From the Unité INSERM 348, Institut
Fédératif de Recherche Circulation-Lariboisière,
Hôpital Lariboisière, 75010 Paris, France and the
¶ Center for Experimental Therapeutics, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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A single gene encodes the human
thromboxane receptor (TP), of which there are two identified splice
variants, Thromboxane (Tx)1
A2 is formed in platelets by the sequential metabolism of
arachidonic acid by cyclooxygenases and TxA2 synthase (1)
following activation by agonists, such as arachidonic acid, thrombin,
collagen, or ADP. Although a weak agonist itself, TxA2 plays an important role in amplifying the response to other, more potent platelet agonists (2). Studies with pharmacological ligands in
human platelets have suggested the presence of two distinct populations
of receptors (3, 4). Distinct functions have been attributed to these
subtypes: (i) aggregation and granule secretion appear to be mediated
by receptors with low affinity for the agonist ligand I-BOP, which are
bound irreversibly by the antagonist GR 32191; and (ii) shape change
appears to be mediated by receptors with high affinity for I-BOP, which
are bound reversibly by GR 32191 (3, 4). Despite these observations,
the molecular basis for this functional segregation of pharmacological
TP subtypes is unknown. TP purified from human platelets consists of a
broad protein band of 57 kDa (5, 6). Initial cloning of the TP, from
megakaryoblastic cell lines and human placental cDNA libraries (referred to as the placental TP or the TP Phosphorylation is an important mechanism in rapid desensitization of
many GPCRs, as exemplified by the Materials--
Ro-43-5054 and Ro-44-9883 were a kind gift of Dr.
B. Steiner (Hoffmann-La Roche, Basel, Switzerland). GR 32191 was kindly provided by Dr. B. Bain (Glaxo, Greenford, Middlesex, United Kingdom). Adenosine 5'-triphosphate, arachidonic acid, bovine Platelet Preparation and Labeling--
Peripheral blood from
healthy volunteers, who had not received any medication for at least 10 days, was collected into ACD-A anticoagulant (National Institute of
Health formula: 0.8% citric acid, 2.2% trisodium citrate,
2H2O, 2.45% glucose) and 1 mM of aspirin
unless otherwise indicated. Informed consent was obtained from all
donors in conformity with the French Etablissement de Transfusion
Sanguine committee. The blood was centrifuged at 120 × g for 20 min at 20 °C, and platelet-rich plasma was
collected, acidified with ACD-A, and further centrifuged at 1200 × g for 20 min. The platelets were washed in a
phosphate-free modified tyrode buffer without calcium (Buffer A, pH
6.8: 136 mM NaCl, 2.7 mM KCl, 12 mM
NaHCO3, 2 mM MgCl2, and 5 mM glucose) in the presence of 0.1 µM
PGE1. Platelets were resuspended at 109/ml in
the same buffer and labeled with 1 mCi/ml of
[32P]orthophosphate for 1 h 30 min at room
temperature (28). After further washing in the same buffer, platelets
were resuspended in the reaction buffer (Buffer A containing 2 mM CaCl2 and 0.4 mM
NaH2PO4, pH 7.4) at 4 × 108/ml and allowed to rest at room temperature for 1 h
before aggregation was performed.
Platelet Activation and Aggregation--
Unless otherwise
indicated, the platelet suspension was incubated at 4 × 108/ml (0.4 ml per sample) in an aggregometer cuvette for 1 min at 37 °C. Different agonists or vehicle were then added, with
constant stirring for 2 min. Platelets were incubated without stirring in the presence or absence of the TP agonist for varied periods of time
(0.5-60 min) in the kinetic experiments. SQ 29548, RGES, RGDS,
Ro-43-5054, and Ro-44-9883 were preincubated for 1 min prior to
platelet activation. PKC inhibitors were incubated for 30 min at
37 °C. Me2SO and ethanol concentrations did not exceed
0.05% and did not modify platelet function or the pattern of
phosphorylation of TP
Samples were further treated to immunoprecipitate the TPs as described
below. The phosphorylation of pleckstrin p-47, a PKC substrate, was
assessed by SDS-PAGE of 20 µl of total platelet lysate (29, 30).
TxB2, the inactive hydrolysis product of TxA2,
was measured in the platelet lysates by enzyme immunoassay (31).
Immunoprecipitation of Human TPs from Platelets or Cells
Overexpressing TPs--
Immunoprecipitation of TPs was performed using
specific polyclonal antibodies for human TP isoforms (27). Briefly,
these antibodies were directed against peptides located at the end of the carboxyl-terminal tail of either TP
In some cases, immunoblot analysis of the TP isoforms was performed.
SDS-polyacrylamide gels were transferred onto nitrocellulose membranes.
The TPs were visualized using the Ab PNGase F
Digestion--
[32P]Pi-labeled platelets
were incubated with 10 nM I-BOP for 2 min. Labeled
HEK-TP Stimulation of Tyrosine Phosphorylation--
Platelets were
exposed to high or low concentrations of I-BOP, thrombin, or
pervanadate, an inhibitor of tyrosine phosphatases, which induced
strong tyrosine phosphorylation. After a 4-min incubation, platelets
were lysed as described above, and tyrosine phosphorylation was assayed
in total platelet lysates or after immunoprecipitation of the TP Immunodetection of TP
We next checked that the broad protein band isolated from platelets
with Ab Phosphorylation of the TP
Low concentrations of I-BOP (
Since previous pharmacological studies suggested the presence of high
and low affinity receptors in human platelets, we investigated the
relevance of these observations in the phosphorylation of TPs in human
platelets. We used a particular TP antagonist, GR 32191, that
dissociates very slowly, if at all, from the low affinity binding sites
in human platelets (4). Fig. 2D illustrates this effect.
When platelets derived from PRP treated with 1 µM of
GR32191, no increase in TP
Further characterization of this phosphorylation showed that okadaic
acid, an inhibitor of serine/threonine phosphatases, resulted in an
increase in TP iPF2 Endogenously Formed TxA2 Phosphorylates TP Heterologous Phosphorylation of the TP Effect of PKC on TP Involvement of Integrin Gp IIb/IIIa in the Phosphorylation of
TP Although mRNA detection for the two recognized human TP
isoforms has been reported in human platelets (13), it is unknown whether either or both are translated to protein. It is also unknown whether these isoforms relate to the high and low affinity forms of the
receptors that have been characterized pharmacologically (4). Using
isoform-specific antibodies, we were able to immunoprecipitate TP In the present studies, Tx analogs induced rapid agonist-induced
phosphorylation of a broad protein band in platelets. Many arguments
support that this broad phosphorylated protein band appears to
correspond to the TP There is presently no information that relates either TP TxA2 originating in platelets from exogenous
(i.e. addition of arachidonic acid) or from endogenous
arachidonic acid (i.e. low concentrations of collagen)
caused phosphorylation of TP Other platelet agonists, such as thrombin, high concentrations of
collagen, PMA, and A23187, also induce TP Phosphorylation of TP Another difference involves the absence of response of this receptor to
the isoprostanes in human platelets. iPF2 In conclusion, we have demonstrated that the TP and
. Both isoforms are rapidly phosphorylated in
response to thromboxane agonists when overexpressed in human embryonic
kidney 293 cells; this phenomenon is only slightly altered by
inhibitors of protein kinase C. Pharmacological studies have defined
two classes of TP in human platelets; sites that bind the agonist I-BOP
with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TP
, but not TP
, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon
activation of platelets for 2 min with I-BOP. This was a rapid
(~30 s) and transient (maximum, 2-4 min) event and was
inhibited by TP antagonists. Both arachidonic acid and low
concentrations of collagen stimulated TP
phosphorylation, which was
blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TP
phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of
these sites with the isoprostane iPF2
-III induced
platelet shape change but not TP
phosphorylation. Heterologous TP
phosphorylation was observed in aspirin-treated platelets exposed to
thrombin, high concentrations of collagen, and the calcium ionophore A
23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TP
was blocked by protein kinase C inhibitors. TP
was the only isoform detectably translated in human platelets. This
appeared to correspond to the activation of the low affinity site
defined by the antagonist GR 32191 and not activated by the high
affinity agonist, iPF2
-III. Protein kinase C played a
more important role in agonist-induced phosphorylation of native TP
in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TP
.
INTRODUCTION
Top
Abstract
Introduction
References
isoform) implied its membership in the G protein-coupled receptor superfamily (GPCR) (7-10). Only one TP gene has been cloned to date (11). However, an
alternatively spliced form of TP, TP
, was cloned from an endothelial cDNA library (12). The mRNA for both splice variants have been demonstrated in platelets (13). Because no pharmacological ligand can
presently distinguish between these two isoforms, it is still unknown
how they relate to the pharmacological subtypes of the TP in human
platelets. We (14, 15) and others (16) have also shown that the
isoprostane, iPF2
-III (formerly known as
8-iso-PGF2
) (17), induces platelet shape
change, calcium mobilization (15) and reversible aggregation at high
concentrations. Although these effects are inhibited by TP antagonists
(18), it is unknown whether the isoprostane acts solely via TPs.
Evidence consistent with the possibility of receptors specific to
iPF2
-III was described in platelets and vascular smooth
muscle cells (14, 19).
2-adrenergic receptors (20, 21). Different kinases can phosphorylate these receptors: for
example, G-protein receptor kinases (GRKs) are receptor-specific kinases that phosphorylate the agonist-occupied receptor, whereas PKC
or PKA can be activated by other ligands and participate in heterologous receptor desensitization (20, 22). Although it is
appreciated that the role of distinct kinases in the phosphorylation of
a particular GPCR may vary according to cell type, study of this
process has largely been confined to heterologous expression systems
(23-25). There are actually few reported studies of agonist-induced phosphorylation of endogenous receptors (26), probably because they are
usually expressed in relatively low abundance. We have previously
described isoform-specific antibodies for TPs (27). Using these
reagents, we now report that only TP
is detected in human platelets.
Upon activation of the platelets with a TP agonist or arachidonic acid
as a source of endogenous TxA2 a rapid, transient, and
PKC-dependent phosphorylation of the TP
occurs. This
involves the low affinity form of the TP, as defined by irreversible binding of GR 32191. Furthermore, TP
may also be phosphorylated in a
PKC-dependent manner in response to platelet activation by thrombin in aspirin-treated platelets. The major role of PKC in rapid,
agonist-dependent phosphorylation of endogenous TP
in platelets contrasts with our previous observations when recombinant TP
was overexpressed in HEK-293 cells (27).
EXPERIMENTAL PROCEDURES
-thrombin (285 units/mg of protein), benzamidine hydrochloride, calcium ionophore
A23187, deoxycholic acid, flurbiprofen, forskolin, leupeptin, phorbol 12-myristate 13-acetate (PMA), H-Arg-Gly-Asp-Ser-OH
(RGDS), H-Arg-Gly-Glu-Ser-OH (RGES), sodium orthovanadate,
sodium fluoride, sodium pyrophosphate, and acetylsalicylic acid
were purchased from Sigma. Hydrogen peroxide
(H2O2) was from Aldrich. Nonidet P-40 was from
BDH (Poole, United Kingdom). Bisindolylmaleimide I or GF109203X,
Ro-31-8220, and okadaic acid were from Calbiochem (San Diego, Ca).
Collagen was from Diagnostica Stago (Asnières, France) and was
prepared according to the manufacturer's instructions. Prostaglandin
(PG) E1, PGE2, iPF2
-III, I-BOP,
U 46619, and SQ 29548 were obtained from Cayman Chemical Co. (Ann
Arbor, MI). PNGase F (500 units/µl) was from New England Biolabs
(Beverly, Ma). [32P]Orthophosphate (~5000-6000
Ci/mmol) was from ICN (Costa Mesa, CA). ECL chemiluminescence reagents,
CNBr-activated Sepharose, and E-Z-SEP® polyclonal kit were
purchased from Amersham Pharmacia Biotech. P-Tyr monoclonal antibody (4 G 10) was from UBI (Lake Placid, Ny). All electrophoresis reagents were
from J. T. Baker (Phillipsburg, NJ).
. The reaction was stopped using 1 volume of
2× radioimmune precipitation buffer (1× radioimmune precipitation
buffer: 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet-P40 (v/v), 0.5% sodium deoxycholate
(w/v), 0.1% SDS (w/v), containing 10 mM sodium fluoride,
10 mM sodium orthovanadate, 25 mM sodium
pyrophosphate, 10 mM ATP, 1 µg/ml leupeptin, 10 µg/ml
soybean trypsin inhibitor, 1 mM benzamidine hydrochloride,
and 0.5 mM phenylmethylsulfonyl fluoride).
or TP
:
NH2-SLSLQPQLTQRSGLQ-COOH (referred to as Ab
) for TP
and NH2-(C)-PFEPPTGKALSRKD-COOH (referred to as Ab
) for
TP
. Immunoaffinity columns with each antibody were prepared as
follows. Briefly, antisera were first partially purified using the
E-Z-SEP® kit and further incubated with CNBr-activated
Sepharose, according to the manufacturer's instructions.
Immunoglobulins derived from nonimmune rabbit serum, coupled to
CNBr-activated Sepharose, were used to preclear the platelet lysates.
After preclearing for 1 h at 4 °C, using 50 µl of normal
rabbit IgG covalently coupled to Sepharose CL-4B, samples were
immunoprecipitated overnight at 4 °C using 50 µl of immunoaffinity
Sepharose for either antibody. The beads were washed four times with 1 ml of radioimmune precipitation buffer and resuspended in 100 µl of
1× Laemmli buffer (4% SDS (w/v), 5% glycerol (v/v), 60 mM Tris, pH 6.8, 2 M urea, and 0.005% bromphenol blue) under nonreducing conditions. Samples were vigorously vortexed for 15 min, centrifuged for 5 min at 10,000 × g, and loaded on SDS-polyacrylamide gels as described
previously (27). Analysis of radioactivity in the samples was performed
using a Fuji BioImaging Analyzer (Fuji, Tokyo, Japan) after the gels
were dried.
or Ab
followed by a donkey
anti-rabbit antibody coupled to horseradish peroxidase (Jackson
ImmunoResearch, West Grove, PA). Positive protein bands were revealed
by ECL according to the manufacturer's instructions.
or HEK-TP
was incubated for 10 min with 300 nM
U 46619 as defined previously (27). TP
or TP
were
immunoprecipitated as described above. Immunoprecipitates were further
denatured for 10 min at 90 °C with SDS 0.5% and
-mercaptoethanol
1% prior to the addition of 1250 units of PNGase F per reaction
according to the manufacturer's instructions. The reaction was carried
on for 1 h at 37 °C and then stopped with 1× Laemmli buffer.
Samples were subjected to SDS-PAGE, and dried gels were exposed to
Biomax MS films.
receptor isoform by immunoblot analysis, using a specific P-Tyr
monoclonal antibody.
RESULTS
in Human Platelets--
We used
polyclonal antibodies raised against specific sequences of TP
or
TP
, to isolate TPs from human platelets. Polyclonal antibodies
specific for TP
(Ab
) were used to immunoprecipitate 1 mg of human
platelet lysate, which corresponds to 0.3 pmol/mg of protein, as
assessed by binding of [3H]SQ 29548. A broad band with a
molecular weight of 50-60 was detected after immunoblot analysis with
the same antibody (Fig. 1A).
Ab
also immunoprecipitated the TP
from HEK-293 cells stably transfected with the corresponding cDNA (Fig. 1A) as
described previously (27). However, immunoprecipitation of 1 mg of
total platelet protein lysate using the TP
isoform-specific
antipeptide antiserum failed to reveal any detectable band (Fig.
1A), although these antibodies were able to
immunoprecipitate the TP
receptor isoform from HEK-293
overexpressing these receptors (Fig. 1A). These antibodies
were able to immunoprecipitate as little as 50 fmol of receptors/mg of
protein from HEK-293 overexpressing either TP
or TP
. Using these
cells, we have previously shown that these antibodies were both
isoform-specific by Western blotting and by immunofluorescence analysis
(data not shown).
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Fig. 1.
Immunodetection of the TP in human
platelets. A, 1 mg of lysate obtained from human
platelets (Plt) or HEK-293 overexpressing TP or TP
(HEK-TP
or HEK-TP
) was immunoprecipitated with Ab
or Ab
linked to Sepharose. This corresponded to 0.3 pmol/mg of protein for
platelets and 1.6 pmol/mg for HEK-TPa and HEK-TPb, respectively.
Samples were subjected to SDS-PAGE, and immunoblot analysis was
performed using corresponding antibodies as described under
"Experimental Procedures." These data are representative of two
similar experiments for HEK-293 cells and at least five experiments for
platelets. B, cell lysates obtained from
[32P]Pi-labeled platelets or HEK-TP
or
TP
activated with Tx analogs were immunoprecipitated using Ab
or
Ab
. Immunoprecipitated samples were further incubated in the absence
or presence of PNGase F for 1 h at 37 °C. Samples were
subjected to SDS-PAGE. Electrophoresis gel was dried and exposed
to Biomax MS films.
was glycosylated. Results were compared with the digestion
of TPs in HEK-293-TP
or TP
. To avoid technical problems
subsequent to deglycosylation of the antibodies used for
immunoprecipitation, we performed these experiments on phosphorylated TPs, which are obtained after activation with TP agonist, as
demonstrated below. Deglycosylation with PNGase F resulted in a shift
of the broad protein band from 50-60 kDa in platelets and from 55-70 kDa HEK-293 cells, to an apparent molecular weight of 28. Deglycosylation of TP
also revealed a shift in the molecular weight
to 32.5. These results indicate that the broad protein band of 50-60
kDa observed in platelets corresponds to glycosylated TP
and that TP
in HEK-293 cells and human platelets are differentially
glycosylated. The difference in the apparent molecular weight between
deglycosylated TP
and TP
corresponds to the difference in the
number of amino acids between the two isoforms (343 amino acids for
TP
and 407 for TP
).
Isoform in Human
Platelets--
Homologous and heterologous desensitization of human
platelets in response to U46619, a TxA2 mimetic, or to
thrombin has been reported previously (32, 33). Incubation of
aspirin-treated platelets with I-BOP, a Tx analog, for increasing
periods of time, resulted in the phosphorylation of a broad protein
band of 50-60 kDa (Fig. 2A).
Phosphorylation was rapid (
0.5 min) but transient (maximum, 2-4
min). We regularly observed a phosphorylated band of 68 kDa in these
samples, with a stronger signal in activated platelets. Detection of
this band was not modified when immunoprecipitation of TP
was
performed in the presence of the specific Ab
-peptide used for
immunization, whereas immunoprecipitation of the broad protein band of
50-60 kDa was completely abolished (data not shown), suggesting that
it is not related to TP receptors.
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Fig. 2.
I-BOP-induced TP
phosphorylation. [32P]Pi-labeled
platelets (0.4 × 109/ml, 0.4 ml) were incubated under
different conditions. The reaction was stopped using 2 volumes of 2×
RIPA. Samples were immunoprecipitated using Ab
-Sepharose and further
subjected to SDS-PAGE as described in legend for Fig. 1. Radioactive
signals were analyzed using a Fuji imaging analyzer. A,
kinetic of TP
phosphorylation. Platelets were incubated with 10 nM I-BOP under nonstirring conditions for increasing
periods of times. Results are representative of two experiments.
B, dose-dependent phosphorylation of TP
was
performed in the presence of I-BOP (0.1-30 nM).
Corresponding aggregation was expressed as percentage of light
transmission. Pleckstrin or p-47 phosphorylation was identified after
autoradiography of SDS-PAGE electrophoresis of 20 µl of total
platelet lysates. These results are representative of three similar
experiments. C, platelets were incubated in the presence or
absence of 10 µM of SQ 29548 for 1 min at 37 °C prior
to the addition of 10 nM I-BOP for 2 min. Data are
representative of 3-5 experiments. D, effect of blocking
low affinity binding sites of TP on TP
phosphorylation using GR
32191. Platelet-rich plasma was treated in the presence or absence of 1 µM of GR 32191 for 1 h at room temperature prior to
platelet washing and labeling. Washed platelets were further incubated
with 10 nM I-BOP. Platelets derived from platelet-rich
plasma untreated with GR 32191 were further incubated in the presence
or absence of 0.1 µM GR 32191 prior to the addition of
I-BOP. p-47 phosphorylation was performed as described for
B. These data are representative of two similar
experiments.
0.3 nM) that induced only
shape change in the absence of platelet aggregation (32, 34) failed to
phosphorylate pleckstrin or induce aggregation (Fig. 2B).
Under these conditions, no phosphorylation of TP
was observed (Fig. 2B). Only higher concentrations of I-BOP (>1-30
nM) induced reproducible phosphorylation of TP
(Fig.
2B). SQ 29548, a TP antagonist, suppressed phosphorylation
of TP
. (Fig. 2C).
phosphorylation was observed with I-BOP
10 nM. TP
in platelets derived from untreated-PRP were
normally phosphorylated by I-BOP and GR 32191 inhibited this
phosphorylation, similarly to SQ 29548.
phosphorylation. Under these conditions, receptor
phosphorylation was sustained for up to 30 min, compared to 4 min in
the absence of okadaic acid (data not shown). Immunoblot analysis of
immunoprecipitated TP
using P-Tyr antibodies did not reveal any
phosphorylation of TP
in platelets activated with I-BOP, thrombin,
or pervanadate, a strong inhibitor of tyrosine-phosphatases (35),
despite marked tyrosine kinase-dependent substrate
phosphorylation (data not shown).
-III Does Not Induce Phosphorylation of the
TP
Receptor--
Previous studies by our group and others have
shown that iPF2
-III induces platelet shape change (14,
16), Ca2+ mobilization, and reversible platelet aggregation
at high concentrations of the agonist (15, 18). All of these effects
were abolished by TP antagonists. However,
iPF2
-III-induced inositol phosphate formation in human
platelets was not blocked by TP antagonists (14). Consistent with this
observation, we failed to observe TP
phosphorylation with 5-50
µM iPF2
-III (Fig.
3A). Although iPF2
-III induced platelet shape change, neither
prolongation of the incubation time (5 min) (Fig. 3A) nor
pretreatment with 1 µM okadaic acid induced significant
TP
phosphorylation as compared with control unstimulated platelets
(data not shown). Thus, iPF2
-III appears to favor
activation of the high affinity sites, which mediates platelet shape
change. In contrast, pretreatment of platelets with 50 µM
of iPF2
-III reduced I-BOP-induced platelet aggregation (60%) and TP
phosphorylation (Fig. 3B), consistent with
a competition between I-BOP and high concentrations of
iPF2
-III for the occupancy of the low affinity sites,
which mediate agonist-induced phosphorylation of TP
in human
platelets. Moreover, activation of HEK-TP
cells with
iPF2
-III resulted in TP
phosphorylation (data not
shown).
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Fig. 3.
Effect of
iPF2 -III on the phosphorylation of
TPs. A, platelets were incubated in the absence or
presence of I-BOP (10 nM) or iPF2
-III (5-50
µM) for 2 or 5 min (iPF2
-III, 50 µM). These data are representative of three experiments.
B, platelets were pretreated with or without 50 µM of iPF2
-III under nonstirring
conditions prior to the incubation with 10 nM I-BOP for 2 min. These results represent two similar experiments. TP
phosphorylation was detected as described in the legend for Fig.
2.
: Effect
of arachidonic acid and low concentrations of collagen--
We next
tested whether endogenously formed TxA2 induces
phosphorylation of the platelet TP
. Addition of arachidonic acid (2.5 µM) to platelets resulted in the formation of
200-400 ng/ml of TxB2 (corresponding to 0.2 × 109 platelets). Under these conditions, TP
was
phosphorylated, to a degree similar to platelets, when incubated with
10 nM I-BOP (Fig. 4). When
platelets were pretreated with 10 µM of SQ 29548 or
flurbiprofen, an inhibitor of cyclooxygenase, TP
phosphorylation was
inhibited, demonstrating that endogenous TxA2 (or
PGH2) formed by cyclooxygenase-1 was responsible for
receptor phosphorylation in response to arachidonic acid (Fig. 4).
Similar results were obtained with a low concentration of collagen
(Fig. 4). These results demonstrate that TP
phosphorylation can
occur in activated platelets via endogenous TxA2
generation. In these samples, platelet aggregation and pleckstrin
phosphorylation were also inhibited by SQ 29548 and flurbiprofen
treatment.
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Fig. 4.
Phosphorylation of TP
receptor by arachidonic acid or low concentration of
collagen. Non-aspirin-treated platelets were incubated in the
absence or presence of 10 µM flurbiprofen
(Flur) or SQ 29548 (SQ) prior to the addition of
2.5 µM of arachidonic acid or 25 µg/ml of collagen.
TP
or p-47 phosphorylation and corresponding aggregation data are
analyzed as described in the legend for Fig. 2. These data are
representative of three similar experiments.
--
Because
heterologous activation of platelets by non-thromboxane agonists may
contribute to the desensitization of TP
(32), we examined the
ability of various agonists to phosphorylate TP
. We utilized
aspirin-treated platelets, thus excluding signaling via endogenous
TxA2 formation. Thrombin, calcium ionophore
A23187 and the active phorbol ester PMA, phosphorylated the TP
(Fig. 5A). In these experiments, the
absence of endogenous TxA2 was verified by measuring
TxB2 in platelet lysates by enzyme immunoassay (data not
shown). Phosphorylation of pleckstrin was also observed (Fig.
5A). In contrast, little phosphorylation of either substrate was obtained with 200 nM PGE1 or
PGE2 or with 10 µM forskolin (Fig.
5A). Although platelet aggregation induced by low
concentrations of collagen is dependent on the formation of
TxA2 (Fig. 4), higher concentrations can bypass this
inhibition. When collagen was used at 100 µg/ml, neither flurbiprofen
nor SQ 29548 prevented platelet aggregation and phosphorylation of the
TP
and pleckstrin (Fig. 5A). Thrombin-induced
phosphorylation of TP
was transient (Fig. 5B) and
resembled kinetics observed with I-BOP-induced phosphorylation (described in Fig. 2A).
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Fig. 5.
Phosphorylation of TP
by heterologous agonist stimuli. A,
[32P]Pi-labeled aspirin-treated platelets
were incubated in the absence or presence of 0.2 units/ml thrombin, 500 nM PMA, 2 µM Ca2+ ionophore
A23187, 200 nM PGE2, 200 nM
PGE1, or 10 µM forskolin (FK) for
2 min. Non-aspirin-treated platelets were incubated in the presence or
absence of 10 µM of Flurbiprofen or SQ 29548 for 1 min at
37 °C prior to the addition of 100 µg/ml of collagen. Platelet
aggregation, TP
or p-47 phosphorylation were assayed on these
samples as described in legend for Fig. 2. B, TP
phosphorylation was assayed in platelets incubated with 0.2 units/ml
thrombin for increasing periods of time (0.5-60 min) under nonstirring
conditions. Data are representative of three similar experiments for
A and B.
Phosphorylation--
Because we observed
that TxA2 and all other agonists tested induced
phosphorylation of TP
and pleckstrin, we utilized specific PKC
inhibitors to address the role of this kinase in TP
phosphorylation. Pretreatment of platelets for 30 min at 37 °C with two structurally distinct but specific PKC inhibitors, GF 109203X and Ro-31-8220, prior
to platelet activation with I-BOP, resulted in a dramatic reduction in
TP
phosphorylation (~80%) (Fig. 6).
Thrombin-induced TP
phosphorylation was also inhibited by GF 109203X
(Fig. 6, right panel). The effectiveness of these molecules
as inhibitors of PKC was assessed by their capacity to inhibit the
PMA-dependent phosphorylation TP
(Fig. 6, right
panel). Our recent studies on the phosphorylation of recombinant
TP isoforms stably expressed in HEK-293 cells showed little involvement
of PKC in response to TxA2 mimetics, although PMA could
readily induce PKC-dependent TP phosphorylation in this
system (27). Thus, agonist-induced phosphorylation of TP
in human
platelets, in contrast to the HEK-293 expression system, appears
largely dependent on PKC.
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Fig. 6.
Effect of PKC inhibition on the
phosphorylation of TP in human platelets.
[32P]Pi-labeled washed platelets (0.4 × 109/ml, 0.4 ml) were incubated for 30 min in the absence or
presence of 5 µM GF 109203X (GF) or
Ro-31-8220 (Ro) prior to activation with 10 nM
I-BOP, 500 nM PMA, or 0.2 units/ml thrombin
(Thr) for 2 min. Analysis of phosphorylated TP
was
performed as described in legend for Fig. 2. The data are
representative of at least five experiments.
--
Activation of platelets by U 46619, a stable Tx analog, has
been shown to result in the association of pp60src with the
cytoskeleton (36). Such events, related to ligand occupancy of
GpIIb/IIIa, may play a role in the phosphorylation of TP
via
"inside-out" signaling (37). Thus, the influence of platelet
aggregation on TP
phosphorylation was investigated. The active
peptide, RGDS, and two peptide mimetics that are antagonists of
GpIIb/IIIa, Ro-43-5054 and Ro-44-9883, were used. The phosphorylation of TP
by I-BOP was unaffected in the presence of 50 µM
RGDS, 0.1 µM Ro-43-5054, or 0.2 µM
Ro-44-9883 (Fig. 7A). Under
these conditions, I-BOP-induced platelet aggregation was totally
inhibited (Fig. 7B). Also, phosphorylation of TP
induced
by I-BOP was unchanged under either stirring or nonstirring conditions
(data not shown). In a few blood donors, we observed a small increase
(~20%) in TP
phosphorylation in nonaggregating conditions (data
not shown). Moreover, TP
phosphorylation induced by low or high
concentrations of collagen was not modified by RGDS (Fig.
7C), thus dissociating platelet TP receptor phosphorylation
from aggregation. These results suggest that engagement of the
GpIIb/IIIa complex is downstream of the events leading to
agonist-induced phosphorylation of TP
.
View larger version (41K):
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Fig. 7.
Effect of platelet aggregation on
TP phosphorylation. A,
[32P]Pi-labeled platelets were pretreated
with 50 µM RGDS (lane 3), 50 µM
RGES (lane 4), 0.1 µM Ro-43-5054 (lane
5), or 0.2 µM Ro-44-9883 (lane 6) prior
to the incubation with 10 nM I-BOP for 2 min. Lane
1 corresponds to unstimulated platelets and lane 2 to
the platelets treated with I-BOP alone. B, aggregation
pattern corresponding to A. C, labeled platelets
were treated with 50 µM of RGDS prior to the addition of
25 or 100 µg/ml of collagen for 2 min. Samples were further analyzed
for TP
receptor phosphorylation. These results summarize four
similar experiments for A and B and two
experiments for C.
DISCUSSION
as
a 50-60-kDa protein band from platelet lysate. TP
could not be
detected. We estimate that
50 fmol/mg of protein of TP
could
be detected with the specific antibodies from binding experiments in
HEK-293 cells transfected with the TP
isoform. These results suggest
that TP
is expressed at very low levels, if at all, in human platelets.
isoform. Thus, (i) the broad protein band is
specifically immunoprecipitated with the Ab
antibodies and migrates
at the same molecular weight as that revealed by immunoblot analysis,
(ii) digestion of immunoprecipitated TP
with PNGase F results in an
apparent molecular weight similar to that obtained from HEK-293 cells
transfected with recombinant TP
, and (iii) the phosphorylation of
the 50-60-kDa protein band is associated with TP receptor activation.
SQ 29548, a TP receptor antagonist, suppress agonist-induced
phosphorylation of this band. Previous results by different groups (5,
38, 39) have detected TP receptors as a broad protein band of 50-57
kDa in human platelets. Other authors have reported a sharp protein
band of 55 kDa obtained from oligodendrocytes, neuronal cells (40), rat
aorta (39), or human platelets (6). This discrepancy may reflect
differential sensitivity of the detection systems involved (ligand
affinity or immunoaffinity purification systems).
or TP
to
the subtypes of TPs that have been defined pharmacologically (4). In
the present studies, rapid agonist-induced phosphorylation of TP
appeared to involve signaling through low affinity binding sites. Thus,
neither low concentrations of the agonist I-BOP, which induce platelet
shape change, nor high agonist concentrations on platelets pretreated
with GR 32191 (which blocks the low affinity sites) caused TP
phosphorylation.
. Thus, endogenous TxA2 (or
PGH2) can bind to and activate the receptor, resulting in
its phosphorylation in a manner similar to that observed using the
synthetic ligand I-BOP.
phosphorylation in
aspirin-treated platelets. It is possible that this phosphorylation relates to homologous or heterologous desensitization of the TP by
other platelet agonists (32, 41). Examples of heterologous phosphorylation of GPCRs include endothelin-dependent
phosphorylation of
1B-adrenoreceptors (42) and
thrombin-dependent phosphorylation of the prostacyclin
receptor (43).Our results suggest that I-BOP, thrombin, and
PMA-induced TP
phosphorylation were dependent on PKC because
specific PKC inhibitors suppressed TP
phosphorylation.
occurs in response to PMA in both platelets
and transfected HEK-293 cells (27). This indicates that PKC
phosphorylation sites are present in TP
. However, the role of this
kinase in mediating agonist-induced TP
phosphorylation differs
between native receptors in human platelets and recombinant TP
stably expressed in HEK-293 (27). Differences in affinities, or in the
relative abundance of the receptors, or in the amounts of the kinases
in different cells could explain this diversity of response.
-III increased
phosphorylation of TP
in the expression system. However, in human
platelets, iPF2
-III failed to cause a
dose-dependent increase in TP
phosphorylation, despite
stimulating inositol phosphate formation as described earlier (44).
isoform exists in
human platelets; TP
is much less abundant, if it is expressed at
all. Phosphorylation of TP
is consistent with the activation of the
low affinity site defined pharmacologically with GR 32191. Our results
suggest that human platelet TP
is phosphorylated by TxA2
analogs and by other platelet agonists, such as thrombin, through
activation of PKCs. Differences in the regulation of the Tx-dependent TP phosphorylation in the HEK-293
overexpressing system, where PKC is of marginal importance, could
derive from differences in cellular contents of kinases and their
affinity for the receptors in the presence of their ligands. Thus,
heterologous expression systems afford sufficient levels of protein to
simplify the study of posttranslational modifications of GPCRs.
However, such observations may not accurately mimic the regulation of
all endogenous receptors in their native milieu.
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ACKNOWLEDGEMENT |
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We thank Dr. Sylviane Lévy-Toledano for helpful advice and continuous support.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (HL 5400), INSERM, and the Ministère de l'Education Nationale (Grant ACC-SV9).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of Dr. Jacques Maclouf,
deceased on July 14, 1998. Jacques Maclouf was a precious mentor and friend.
§ A. Habib was supported by fellowships from Association Sanofi Thrombose pour la Recherche and the Fondation pour la Recherche Médicale. To whom correspondence should be addressed. E-mail: aida.habib{at}inserm.lrb.ap-hop-paris.fr.
Robinette Foundation Professor of Cardiovascular Medicine.
The abbreviations used are:
Tx, thromboxane; TP, TxA2 receptor; PG, prostaglandin; GPCR, G-protein coupled
receptor; PKC, protein kinase C; HEK-293, human embryonic kidney 293 cells; HEK-TP or HEK-TP
, HEK-293 cells stably overexpressing
TP
or TP
, respectively; PMA, phorbol 12-myristate 13-acetate; p-47, pleckstrin; PAGE, polyacrylamide gel electrophoresis; RIPA, radioimmune precipitation buffer; I-BOP, [1S-1
,2
(5z),3
(1E,3S*),4
]-7-[3-(3-hydroxy-4-(4'-iodiphenoxy)-1-butanyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-hepatonoic acid.
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REFERENCES |
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