereas 2 mM ADP and 8-bromo-ADP (Fig. 7b, lanesB and D) considerably reduced the
intensity of the [
-P]8-BDB-TADP-labeled
aggregin, ATP-
-S, adenosine, AMP, and GDP (Fig. 7c, lanesE-H), at the
same concentration, minimally protected aggregin from labeling by
[-P]8-BDB-TADP (Fig. 7c).
Platelets previously exposed to 2 mMp-chloromercuribenzenesulfonate (a sulfhydryl modifying
reagent) were not labeled by [
-P]8-BDB-TADP (Fig. 7c, lane I). This result is consistent
with our previous demonstration that aggregin contains lysine and
cysteine residues at the ADP-binding domain in aggregin and that
covalent and simultaneous modification of such residues by o-phthaladehyde leads to loss of ADP-induced platelet
responses(40) . Labeling of platelets by 8-BDB-TADP followed by
chemical reduction by NaB[
H] of the
keto groups in the label covalently attached to platelets incorporated
tritium into a 100 kDa protein band corresponding to aggregin.
Incorporation of the label into aggregin was completely blocked when
labeling was carried out by preincubating platelets with 10 mM ADP or 10 mM ATP (Fig. 8). The results are
consistent with the fact that [H]FSBA,
[-P]8-BDB-TADP, and 8-BDB-TADP and
NaB[
H] covalently and selectively
label the same ADP-binding protein, aggregin, on the platelet surface.
-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.
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.
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.
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.
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) .
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.
)-ADP, 2-azidoadenosine-5`-diphosphate;
AzPET-ADP, 2-(p-azidophenyl)ethylthio-adenosine-5`-diphosphate;
ATPS, adenosine-5`-(1-thiotriphosphate); A23187, a calcium
ionophore; U46619, 9,11-dideoxy-9,11-methanoepoxy
prostaglandin F; PMA, phorbol-12-myristate-13-acetate; p-APMSF, p-aminophenylmethylsulfonylfluoride; GPIIb,
glycoprotein IIb; GPIIIa, glycoprotein IIIa; PAGE, polyacrylamide gel
electrophoresis; MES, 4-morpholineethanesulfonic acid.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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