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Am J Physiol Cell Physiol 279: C1760-C1771, 2000;
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Vol. 279, Issue 6, C1760-C1771, December 2000

Epinephrine correction of impaired platelet thromboxane receptor signaling

Patricia C. Dunlop, Linda A. Leis, and Gerhard J. Johnson

Hematology/Oncology Section, Department of Medicine, Veterans Administration Medical Center and University of Minnesota, Minneapolis, Minnesota 55417


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study evaluated the mechanism of epinephrine potentiation of platelet secretion induced by thromboxane A2 (TXA2). Dog platelets that do not secrete in response to TXA2 alone (TXA2-) were compared with dog platelets that do secrete (TXA2+) and with human platelets. TXA2- platelets had impaired TXA2 receptor (TP receptor)-G protein coupling, indicated by 1) impaired stimulated GTPase activity, 2) elevated basal guanosine 5'-O-(3-thiotriphosphate) binding, and 3) elevated Galpha q palmitate turnover that was corrected by preexposure to epinephrine. Kinetic agonist binding studies revealed biphasic dog and human platelet TP receptor association and dissociation. TXA2- and TP receptor-desensitized TXA2+ dog and human platelets had altered ligand binding parameters compared with untreated TXA2+ or human platelets. These parameters were reversed, along with impaired secretion, by epinephrine. Basal phosphorylation of TXA2- platelet TP receptors was elevated 60% and was normalized by epinephrine. Epinephrine potentiates platelet secretion stimulated by TXA2 by reducing basal TP receptor phosphorylation and facilitating TP receptor-G protein coupling in TXA2- platelets and, probably, in normal platelets as well.

G proteins; dogs; platelet activation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPINEPHRINE BINDS to alpha 2-adrenergic receptors (alpha 2-AR) and stimulates variable human platelet aggregation and secretion, and it potentiates other agonist-induced platelet activation in human and dog platelets (2, 21, 22, 26, 41). The mechanism of epinephrine-induced platelet activation has been the subject of numerous studies and considerable debate (see Ref. 41 for review). Previous investigations indicated that epinephrine did not function as a single platelet agonist but, rather, that it enhanced the activation initiated by other agonists (2, 26, 41). Banga et al. (2) found that epinephrine promoted human platelet aggregation and secretion by increasing thromboxane A2 (TXA2) formation, via activation of phospholipase A2, and by facilitating TXA2-mediated signaling. The mechanism responsible for the latter effect was not defined, but it was suggested that epinephrine potentiated platelet activation either by enhancing the binding of agonists to TXA2 receptors (TP receptors) or by the coupling of TP receptors to phosphoinositide-specific phospholipase C (PLC) (2). This hypothesis was of interest to us because of our previous observation that dog platelets, which showed little or no response to TXA2 alone, had impaired signal transduction via G proteins from TP receptors to PLC (18), but they aggregated and secreted their granular contents if exposed to epinephrine before TXA2 (6, 18, 21, 22).

Most dogs, whether random or purpose bred, have blood platelets that form TXA2, but they have very impaired secretion and aggregation in response to TXA2 (5, 6, 18, 21, 22). These TXA2-insensitive (TXA2-) dog platelets also have impaired responses to TXA2 analogs such as U-46619 or I-BOP {[1S-[1alpha ,2alpha (Z),3beta (1E,3S*),4alpha ]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo (2.2.1)hept-2-yl]-5-heptenoic acid} (18). However, some mixed breeds, and a few purpose-bred dogs, have TXA2-sensitive (TXA2+) platelets (6, 18, 21) that secrete and aggregate in response to TXA2 or TXA2 analogs as do human platelets. Each dog's platelet response to TXA2 is consistent and genetically determined (20).

TP receptors belong to the family of G protein-coupled receptors (GPCRs) that activate effectors via G proteins (16). Agonist binding to platelet TP receptors activates Galpha 13, which results in platelet shape change (24), and Galpha q, which in turn activates PLC beta -isoforms (PLC-beta ) (3, 19, 17, 34). Activation of PLC-beta liberates two important intracellular messengers: D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG). The subsequent elevation of the cytosolic ionized calcium concentration by IP3 and activation of protein kinase C by DG lead to platelet secretion. Previous investigation of the mechanism responsible for TXA2- platelets implicated defective signaling from TP receptors via G proteins to PLC-beta (18). Subsequent study found no mutation in Galpha q in TXA2- platelets (19). Therefore, we sought evidence of an alternative mechanism.

An important characteristic of dog TXA2- platelets is the reversibility of their functional defect by epinephrine. Exposure of TXA2- platelets to epinephrine before exposure to TXA2 or TXA2 analogs results in aggregation and secretion comparable to that observed in TXA2+ platelets (6, 18, 21, 22). Therefore, we studied the effects of epinephrine on several aspects of dog TXA2- platelet TP receptor signal transduction including Gq function, PLC-beta activation, TP receptor kinetic agonist binding, and phosphorylation. Because we observed that human platelets with homologously desensitized TP receptors had their defective activation of PLC-beta corrected by exposure to epinephrine in a manner similar to that of dog TXA2- platelets, we also studied the mechanism of epinephrine correction of human platelets. These studies indicated that epinephrine corrects the signaling defect of dog TXA2- platelets and desensitized human platelets by facilitating Gq-TP receptor coupling. In TXA2- platelets the corrective effect of epinephrine appears to be mediated by dephosphorylation of hyperphosphorylated TP receptors.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

[125I]BOP and [127I]BOP were obtained from Cayman Chemical (Ann Arbor, MI). U-46619 (9,11-dideoxy-9alpha ,11alpha -methanoepoxy-prostaglandin F2alpha ) was a gift from Upjohn (Kalamazoo, MI). 5-Hydroxy-3-indolyl ([1-14C]ethyl-2-amine) creatinine sulfate ([14C]5-HT), guanosine 5'-[gamma -35S]-triphosphate, and the [3H]IP3 assay system were purchased from, and [3H]U-46619 was prepared by, Amersham (Arlington Heights, IL). Guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) and oxymetazoline were obtained from Sigma Chemical (St. Louis, MO), and epinephrine was from Parke Davis (Morris Plains, NJ). Carrier-free H3[32P]O4 was obtained from ICN Biomedical (Irvine, CA). The Gamma Prep G kit was obtained from Promega (Madison, WI). All other chemicals were the best reagent grade available from commercial suppliers.

Antisera

Antisera N345 and N432 were commercially produced (HRP, Dover, PA) in rabbits by injection of synthetic peptides coupled to keyhole limpet hemocyanin (Pierce conjugation protocol). The dilution of whole antisera used is designated for each experiment. Antiserum N345 recognizes an internal sequence in Galpha q (19), and N432 recognizes a third TP receptor internal loop sequence (HGQEAAQQRPRDSEV).

Subjects

Dogs with well-characterized TXA2- or TXA2+ platelets were maintained in an American Association for Accreditation of Laboratory Animal Care-certified animal care facility. Human subjects were normal healthy volunteers who had taken no medication within the previous week. This study was approved by the Animal Studies and Human Studies Subcommittees of the Research Committee of the Minneapolis Veterans Affairs Medical Center.

Platelet Preparation

Platelets were prepared as described previously (18) and were resuspended with HEPES citrate buffer for binding assays. For receptor phosphorylation and IP3 generation studies, the resuspension buffer was HEPES Tyrode buffer (18). In [14C]5-HT secretion studies, the resuspension buffer was modified Lindon's buffer (18). Platelet membranes were prepared by the method of Baldassare et al. (1).

GTPase Activity

Platelet membrane GTPase activity was assayed by standard methods (18).

Binding of [35S]GTPgamma S to Platelet Membranes

Binding experiments were conducted as described by Gachet et al. (11). Aliquots of platelet membranes, resuspended via sonication, were diluted to ~6 mg/ml. The reaction mixture for measuring [35S]GTPgamma S binding contained 50 mM Tris · HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 µM GDP, 0.3 µM GTPgamma S (2.0-4.0 µCi [35S]GTPgamma S), buffer or agonist (12 nM I-BOP), and ~900 µg of membrane protein in a volume of 1,200 µl. The reaction was started by the addition of the membrane suspension, and duplicate samples (75 µl) were removed immediately and at 2, 4, 6, 10, 30, and 60 min after addition and diluted into 10 ml of wash buffer. The reaction was terminated by rapid filtration through 0.45-µm nitrocellulose filters under vacuum, and the filters were washed with 10 ml of buffer. Radioactivity bound to the membrane filters was determined via scintillation counting. Nonspecific binding, determined in the presence of 266 µM cold GTPgamma S, amounted to ~2.5% of the added [35S]GTPgamma S and was subtracted from the total bound radioactivity to determine specific activity. The intra-assay variation between duplicates was <5% of the mean.

[3H]Palmitate Labeling

Palmitate exchange in platelets was determined by the method of Hallak et al. (15). Platelets (3 × 109) suspended in 2 ml of acylation buffer (140 mM NaCl, 2.5 mM KCl, 0.1 mM MgCl2, 10 mM NaHCO3, 0.5 mM NaH2PO4, 5.5 mM glucose, and 10 mM HEPES, pH 7.4, containing 3.6 mg/ml fatty acid-free BSA, 1 U/ml apyrase, and 0.3 µM PGE1). [3H]palmitate (1 mCi) was dried under nitrogen and dissolved in 1 ml of acylation buffer. Both palmitate and cells were incubated at 37°C for 3 min before the reaction was initiated. For stimulated exchange reactions, 30 µl (1:200 dilution) of stock I-BOP were added to the palmitate tube (final concentration 12.5 nM I-BOP). The reaction was initiated by combining the cells with the palmitate, and 315-µl aliquots were withdrawn at 2, 5, 9, 15, 24, and 60 min, diluted into 3.75 ml of HEPES citrate containing 25 µl of 0.1 M EDTA, and centrifuged at 1,700 rpm for 10 min. The supernatant was removed, and 90 µl of SDS disruption buffer (50 mM NaPO4, pH 8.0, 0.5% SDS, and 2 mM EDTA) were added to the pellet. The cells were vortexed, heated at 90°C for 5 min, and cooled, and 30 µl of 4× RIPA (200 mM NaPO4, pH 7.2, 4% deoxycholate, 4% Triton, 0.6 M NaCl, 2% SDS, and 8 mM EDTA) plus inhibitors (1% aprotinin, 200 µg/ml leupeptin) were added. Each sample (~150 µl) was clarified, with 2 µl of preimmune sera and 25 µl of protein A-Sepharose, for 3 h before reaction with Galpha q antisera N345 (1:25 dilution) overnight in the cold. The following morning, 100 µl of 20% protein A-Sepharose were added, and the samples were mixed for 3 h at room temperature and centrifuged. The recovered immunoprecipitate was washed three times with washing buffer and then suspended in 40 µl of wash buffer plus 0.02% NaN3. Immediately before gel electrophoresis, SDS samples were prepared with low mercaptan (0.15% beta -mercaptoethanol) 3× sample buffer. Mini-gels (10.5%) were loaded with one-third total sample volume, electrophoresed, and transblotted to polyvinylidene difluoride membranes. The membranes were dried, sprayed with EN3HANCE, and exposed for 3 wk at -70°C on Reflection film (NEN). Palmitate labeling was evaluated through densitometric scanning of the radiogram and is expressed in arbitrary units.

IP3 Formation and Platelet [14C]5-HT Secretion

IP3 formation was assayed by RIA (Amersham), and [14C]5-HT secretion was measured as previously described (18).

[125I]BOP Binding

Aliquots of washed platelets (0.02-1 × 109 platelets/ml) in HEPES citrate buffer (pH 7.4) were incubated with [125I]BOP (1-2 µCi) in combination with unlabeled drug over a 100-fold concentration range (0.3-30 nM) at room temperature (~20°C). At multiple time points (association: 0.2-30 min; dissociation after 30-min incubation: 1-45 min), the binding reaction was terminated by the removal of cell aliquots that were immediately added to 10 ml of ice-cold HEPES buffer, followed by rapid filtration through Whatman GF/C glass fiber filters under reduced pressure. The filters were then washed twice with 10 ml of ice-cold buffer and counted. The entire filtration was complete in <15 s. Nonspecific binding was determined in the presence of 1 µM I-BOP and was generally <15% of the total binding. In experiments with U-46619 or epinephrine pretreatment, cells were incubated with U-46619 (1.43 µM [3H]U-46619) or epinephrine (1-2 µM) for 30 min at room temperature, washed twice, and resuspended in HEPES citrate buffer before agonist (1-2 nM [125I]BOP) binding. The residual U-46619 concentration never exceeded 14 nM.

Phosphorylation of TP Receptors

The method of Carlson et al. (4) was employed. Briefly, platelets (2.5 × 109 platelets/ml) were labeled with 0.8 mCi/ml H3[32P]O4 for 90 min at 30°C, washed by dilution with HEPES citrate buffer, spun, and resuspended in HEPES Tyrode buffer to 1.8 × 109 platelets/ml. Phosphorylation reactions were conducted at room temperature with 660 µl of labeled cells to which were added (at time 0) buffer, U-46619 (1.43 µM), epinephrine (1 µM), or epinephrine plus U-46619. To inhibit TXA2 formation, indomethacin (5 µM) was added to some samples 3 min before the beginning of the reaction. After 1 and 5 min, 300-µl samples were removed, 30 µl of 0.1 M EDTA were added to each sample, and the samples were spun at 2,000 rpm for 5 min. Supernatants were removed, 90 µl of stopping buffer plus inhibitors (50 mM NaPO4, pH 8.0, 0.5% SDS, 2 mM EDTA, 1 mM DTT, 5 mM NaF, 10 mM Na4P2O7, 2 mM Na3VO4, 1% aprotinin, 0.5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) were added to the pellets, and the pellets were vortexed until dissolved and then heated to 90°C for 3 min. To 90 µl of sample, 30 µl of 4× RIPA plus inhibitors were added, and the mixture was allowed to cool on ice for 30 min. Preimmune sera (1:50) was added, allowed to react for 1 h at 4°C, cleared with 20 µl of protein A-Sepharose for 2 h at 4°C, and centrifuged. Sample supernatant (100 µl) was removed, and TP receptor antisera N432 (1:10 dilution) was added before overnight incubation at 4°C. Protein A-Sepharose (100 µl) was used to capture the immunoprecipitate, and the material was collected by centrifugation. Immunoprecipitates were washed twice, resuspended in 40 µl of buffer plus 20 µl of 3× SDS sample buffer, and boiled for 4 min. Samples were run on 12% mini-gels, transblotted, and exposed to X-OMAT film at -70°C for 3-7 days. Phosphorylation was evaluated by densitometry and is expressed in arbitrary units.

Miscellaneous

The protein concentration was determined by dye binding using the Coomassie blue dye reagent (Bio-Rad, Hercules, CA) with ovalbumin as the standard.

Calculations

Analysis of the binding association and dissociation parameters under nonequilibrium conditions was done with the use of the computer program KINETIC (BioSoft). Statistical analysis was performed with the program Statworks using the Student's t-test. Data are expressed as means ± SE unless otherwise specified.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biochemical Studies

To define the mechanism of the corrective effect of epinephrine on TXA2- platelets, several G protein-related parameters were evaluated.

TP receptor-stimulated GTPase activity. Basal GTPase activities of TXA2- and TXA2+ platelet membranes were not significantly different from each other [42.7 ± 2.1 (n = 13) vs. 46.1 ± 2.6 pmol · min- · mg-1 (n = 10)]. The addition of U-46619 to TXA2+ membranes significantly increased GTPase activity (6.9 ± 1.2 pmol · min- · mg-1), but TXA2- membrane GTPase activity increased only slightly (2.3 ± 1.0 pmol · min- · mg-1) (Fig. 1, whole cells + buffer, top).


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  		Fig. 1.   U-46619-stimulated platelet membrane GTPase activity. Dog platelet membranes that do (TXA2+) and do not secrete in response to thromboxane A2 alone (TXA2-) were prepared after exposure of intact platelets (1 × 109 cells/ml) to either buffer (top) or 1 µM epinephrine (Epi; middle and bottom) for 30 min at room temperature. Dark-shaded bars indicate presence of U-44619; light-shaded bars indicate buffer without U-46619. The membranes were centrifuged and resuspended in buffer. Basal and agonist (1.43 µM U-46619)-stimulated GTPase activity was determined via liquid scintillation counting of intracellular 32P concentration generated from 0.4 µM [gamma -32P]GTP, at 37°C, after Norit A-charcoal removal of unreacted nucleotide. The results are expressed as percent change from basal values (means ± SE; n = 6-13 experiments). *P < 0.01 compared with control; dagger P < 0.03 compared with control; Dagger P < 0.01 compared with Epi.

TXA2+ membranes prepared from platelets exposed to epinephrine before membrane preparation demonstrated a nonsignificant decrease in GTPase activity (1.8 ± 2.4 pmol · min- · mg-1), while the GTPase activity of TXA2- membranes similarly prepared decreased significantly (4.9 ± 2.0 pmol · min- · mg-1) (Fig. 1, whole cells + Epi, middle). Subsequent addition of U-46619 to these membranes significantly elevated the GTPase activity of TXA2+ (9.8 ± 1.8 pmol · min- · mg-1) and TXA2- membranes (6.1 ± 1.6 pmol · min- · mg-1) (Fig. 1, whole cells + Epi, bottom). The increase in GTPase activity stimulated by U-46619 in TXA2- membranes obtained from platelets exposed to epinephrine before membrane preparation was approximately equal to that of buffer-exposed TXA2+ membranes (Fig. 1).

In contrast to these results, the addition of epinephrine directly to TXA2+ or TXA2- membranes resulted in a slight increase, rather than a decrease, in GTPase activity, and the addition of U-46619 to these membranes did not result in an additional increase in GTPase activity in either type of dog platelet (data not shown).

TP receptor-stimulated GTPgamma S binding. To further evaluate the effect of TP receptor agonist binding on G protein activation, we studied GTPgamma S binding to platelet membranes in the absence and presence of the TXA2 analog I-BOP (Fig. 2). I-BOP increased mean [35S]GTPgamma S binding to TXA2+ membranes at 30 min (P < 0.03, n = 5). I-BOP did not increase binding to TXA2- membranes; however, the mean basal binding of TXA2- compared with TXA2+ platelet membranes was significantly elevated at 30 min (P < 0.02, n = 5). Exposure of TXA2- platelets to epinephrine before membrane preparation decreased the basal association of [35S]GTPgamma S with TXA2- membranes, and subsequent addition of I-BOP to these membranes resulted in a significant increase in mean [35S]GTPgamma S binding at 30 min (P < 0.05, n = 2), comparable to that of TXA2+ membranes exposed to I-BOP alone. The similarity of the net I-BOP-induced increase in GTPgamma S binding of TXA2- platelets that had been preexposed to epinephrine to that of TXA2+ platelets without epinephrine preexposure is illustrated in Fig. 2C.


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  		Fig. 2.   I-BOP-stimulated platelet membrane guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) binding. Dog TXA2+ and TXA2- platelet membranes were prepared after exposure of intact platelets to either buffer or 1 µM Epi for 30 min at room temperature. Basal and agonist (12.5 nM I-BOP)-stimulated binding of [gamma -35S]GTP to platelet membranes (~56 µg membrane protein/sample) was determined at room temperature over a 75-min time course in TXA2+ (A) and TXA2- (B) platelets. C: net I-BOP-stimulated increase in specific binding to TXA2+ membranes prepared from platelets not exposed to Epi [TXA2+(-Epi)] and to TXA2- membranes prepared with [TXA2-(+Epi)] and without exposure of intact platelets to Epi [TXA2-(-Epi)]. open circle , Platelets exposed to buffer; , platelets exposed to I-BOP; , platelets pretreated with Epi; , platelets pretreated with Epi followed by I-BOP. The data presented were obtained in 1 study representative of 2-5 studies performed.

TP receptor-stimulated palmitate exchange. G protein alpha -subunits are palmitoylated at their NH2-terminal cysteines (44), and they undergo increased palmitate exchange on receptor activation. To determine whether impaired agonist-stimulated GTPase activity in TXA2- platelets was attributable to a failure to activate Galpha q, [3H]palmitate exchange labeling of Galpha q subunits of TXA2+ and TXA2- platelets was carried out. The results (Fig. 3) closely parallel those obtained from the study of GTPgamma S binding. In TXA2+ platelets, I-BOP stimulated an increased level of palmitate exchange (P < 0.04, n = 2), but in TXA2- platelets, no increase was observed. Exposure of TXA2- platelets to epinephrine before the addition of palmitate significantly decreased basal palmitate turnover compared with control cells (P < 0.05, n = 3), and the addition of I-BOP to these platelets resulted in significantly increased palmitate turnover (P < 0.03, n = 3). The similarity of the net increase in palmitate exchange of TXA2- platelets that had been preexposed to epinephrine to that of TXA2+ platelets without epinephrine preexposure is illustrated in Fig. 3C.


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  		Fig. 3.   I-BOP-stimulated platelet Galpha q-[3H]palmitate exchange. Basal and agonist (12.5 nM I-BOP)-stimulated Galpha q-[3H]palmitate exchange in dog TXA2+ (A) and TXA2- platelets (B) was determined in the presence or absence of 1 µM Epi at room temperature over a 60-min time course. The data are expressed as arbitrary radiogram density units. C: net I-BOP-stimulated increase in [3H]palmitate exchange in TXA2+ platelets not exposed to Epi and TXA2- platelets with and without exposure to Epi. open circle , Platelets exposed to buffer; , platelets exposed to I-BOP; , platelets pretreated with Epi; , platelets pretreated with Epi followed by I-BOP. The data presented were obtained in 1 study representative of 2-3 studies performed.

Effector activation subsequent to receptor agonist binding. The results of the biochemical studies reported above indicated that TP receptor-stimulated G protein activation was impaired in TXA2- platelets and that it was corrected by epinephrine. To evaluate the functional consequences of epinephrine exposure, we observed its effect on activation of PLC-beta by assaying IP3 formation in platelets with intact cyclooxygenase activity.

In TXA2+ platelets, significant IP3 formation was observed in response to U-46619 alone and epinephrine alone, while in TXA2- platelets, a much lower response to both U-46619 and epinephrine was observed (Fig. 4). The critical observation was the significant difference between the U-46619-stimulated responses of TXA2+ vs. TXA2- platelets. When U-46619 was added to platelets that had been preexposed to epinephrine, IP3 formation was significantly increased in both TXA2+ and TXA2- platelets (Fig. 4), and the results were not statistically different. IP3 formation in response to U-46619 was increased approximately threefold in TXA2- platelets that had been preexposed to epinephrine.


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  		Fig. 4.   Inositol trisphosphate (IP3) production stimulated by U-46619, Epi, and U-46619 plus Epi. IP3 production was measured by RIA in dog TXA2+ and TXA2- intact platelets 20 s after the addition of 715 nM U-46619 (solid bars), 1 µM Epi (hatched bars), or 1 µM Epi followed by 715 nM U-46619 (crosshatched). The data are presented as pmol IP3/2 × 108 platelets produced above buffer control (means ± SE; n = 5). Basal IP3 production was 1.59 ± 0.6 in TXA2+ platelets and 1.07 ± 0.1 in TXA2- platelets. *P < 0.01 compared with control. dagger P < 0.01 compared with TXA2+ with U-46619. Dagger P < 0.03 compared with Epi.

TP Receptor Kinetic Agonist Binding

To further elucidate the mechanism responsible for TXA2- platelets, we studied [125I]BOP kinetic agonist binding to TP receptors of intact TXA2+ and TXA2- dog platelets, and we compared the results with those obtained using human platelets.

[125I]BOP association kinetics. Studies of the time course of [125I]BOP binding to TXA2+ and TXA2- platelets and human platelets, performed at six different concentrations (range: 0.3-30 nM; see MATERIALS AND METHODS), yielded data that were resolved by the computer program KINETIC to two exponential phases of binding (Fig. 5A). The rate constants for the fast and slow components of binding were determined from linear regression of the secondary plots of the observed rate constant (kobs) vs. agonist concentration (Fig. 5B) and were analyzed according to the mass action equation: kobs = k- + k+[A], where the dissociation constant (Kd) is defined as k-/k+ (dissociation rate constant/association rate constant) and [A] is the agonist concentration.


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  		Fig. 5.   [125I]BOP binding kinetics. Binding studies (association, dissociation) were carried out at various concentrations over a 100-fold concentration range (0.3-30 nM) in 7 studies of TXA2+, 11 studies of TXA2-, and 5 studies of human platelets. Specific binding of [125I]BOP was determined (22°C) as outlined in MATERIALS AND METHODS. Equilibrium bound [125I]BOP dissociation was initiated with 1,000-fold cold I-BOP. A: association. Two representative plots (0.3 and 30 nM [125I]BOP) show the dependence of association on concentration. B: dependence of amplitude and rate constants of [125I]BOP association kinetics on ligand concentration. Plots show the observed rate constant of the fast component (kobs,fast) and the slow component of binding (kobs,slow) vs. [125I]BOP concentration. Values for the fast component kinetics (left) are as follows: TXA2+ platelets (), Kd1 = 0.69 nM, k-1 = 0.404 min-1, and k1 = 5.87 × 108 M-1 · min-1; TXA2- platelets (open circle ), Kd1 = 0.91 nM, k-1 = 0.837 min-1, and k1 = 9.22 × 108 M-1 · min-1; and human platelets (), Kd1 = 0.29 nM, k-1 = 0.208 min-1, and k1 = 7.29 × 108 M-1 · min-1. Values for the slow component kinetics (right) are as follows: TXA2+ platelets (), Kd2 = 3.2 nM, k-2 = 0.212 min-1, and k2 = 0.66 × 108 M-1 · min-1; TXA2- platelets (open circle ), Kd2 = 4.05 nM, k-2 = 0.259 min-1, and k2 = 0.64 × 108 M-1 · min-1; and human platelets , Kd2 = 8.94 nM, k-2 = 0.148 min-1, and k2 = 0.17 × 108 M-1 · min-1. Values were determined with the equilibrium dissociation constant (Kd) equal to k-/k+ from the results of nonlinear least-squares analysis of association profiles with KINETIC and are presented as means ± SD; n = 3-5. C: dissociation. Two representative plots (1.0 and 30 nM [125I]BOP) show the independence of dissociation rates from I-BOP concentration. For 1 nM [125I]BOP, the ligand dissociation (R0) was 37,950 cpm with nonspecific binding of 720 cpm and specific activity of 986 cpm/fmol (0.13 mg protein). For 30 nM [125I]BOP, R0 was 18,600 cpm with nonspecific binding of 2,700 cpm and specific activity of 42 cpm/fmol (0.39 mg protein).

TXA2+ platelet TP receptor association parameters yielded a calculated equilibrium dissociation constant (Kd1) of 0.69 nM for the fast component of binding and a Kd2 of 3.20 nM for the slow component. The amplitude of the fast component of binding (binding state distribution) seen in TXA2+ platelets increased with increasing agonist concentration to ~50% of maximum at ~1 nM I-BOP and remained at this value over the rest of the concentration range evaluated. TXA2- platelet TP receptor association parameters yielded a Kd1 of 0.91 nM and a Kd2 of 4.05 nM. TXA2- platelets exhibited reduced binding amplitude relative to TXA2+ platelets (maximum: 33%). For both TXA2+ and TXA2- platelets, the Kd1 values calculated for the fast component of binding were higher (Kd1 = 0.69 and 0.91 nM) than the high-affinity equilibrium Kd values (0.22 and 0.21 nM) that we previously observed (18).

Human platelet association parameters resulted in a Kd1 of 0.29 nM for the fast component of binding and a Kd2 of 8.94 nM for the slow component of binding, values that were not significantly different from those derived from prior equilibrium binding studies (0.25 and 6 nM, respectively) (18). In human platelets the amplitude of the fast component of binding was ~50% for agonist concentrations <4 nM, but subsequently it decreased with increasing substrate concentration as the Kd for the numerous low-affinity receptors was approached (18).

Because the kinetically calculated Kd1 values for both types of dog platelet were higher than expected from studies of equilibrium binding, and because comparison of the graphically determined kinetic constants (Fig. 5B) suggested that the disparity between the calculated Kd values and those observed on equilibrium binding was attributable to faster k-1 values (y-intercept values), we conducted kinetic dissociation studies to directly measure dissociation constants.

[125I]BOP dissociation kinetics. [125I]BOP dissociation rates at equilibrium (30 min at room temperature) were measured from the same ligand reaction samples used in the association studies. Biphasic kinetic patterns were observed in all three platelet types, and the rate constants were independent of agonist concentration (Fig. 5C). More than 95% of [125I]BOP-specific binding dissociated by 150 min, indicating that virtually all of the binding was reversible.

TXA2+ platelets (n = 4) exhibited a rapid dissociation of 34% of the total binding with a half-time of 3.7 min (k-1 = 0.185 min-1), while the residual 66% had a slow dissociation half-time of 14.7 min (k-2 = 0.047 min-1). TXA2- platelets (n = 8) exhibited 68% rapid dissociation with a half-time of 5.1 min (k-1 = 0.135 min-1), while the remainder dissociated with a slow dissociation half-time of 19.3 min (k-2 = 0.036 min-1). Human platelets (n = 5) demonstrated dissociation parameters similar to those of TXA2+ platelets (30% rapid dissociation, half-time of 3.8 min, k-1= 0.182 min-1; 70% slow dissociation, half-time of 13.1 min, k-2 = 0.053 min-1).

Analysis of paired association/dissociation parameters. To further evaluate TP receptor kinetic binding parameters, we performed multiple studies with agonist concentrations of 1-2 nM (to allow comparison of dog and human platelet results with minimal interference from the abundant human low-affinity receptors), and we calculated association constants (ka) using the relationship ka = [ka(obs) - k-a]/[L], where [L] is the ligand concentration. Dissociation parameters were determined concurrently. An important result of the analysis of these data was recognition of the fact that the association/dissociation relationships expected from mass action kinetics were not observed. Instead, we observed a proportional relationship between rapidly associating and slowly dissociating TP receptors of human and both dog platelet types (Fig. 6). TXA2- platelets demonstrated significantly less fast association binding (Re1) and slow bound ligand dissociation (R02), but total binding was not significantly different from that of TXA2+ or human platelets (Fig. 6).


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  		Fig. 6.   Kinetic [125I]BOP binding to unstimulated platelets. A: fast (Re1) and slow ligand association binding (Re2). B: fast (R01) and slow bound ligand dissociation (R02). Values for total (slow + fast) binding association and dissociation are 231.1 and 248.0 fmol/mg protein for TXA2+ platelets, 275.1 and 298.1 fmol/mg protein for TXA2- platelets, and 264.3 and 304.7 fmol/mg protein for human platelets, respectively. The data are derived from studies performed with 1-2 nM [125I]BOP and are presented as means ± SE; n = 3-5. *P < 0.05 compared with TXA2+.

The bimolecular rate constants were all less than that expected for a simple diffusion-controlled reaction and were comparable to those seen for other platelet GPCRs (32). The fast association rate constants (k1) for TXA2+ and human platelets were very similar, while the TXA2- platelet k1 was significantly greater (Table 1). These data are similar to those previously reported for kinetic [125I]BOP binding to intact platelets, although a direct comparison is not possible because the data were analyzed as one site rather than two (9).

                              
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  		Table 1.   [125I]BOP binding parameters

Effects of homologous or heterologous agonists on [125I]BOP binding. We evaluated the binding distribution and rate constants for [125I]BOP binding as functions of prior exposure to U-46619 (homologous agonist), epinephrine (heterologous agonist), or both to correlate change in the TP receptor functional state with change in the fast and slow components of binding.

After prior U-46619 incubation of TXA2+ platelets, significant changes were noted in several I-BOP binding parameters (Table 1). The fast rate constant (k1) increased and the proportion bound (Re1) decreased, while the slow constant (k2) decreased and the proportion bound (Re2) increased (Table 1). The amplitude of fast dissociation (R01) increased, and that of slow dissociation (R02) decreased significantly. Total binding (Re1 + Re2) decreased 56 fmol/mg. Human platelet parameters, after exposure to U-46619, were similar to those observed with TXA2+ platelets (Table 1).

In TXA2- platelets, U-46619 exposure resulted in a reduction in total binding of 103.1 fmol/mg and changes in association parameters that were similar to those seen in TXA2+ and human platelets, with two notable exceptions. In TXA2- platelets, Re2 and R01 decreased, whereas these parameters increased in TXA2+ and human platelets.

Prior exposure of TXA2+ and human platelets to 1 µM epinephrine resulted in no significant changes in association or dissociation parameters. In contrast, TXA2- platelets exposed to epinephrine showed significant increases in both association (Re1) and dissociation (R02), together with significant changes in their rate constants. Total binding increased ~34%. The greatest parameter change observed was in the dissociation profile. This was analyzed as a statistically superior single form of dissociation (Table 1). These data suggest that epinephrine directly modulated TXA2- platelet TP receptor agonist binding.

To determine whether the effect of epinephrine was mediated via alpha 2-AR, we carried out [125I]BOP binding experiments in the presence of 160 µM oxymetazoline, an alpha 2-AR antagonist. No change in platelet binding of [125I]BOP was observed with oxymetazoline alone; however, the effects of epinephrine on TXA2- platelet binding of I-BOP were eliminated in the presence of oxymetazoline (data not shown).

Finally, we investigated whether the effects of prior incubation with U-46619 on [125I]BOP binding could be reversed by subsequent exposure to epinephrine. Agonist binding rates, elevated after U-46619 exposure, were significantly decreased by epinephrine treatment to rates comparable to those observed in TXA2-, TXA2+, and human platelets subsequent to epinephrine alone or to control TXA2+ and human platelets (Table 1). Total agonist binding (Re1 + Re2) increased in all three platelet types with statistically significant increases in Re1 in TXA2-, TXA2+, and human platelets compared with the U-46619 values. The dissociation profiles similarly demonstrated significant increases in R02 (82.2-117.3 fmol/mg) in each platelet type. After epinephrine treatment of U-46619-desensitized platelets, Re1 did not increase to control or epinephrine-treated values, suggesting that U-46619 pretreatment had permanently altered total binding capability. However, the dissociation distributions (R01:R02) seen in U-46619-pretreated platelets were significantly shifted after epinephrine treatment to values comparable to those seen in functional TXA2+ and human controls. Moreover, total binding increased after epinephrine treatment comparable to the total increase observed after epinephrine treatment of control platelets.

Evidence that the changes in TP receptor binding distributions are related to functional consequences was obtained from studies of platelet [14C]5-HT secretion. TXA2- platelets, which demonstrated a minimal secretory response to agonist alone, secreted strongly after exposure to epinephrine followed by I-BOP. TXA2+ and human platelet secretion, eliminated by U-46619 desensitization, was restored after epinephrine exposure (data not shown).

TP Receptor Phosphorylation

Because our kinetic binding studies revealed that homologously desensitized TP receptors of TXA2+ and human platelets had impaired agonist binding similar to that observed in TXA2- platelets, and previous studies indicated that TP receptors were phosphorylated after desensitization (14, 23, 35), we searched for evidence that TXA2- platelet TP receptors might be constitutively phosphorylated. Platelet proteins and nucleotide pools were labeled to equilibrium with H3[32P]O4, and the platelets were exposed to U-46619 alone, epinephrine alone, epinephrine in the presence of indomethacin, or epinephrine plus U-46619.

Basal TP receptor phosphorylation and effect of epinephrine. Basal phosphorylation of TP receptor protein in TXA2- platelets was significantly higher (160%) than that observed for TXA2+ platelets (Fig. 7). Exposure to epinephrine alone increased phosphorylation of TXA2+ platelet TP receptor protein, but this increase was indomethacin sensitive, indicating that the increase was attributable to TXA2. Epinephrine decreased TXA2- platelet TP receptor phosphorylation to a level comparable to the basal state of TXA2+ platelets, and this change was not affected by indomethacin (Fig. 7).


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  		Fig. 7.   Basal and agonist-stimulated TXA2 receptor (TP receptor) phosphorylation. Platelets were labeled with H3[32P]O4, and TP receptor phosphorylation was determined in the absence (control, open bars) or presence of 1.43 µM U-46619 (solid bars), 1 µM Epi (hatched bars), or both (crosshatched bars). To inhibit TXA2 formation, 5 µM indomethacin plus Epi (shaded bars) was added to some samples before the reaction. Phosphorylation was evaluated by densitometry scanning and is expressed in arbitrary units as a percentage of the basal phosphorylation observed in TXA2+ platelets, presented as means ± SE; n = 3. Variances in TP receptor protein loading were corrected by densitometric analysis of amido black-stained immunoblots. *P < 0.03 compared with TXA2+ control. dagger P < 0.01 compared with TXA2- control. Dagger P < 0.02 compared with Epi.

Agonist-stimulated TP receptor phosphorylation. The elevated basal level of TXA2- platelet TP receptor phosphorylation did not increase further on exposure to U-46619 alone; however, exposure of TXA2+ and TXA2- platelets to epinephrine before U-46619 treatment resulted in a significant increase in TP receptor phosphorylation of both types of platelets (Fig. 7). The increase in phosphorylation of TXA2- platelets over the basal level observed after exposure to epinephrine was nearly equal to that of TXA2+ platelets after exposure to U-46619 alone. TP receptor-linked Galpha q and Galpha 13, which are phosphorylated on agonist stimulation (25, 29, 33, 43), were quantitatively similar in TXA2- and TXA2+ platelets, and they migrated at lower molecular weights than TP receptors on the gels we utilized (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current study yielded additional evidence of impaired receptor-Gq coupling in TXA2- platelets (e.g., diminished GTPase stimulation by TP receptor agonists and elevated basal levels of GTPgamma S binding and palmitate turnover, with the latter attributable to Galpha q). On activation, G protein alpha -subunits (Galpha ) exchange GDP for GTP, separate from beta gamma -subunits (Gbeta gamma ), and some Galpha undergo increased palmitate turnover (44). They subsequently hydrolyze GTP to GDP by the intrinsic GTPase activity of Galpha . Galpha q in TXA2- platelets appears to be cycling at an increased rate without being effectively linked to TP receptors. These results could be due to mutant Galpha q or mutant TP receptors, such as the beta -adrenergic mutants previously described (39, 46). However, Galpha q is not mutated (19); recent studies in our laboratory indicated that the TP receptor is not mutated (unpublished observation); and PLC-beta activation in the absence of TP receptor agonists (expected from a constitutively activated receptor) is not present in TXA2- platelets (18). In contrast to the impaired signal transduction via Galpha q, TXA2- platelet signaling via Galpha 13 is intact since Galpha 13 is not deficient, and shape change is normal (21, 22).

An important observation made in the biochemical studies of dog platelets is that the basal level of GTP-ase activity, which was higher than that of human platelets, was reduced in both types of dog platelets following the addition of epinephrine, but the reduction seen in TXA2- platelets was approximately three times greater than that in TXA2+ platelets. This reduction in basal GTPase activity was followed by an agonist-stimulated rise in activity that was comparable to that in TXA2+ platelets. Thus we observed increased agonist-receptor-coupled GTPase stimulation, after epinephrine exposure of TXA2- platelets, that was indicative of Galpha q heterotrimer coupling. Subsequent studies of IP3 formation demonstrated restoration of second messenger formation. When TXA2- platelets were pretreated with epinephrine, the agonist-stimulated IP3 rise was threefold higher than that observed with epinephrine alone, indicating significantly greater PLC-beta stimulation. That degree of stimulation was more likely the result of Galpha q stimulation of PLC-beta 3 than Gbeta gamma stimulation of PLC-beta 2 (40). Thus these observations strongly suggest that epinephrine restored productive TP receptor-Gq interaction in TXA2- platelets.

Because G protein activation is dependent on heterotrimer association with receptors (7), and agonist binding to GPCRs is influenced by receptor-G protein association (8, 32, 37, 46), we studied the kinetic binding characteristics of untreated and homologously desensitized TXA2- and TXA2+ platelet TP receptors in the absence and presence of epinephrine. As a control, we studied intact human platelets. These studies yielded data that closely paralleled those obtained in prior studies of agonist binding to alpha 2-AR (31, 32).

Three basal-state TP receptor binding parameters distinguished biochemically unresponsive TXA2- platelets from TXA2+ or human platelets. First, the elevated fast association rate (k1) correlated with a reduced proportion of bound ligand (Re1) and an elevated proportion of ligand bound by slow association (Re2). Second, the proportion of ligand undergoing fast dissociation (R01) was increased, and that involved with slow dissociation (R02) was decreased. Third, TXA2- platelet TP receptor binding parameters were comparable to TXA2+ and human platelet parameters when TXA2- platelets were exposed to epinephrine, and secretion followed the addition of agonist. After homologous desensitization of TXA2+ and human platelets, fast association rates increased significantly toward those seen with simple diffusion-controlled binding. The similarity of the association rate of TXA2- platelets to that of desensitized platelets, previously shown to manifest receptor-G protein uncoupling (30), was further evidence that a similar state existed in TXA2- platelets. The altered binding parameters and impaired function of desensitized platelets were restored by epinephrine. Thus fast-affinity TP receptor binding parameters correlated with biochemical efficacy.

Although epinephrine treatment of U-46619-desensitized platelets reversed the parameter shifts and restored biochemical responsiveness, the increased fast component of association that resulted was not equal to the sum of the loss with desensitization plus the gain from epinephrine exposure. Rather, it was similar to the increase observed with exposure to epinephrine alone. This suggested that only some of the TP receptors were restored to fast association status after epinephrine exposure or that they were derived from "spare" receptors (10). The apparently nonrecoverable decrease in the total binding capability (Re1 + Re2) following homologous desensitization was not due to internalization, since only ~1% residual [3H]U-46619 was detected, and internalization of TP occurs only after hours of agonist exposure (10, 30, 36, 45).

TP receptors are phosphorylated in the absence of agonists, and agonist binding to TP receptors stimulates time-dependent and concentration-dependent increased phosphorylation (13, 14, 35). Agonist-induced receptor phosphorylation plays an important role in TP receptor desensitization, but the influence of basal phosphorylation on receptor binding has not been evaluated. The proximal cause of impaired Gq-TP receptor interaction in TXA2- platelets appears to be an elevated state of basal TP receptor phosphorylation. Because epinephrine resulted in a change in TP receptor binding parameters in both TXA2- and homologously desensitized TXA2+ and human platelets, and because it decreased the elevated phosphorylation and the elevated GTPgamma S binding and palmitate cycling of TXA2- TP receptors, our studies suggest that basal TP receptor phosphorylation is a regulatory mechanism for TP receptor agonist binding. Epinephrine might influence TP receptor efficacy via Gbeta gamma liberated from alpha 2-AR (38), but the mechanism is unknown.

Agonist binding to GPCRs has been characterized by cyclic agonist-receptor-G protein ternary complex models (12, 32, 39, 42). The allosteric ternary complex model (28, 39) of agonist binding to receptors includes spontaneous isomerization (isomerization constant, J) between a resting (constrained) state, R, and an activated (relaxed) state, R*, that favors agonist binding and permits G protein interaction. Agonists may bind preferentially to the R* receptor conformation, or to other intermediate conformations (12, 27), resulting in an increased proportion of receptors in an activated state and stabilization of the agonist-bound receptor-G protein complex (12). The increased basal phosphorylation that we observed in TXA2- platelets could result in a shift of receptor equilibrium toward the R configuration by impairing receptor conformational change. Conversely, the reduction in phosphorylation observed after epinephrine exposure could shift the equilibrium toward R*. Other investigators have hypothesized that high- and low-affinity TP receptors might be the result of different levels of phosphorylation (35).

Thus phosphorylation may control the binding state of normal TP receptors by influencing the J constant of receptor isomerization. Epinephrine may potentiate TP receptor-mediated activation of normal as well as TXA2- platelets by reducing the basal state of receptor phosphorylation. The expected result would be an increase in fast-association ligand binding and G protein coupling that would be expressed as increased agonist potency. The data available are not sufficient to confirm this speculation, but the strong parallels between the kinetic TP receptor binding characteristics of desensitized TXA2+ and human platelets and those of TXA2- platelets, plus the effects of epinephrine on TP receptor binding and function, provide support for this hypothesis. Basal receptor phosphorylation is a potentially important control mechanism for GPCRs that merits further study.


    ACKNOWLEDGEMENTS

This work was supported by the Merit Review Program of the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: G. J. Johnson, Hematology/Oncology (111E), VA Medical Center, 1 Veterans Drive, Minneapolis, MN, 55417 (E-mail: johns337{at}tc.umn.edu).

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.

Received 28 March 2000; accepted in final form 6 July 2000.


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Am J Physiol Cell Physiol 279(6):C1760-C1771



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