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Originally published In Press as doi:10.1074/jbc.M800358200 on May 14, 2008

J. Biol. Chem., Vol. 283, Issue 27, 18493-18504, July 4, 2008
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The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine*

Magnus Grenegård{ddagger}1, Karin Vretenbrant-Öberg§, Martina Nylander§, Stéphanie Désilets{ddagger}, Eva G. Lindström{ddagger}2, Anders Larsson||, Ida Ramström{ddagger}, Sofia Ramström§23, and Tomas L. Lindahl§

From the {ddagger}Department of Medicine and Health, Division of Drug Research and §Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Cardiovascular Inflammation Research Center, Linköping University, Linköping SE-581 85 Sweden and the ||Department of Medical Sciences, University Hospital, Uppsala SE-75105, Sweden

Received for publication, January 14, 2008 , and in revised form, May 14, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via {alpha}2A-adrenergic receptors to provoke aggregation, secretion, and Ca2+ mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X1, P2Y1, and P2Y12 (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with {alpha}2A-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X1-receptor and the {alpha}2A-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon injury of a vessel wall, subendothelial structures such as collagen and tissue factor are exposed to the blood. Collagen activates platelets directly, whereas tissue factor initiates the coagulation cascade and thereby induces formation of thrombin, which is considered to be the most powerful platelet activator. Two G-protein-coupled thrombin receptor subtypes designated protease-activated receptors 1 (PAR1)4 and PAR4 are expressed on the surface of human platelets. Thrombin acts on these receptors by cleaving the N-terminal extension to create a new N terminus that functions as a tethered ligand. The subsequent signal transduction pathway comprises phosphorylation cascades and rises in the cytosolic concentration of Ca2+ ([Ca2+]i). The biphasic Ca2+ response induced by thrombin can be separated into two discrete components, a rapid spike response, caused mainly by PAR1 activation, followed by a slower and prolonged response of PAR4, possibly caused by slower uncoupling from G-protein signaling (1, 2). Both PAR1 and PAR4 activate the G{alpha}q and G{alpha}12/13 proteins (3), but recent publications have indicated differences in the signal transduction pathways (4, 5). However, the precise role of PAR4 signaling in platelet activation is still unclear. For instance, it is assumed that PAR1 is the high affinity thrombin receptor, whereas PAR4 may contribute to platelet activation only at higher thrombin concentrations (6). Furthermore, cleavage of PAR1 alone is sufficient to cause complete release of ADP from dense granules (7).

The response of platelets to a primary stimulus is amplified (both in vitro and in vivo) by different feedback loops, such as the release of ADP from the dense granules. ADP binds to two subtypes of G-protein-coupled purinergic receptors, designated P2Y1 and P2Y12 (8), and antagonists to purinergic receptors are now used clinically to control misdirected platelet activation. During dense granule secretion, ATP is concomitantly released to the extracellular environment, and platelets express ATP-gated Ca2+ channels called P2X1 receptors. Nevertheless, compared with ADP, the role of ATP in platelet activation is less well established.

Circulating hormones such as epinephrine also participate in the interplay between different platelet activators. Epinephrine causes aggregation in platelet-rich plasma (PRP), but many investigators consider that epinephrine should be classified as a "potentiating" stimulus (9, 10). For example, it has been shown that epinephrine and thrombin collaborate to cause powerful activation of platelets. However, research has not yet revealed the significance of different thrombin receptor subtypes and the importance of a secondary release of ADP and ATP in the cross-talk between thrombin and epinephrine. Epinephrine acts by binding to the Gz-coupled {alpha}2A-adrenergic receptor (Gz is a Gi variant), which inhibits cyclic AMP production (11). From a mechanistic perspective, epinephrine has been found to potentiate the Ca2+ mobilization induced by thrombin and other agonists (12). Activation of Syk tyrosine kinase, protein kinase C (PKC), and extracellular signal-regulated kinase 2 (ERK2) have also been implicated in the interaction between thrombin and other platelet activators (12–15).

The aim of the present study was to elucidate the relative roles of PAR1 and PAR4 in the thrombin-epinephrine cross-talk, and we hypothesized that PAR4 might be more important than PAR1 in that context, possibly because of more prolonged signal transduction. Indeed, we found that PAR4, but not PAR1, interacted synergistically with the {alpha}2A-adrenergic receptor to cause pronounced platelet activation. Intriguingly, we also noted that released ATP had a significant impact on platelet aggregation. This suggests both a vital role for PAR4 and an unexpectedly strong effect of ATP in the process of platelet activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The peptides TRAP-6 (SFLLRN) and AYPGKF, which are agonists of the thrombin receptor subtypes PAR1 and PAR4, respectively, were synthesized by the Biotechnology Centre of Oslo, Oslo University, Norway. The PAR4-blocking polyclonal chicken antibody was directed against a peptide that spans the thrombin cleavage site, which has the sequence GGDDSTPSILPAPRGYPGQVC. The peptide was synthesized by the Biotechnology Center of Oslo and used to immunize chickens. Chicken antibodies, in contrast to mammalian antibodies, do not cause platelet activation per se and are less likely to produce artifacts (16). Briefly, three laying white leghorn hens were immunized with the PAR4 peptide (2.0 mg) conjugated to hemocyanin (6.6 mg; obtained from Concholepas concholepas). The hens received one immunization with Freund's complete adjuvant and three booster injections with incomplete adjuvant. After the immunization period the eggs from the three hens were collected, and the antibodies were purified from the egg yolk by the polyethylene glycol method (17).

Innovagen (Lund, Sweden) synthesized the cell-penetrating pepducin P4pal-i1 (palmitate-NH-ATGAPRLST), which resembles the first intracellular loop of PAR4 and can, therefore, selectively inhibit PAR4 activation by interfering with binding of the G-protein (18). The PAR1 antagonist SCH79797 dihydrochloride (N3-Cyclopropyl-7-[4-(1m-ethylethyl) phenyl] methyl-7H-pyrrolo [3,2-f]quinazoline-1,3-diamine dihydrochloride) was obtained from Tocris Cookson Ltd. (Bristol, UK). The polyclonal rabbit antibody (#9271) directed against serine 437-phosphorylated Akt was purchased from Cell Signaling Technology (Danvers, MA), and the phycoerythrin-conjugated SPAN12 antibody was from Immunotech (Marseilles, France). The lysing solution came from Diapensia HB (Linköping, Sweden).

Cangrelor (formerly AR-C69931MX; N6-(2-methyl-tioethyl)-2-(3,3,3-trifluoro propylthio)-β,{gamma}-dichloromethylene ATP tetrasodium salt) was kindly provided by AstraZeneca (Dr. Michael Wayne, Wilmington, DE) and the Medicines Co. We also used the following drugs (all from Sigma): ADP, aspirin, apyrase, {alpha},β-MeATP, epinephrine, fura-2, ionomycin, LY 294002, luciferin/luciferase bioluminescent kit, MRS2159, MRS2179, NF449, prazosine, Ro318220, thrombin, UK 14.304, and yohimbine.

Isolation of Human Platelets—Human blood was collected from healthy volunteers and immediately mixed with an acid-citrate-dextrose (ACD) solution (5 volumes blood, 1 volume ACD) comprising 85 mM sodium citrate, 71 mM citric acid, and 111 mM glucose. The blood was centrifuged for 20 min at 220 x g to obtain PRP. Aspirin (100 µM) and apyrase (1 units/ml) were added to the PRP to prevent platelet activation by thromboxane A2 and ADP during the isolation procedure; in some experimental settings aspirin was excluded from the isolation protocol. The PRP was subsequently centrifuged for 20 min at 480 x g to obtain a platelet pellet. The supernatant was removed, and the platelets were gently resuspended in HEPES buffer (pH 7.4) composed of 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM HEPES, 1 units/ml apyrase, and 10 mM glucose. The platelet suspensions were stored in plastic tubes at room temperature and were used within 3 h of preparation. The extracellular Ca2+ concentration was adjusted to 1 mM immediately before each measurement.

Experimental Designs—Platelets were exposed to various concentrations of thrombin (0.21–21 nM) and, after 3 min, also to epinephrine (0.1–10 µM). In one experimental design, the time of incubation with thrombin (before adding epinephrine) was varied from 15 s to 5 min.

Platelets were also exposed to the PAR1- and PAR4-activating peptides SFLLRN (0.3–12.5 µM) and AYPGKF (30–300 µM) followed by 10 µM epinephrine. Another method used to evaluate the significance of PAR4 and PAR1 activation was to initially incubate platelets with a PAR4-blocking antibody (10 µg/ml), an unspecific IgY antibody (10 µg/ml), the PAR4-specific inhibitor pepducin P4pal-i1 (10 µM), or the PAR1 antagonist SCH79797 (5 µM) for 5 min and then expose them to thrombin and epinephrine.

The role of secondary release of ADP was evaluated by preincubating platelets for 3 min with the P2Y1 antagonist MRS2179 (10–20 µM) and/or the P2Y12 antagonist cangrelor (10–100 nM). Correspondingly, released ATP was analyzed by pretreating platelets for 3 min with the P2X1 antagonists MRS2159 (0.01–10 µM) and NF449 (0.1–10 µM).

Measurement of Cytosolic Ca2+—Platelets were loaded with fura-2 by incubating PRP with 4 µM fura-2-acetoxymethylester (from a 4 mM stock solution dissolved in DMSO) for 45 min at 20 °C, and they were subsequently pelleted and resuspended as described under "Isolation of human platelets," above. Before each measurement, 2 ml of platelet suspension (1–2 x 108/ml) was incubated at 37 °C for 5 min and then exposed to different drugs (see "Experimental designs," above). Fluorescence signals from platelet suspensions were recorded using a Hitachi F-2000 fluorescence spectrofluorometer specially designed to measure [Ca2+]i. Fluorescence emission was determined at 510 nm, with simultaneous excitation at 340 and 380 nm. [Ca2+]i was calculated according to the general equation reported by Grynkiewicz et al. (19): [Ca2+]i = Kd(R – Rmin)/(Rmax – R) (Fo/Fo). Maximum and minimum ratios were determined by adding 0.1% Triton X-100 and 25 mM EGTA, respectively.


Figure 1
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  		FIGURE 1.
Ca2+ responses in thrombin- and epinephrine-stimulated platelets. A, fura-2-loaded platelets were stimulated with thrombin (0.21–21 nM; THR) and 3 min later exposed to 10 µM epinephrine (EPI), as indicated by the arrows. Treatment of platelets with epinephrine alone did not change the [Ca2+]i (trace to the left). Inset, platelets were pre-stimulated with ADP (10 µM), platelet-activating factor (PAF, 1 µM), or ionomycin (IONO, 50 nM) and subsequently exposed to epinephrine (10 µM) as indicated. B, Ca2+ mobilization by epinephrine in platelets pre-activated with different concentrations of thrombin. C, summarized dose-response effects of epinephrine on platelets pre-stimulated with thrombin. The results in the bar graphs in B and C represent the thrombin- and epinephrine-induced peak rises in [Ca2+]i and are expressed as the means ± S.E. (n = 6–7). Control experiments using ADP, platelet-activating factor and ionomycin: n = 3–4.

 
Measurement of Platelet Aggregation—Aliquots (0.5 ml) of platelet suspensions (2.5 x 108 platelets/ml) were preincubated at 37 °C for 2 min. Thereafter, platelet aggregation was induced by adding thrombin, PAR-activating peptides, and epinephrine. Changes in light transmission were recorded using a Chronolog Dual Channel lumi-aggregometer (Model 560, Chrono-Log, Haverston, PA).

Measurement of Dense Granule Secretion—The amount of liberated ATP in platelet suspensions (0.5-ml aliquots; 2.5 x 108 platelets/ml) was registered using a luciferin/luciferase bioluminescent kit. Secretion of ATP was induced by adding thrombin or PAR-activating peptides alone or combined with epinephrine. The ATP-dependent increase in bioluminescence was recorded in the Chronolog lumi-aggregometer.

Analysis of Serine-phosphorylated Akt—Isolated platelets (1–2 x 109/ ml; 100-µl aliquots) were prewarmed at 37 °C for 3 min and then incubated with SFLLRN, AYPGKF, or thrombin for 5 min. The reaction was stopped by mixing platelet suspensions (1:2 v/v) with Laemmli buffer (Bio-Rad; 62.5 mM Tris-HCl, 25% glycerol, 2% SDS, 0.01% bromphenol blue, and 5% mercaptoethanol (pH 6.8)) and subsequently heated at 95 °C for 5 min. Thereafter, the samples were stored at –70 °C until used.

To analyze serine 473-phosphorylated Akt, the samples were again heated at 95 °C for 5 min and then separated by 7.5% SDS-PAGE using a Mini-PROTEAN II electrophoresis cell (Bio-Rad). The proteins were transferred to polyvinylidene difluoride membranes (Millipore Corp.) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). To minimize unspecific binding, the membranes were blocked overnight at 4 °C with 5% (w/v) dry milk and 0.1% (v/v) Tween 20 in phosphate-buffered saline (pH 7.4) composed of 10 mM phosphate buffer and 150 mM NaCl. To detect of phosphorylated Akt, a polyclonal rabbit antibody against the serine 437 position of Akt and a secondary horseradish peroxidase-conjugated antibody were used at dilutions of 1:1000 (Cell Signaling Technology). The membranes were rinsed in phosphate-buffered saline supplemented with 0.1% (v/v) Tween 20 between incubations and then analyzed using ECL Western blotting detection reagents (Amersham Biosciences) in a LAS-1000 Imaging Analyzer (Fuji Photo Film, Tokyo, Japan).

Statistical Analysis—The results are expressed as the mean values ± S.E. Statistical significance was determined by applying Student's t test or analysis of variance followed by Dunette's post hoc test for multiple comparisons, as indicated. Data were analyzed using GraphPad Prism4TM (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synergistic Interaction between Thrombin and Epinephrine in Mobilizing Cytosolic Ca2+—First, we evaluated the effect of the interaction between thrombin and epinephrine on the mobilization of cytosolic Ca2+. In fura-2-loaded, aspirin-treated platelets, thrombin caused a concentration-dependent increase in [Ca2+]i that was detectable at 0.21 nM and reached a maximum at around 7–21 nM (Fig. 1A). Epinephrine alone did not cause Ca2+ mobilization. However, in platelets pre-stimulated with thrombin, epinephrine (0.1–10 µM) induced a significant rise in [Ca2+]i (original traces in Fig. 1A; results are summarized in Figs. 1, B and C). Notably, the capacity of epinephrine to mobilize cytosolic Ca2+ was virtually independent of the concentration of thrombin (see Figs. 1, A and B). Hence, the magnitude of the epinephrine-induced rise in [Ca2+]i was about 100 nM in platelets pre-stimulated with low, intermediate, and high concentrations of thrombin.


Figure 2
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  		FIGURE 2.
The Ca2+-mobilizing effect of PAR-activating peptides and epinephrine. A, original traces of fura-2-loaded platelets exposed to the PAR1-activating peptide SFLLRN (0.3–12.5 µM) and the PAR4-activating peptide AYPGKF (30–300 µM). EPI (10 µM) was introduced 3 min after the PAR-activating peptides. The two traces to the right show the Ca2+-mobilizing capacity of epinephrine introduced 1 or 3 min after THR (0.7 nM). Inset, platelets stimulated with thrombin (0.7 nM) and epinephrine (10 µM) in the absence (Ctrl) and presence of the PAR4-specific inhibitor pepducin P4pal-i1 (10 µM). B, summary of the peak rise in [Ca2+]i induced by SFLLRN and AYPGKF. C, the peak rises in [Ca2+]i induced by epinephrine in platelets pre-stimulated with SFLLRN or AYPGKF. D, time study of the peak rises in [Ca2+]i induced by epinephrine (10 µM) in platelets pre-stimulated with 0.7 nM thrombin ({blacksquare}), 100 µM AYPGKF ({square}), or 12.5 µM SFLLRN ({circ}). Epinephrine was added from 15 s to 5 min after thrombin or the PAR-activating peptides. The data in bar graphs B–D represent the means ± S.E. (n = 4–6).

 
As shown in the inset in Fig. 1, epinephrine did not increase the [Ca2+]i in platelets pretreated with ADP (10 µM), platelet-activating factor (1 µM), or the Ca2+-ionophore ionomycin (50 nM). Moreover, a combination of activators (e.g. ADP combined with 10 µM lysophosphatidic acid) was not followed by an epinephrine-induced Ca2+ response (not shown). Thus, the Ca2+-mobilizing capacity of epinephrine in platelets seems to require pre-stimulation with thrombin.

Involvement of PAR1 and PAR4 in Epinephrine-induced Ca2+ Mobilization—To clarify the relative roles of different subtypes of thrombin receptors, we treated platelets with the PAR1- and PAR4-activating peptides (SFLLRN and AYPGKF, respectively) followed by epinephrine (Figs. 2, A and B). Both SFLLRN (0.3–12.5 µM) and AYPGKF (30–300 µM) dose-dependently increased the [Ca2+]i. However, only in AYPGKF-pretreated platelets did subsequent exposure to epinephrine (10 µM; added 3 min after the PAR-activating peptides) cause a significant increase in [Ca2+]i (traces shown in Fig. 2A; results summarized in Fig. 2C). Moreover, preincubation of platelets with PAR4-blocking antibodies (10 µg/ml) or the PAR4-specific inhibitor pepducin P4pal-i1 (10 µM) antagonized the epinephrine-induced rise in [Ca2+]i in platelets preactivated with thrombin (results are summarized in Table 1; original traces are shown in the inset in Fig. 2A). Irrelevant isotype antibodies did not influence the effect of epinephrine (Table 1). SPAN-12 is an antibody that can only bind to the platelet PAR1 receptor when it is not cleaved by thrombin (20). Furthermore, in a recent study, we found that binding of SPAN-12 to platelets was not influenced by the PAR4-blocking antibody, which indicates that those antibodies did not affect thrombin-induced cleavage of PAR1 (21). It should be mentioned that, because of its autofluorescence properties, the PAR1 antagonist SCH79797 could not be used in this experimental design. The role of adrenergic receptors was evaluated by pretreating platelets with the {alpha}2-adrenergic receptor antagonist yohimbine (1 µM) or the {alpha}1-adrenergic receptor antagonist prazosine (1 µM) and then exposing them to thrombin and epinephrine. Yohimbine, but not prazosine, abolished the epinephrine-induced Ca2+ mobilization (data not shown), which implies that the effect of epinephrine is mediated by {alpha}2A-adrenergic receptors.


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  		TABLE 1
Effects of PAR4 inhibitors

Rises in [Ca2+]i in platelets stimulated by exposure to THR and 3 min later also EPI (10 µM) are shown. In some experiments platelets were pretreated with PAR4-blocking antibodies (10 µg/ml; PAR4 ab), irrelevant control antibodies, or the PAR4-specific inhibitor pepducin P4pal-i1 (10 µM). The data represent the means ± S.E. (n = 3). Statistical significance was determined using Student's t test.

 
We also found that the Ca2+-mobilizing effect of epinephrine in thrombin-pretreated platelets was strictly time-dependent. More exactly, the magnitude of the Ca2+ response gradually increased and reached a maximum level when epinephrine was introduced 3 min after thrombin (Figs. 2, A and D). Interestingly, AYPGKF-pretreated platelets responded more rapidly to epinephrine. As shown in Fig. 2D, the maximum magnitude of the epinephrine-induced Ca2+ response was observed only 1 min after the introduction of the PAR4-activating peptide. In SFLLRN-pretreated platelets, epinephrine induced a detectable Ca2+ response when the time gap was reduced to 1 min (Fig. 2D). However, the response induced by epinephrine was always much smaller than that caused by pre-exposing platelets to the PAR4-activating peptide AYPGKF or thrombin. Taken together, these results show that pre-stimulation of PAR4, but not PAR1, is necessary for epinephrine-induced Ca2+ mobilization in aspirin-treated platelets.


Figure 3
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  		FIGURE 3.
Effects of the PKC inhibitor Ro31220 on Ca2+ mobilization. A, original traces of fura-2-loaded platelets stimulated with the PAR4-activating peptide AYPGKF (100 µM) followed by the PAR1-activating peptide SFLLRN (12.5 µM) and finally EPI (10 µM). SFLLRN and epinephrine were introduced 1 and 3 min, respectively, after stimulation with AYPGKF, as indicated by the arrows. In some experiments the platelets were pretreated for 3 min with the PKC inhibitor Ro318220 (0.3 µM; RO). The traces shown represent one of five separate experiments. B, summarized effects of the PKC inhibitor Ro318220 on epinephrine-induced peak rises in [Ca2+]i in platelets. Platelet suspensions were treated with Ro318220 for 3 min, after which THR (7 nM), SFLLRN (12.5 µM), or AYPGKF (100 µM) was added. Epinephrine (10 µM) was introduced 3 min later. The results are expressed as means ± S.E. (n = 4–8). Statistical significance was determined using Student's t test (**, p < 0.01). Ctrl, control.

 
Effect of PAR1 Activation on the Interplay between PAR4 and the {alpha}2A-Adrenergic Receptor—We found that pre-exposure of platelets to SFLLRN (12.5 µM) for 1 min significantly reduced subsequent Ca2+ responses triggered by AYPGKF (100 µM) and epinephrine (10 µM). Notably, this antagonizing effect was abolished when the time elapsed between SFLLRN and AYPGKF treatment was increased to 3 min (data not shown). In a slightly modified protocol, SFLLRN was introduced 1 min after AYPGKF and consequently 2 min before epinephrine. Even in this experimental design, activation of PAR1 significantly reduced the rise in [Ca2+]i caused by epinephrine (Fig. 3A). Specifically, the peak epinephrine-induced rise in [Ca2+]i was 71.6 ± 14.3 nM in platelets pretreated with AYPGKF alone and 43.3 ± 12.1 nM in those exposed to AYPGKF followed by SFLLRN (n = 5, p < 0.01). Thus, the results indicate that PAR1 signaling temporarily antagonizes the interplay between PAR4 and epinephrine. PKC has been implicated in the phenomenon of receptor desensitization (13, 22), and therefore, we analyzed involvement of this signaling molecule in the antagonistic action of PAR1 on the epinephrine-induced increase in [Ca2+]i. Preincubation of the platelets with the PKC inhibitor Ro318220 (0.3 µM) abolished the suppressive effect of SFLLRN on epinephrine-induced Ca2+ mobilization (Fig. 3A). Furthermore, such treatment with Ro318220 significantly enhanced the epinephrine-induced Ca2+ response in platelets pre-stimulated with thrombin or SFLLRN (Fig. 3B). However, the latter effect was not observed in AYPGKF preactivated platelets. Together, these findings indicate that PAR1-mediated activation of PKC transiently inhibits the interaction between PAR4 and the {alpha}2A-adrenergic receptor.


Figure 4
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  		FIGURE 4.
Platelet aggregation induced by thrombin and epinephrine. A, original traces of platelet suspensions exposed to subthreshold concentrations of THR (0.21 nM) and EPI (10 µM), as indicated by the arrows. In some experiments the platelets were pretreated with the PAR-1 antagonist SCH79797 (5 µM; SCH), the PAR4-specific pepducin P4pal-il (10 µM) or the PAR4-blocking antibodies (PAR4 ab; 10 µg/ml) for 5 min before introducing thrombin and epinephrine. The trace to the right shows that the PAR1-activating peptide SFLLRN (12.5 µM) induced a strong aggregation response in the presence of the PAR4-blocking antibodies. Inset, SCH79797 (5 µM) abolished platelet aggregation induced by SFLLRN (12.5 µM), whereas the PAR4-blocking antibodies and the PAR4-specific pepducin P4pal-il eliminated aggregation caused by AYPGKF (100 µM). Ctrl, control. B, summarized effects of thrombin and epinephrine on platelet aggregation. Platelets were pretreated for 3 min with subthreshold concentrations of thrombin, and epinephrine was introduced 3 min later. C, summarized effects of SCH79797, the PAR4-blocking antibodies, and P4pal-il on thrombin/epinephrine-induced platelet aggregation. The platelets were treated with the inhibitors for 5 min after which a subthreshold concentration of thrombin was added, and epinephrine (10 µM) was introduced 3 min later. Bar graphs B and C show the maximum aggregation induced by thrombin and epinephrine (means ± S.E., n = 3–8). Statistical significance was tested by analysis of variance (**, p < 0.01).

 
Synergistic Interaction between PAR4 and the {alpha}2A-Adrenergic Receptor on Platelet Aggregation—We also evaluated the effect of epinephrine added together with low concentrations of thrombin and PAR1- and PAR4-activating peptides on aggregation in aspirin-treated platelets. The aggregation response to thrombin alone was detectable down to a concentration of about 0.21 nM. In contrast, epinephrine alone did not induce platelet aggregation. Preincubation of platelets for 3 min with a subthreshold concentration of thrombin (i.e. a dose that did not directly provoke aggregation, 0.07–0.21 nM) markedly amplified aggregation subsequently induced by 10 µM epinephrine (original traces shown in Fig. 4A; results are summarized in Fig. 4B). Platelets stimulated with thrombin (0.21 nM) and pretreated with PAR4-blocking antibodies (10 µg/ml) or the PAR4-specific inhibitor pepducin P4pal-i1 (10 µM) exhibited no aggregation upon subsequent exposure to epinephrine (Figs. 4, A and C). Adding a higher dose of thrombin (not shown) or the PAR1-activating peptide SFLLRN easily overcame the inhibition induced by the PAR4-blocking antibodies or P4pal-i1 (Fig. 4A; traces to the right). Furthermore, both the PAR4-blocking antibodies and P4pal-i1 abolished aggregation responses induced by high concentrations (100–300 µM) of AYPGKF (Fig. 4A, inset). On the other hand, the platelets reacted normally to subsequent addition of 12.5 µM SFLLRN. Incubation with irrelevant isotype antibodies did not influence the thrombin/epinephrine-induced aggregation (data not shown). In addition, pretreatment of platelets with the PAR1 antagonist SCH79797 (5 µM) did not affect the aggregation response induced by a low concentration of thrombin combined with epinephrine. SCH79797 eliminated platelet aggregation induced by 12.5 µM SFLLRN but did not affect subsequent AYPGKF (300 µM)-induced aggregation (Fig. 4A, inset). We also found that epinephrine produced prompt aggregation of aspirin-treated platelets that had been pre-exposed to a subthreshold concentration of AYPGKF (Fig. 5A). In contrast, a subthreshold concentration of SFLLRN combined with epinephrine did not cause platelet aggregation. However, when we used platelets that were not exposed to aspirin during the isolation procedure, epinephrine induced essentially the same aggregation response when it was added 3 min after AYPGKF or SFLLRN (Fig. 5A). Epinephrine alone did not induce aggregation of platelets that either were or were not treated with aspirin. Concomitant exposure to SFLLRN and epinephrine caused a minor aggregation response (25.1 ± 9.1% aggregation, n = 3) in aspirin-treated platelets (Fig. 5, inset), but this effect was abolished when the time between addition of the activators was increased to 15 s. No such time dependence was observed when platelets were cotreated with AYPGKF and epinephrine. Therefore, the results concerning aggregation concur with the findings regarding Ca2+ mobilization (see Fig. 2). The outcomes of the experiments using the PAR-activating peptides combined with epinephrine are summarized in Figs. 5, B and C, which shows that PAR4 and {alpha}2A-adrenergic receptors interact synergistically to produce strong platelet aggregation independently of functional cyclooxygenase activity. This effect was not observed when the PAR1-activating peptide SFLLRN was combined with epinephrine.


Figure 5
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  		FIGURE 5.
Effects of the PAR-activating peptides and epinephrine on platelet aggregation. A, original traces of platelet suspensions exposed to subthreshold concentrations of the PAR4-activating peptide AYPGKF or the PAR1-activating peptide SFLLRN followed by EPI, as indicated by the arrows. For comparison, one trace shows aggregation induced by a high concentration of SFLLRN. The two traces to the right illustrate the effect of epinephrine introduced to non-aspirin-treated platelets 3 min after the addition of a subthreshold concentration of either AYPGKF or SFLLRN. Inset, time studies of aggregation provoked in aspirin-treated platelets by 10 µM epinephrine introduced simultaneously or 15 or 60 s after AYPGKF or SFLLRN. B, summarized effects of AYPGKF/SFLLRN alone or together with epinephrine on aggregation of aspirin-treated platelets. C, summarized effects of AYPGKF/SFLLRN alone or together with epinephrine on aggregation of non-aspirin-treated platelets. The bar graphs show the maximum aggregation induced by AYPGKF, SFLLRN, and epinephrine (means ± S.E., n = 3–4).

 
The Role of Phosphatidyl Inositide 3-Kinase Activation in the Cross-talk between PAR4 and the {alpha}2A-Adrenergic Receptors—Activation of phosphatidyl inositide 3-kinase (PI3-kinase) has been increasingly recognized as an important molecular event in platelet activation. Therefore, we performed Western blot analyses using antibodies against serine 473-phosphorylated Akt to clarify the role of PI3-kinase in the thrombin-epinephrine cross-talk. As shown in Fig. 6A, a low, subthreshold concentration of the PAR4-activating peptide AYPGKF (30 µM), but not the PAR1-activating peptide SFLLRN (1.2 µM), induced an increase in serine-phosphorylated Akt. When added in higher concentrations (which directly provoked platelet aggregation and secretion), both AYPGKF (300 µM) and SFLLRN (12.5 µM) caused Akt phosphorylation.

The addition of the PI3-kinase inhibitor LY294002 (1–10 µM; 8 min) had a significant impact on platelet aggregation induced by a subthreshold concentration of thrombin (0.21 nM) or AYPGKF (30 µM) followed by epinephrine. More specifically, pretreatment with LY294002 significantly decreased the aggregation induced by the thrombin/epinephrine and the AYPGKF/epinephrine combinations (traces shown in Fig. 6B; results are summarized in the inset bar graph). On the other hand, LY294002 did not affect platelet aggregation induced by higher concentrations of thrombin or AYPGKF (not shown).


Figure 6
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  		FIGURE 6.
The role of PI3-kinase in thrombin-epinephrine cross-talk. A, Western blot demonstrating phosphorylation of serine 473 on Akt. Platelet suspensions were treated with the PAR4-activating peptide AYPGKF (30 or 300 µM) or the PAR1-activating peptide SFLLRN (1.25 or 12.5 µM) for 5 min, and the reaction was subsequently stopped by adding Laemmli buffer. Untreated suspensions were analyzed in parallel. The illustrated blot is representative of four separate analyses. B, original traces of aggregation (top) and Ca2+ (below) measurements. Platelet aggregation was induced by adding THR (0.21 nM) or the PAR4-activating peptide AYPGKF (30 µM) followed by EPI (10 µM). Ca2+ responses were provoked by adding 0.7 nM thrombin or 300 µM AYPGKF followed by 10 µM epinephrine. Platelets were pretreated for 5 min with the PI3-kinase inhibitor LY294002 (10 µM; LY). Untreated platelets were analyzed in parallel (Ctrl). Inset, summarized effect of LY294002 (10 µM) on platelet aggregation induced by a low concentration of thrombin (0.21 nM) or AYPGKF (30 µM) combined with epinephrine (10 µM). The results represent the maximum aggregation (means ± S.E., n = 6). Statistical significance was determined using Student's t test (*, p < 0.05).

 
Our Ca2+ measurements showed that pretreatment with LY294002 significantly reduced the rise in [Ca2+]i elicited by epinephrine (10 µM) in platelets pre-stimulated with AYPGKF (300 µM) or thrombin (0.7 nM) (traces shown in Fig. 6C; results are summarized in Table 2). However, LY294002 did not have a significant effect on the initial thrombin- or AYPGKF-induced peak rise in [Ca2+]i (Table 2). Together, these results suggest that PI3-kinase plays an important role in the cross-talk between PAR4 and the {alpha}2A-adrenergic receptor.


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  		TABLE 2
Effects of the PI3-kinase inhibitor LY294002

Rises in [Ca2+]i in platelets caused by exposure to THR (0.7 nM) or the PAR4-activating peptide AYPGKF (300 µM) and 3 min later also EPI (10 µM) are shown. In some experiments platelets were pretreated with the PI3-kinase inhibitor LY294002 (1-10 µM) for 3 min before introducing the activators. The data represent the means ± S.E. (n = 4). Statistical significance was tested by analysis of variance.

 
The Role of ADP and ATP in the Cross-talk between PAR4 and the {alpha}2-Adrenergic Receptors—There is considerable evidence that secondary release of ADP is an important factor in platelet activation, whereas less is known about the role of ATP in that context. We observed that thrombin dose-dependently stimulated release of ATP from platelet suspensions (original traces shown to the left in Fig. 7A; results are summarized in Table 3). Similar to the aggregation responses, subthreshold concentrations of thrombin synergized with epinephrine to provoke secretion of ATP (Figs. 7A, left, and B). Epinephrine alone did not induce dense granule secretion. These findings indicate that dense granule constituents like ATP and ADP are liberated during the interplay between thrombin and epinephrine. Correspondingly, epinephrine induced detectable ATP secretion when platelets were pretreated with low concentrations of the PAR4-activating peptide AYPGKF (Table 3). However, secretion was not elicited by epinephrine added to platelets after they were pretreated with low concentrations of SFLLRN (Table 3).


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  		TABLE 3
ATP release from platelets in response to thrombin, PAR-activating peptide, and epinephrine

Platelet ATP secretion was induced by THR, the PAR-activating peptide AYPGKF, and SFFLRN alone or in combination with EPI (10 µM). Epinephrine was added 3 min after thrombin/AYPGKF/SFLLRN. The data represent the means ± S.E. (n = 5-8).

 
To clarify the importance of ADP on thrombin/epinephrine-induced platelet aggregation, we incubated platelet suspensions with the P2Y1 receptor antagonist MRS2179 (10–20 µM), the P2Y12 receptor antagonist cangrelor (10–100 nM), or a combination of the two. MRS2179 together with cangrelor did not affect the aggregation response induced by a subthreshold concentration of thrombin (0.21 nM) followed by 10 µM epinephrine (Figs. 7A, middle traces, and C). Control experiments showed that treatment with MRS2179 (10 µM) or cangrelor (10 nM) alone nearly abolished the aggregation induced by 1 µM ADP (Fig. 7A, middle inset). Hence, in suspensions of aspirin-treated platelets, secondary release of ADP did not contribute to the strong aggregation response provoked by a low concentration of thrombin combined with epinephrine.

To clarify the role of ATP in thrombin/epinephrine-induced platelet aggregation, we incubated cell suspensions with the P2X1 receptor antagonists MRS2159 (0.01–1 µM) and NF449 (0.1–10 µM). Most interestingly, both of those compounds abolished the aggregation response (original traces shown in Fig. 7A, middle; dose-response effects are summarized in Fig. 7D). To further establish the importance of a secondary release of ATP, we replaced epinephrine with the stable ATP analogue {alpha},β-MeATP (2 µM). In platelet suspensions pretreated with a subthreshold concentration of thrombin (0.21 nM), the addition of {alpha},β-MeATP induced aggregation (Fig. 7A, left inset). The maximum aggregation response to thrombin/{alpha},β-MeATP was around 30–40%, which can be compared with >80% aggregation induced by thrombin/epinephrine (see Fig. 4B). This indicates that {alpha},β-MeATP could not fully substitute for epinephrine. It should also be noted that aggregation was not provoked by epinephrine combined with {alpha},β-MeATP (Fig. 7A, left inset).

In our final experiments we evaluated the role of ATP in the epinephrine-induced Ca2+ mobilization. Preincubation of platelets for 3 min with 1 µM MRS2159 significantly reduced the epinephrine-provoked peak rise in [Ca2+]i in cell suspensions pretreated with 0.7 nM thrombin (Fig. 7A, original traces to the right; results are summarized in right inset). Fig. 7A shows that the P2X1 receptor antagonist had only a negligible effect on the primary thrombin-induced Ca2+ response. Furthermore, MRS2159 did not inhibit the epinephrine-induced Ca2+ response in platelets pretreated with a high concentration of thrombin (7 nM). In summary, these results imply a pivotal role for released ATP, but not ADP, in the interplay between PAR4 and the {alpha}2A-adrenergic receptor in aspirin-treated platelets.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The interplay between different activators that enhances the response exhibited by platelets has been studied extensively in recent decades. In accordance with previous findings (12), our results show that epinephrine acts via {alpha}2A-adrenergic receptors to induce Ca2+ mobilization and pronounced aggregation in platelets preactivated with thrombin. However, we found that these effects could be provoked by surprisingly low concentrations of thrombin and relatively low concentrations of epinephrine. Furthermore, other combinations of activators (e.g. platelet-activating factor/epinephrine and ADP/epinephrine; see the inset in Fig. 1) did not magnify the Ca2+ response induced by the second activator, and indeed it is well known that repeated stimulation of platelet rapidly induces homologous and heterologous receptor desensitization (13). Consequently, we conclude that the epinephrine-induced rise in [Ca2+]i is far from being a general receptor-dependent phenomenon and that it requires pre-activation of platelets with low concentrations of thrombin. Together, our data suggest that the interaction between thrombin and epinephrine may be an important event in hemostasis and thrombosis. Many research groups have in fact illustrated the pathophysiological role of epinephrine as exemplified by the results of Wallén et al. (23) showing that both exercise and epinephrine infusion could enhance thrombin-induced fibrinogen binding and aggregability of platelets.

In agreement with previous reports (1, 2), we found that the PAR1-activating peptide SFLLRN induced a rapid peak rise in [Ca2+]i, and the PAR4-activating peptide AYPGKF also caused an increase in [Ca2+]i, but it was slower and more prolonged. A biphasic model of thrombin-induced platelet activation has been proposed in which PAR1 is initially activated by a low concentration of thrombin (1), possibly aided by binding to glycoprotein Ib{alpha}. Notably, picomolar concentrations of thrombin have been observed to induce 50% of maximum PAR1 cleavage (24). On the other hand, it is assumed that PAR4 is a low affinity thrombin receptor that is cleaved and activated by higher concentrations of thrombin (6). In comparison, we found that the effect of epinephrine on aggregation was marked even in platelets pretreated with nanomolar concentrations of thrombin. We also noted that inhibitors of PAR4, but not PAR1, abolished the effect of epinephrine. Furthermore, epinephrine induced a strong aggregation response in platelets pretreated with subthreshold concentrations of the PAR4-activating peptide AYPGKF. This effect was not seen in platelets pretreated with the PAR1-activating peptide SFLLRN. However, when aspirin was excluded, platelets pre-exposed to AYPGKF and those pretreated with SFLLRN exhibited nearly the same response to epinephrine. Hence, the present study strongly suggests that PAR4 activation is crucial for the synergistic effects of epinephrine and thrombin in platelets with inactivated cyclooxygenase 1. It has previously been shown that the PAR1-activating peptide YFLLRNP synergizes with epinephrine to induce aggregation in aspirin-treated platelets (25). However, in contrast to the peptide SFLLRN, YFLLRNP is a partial agonist of PAR1, which might explain the difference in platelet responsiveness to added epinephrine. Based on our results, we suggest that PAR4 is activated by nanomolar concentrations of thrombin. This conclusion has also been drawn by Covic et al. (7), who found that antibodies against PAR4 blocked 68% of the aggregation induced by 3 nM of thrombin. To the best of our knowledge there are no other publications concerning this phenomenon, possibly because so far only a few research groups have been able to selectively block PAR4. We also propose that activation of just a minor fraction of the total number of PAR4 may be sufficient to transform platelets to a highly epinephrine-sensitive state. In this regard it was recently shown that PAR4, but not PAR1, interacted synergistically with purinergic receptors to induce platelet aggregation (26). Hence, we suggest that PAR4 may be more important than PAR1 in long-lasting cross-talk between thrombin and other platelet activators.

It has been proposed that decreased production of cyclic AMP and activation of Syk tyrosine kinase and PKC are important signaling events in the thrombin-epinephrine cross-talk (12, 15). Our results imply that PKC activation is involved in the interaction between PAR1 and epinephrine. Specifically, we found that the PAR1-activating peptide SFLLRN counteracted the epinephrine-induced increase in [Ca2+]i in AYPGKF-pretreated platelets and that effect was abolished by the PKC inhibitor Ro318220 (see Fig. 3). Thus, our data indicate that thrombin-induced PKC activation is more closely related to PAR1 signaling. In accordance with that assumption, it was recently found that PAR1, but not PAR4, is associated with activation of phospholipase D (4), and such an effect would presumably lead to increased production of the endogenous PKC activator diacylglycerol. Notably, we observed that the inhibitory influence of PAR1 was transient, which might explain the results showing time dependence of epinephrine-induced Ca2+ responses in thrombin-pretreated platelets (i.e. the magnitude of the Ca2+ response was gradually increased by prolonging incubation with thrombin; see Fig. 2D). Together, our results suggest that some aspects of PAR1 signaling decrease and other aspects increase the effect of epinephrine-induced platelet activation. Activation of PKC isoforms, probably followed by heterologous receptor desensitization, may diminish the responsiveness of platelets to epinephrine. Conversely, the signal transduction pathway(s) responsible for the synergistic interplay will be determined by whether or not cyclooxygenase 1 is functional.


Figure 7
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  		FIGURE 7.
The role of ADP and ATP in thrombin-epinephrine cross-talk. A, original traces of platelet suspensions exposed to THR (0.21–7 nM) alone or combined with EPI (10 µM). The traces to the left show ATP liberation induced by thrombin and epinephrine. The middle traces show platelet aggregation provoked by thrombin (0.21 nM) and epinephrine (10 µM) combined (Ctrl). In some experiments the platelets were pretreated with the P2X1 receptor antagonist MRS2159 or the P2Y1/P2Y12 receptor antagonists MRS2179 (20 µM) and cangrelor (100 nM; CAN). Middle inset, effect of MRS2179 (10 µM) and cangrelor (10 nM) on aggregation induced by ADP (1 µM). Left inset, platelet aggregation caused by thrombin (0.21 nM) combined with epinephrine (10 µM) or {alpha},β-MeATP (2 µM; Me-ATP). The traces to the right show Ca2+ responses induced by 0.7 nM thrombin followed by 10 µM epinephrine (Ctrl). In some experiments the platelets were pretreated with the P2X1 receptor antagonist MRS2159 (1 µM). Right inset, summarized effects of MRS2159 (1 µM) on epinephrine-induced peak rises in [Ca2+]i in platelets. The platelets were pre-activated with 0.7 or 7 nM thrombin (means ± S.E., n = 5–6). Statistical significance was determined using Student's t test (**, p < 0.01). B, summarized effects of thrombin and epinephrine on platelet ATP secretion (means ± S.E., n = 6–8). Statistical significance was tested by analysis of variance (*, p < 0.05; **, p < 0.01). C, impact of the combination of MRS2179 (20 µM) and cangrelor (100 nM) on thrombin/epinephrine-induced platelet aggregation (means ± S.E., n = 6). D, dose-response effects of the P2X1 receptor antagonists MRS2159 and NF449 on thrombin/epinephrine-induced platelet aggregation (means ± S.E., n = 3–5). The results in C and D represent the maximum aggregation.

 
There is increasing support for the idea that activation of the PI3-kinase/Akt signaling pathway is an essential event in platelet activation (27, 28). We observed significant inhibition of epinephrine-induced Ca2+ mobilization and aggregation in platelet suspensions pretreated with the PI3-kinase inhibitor LY294002. Moreover, subthreshold concentrations of AYPGKF induced PI3-kinase activation, as shown by Western blot analysis of serine-phosphorylated Akt, and this effect was not provoked by a subthreshold concentration of SFLLRN. Therefore, we conclude that PAR4-mediated PI3-kinase activation is involved in platelet stimulation brought about by adding epinephrine. It was recently reported that Akt phosphorylation induced by PAR1 (but not by PAR4) depends on the release of ADP and its action on P2Y12 receptors (29). This may explain the difference we observed when using low "non-secretory" doses of the PAR-activating peptides. As in other studies (29, 30), we found that higher concentrations of both PAR1- and PAR4-activating peptide (i.e. concentrations that directly provoked platelet secretion and aggregation) induced serine phosphorylation of Akt. It should also be noted that LY294002 reduced, but did not abolish, epinephrine-induced Ca2+ mobilization and aggregation in platelets pretreated with thrombin or AYPGKF. Consequently, other signaling pathways and mechanisms must be involved in the thrombin-epinephrine cross-talk. Additional work is needed to determine the significance of other quantitative differences and/or unknown qualitative differences between PAR1 and PAR4 signaling. In this regard, Harper and Sage (31) recently reported that Ca2+ mobilization induced by PAR1 and PAR4 is to some extent regulated by different mechanisms.

We found that epinephrine caused dense granule secretion in platelets pretreated with low concentrations of thrombin and AYPGKF. Most intriguingly, our results also revealed that ATP, but not ADP, was crucial for the aggregation response. The unexpected lack of effect of ADP might have arisen because stimulation of {alpha}2A-adrenergic receptor substituted for, and thus reduced, the importance of secondary activation of P2Y12. However, further research is needed to determine the exact mechanism(s) underlying the lack of contribution of P2Y1/ P2Y12 receptors. It has been suggested that ATP and its P2X1 receptor contribute to collagen-induced shape change, Ca2+ mobilization, and ERK2 phosphorylation in platelets (32, 33). ATP may also potentiate the effect of other platelet activators (34, 35). However, ATP alone is a very weak platelet stimulator, and the P2X1 receptor can be rapidly desensitized (33). Consequently, it is noteworthy that two weak activators collaborate with PAR4 to produce powerful platelet aggregation. Based on the present results, we conclude that epinephrine elicited dense granule secretion and released ATP via P2X1 receptors concomitantly with signaling from the {alpha}2A-adrenergic receptor provoked aggregation. These events require activation of only a few numbers of surface expressed PAR4.

The P2X1 receptor is an ATP-gated Ca2+ channel, and our data showed that ATP contributed significantly to epinephrine-induced Ca2+ mobilization. However, the role of ATP was less prominent when platelets were pre-stimulated with a high concentration of thrombin followed by epinephrine (see Fig. 7), which implies that the epinephrine-induced Ca2+ mobilization has at least two components; at low thrombin concentrations, the addition of epinephrine provokes secretion, and liberated ATP causes a rise in [Ca2+]i. At higher thrombin concentrations, ATP is released instantly, but the subsequent addition of epinephrine still leads to Ca2+ mobilization. The precise mechanism underlying the latter Ca2+ response is beyond the scope of this investigation, although it is possible that it is related to up-regulation of PARs.

Aspirin is used extensively and worldwide to manage cardiovascular diseases. In addition, particularly in clinical research, much attention has been focused on "aspirin resistance," a term used somewhat indiscriminately to mean resistance measured by applying one or more methods to analyze platelet activation, failure to inhibit cyclooxygenase 1, or treatment failure. In relation to that, our study has revealed a new mechanism in which ATP and its P2X1 receptor results in complete circumvention of the action of aspirin on epinephrine-facilitated thrombin stimulation of platelets. Nonetheless, the specific physiological and/or pathophysiological significance of our findings remains to be determined.

In conclusion, we found that in aspirin-treated platelets the synergistic interplay of thrombin and epinephrine depends primarily on occupancy of PAR4 and the {alpha}2A-adrenergic receptor, and we also noted that ATP (via its P2X1 receptor) is a key mediator during the cross-talk between those two receptors. This means that ATP can be a surprisingly effective inducer of platelet aggregation. Moreover, we suggest that PAR4-induced activation of PI3-kinase participates in the signaling cross-talk that leads to a more powerful platelet response. Together, these findings imply that PAR4 plays an important and unique role in platelet signaling and activation.


   FOOTNOTES
 
* This study was supported by the strategic research area "Cardiovascular Inflammation Research Center," sponsored by the County Council of Östergötland and Linköping University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Supported by Swedish Research Council Project K2007-64X-15060-04-3. Back

3 Supported by Forsknings-och forskarutbildningsnämnden at Linköping University. Back

1 To whom correspondence should be addressed. Tel.: 46-13-221082; Fax: 46-13-149106; E-mail: Magnus.Grenegard{at}imv.liu.se.

4 The abbreviations used are: PAR, protease-activated receptor; [Ca2+]i, cytosolic Ca2+ concentration; ERK2, extracellular signal-regulated kinase 2; PI3-kinase, phosphatidyl inositide 3-kinase; PKC, protein kinase C; PRP, platelet-rich plasma; EPI, epinephrine; THR, thrombin. Back


   ACKNOWLEDGMENTS
 
We are grateful to PatriciaÖdman for linguistic revision of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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