Phosphorylation of the Thromboxane Receptor alpha , the Predominant Isoform Expressed in Human Platelets*

Aïda HabibDagger §, Garret A. FitzGeraldparallel , and Jacques Macloufdagger Dagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
References

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha  and beta . 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 TPalpha , but not TPbeta , 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 TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha -III induced platelet shape change but not TPalpha 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 TPalpha was blocked by protein kinase C inhibitors. TPalpha 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, iPF2alpha -III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha .

    INTRODUCTION
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Abstract
Introduction
References

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 TPalpha 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, TPbeta , 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, iPF2alpha -III (formerly known as 8-iso-PGF2alpha ) (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 iPF2alpha -III was described in platelets and vascular smooth muscle cells (14, 19).

Phosphorylation is an important mechanism in rapid desensitization of many GPCRs, as exemplified by the beta 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 TPalpha 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 TPalpha occurs. This involves the low affinity form of the TP, as defined by irreversible binding of GR 32191. Furthermore, TPalpha 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 TPalpha in platelets contrasts with our previous observations when recombinant TPalpha was overexpressed in HEK-293 cells (27).

    EXPERIMENTAL PROCEDURES

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 alpha -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, iPF2alpha -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).

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 TPalpha . 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).

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 TPalpha or TPbeta : NH2-SLSLQPQLTQRSGLQ-COOH (referred to as Abalpha ) for TPalpha and NH2-(C)-PFEPPTGKALSRKD-COOH (referred to as Abbeta ) for TPbeta . 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.

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 Abalpha or Abbeta 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.

PNGase F Digestion-- [32P]Pi-labeled platelets were incubated with 10 nM I-BOP for 2 min. Labeled HEK-TPalpha or HEK-TPbeta was incubated for 10 min with 300 nM U 46619 as defined previously (27). TPalpha or TPbeta were immunoprecipitated as described above. Immunoprecipitates were further denatured for 10 min at 90 °C with SDS 0.5% and beta -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.

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 TPalpha receptor isoform by immunoblot analysis, using a specific P-Tyr monoclonal antibody.

    RESULTS

Immunodetection of TPalpha in Human Platelets-- We used polyclonal antibodies raised against specific sequences of TPalpha or TPbeta , to isolate TPs from human platelets. Polyclonal antibodies specific for TPalpha (Abalpha ) 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). Abalpha also immunoprecipitated the TPalpha 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 TPbeta isoform-specific antipeptide antiserum failed to reveal any detectable band (Fig. 1A), although these antibodies were able to immunoprecipitate the TPbeta 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 TPalpha or TPbeta . 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 TPalpha or TPbeta (HEK-TPalpha or HEK-TPbeta ) was immunoprecipitated with Abalpha or Abbeta 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-TPalpha or TPbeta activated with Tx analogs were immunoprecipitated using Abalpha or Abbeta . 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.

We next checked that the broad protein band isolated from platelets with Abalpha was glycosylated. Results were compared with the digestion of TPs in HEK-293-TPalpha or TPbeta . 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 TPbeta 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 TPalpha and that TPalpha in HEK-293 cells and human platelets are differentially glycosylated. The difference in the apparent molecular weight between deglycosylated TPalpha and TPbeta corresponds to the difference in the number of amino acids between the two isoforms (343 amino acids for TPalpha and 407 for TPbeta ).

Phosphorylation of the TPalpha 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 TPalpha was performed in the presence of the specific Abalpha -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 TPalpha 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 Abalpha -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 TPalpha 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 TPalpha 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 TPalpha 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.

Low concentrations of I-BOP (<= 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 TPalpha was observed (Fig. 2B). Only higher concentrations of I-BOP (>1-30 nM) induced reproducible phosphorylation of TPalpha (Fig. 2B). SQ 29548, a TP antagonist, suppressed phosphorylation of TPalpha . (Fig. 2C).

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 TPalpha phosphorylation was observed with I-BOP 10 nM. TPalpha in platelets derived from untreated-PRP were normally phosphorylated by I-BOP and GR 32191 inhibited this phosphorylation, similarly to SQ 29548.

Further characterization of this phosphorylation showed that okadaic acid, an inhibitor of serine/threonine phosphatases, resulted in an increase in TPalpha 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 TPalpha using P-Tyr antibodies did not reveal any phosphorylation of TPalpha 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).

iPF2alpha -III Does Not Induce Phosphorylation of the TPalpha Receptor-- Previous studies by our group and others have shown that iPF2alpha -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, iPF2alpha -III-induced inositol phosphate formation in human platelets was not blocked by TP antagonists (14). Consistent with this observation, we failed to observe TPalpha phosphorylation with 5-50 µM iPF2alpha -III (Fig. 3A). Although iPF2alpha -III induced platelet shape change, neither prolongation of the incubation time (5 min) (Fig. 3A) nor pretreatment with 1 µM okadaic acid induced significant TPalpha phosphorylation as compared with control unstimulated platelets (data not shown). Thus, iPF2alpha -III appears to favor activation of the high affinity sites, which mediates platelet shape change. In contrast, pretreatment of platelets with 50 µM of iPF2alpha -III reduced I-BOP-induced platelet aggregation (60%) and TPalpha phosphorylation (Fig. 3B), consistent with a competition between I-BOP and high concentrations of iPF2alpha -III for the occupancy of the low affinity sites, which mediate agonist-induced phosphorylation of TPalpha in human platelets. Moreover, activation of HEK-TPalpha cells with iPF2alpha -III resulted in TPalpha phosphorylation (data not shown).


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Fig. 3.   Effect of iPF2alpha -III on the phosphorylation of TPs. A, platelets were incubated in the absence or presence of I-BOP (10 nM) or iPF2alpha -III (5-50 µM) for 2 or 5 min (iPF2alpha -III, 50 µM). These data are representative of three experiments. B, platelets were pretreated with or without 50 µM of iPF2alpha -III under nonstirring conditions prior to the incubation with 10 nM I-BOP for 2 min. These results represent two similar experiments. TPalpha phosphorylation was detected as described in the legend for Fig. 2.

Endogenously Formed TxA2 Phosphorylates TPalpha : Effect of arachidonic acid and low concentrations of collagen-- We next tested whether endogenously formed TxA2 induces phosphorylation of the platelet TPalpha . 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, TPalpha 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, TPalpha 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 TPalpha 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 TPalpha 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. TPalpha 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.

Heterologous Phosphorylation of the TPalpha -- Because heterologous activation of platelets by non-thromboxane agonists may contribute to the desensitization of TPalpha (32), we examined the ability of various agonists to phosphorylate TPalpha . We utilized aspirin-treated platelets, thus excluding signaling via endogenous TxA2 formation. Thrombin, calcium ionophore A23187 and the active phorbol ester PMA, phosphorylated the TPalpha (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 TPalpha and pleckstrin (Fig. 5A). Thrombin-induced phosphorylation of TPalpha 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 TPalpha 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, TPalpha or p-47 phosphorylation were assayed on these samples as described in legend for Fig. 2. B, TPalpha 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.

Effect of PKC on TPalpha Phosphorylation-- Because we observed that TxA2 and all other agonists tested induced phosphorylation of TPalpha and pleckstrin, we utilized specific PKC inhibitors to address the role of this kinase in TPalpha 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 TPalpha phosphorylation (~80%) (Fig. 6). Thrombin-induced TPalpha 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 TPalpha (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 TPalpha 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 TPalpha 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 TPalpha was performed as described in legend for Fig. 2. The data are representative of at least five experiments.

Involvement of Integrin Gp IIb/IIIa in the Phosphorylation of TPalpha -- 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 TPalpha via "inside-out" signaling (37). Thus, the influence of platelet aggregation on TPalpha 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 TPalpha 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 TPalpha 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 TPalpha phosphorylation in nonaggregating conditions (data not shown). Moreover, TPalpha 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 TPalpha .


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Fig. 7.   Effect of platelet aggregation on TPalpha 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 TPalpha receptor phosphorylation. These results summarize four similar experiments for A and B and two experiments for C.


    DISCUSSION

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 TPalpha as a 50-60-kDa protein band from platelet lysate. TPbeta could not be detected. We estimate that >=  50 fmol/mg of protein of TPbeta could be detected with the specific antibodies from binding experiments in HEK-293 cells transfected with the TPbeta isoform. These results suggest that TPbeta is expressed at very low levels, if at all, in human platelets.

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 TPalpha isoform. Thus, (i) the broad protein band is specifically immunoprecipitated with the Abalpha antibodies and migrates at the same molecular weight as that revealed by immunoblot analysis, (ii) digestion of immunoprecipitated TPalpha with PNGase F results in an apparent molecular weight similar to that obtained from HEK-293 cells transfected with recombinant TPalpha , 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).

There is presently no information that relates either TPalpha or TPbeta to the subtypes of TPs that have been defined pharmacologically (4). In the present studies, rapid agonist-induced phosphorylation of TPalpha 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 TPalpha phosphorylation.

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 TPalpha . 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.

Other platelet agonists, such as thrombin, high concentrations of collagen, PMA, and A23187, also induce TPalpha 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 alpha 1B-adrenoreceptors (42) and thrombin-dependent phosphorylation of the prostacyclin receptor (43).Our results suggest that I-BOP, thrombin, and PMA-induced TPalpha phosphorylation were dependent on PKC because specific PKC inhibitors suppressed TPalpha phosphorylation.

Phosphorylation of TPalpha occurs in response to PMA in both platelets and transfected HEK-293 cells (27). This indicates that PKC phosphorylation sites are present in TPalpha . However, the role of this kinase in mediating agonist-induced TPalpha phosphorylation differs between native receptors in human platelets and recombinant TPalpha 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.

Another difference involves the absence of response of this receptor to the isoprostanes in human platelets. iPF2alpha -III increased phosphorylation of TPalpha in the expression system. However, in human platelets, iPF2alpha -III failed to cause a dose-dependent increase in TPalpha phosphorylation, despite stimulating inositol phosphate formation as described earlier (44).

In conclusion, we have demonstrated that the TPalpha isoform exists in human platelets; TPbeta is much less abundant, if it is expressed at all. Phosphorylation of TPalpha is consistent with the activation of the low affinity site defined pharmacologically with GR 32191. Our results suggest that human platelet TPalpha 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.

    ACKNOWLEDGEMENT

We thank Dr. Sylviane Lévy-Toledano for helpful advice and continuous support.

    FOOTNOTES

* 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.

dagger 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.

parallel 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-TPalpha or HEK-TPbeta , HEK-293 cells stably overexpressing TPalpha or TPbeta , respectively; PMA, phorbol 12-myristate 13-acetate; p-47, pleckstrin; PAGE, polyacrylamide gel electrophoresis; RIPA, radioimmune precipitation buffer; I-BOP, [1S-1alpha ,2beta (5z),3alpha (1E,3S*),4alpha ]-7-[3-(3-hydroxy-4-(4'-iodiphenoxy)-1-butanyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-hepatonoic acid.
    REFERENCES
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Abstract
Introduction
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