JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 JPET FastThis Forward.article has not Published been copyedited on and November formatted. The 8, final 2010 version as DOI:10.1124/jpet.110.174821may differ from this version. JPET #174821

Title page

Prostaglandin E2 differentially modulates human function through the EP2 and EP3

receptors

Giovanna Petrucci, Raimondo De Cristofaro, Sergio Rutella, Franco O. Ranelletti, Davide

Pocaterra, Stefano Lancellotti, Aida Habib, Carlo Patrono and Bianca Rocca

Downloaded from

Departments of Pharmacology (GP, DP, BR, CP), Medicine (RDC, SL), Pathology (FOR), and

Hematology (SR), Catholic University School of Medicine, Rome, Italy; IRCCS San Raffaele jpet.aspetjournals.org Pisana, Rome, Italy (SR) and American University of Beirut, Beirut, Lebanon (AH).

at ASPET Journals on September 27, 2021

1

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Running title page

Running title: and PGE2 receptors

Corrisponding Autor:

Bianca Rocca, MD

Department of Pharmacology, Catholic University School of Medicine

Largo Francesco Vito 1

00168 Rome, Italy Downloaded from

Phone +39 06 30154253

Fax +39 06 3050159 jpet.aspetjournals.org Email: [email protected]

Document statistics: at ASPET Journals on September 27, 2021 Text Pages: 22 (excluding references)

Tables: 1

Figures: 10

References: 41

Words in Abstract: 249

Words in Introduction: 620

Words in Discussion: 1682

List of Abbreviations

PG: ; COX: ; EP: PGE2 receptors; AA: ; ADP: adenosine diphosphate; cAMP: cyclic adenosine monophosphate; VASP: vasodilator-stimulated phosphoprotein; DAB: 3,3´-diaminodbenzidine; PE: phycoerythrin; FITC: fluorescein isothiocyanate; TRITC: tetramethyl rhodamine isothiocyanate; 11d-16dm PGE2: 11-deoxy-16,16- dimetyl PGE2; PRP: platelet rich plasma; LTA: light transmittance aggregation; LT: lag time

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Abstract

Activated human platelets synthesize prostaglandin (PG) E2, although at lower rate than A2. PGE2 acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized as compared to thromboxane. We studied the effect of PGE2 and its analogs on in vitro human platelet function, and platelet and megakaryocyte EP expression.

Platelets pre-incubated with PGE2 or its analogs were stimulated with agonists and studied by

optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow Downloaded from method, platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin and microaggregates were investigated by flow cytometry. PGE2 at nM concentrations dose-dependently increased the jpet.aspetjournals.org slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC50: 25.6±6 nM,

Emax of 100±19% increase vs. vehicle-treated), without affecting final maximal aggregation. PGE2 stabilized reversible aggregation induced by low ADP concentrations (EC50 37.7±9 nM). The EP3 at ASPET Journals on September 27, 2021 agonists, 11-deoxy-16,16-dimetyl PGE2 (11d-16dm PGE2) and enhanced the secondary wave of ADP-induced aggregation, with EC50 of 48.6±10 nM (Emax 252±51%) and 5±2 nM (Emax

300±35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes

(IC50 40±20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE2 alone raised

2+ 2+ intraplatelet Ca and enhanced ADP-induced Ca increase. 11d-16dm PGE2 and 17-phenyl-trinor

PGE2 (EP3>EP1 agonist) at nM concentrations counteracted PGE1-induced VASP phosphorylation, induced platelet microaggregates, and P-selectin expression. EP1, EP2, EP3 and EP4 were expressed on human platelets and megakaryocytes.PGE2 through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel strategies based on EP modulation.

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Introduction

Activated human platelets synthesize and release prostaglandin (PG) E2 during whole blood clotting, although at concentrations approximately 30-fold lower as compared to thromboxane (TX)

A2 (Patrignani et al, 1982). TXA2, by binding the PGH2/TXA2 receptors (TPs) promotes pro- hemostatic responses such as vasoconstriction and platelet aggregation (Patrono and Rocca, 2010).

PGE2 can bind at least 4 structurally different receptor subtypes (EP1-4) resulting in diverse and

often opposite final biological responses (Narumiya et al, 1999; Tsuboi et al, 2002). The role of Downloaded from

PGE2 in inflammation, pain, fever, gastroprotection and labor is also well established (Narumiya et al, 1999; Tsuboi et al, 2002) and can be pharmacologically modulated by selective and/or non- jpet.aspetjournals.org selective cyclooxygenase (COX) inhibitors (Patrono and Rocca, 2009) or by PGE2 synthetic analogs, as in the case of for gastroprotection or early pregnancy termination (Rocca,

2006). at ASPET Journals on September 27, 2021 The role of PGE2 in hemostasis and, possibly, thrombosis has emerged relatively more recently, mainly from the study of EP-deleted mice. Indeed, EP3-deleted mice display lower platelet response to sub-threshold concentrations of agonists and are less susceptible to experimental thrombosis than wild-type mice (Fabre et al, 2001; Ma et al, 2001; Gross et al, 2007). Messenger

RNAs for EP2, EP4 and EP3 subtypes have been isolated from human platelets (Paul et al, 1998).

Early studies of PGE2 showed its capacity to potentiate platelet aggregation in response to subthreshold concentrations of agonists (Shio and Ramwell, 1972; Bruno et al, 1974; Vezza et al,

1993). Upon the pharmacological characterization of EPs, EP3 receptor agonists have been shown to potentiate human platelet aggregation in response to low concentrations of various agonists

(Matthews and Jones, 1993; Heptinstall et al, 2008; Iyu et al, 2010) and an EP3 antagonist has recently started early clinical development (Singh et al, 2010). However, human EP3 receptor has many splice variants which have different signaling pathways (Kotani et al, 1995; Schmid et al,

1994), and mRNAs for at least four EP3 splicing variants have been isolated from human platelets

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(Paul et al, 1998). Moreover, a ‘dual’ effect of PGE2 on platelet response, ie, activatory at nM concentrations and inhibitory at μM concentrations was repeatedly observed in early studies

(Salzman et al, 1972; Bruno et al, 1974; Andersen et al, 1980; Tynan et al, 1984; Vezza et al, 1993;

Shio and Ramwell, 1997). At least in mice, the latter inhibitory effect has been attributed to heterologous activation of the receptor (Fabre et al, 2001), but it remains unclear whether this heterologous activation occurs in humans or the inhibitory effect results from different

EP activation (Tynan et al, 1984; Gray and Heptinstall, 1985). Downloaded from

In many biological systems, within the same tissue or cell, such as T-cells, mast cells, human fibroblasts, ovary cells, PGE2 can act as positive and negative modulator eliciting different jpet.aspetjournals.org final responses, depending on its concentration in the microenvironment, type of available EPs, different affinities for the EPs present in the system at a given concentration of the agonist (Tsuboi et al, 2002; Rocca, 2006; Hoshikawa et al, 2009; Markosyan and Duffy, 2009; Li et al, 2010). at ASPET Journals on September 27, 2021

Similarly, platelets might be modulated by PGE2 in a more complex way, through different EP subtypes and variants.

The present study was aimed at characterizing: i) the effect of PGE2 on human platelet function in response to different agonists used over a range of concentrations, ii) the effects of selective EP activation, iii) the EP protein expression pattern both in platelets and megakaryocytes, iv) the effects of EP agonists on intraplatelet calcium, vasodilator-stimulated phosphoprotein

(VASP) phosphorylation, microaggregate formation, and P-selectin expression. The results of these studies suggest that PGE2 acts as a fine tuner of platelet function primarily through its interaction with EP2 and EP3, allowing diverse pharmacological strategies potentially useful in cardiovascular treatment and prevention.

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Methods

Materials

Collagen and adenosine diphosphate (ADP) were purchased from Mascia Brunelli (Milan,

Italy), phycoerythrin (PE)-conjugated anti-CD61and isotype- and fluorochrome-matched irrelevant mouse IgGs were purchased from eBioscience (San Diego, CA), FITC-conjugated anti-CD62P (P- selectin) was purchased from Caltag Laboratories (Burlingame, CA), anti-CD61 monoclonal

antibody was obtained from Dako Cytomation (Glostrup, Denmark), affinity purified rabbit Downloaded from polyclonal antibodies against EP2, EP3 and EP4 receptors were purchased from Cayman Chemical

(Ann Arbor, MI), rhodamine (TRITC)-conjugated goat anti-mouse IgG was purchased from jpet.aspetjournals.org Jackson ImmunoResearch Laboratories (West Groove, PA), FITC-conjugated goat anti-rabbit IgG were obtained from VectorLaboratories (Burlingame, CA), Rapide Decalcificant Osseux (RDO) was purchased from Eurobio (Les Ulis, France), biotinylated polyvalent antibody-streptavidin at ASPET Journals on September 27, 2021 peroxidase kit and DAB substrate kit were purchased from Scy-Tek Laboratoires (Logan, UT),

Platelet VASP-P2Y12 kit was obtained from Biocytex (Marseille, France), FURA-2 acetoxy-methyl ester from Sigma (St. Louis, MO), 11-deoxy-16,16-dimethyl PGE2, 17-phenyl-trinor PGE2,

AH6809, butaprost, sulprostone, CAY10580, , indomethacin, SC-560 were all obtained from

Cayman Chemical. PGE2, its stable analogs and COX inhibitors (aspirin, indomethacin, SC-560) were dissolved in ethanol at a concentration at least 500-1000x as compared to the final working concentration.

Blood and bone marrow collection

Venous blood was obtained following informed consent by healthy volunteers from the laboratory Personnel who denied taking any in the preceding 14 days. Blood was taken by forearm venipuncture and dispensed into polystyrene tubes containing tri-sodium citrate (0.38%

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w/v final concentration). Bone marrow slides were obtained from the archives of the Pathology

Department from post-mortem tissues of subjects who died from accidental causes.

Platelet aggregation

For platelet-rich plasma (PRP) preparation, citrated whole blood was centrifuged at 800 rpm for 15 min and the supernatant PRP was removed. Platelet aggregation was measured in PRP by

standard light-transmittance aggregometry (LTA) using a PACKS-4 aggregometer (Helena Downloaded from

Laboratories, Beaumont, TX) in response to ADP, collagen or arachidonic acid (AA), as indicated.

In a typical experiment, 1000x stock solutions of PGE2 or its analogs (as indicated) in ethanol were jpet.aspetjournals.org added to PRP at 1:1000 dilution to the indicated final concentration, in control samples an equivalent volume of ethanol was added, thus, in each sample the final concentration of ethanol was always 0.001%. In preliminary experiments versus saline alone, this ethanol concentration did not at ASPET Journals on September 27, 2021 modify platelet response patterns. After 90 sec of incubation of PRP with PGE2 or its analogs under stirring 150 g at 37°C, the platelet agonist was added. The following parameters of the aggregation tracings were considered for the analyses: the maximal extent of aggregation (Tmax) expressed in percentage of light transmittance, the slope of the aggregation tracings as an index of aggregation velocity (expressed in mm/min) and the lag-time between agonist addition and onset of aggregation in the presence of collagen (expressed in sec). In experiments with lower ADP concentrations giving a single-phase, fully-reversible aggregation, the % of light transmittance 240 sec after agonist addition was used to analyze experimental data.

In experiments using cyclooxygenase inhibitors, PRPs were pre-incubated with each inhibitor for 20 min at 37°C and then LTA experiments were performed as described above.

Flow cytometry analyses

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In a typical experiment investiganting P-selectin expression on platelet surface, aliquots of citrated whole blood were incubated with different concentrations of PGE2 analogs, as indicated, diluted from 1000x stock solutions, or with a similar volume of ethanol as control. After mixing and

5 min incubation without stirring, samples were stained with the primary fluorescent antibody: anti-

P-selectin (1:20 final dilution), anti-CD61 (1:20 final dilution) or isotype- and fluorochrome- matched irrelevant mouse IgGs as negative control, for 1 hour at 4°C. The platelet population was

identified on a Log scale on the basis of forward and side scatter distribution and CD61 positivity, Downloaded from and 30,000 CD61-positive platelets were acquired and analyzed using a FACS Canto® flow cytometer (BD Biosciences, San Josè, CA). Data were analyzed as mean fluorescence intensity jpet.aspetjournals.org (MFI) using the FACS Diva® software package (BD Biosciences).

VASP phosphorylation was assessed in citrated whole blood by flow cytometry using the

Platelet VASP-P2Y12 kit according to the manufacturer’s instructions. In a typical experiment using at ASPET Journals on September 27, 2021 PGE2 analogs, citrated whole blood aliquots were pre-incubated with different concentrations of each compounds, as indicated, or vehicle for 5 min without stirring and then processed according to the manufacturer’s instructions, with or without ADP stimulation, as indicated.

Immunohistochemistry

Immunohistochemistry was performed on washed platelets as previously described (Rocca et al, 2002), using anti-CD61 (1:50 final dilution), anti-EP2 (1:100), anti-EP3 (1:150) and anti-EP4

(1:300) antibodies. After incubation with the anti-CD61 antibody, immuno-detection was performed using a goat anti-mouse TRITC-conjugate. Primary anti-EP2, anti-EP3 and anti-EP4 antibodies were detected with a goat anti-rabbit FITC-conjugate. Bone marrow biopsies were fixed 12 hr in

4% formalin, decalcified in rapid decalcificant for 4 hr, embedded in paraffin, cut at 3μm, mounted on polarized slides and dewaxed. Slides were rehydrated, washed in PBS containing 0.1% Triton X for 2 min and treated with 3% H2O2 for 5 min to block endogenous peroxidase. The antigen

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retrieval was performed with 0.01M citrate buffer pH 6 in a microwave oven at 750W for 10 min.

Bone marrow slides were incubated overnight at 4°C with one of the following primary antibodies: anti-EP2 (1:100), anti-EP3 (1:150) and anti-EP4 (1:300) or normal goat serum as negative control.

For immunoperoxidase, biotinylated polyvalent antibody-streptavidin peroxidase kit was used and developed with the DAB substrate kit. Slides were lightly counterstained with hematoxylin.

Specimens were observed and digitalized by a Zeiss Axioskop (Zeiss, Jena, Germany) equipped

with an intensified charge-coupled device (CCD) camera system (Photometrics, Tucson, AZ). Downloaded from

Ca2+ mobilization jpet.aspetjournals.org Intracellular Ca2+ was measured in platelets labeled with the fluorescent dye FURA-2 using a Cary-Eclipse Fluorescence Spectrophotometer (Varian, Palo Alto, CA), equipped with RX2000

Rapid Mixing Stopped-Flow Unit (Applied Photophysics Ltd, Leatherhead, UK) as previously at ASPET Journals on September 27, 2021 described (Sage et al, 1990). Platelet suspensions were excited at 360 nm and the emitted light was measured at 510 nm, using slits of 10 nm. PRP samples were first incubated with FURA-2 at 37 °C for 30 min, then washed by gel-filtration chromatography, resuspended in Tyrode buffer (HEPES

10 mM, NaCl 135 mM, KCl 3.5 mM, Glucose 5.5 mM, BSA 0.1%, pH 7.4) and adjusted at a final concentration of 130x103 platelets/μL. Three ml of FURA-2 loaded platelets were injected into a cell holder and 3 ml solutions of agonists in Tyrode buffer were simultaneously injected through another syringe into the same cell holder. The tubes carrying the solutions into the mixing chamber passed through a flexible jacket, whose temperature was controlled by a Haake water circulator and a Varian Peltier apparatus. The syringe pistons were moved by compressed air set at 3 Bar and 150

μl of each solution entered the chamber before flow was stopped. For each measurement ADP with or without different PGE2 analogs or vehicle were used as indicated. Based on preliminary experiments to identify the ADP concentration (from 1.25 to 10 μM) which better enabled to study different parameters of the kinetics of intraplatelet Ca2+, 2.5 μM ADP (final concentration) was

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selected. Fluorescence was recorded every 50 ms and analyzed by Cary Eclipse kinetics application.

The [Ca2+] was calculated using the general formula:

2+ Ca = Kd (Fobs-Fmin./ (Fmax.-Fobs) (1)

2+ where Kd is the dissociation constant for Ca binding to the indicator, Fobs is the fluorescent unit, and the Kd calculated for FURA 2 is 224 nM (Grynkewitz et al, 1985), whereas Fmax and Fmin were determined for any data set as previously detailed (Grynkewitz et al, 1985). In the kinetics of

2+ intraplatelet Ca changes three sequential phases were identifiable on the tracings: 1) a lag time Downloaded from

(LT); 2) the raise in cytoplasmic [Ca2+]; 3) a slower decrease in intraplatelet Ca2+ returning to baseline. In our experiments we analyzed the first two phases, eg LT and [Ca2+] raise as follows: LT

2+ jpet.aspetjournals.org was defined as the extrapolation of the maximum velocity (Vmax) of -induced Ca increase

2+ 2+ to its basal value at t = 0 (Ca0 ). The [Ca ] at t = 0 was obtained by linear regression of the first 20 points of the kinetic curve. The maximum velocity of Ca2+ increase was computed by linear at ASPET Journals on September 27, 2021 regression of a 20 experimental points centered around a given Ca2+ concentration, as previously detailed (Grynkewitz et al, 1985). In the fitting procedure, only the ascending part along with the

2+ plateau value of Ca concentration was analyzed to compute Vmax. The time required to reach Vmax

2+ was defined as tmax. When Vmax, Ca0 and tmax were calculated, on the basis of simple trigonometric relations, the LT value was calculated as follows:

2+ 2+ LT = tmax - (Catmax - Ca0 ) Vmax, (2)

2+ 2+ where Catmax is the value of intraplatelet Ca concentration at Vmax. Only the ascending part

2+ along with the plateau value of Ca concentration was analyzed to compute Vmax.

Statistical analysis

Data were first checked for normality of distribution. Differences versus control samples were analyzed with paired or non-paired t test as appropriate. Data are reported as mean ± standard deviation (SD) or standard error (SE) as indicated. Significance was defined as p < 0.05. Analyses

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were performed using SPSS (version 13.0, SSPS Inc., Chicago, IL) and SigmaStat 3.1 (Systat

Software Inc., Hounslow, UK). Grafit software (Erithacus Software, Staines, UK) was used for fittings of the dose-response curves and for calculating the IC50, EC50 or Emax values. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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Results

Platelet aggregation and EP expression

We first tested the effects of pre-incubation with PGE2 (0.2 nM to 150 μM) on LTA induced by increasing concentrations of three different agonists, i.e. ADP, collagen and arachidonic acid

(AA). PGE2 was added to PRPs 90 sec before each agonist. LTA continuously recorded during this pre-incubation under stirring did not detect any optical signal variation.

Based on preliminary experiments, we selected 3 different ranges of ADP concentrations, Downloaded from chosen to generate three distinct patterns of aggregation: lower (2-4 μM) concentrations generating a small monophasic fully reversible aggregation, intermediate (6-8 μM) concentrations generating jpet.aspetjournals.org two distinct primary and secondary phases of aggregation, and a high (20 μM) concentration giving a monophasic, complete and irreversible aggregation. This was designed to selectively explore the effects of PGE2 on primary and secondary ADP-induced phases of aggregation (Andersen et al, at ASPET Journals on September 27, 2021 1980; Hechler et al, 2005) in spite of the individual variability in the sensitivity to ADP and in spite of different commercial ADP preparations. In fact the two sequential phases of ADP-induced aggregation are known to reflect different components of response to ADP: a P2Y1-dependent, reversible, phase and a P2Y12- and release reaction-dependent irreversible phase (Hechler et al,

2005). Furthermore in some experiments we used also 5 μM ADP which was consistently giving a stable primary wave of aggregation.

At the highest (20 μM) ADP concentrations, we did not observe any effect of PGE2 pre- incubation up to 400 nM, on both Tmax and slope of aggregometric traces (data not shown). When the intermediate (mainly 8 μM) ADP concentration was used resulting in a two-wave aggregation,

PGE2 dose-dependently and selectively increased the slope of the secondary phase of aggregation with an EC50 of 25.6±6 nM (Emax 100±19% slope increase vs. vehicle-treated samples) (Figure 1

A and B) without affecting either Tmax or the primary phase of aggregation. At the lowest ADP concentrations generating a small, reversible aggregation, PGE2 dose-dependently stabilized

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aggregation by reverting disaggregation and originated a small secondary wave of aggregation at the highest concentrations (approximately 200 nM, Figure 1C) without modifying the slope and

Tmax of the primary wave. The EC50 of PGE2 in blunting reversible aggregation was 37.7±9 nM

(Emax 137±26% increase of aggregation measured 4 min after agonist addition, vs. vehicle-treated samples) (Figure 1 C and D), being close to the one accelerating secondary aggregation.

Aspirin blocks the platelet synthesis of TXA2, which enhances platelet release reaction and

stabilizes aggregates. Thus, aspirin is known to blunt the secondary phase of ADP-induced Downloaded from aggregation, resulting in reversible aggregation (Shio and Ramwell, 1972). Given the effect of

PGE2 on enhancing the secondary wave of ADP-induced aggregation and on stabilizing reversible jpet.aspetjournals.org aggregation (Figure 1 and 2 A-B), we explored whether exogenous PGE2 was able to partially revert the effect of 50 μM aspirin added in vitro. At this concentration aspirin suppresses by >99%

TXA2 generation from platelets in vitro (Dragani et al, 2010). As expected, 50 μM aspirin inhibited at ASPET Journals on September 27, 2021 the secondary aggregation wave induced by 8 μM ADP (Figure 2A) and reverted stable primary aggregation induced by 5 μM ADP (Figure 2B). When 200 nM PGE2 was added to aspirin- pretreated samples, the effect of aspirin on ADP-induced aggregation was partially reverted

(percentage of light transmittance measured at 4 min after agonist addition: 16±17% with aspirin alone versus 24.3±17.3% with aspirin plus 200 nM PGE2, p=0.003 for paired samples, n=6; and

Figure 2A-C). Similar effects were observed by pre-treating PRP with 20 μM indomethacin or 20

μM SC-560, a selective COX-1 inhibitor (data not shown).

PGE2 in the high nM and μM range (starting from 500 nM) dose-dependently inhibited both

Tmax and slopes of ADP-induced aggregation independently of ADP concentrations, with IC50 of

5±2 μM for slope reduction and of 15±8 μM for Tmax reduction (Figure 3 A-D).

We also studied collagen-induced platelet aggregation. As shown in Figure 3E, at the lowest collagen concentration (1.25 μg/ml), PGE2 significantly increased Tmax (37±18.4% in vehicle- treated samples versus 49.7±18.5% in 200 nM PGE2-pretreated samples, n=7, p=0.031) without

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affecting slope (25.6±5 vs. 27.6±12 mm/min in vehicle-treated vs 200 nM PGE2, n=7, p=0.68), and there was a non-significant trend toward a reduction of the lag interval (123±12 sec vs. 111±25 sec in vehicle-treated vs 200 nM PGE2, respectively, n=7, p=0.10). At higher collagen concentrations

(2.5-5 μg/ml), PGE2 preincubation in the nM range did not significantly affect Tmax or slopes (data not shown), and was associated with a non-significant shortening of the lag intervals (73.13±13 sec and 68.3±13.6 sec in vehicle- and PGE2-treated samples, respectively, n=9, p=0.25). Micromolar

concentrations of PGE2 dose-dependently inhibited collagen-induced aggregation independently of Downloaded from agonist concentrations, reducing both Tmax and slope. Finally, pre-incubation up to 200 nM concentrations of PGE2 did not affect AA-induced aggregation to any statistically significant extent jpet.aspetjournals.org (data not shown).

We next used different PGE2 analogs to study the contribution of each EP to the net effect on platelets observed with PGE2. The 11-deoxy-16,16-dimethyl PGE2 (11d-16dm PGE2) dose- at ASPET Journals on September 27, 2021 dependently and selectively accelerated the secondary phase of 8 μM ADP-induced aggregation with an EC50 of 48.6±10 nM (Figure 4A,C), and the enhancement of the secondary wave of aggregation was greater than the one observed with PGE2, as indicated by the corresponding Emax values (252±51 vs. 100±19 % increase, respectively, p<0.01). Moreover, 11d-16dm PGE2 was also able to trigger a secondary aggregation, dose-dependently, when lower concentrations of ADP were used, with an EC50 of 43.5±7.1 nM (Figure 4B). Similar effects were observed with sulprostone, which enhanced the secondary ADP-induced aggregation phase with an EC50 of 5±2 nM and an

Emax of 300±35%. In addition, sulprostone partially reverted the effect of aspirin on ADP-induced aggregation (Figure 2D). 11d-16dm PGE2 did not modify aggregation induced by the highest concentrations of ADP (20 μM). In response to 2.5 and 5 μg/ml collagen, increasing concentrations of 11d-16dm PGE2 did not affect Tmax or slopes, but the lag time was dose-dependently shortened, with an IC50 of 36±23 nM, and the shape change was abolished between 50 and 70 nM (Figure 4D).

At variance with μM concentrations of PGE2, both sulprostone and 11d-16dm PGE2 at

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concentrations ≥500 nM induced a full irreversible platelet aggregation in the absence of other agonists (data not shown). Based on Kd and Ki values, sulprostone and 11d-16dm PGE2 act preferentially as EP3 agonists (Kiriyama et al, 1997; Abramovitz et al, 2000).

Butaprost, a preferential EP2 agonist (Kiriyama et al, 1997; Abramovitz et al, 2000), dose- dependently and selectively inhibited the secondary wave of 8 μM ADP-induced aggregation

(Figure 5A and B) with an IC50 of 40±20 nM. Moreover butaprost reduced the Tmax of 2-4 μM Downloaded from ADP-induced reversible aggregation with a similar IC50 of 30.4±17 (Figure 5C). When used before

2.5 μg/ml collagen, butaprost significantly reduced the slope, with a significant effect at 1 μM:

68.5±12 vs. 42.3±15.5 mm/min in vehicle- and 1 μM butaprost-treated samples, respectively, n=4, jpet.aspetjournals.org p=0.01. We also observed a non-significant prolongation of lag times and reduction of Tmax

(Figure 5D). The CAY10580, an EP4 agonist (Billot et al, 2003), had only modest effects on platelet response. A statistically significant reduction in slope and Tmax of ADP-induced at ASPET Journals on September 27, 2021 aggregation was detectable only at the highest concentrations of 1μM: 23.9±9% reduction in Tmax and a 23.8±8 % reduction on slope, p=0.036 (n=5).

In light of the above functional data, we investigated EP protein expression by immunohistochemistry in peripheral platelets and bone marrow megakaryocytes. These studies revealed an immunoreactivity for EP1 (not shown), EP2, EP3 and EP4 in platelets (Figure 6) and megakaryocytes (Figure 7). Interestingly, the pattern of positivity of the EPs in megakaryocytes was different, with EP3 positivity showing a reinforcement at the periphery of the cytoplasm, where pro- platelets are formed and released, while the EP2 and EP4 showed a more diffuse cytoplasmic positivity.

Ca2+ movements

2+ Due to the activating effects of 11d-16dm PGE2, we investigated intra-platelet Ca movement, a known pro-aggregator signal. The kinetics of rapid Ca2+ movements was continuously

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recorded by the stopped flow method in washed platelets treated with: vehicle, 2.5 μM ADP alone,

11d-16dm PGE2 alone (10 nM to 1 μM), ADP plus 100 nM 11d-16dm PGE2, 500 nM butaprost or

CAY10580 with or without ADP, and of 100 nM 11d-16dm PGE2 plus the EP3-III antagonist

AH6809 (50 μM) (Abramovitz et al, 2000).

Intraplatelet Ca2+ increase displays a complex kinetics following a reaction of A→B→C type (Sage et al, 1990) and the complex Ca2+ rise induced by ADP is associated with a very rapid influx by receptor-operated channels in the plasma membrane. The delayed phase of the ADP- Downloaded from evoked intraplatelet Ca2+ rise likely results from the release of Ca2+ from intracellular stores (Sage et al, 1990). Notably, the timing of the delayed ADP-evoked event is modulated by intra-cytosolic jpet.aspetjournals.org Ca2+ concentration, being more rapid in onset when internal [Ca2+]i is high (Sage et al, 1990).

Figure 8A, shows indeed that both the Vmax and the maximum calcium concentration values are progressively higher as an inverse function of the LT values, or, conversely, as a direct function of at ASPET Journals on September 27, 2021 the rapid increase in the intraplatelet Ca2+ (inset Figure 8A-B and Table 1).

2+ The 11d-16dm PGE2 dose-dependently increased cytoplasmic Ca (Figure 8A), with an

EC50 of 39±3 nM and it strongly synergized with 2.5 μM ADP in further augmenting intra-platelet

2+ Ca to values of about 1μM (Figure 8B). The AH6809 blunted the effect of 11d-16dm PGE2 while neither butaprost nor CAY10580, alone at different concentrations or in combination with ADP, had any effect on Ca2+ movements (data not shown).

VASP-P phosphorylation, microaggregates and P-selectin expression

We next investigated the effects of different PGE2 analogs on the phosphorylation of platelet

VASP (VASP-P), surface P-selectin expression and platelet microaggregates identified on the basis of forward and side scattering (Fox et al., 2004) together with CD61 positivity.

Pre-incubation of whole blood samples with 200 nM 11d-16dm PGE2 or PGE2 alone decreased platelet VASP-P induced by PGE1 (Figure 9A). We also tested another compound, the

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17-phenyl-trinor PGE2 (EP3>EP1 agonist; Kiriyama et al, 1997), which gave results similar to 11d-

16dm PGE2. At variance with 11d-16dm PGE2, PGE2 or 17-phenyl-trinor PGE2, butaprost added alone to whole blood dose-dependently increased platelet VASP-P as compared to controls (Figure

9B), and counteracted the de-phosphorylating effect of ADP on VASP when added to samples treated with PGE1 and ADP (Figure 9C). The CAY10580 up to 2 μM did not cause any variation of

VASP-P, nor modified the effect of ADP in ADP/PGE1 treated samples (data not shown).

Based on forward (FCS) and side (SSC) scatter parameters of flow cytometry plots together Downloaded from with CD61 positivity, both 11d-16dm PGE2 and 17-phenyl trinor PGE2 alone caused a shift in the

CD61 positive platelet population gate as compared to controls, which is likely due to platelet jpet.aspetjournals.org microggregate formation (Fox et al, 2004) (Figure 10A-D). This effect was dose-dependent and visible on flow cytometry FCS/SSC plots starting from 20 nM 11d-16dm PGE2 and 40 nM 17- phenyl trinor PGE2. No shift in the platelet population gate was observed when butaprost of at ASPET Journals on September 27, 2021 CAY10580 were used up to 2μM (data not shown).

Given the formation of platelet microaggregates, we checked whether 11d-16dm PGE2 or

17-phenyl trinor PGE2 alone were able to induce markers of activation on platelet surface and investigated surface P-selectin (CD62p) expression on non-permeabilized platelets. Both compounds added alone dose-dependently increased P-selectin expression on platelets as compared to vehicle-treated samples (Figure 10E). Butaprost and the CAY10580 at concentrations up to 2

μM, did not induce P-selectin exposure on platelet membranes (data not shown). Finally, AH6809 up to 50 μM did no have any effect on VASP-P nor P-selectin induction (data not shown).

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Discussion

The present work explored the effects of PGE2 in modulating human platelet responsiveness.

Different PGE2 analogs were used to dissect the relative contribution of individual receptor subtypes. Moreover, we provided the first evidence for the presence of several EP proteins in both platelets and megakaryocytes.

We have shown a selective role for nM concentrations of PGE2 in amplifying the secondary

phase of ADP-induced aggregation and stabilizing reversible aggregation, by using increasing Downloaded from concentrations of ADP. Although a role for PGE2 as positive modulator of platelet aggregation has been reported in mice (Fabre et al, 2001; Gross et al, 2007) and humans (Bruno et al, 1974; Vezza jpet.aspetjournals.org et al, 1993; Matthews and Jones 1993), this is the first report identifying the specific phase of platelet response which is modulated by PGE2 as a function of a range of agonist concentrations.

Previous studies on mice used sub-threshold, non-aggregating concentrations of ADP. More recent at ASPET Journals on September 27, 2021 studies using a whole blood method based on platelet counting (Heptinstall et al, 2008) investigated a single, low ADP concentration and could not detect any direct effect of PGE2 (from 0.01 to 10

μM) on platelets, thus the role of PGE2 was indirectly extrapolated by studying the effect of EP antagonists in PGE2-stimulated samples. We also showed for the first time, a positive effect of

PGE2 on collagen-induced aggregation, at low and intermediate collagen concentrations. Based on our data, nM PGE2 acts as positive platelet modulator, stabilizing transient aggregation (low ADP or collagen concentrations) and accelerating the completion of aggregation (eg slope increase of

ADP-induced secondary aggregation, shortening lag time of collagen-induced aggregation).

Consistently, PGE2 counteracted, at least in part, the inhibition exerted by aspirin, again stabilizing and amplifying aggregation.

We used a series of PGE2 analogs to try to identify the receptor(s) mediating PGE2-evoked responses. Both 11d-16dm PGE2 and sulprostone reproduced the positive pattern of PGE2 on the amplification/stabilization of platelet aggregation, but more effectively, as shown by the Emax for

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accelerating the secondary ADP-induced phase, the shortening of the lag interval in collagen- induced aggregation and their capacity of triggering a complete, irreversible aggregation in the high nM/low μM range. Based on Ki values, sulprostone is approximately 30 to 300-fold more selective for EP3 versus EP1, while 11d-16dm is approximately 20-fold more selective for EP3 versus EP4

(Kiriyama et al, 1997; Abramovitz et al, 2000). Considering the rank order of potency for accelerating the secondary phase of ADP-induced aggregation relatively to PGE2 EC50, sulprostone

had a value of 0.19, while 11d-16dm of 1.8. This hierarchy (sulprostone>PGE2>11d-16dm PGE2) Downloaded from and the characteristics of each compound (Kiriyama et al, 1997; Abramovitz et al, 2000), support

EP3 as a mediator of the activating effects of PGE2. The data of 17-phenyl-trinor PGE2 (EP3>EP1 jpet.aspetjournals.org agonist based on Ki values, Kiriyama et al, 1997;) mimicking 11d-16dm PGE2 on VASP-P, microaggregates and P-selectin expression further support this hypothesis. Our results are consistent with and extend previous data (Iyu et al, 2010; Heptinstall et al, 2008), but we used diverse PGE2 at ASPET Journals on September 27, 2021 analogs, calculated the EC50s, ranked potency profiles on specific effects (enhancement of the secondary aggregation, stabilization of aggregates), and explored different platelet agonists at various concentrations. Furthermore, this is the first description of 11d-16dm PGE2-responsive receptors on platelets. This compound likely identifies a specific, pharmacologically-defined subtype of the EP3, which was first identified in erythroleukemia, a megakaryocyte-related cell line

2+ (Feoktistov et al, 1997). Consistently with data on erythroleukemia, 11d-16dm PGE2 increased Ca in platelets, and EC50s were similar in the two cell types (28 nM in erythroleukemia cells, 39 nM in platelets). In our experimental setting 11d-16dm PGE2 not only enhanced ADP-induced intraplatelet

Ca2+ raise, but it also dose-dependently increased intraplatelet Ca2+ when used alone. This is the first report of an EP3 agonist raising alone intraplatelet Ca2+, while previous studies described synergizing effects with other agonists (Heptinstall et al, 2008). Possibly, continuous recording in stopped flow is a more sensitive technique as compared to flow-cytometry-based single measurements. The kinetics of Ca2+ increase in platelets was similar to cells transfected with human

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EP3 splice variants (An et al, 1994; Schmid et al, 1995). Moreover, the kinetics of Ca2+ increase induced by 11d-16dm PGE2 alone in platelets resembled the one of ADP, possibly reflecting similar signaling.

EP3 splice variants have been described to signal not only through Ca2+ increase but also through cAMP reduction (An et al, 1994; Schmid et al, 1995; Kotani et al, 1995). Consistently, 11d-

16dm PGE2 and 17-phenyl-trinor PGE2 were able to counteract PGE1-induced increase in platelet

VASP-P. Similar findings have been reported using sulprostone (Heptinstall et al, 2008). Platelet Downloaded from

VASP phosphorylation is due to an increase in cAMP. Thus EP3 stimulation appears also to counteract cAMP increase, thus increasing platelet reactivity. Based on our data, different EP3 jpet.aspetjournals.org variants might operate in platelets increasing intraplatelet Ca2+ and/or reducing cAMP.

Interestingly, only some human EP3 splice variants increase Ca2+, while other signal through cAMP

(An et al, 1994; Schmid et al, 1995). In our experimental setting, AH6809 (EP3-III antagonist, at ASPET Journals on September 27, 2021 2+ Kiriyama et al, 1997;) antagonized 11d-16dm PGE2–induced Ca increase but not VASP-P or P- selectin expression. Consistently, mRNAs for 4 EP3 splice variants (Ib, III, III and IV), have been isolated in platelets. Altogether these data might indicate that different EP3 variants potentiate platelet function.

Both 11d-16 dm PGE2 and 17-phenyl-trinor PGE2 shifted the FSC/SSC of the CD61 positive gate at flow cytometry, which can be accounted for by microaggregate formation (Fox et al, 2004).

Flow cytometry, being more sensitive than LTA, possibly detected early microaggregates at nM concentrations of EP3 agonists, while LTA could only detect macroaggregates at higher (high nM) concentrations. In agreement with an activatory pattern of PGE2/EP3, platelet membrane P-selectin was induced by 11d-16dm PGE2 and 17-phenyl-trinor PGE2. This can be a relevant mechanism bridging platelets in between coagulation and inflammation. PGE2 is synthesized at sites of inflammation and platelet P-selectin mediates platelet-leukocyte interaction (Dixon et al, 2006).

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Indeed, we observed an enrichment in CD61 positivity in the FSC/SSC gate of leukocytes when the

EP3 agonists were used (data not shown).

In our experiments, the EP2 agonists butaprost exerted a negative modulation of platelet function, on the same phase of ADP- and collagen-induced platelet aggregation enhanced by EP3 activation. A negative effect of butaprost on PAF-induced platelet aggregation has also been reported (Matthews and Jones, 1993; Iyu et al, 2010). Butaprost did not modify intraplatelet Ca2+,

with or without ADP, while it promoted VASP-P indicating a cAMP-mediated effect, which is Downloaded from consistent with previous data on different cells (Regan et al, 1994). Based on Emax values, sulprostone and 11d-16dm PGE2 were more effective than PGE2 in accelerating the secondary jpet.aspetjournals.org phase of ADP-induced aggregation, and, at variance with the inhibitory effect of PGE2, at the highest concentrations (>500 nM) triggered aggregation in the absence of other agonists. Thus, together with the data of butaprost, our results might indicate that the net response to nM PGE2 at ASPET Journals on September 27, 2021 concentrations might result from the balance between EP2 and EP3 (variants) activation.

Consistently, based on Kd values, PGE2 has higher affinity for EP3 as compared to EP2

(approximately 5 to 10-fold difference, Kiriyama M et al, 1997; Abramovitz et al, 2000). Thus an

EP3-driven response might be triggered by lower PGE2 levels, while EP2 might prevail at higher concentrations. Based on previous (Gray et al, 1991) and our data, and on the lower affinity of

PGE2 for the EP2 as compared to the EP3 (Kiriyama M et al, 1997; Abramovitz et al, 2000), it is also conceivable that the platelet-inhibiting effect of μM concentrations of PGE2 can be mediated by the EP2 rather than by an heterologous IP stimulation, at least in humans. At variance with recent reports (Kuriyama et al, 2010), we could not detect any major effect of EP4 stimulation, even though EP4 was present in platelets and megakaryocytes.

PGE2 synthesized during in vitro whole blood clotting, which represents the maximal biosynthetic capacity of platelet COX-1 activity, reaches levels of approximately 10 ng/ml corresponding to 28 nM (Patrignani et al, 1982). This concentration is surprisingly close to the

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EC50s we observed for the activatory effects of PGE2 on platelets. The platelet origin of PGE2 synthesized in this experimental setting, at least under physiological conditions, is suggested by its virtually-complete suppression by low-dose aspirin administered in vivo (7±4.3 ng/ml, n=18 healthy subjects vs. 0.2±0.1 ng/ml, n=21 healthy subjects on aspirin, unpublished data). Thus, during platelet activation, the amount of released PGE2 is likely to fall within the range required for amplifying platelet function. In addition, PGE2 could have a different cellular origin at sites of

inflammation, thus platelet function could be also modulated by PGE2 released from inflammatory Downloaded from cells. Based on our data, it is conceivable that pharmacological strategies based on selective TXA2 receptor blockade or inhibition of TX-synthase, might be less effective than expected, because of jpet.aspetjournals.org unopposed PGE2/EP3 pathway. Indeed, recently a TP antagonist (Terutroban) failed to demonstrate superiority versus low-dose aspirin in a large phase 3 trial (Patrono and Rocca, 2010).

The presence of several EPs on the same cell, exerting different effects, is common for PGE2 at ASPET Journals on September 27, 2021 (Rocca 2006; Hoshikawa et al, 2009; Markosyan and Duffy, 2009). Possibly, the variable PGE2 concentration within the microenvironment can elicit an activatory or inhibitory modulation, finely tuning platelet responsiveness. Thus, the EP3 blockers in clinical development (Singh et al, 2010) might have the advantage of leaving the inhibitory PGE2/EP2 pathway unopposed. Selective agonism of platelet EP2 deserves further investigation as a potential adjuvant strategy for the prevention of atherothrombosis. Finally, the strong positivity of EPs in megakaryocytes might also suggest a role in megakaryopoiesis. Indeed PGE2 is the prevalent eicosanoid during megakaryocyte maturation (Rocca et al, 2002). Although we detected EP1 protein in platelets and megakaryocytes, the lack of commercially-available specific agonists precluded further characterization of its role in platelet function. In conclusion, we have shown that the net effect of PGE2 at low concentrations consists of enhancing stabilization and amplification of platelet response. This appears to result from a complex balance between EP3 variants- and EP2-mediated responses which positively and

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negatively modulate platelet aggregation, respectively. These findings should help guiding the selection of novel antithrombotic stratesies based on platelet EP modulation.

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Acknowledgements

The contribution of the late Dr. Nicola Maggiano to this paper is gratefully acknowledged, and will be always remembered among his colleagues and friends.

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Author Contribution

GP performed immunohistochemistry and platelet aggregation studies. RDC contributed to designing the study and performed kinetic analyses of Ca2+ mobilization experiments. SR performed flow cytometry experiments and analyses. FOR performed immunohistochemistry, and participated to the design of the phenotypic study. DP performed Ca2+ mobilization and VASP-P experiments. SL performed Ca2+ mobilization experiments. AH contributed to P-selectin

experiments and platelet aggregation planning. CP contributed to designing and discussing Downloaded from experiments and writing the manuscript. BR contributed to design of the project , performed experiments of platelet aggregation, statistical analyses of the data and contributed to write the jpet.aspetjournals.org manuscript. at ASPET Journals on September 27, 2021

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References

Abramovitz M, Adam M, Boie Y, Carriere M, Denis D, Godbout C, Lamontagne S,

Rochette C, Sawyer N, Tremblay NM, Belley M, Gallant M, Dufresne C, Gareau Y, Ruel R, Juteau

H, Labelle M, Ouimet N and Metters KM (2000) The utilization of recombinant receptors to determine the affinities and selectivities of and related analogs. Biochim

Biophys Acta 1483:285-293.

An S, Yang J, So SW, Zeng L, Goetzl EJ (1994) Isoform of the EP3 subtype of human Downloaded from receptor transduce both intracellular calcium and cAMP signals. Biochemistry

33:14496-14502 jpet.aspetjournals.org Andersen NH, Eggerman TL, Harker LA, Wilson CH and De B (1980) On the multiplicity of platelet prostaglandin receptors. I. Evaluation of competitive antagonism by aggregometry.

Prostaglandins 19:711-735. at ASPET Journals on September 27, 2021 Billot X, Chateauneuf A, Chauret N, Denis D, Greig G, Mathieu MC, Metters KM, Slipetz

DM and Young RN (2003) Discovery of a potent and selective agonist of the prostaglandin EP4 receptor. Bioorg Med Chem Lett 13:1129-1132.

Bruno JJ, Taylor LA and Droller MJ (1974) Effects of prostaglandin E2 on human platelet adenyl cyclase and aggregation. Nature 251:721-723.

Dixon DA, Tolley ND, Bemis-Standoli K, Martinez ML, Weyrich AS, Morrow JD, Prescott

SM and Zimmerman GA (2006) Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling. J Clin Invest 116:2727-2738.

Dragani A, Pascale S, Recchiuti A, Mattoscio D, Lattanzio S, Petrucci G, Mucci L, Ferrante

E, Habib A, Ranelletti FO, Ciabattoni G, Davi G, Patrono C and Rocca B (2010) The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy. Blood 115:1054-1061.

26 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Fabre JE, Nguyen M, Athirakul K, Coggins K, McNeish JD, Austin S, Parise LK,

FitzGerald GA, Coffman TM and Koller BH (2001) Activation of the murine EP3 receptor for

PGE2 inhibits cAMP production and promotes platelet aggregation. J Clin Invest 107:603-610.

Feoktistov I, Breyer RM and Biaggioni I (1997) Prostanoid receptor with a novel pharmacological profile in human erythroleukemia cells. Biochem Pharmacol 54:917-926.

Fox SC, Sasae R, Janson S, May JA and Heptinstall S (2004) Quantitation of platelet

aggregation and microaggregate formation in whole blood by flow cytometry. Platelets 15:85-93. Downloaded from

Gray SJ and Heptinstall S (1985) The effects of PGE2 and CL 115,347, an antihypertensive

PGE2 analogue, on human blood platelet behaviour and vascular contractility. Eur J Pharmacol jpet.aspetjournals.org 114:129-137.

Gray SJ and Heptinstall S (1991) Interaction between prostaglandin E2 and inhibitors of platelet aggregation which act through cyclin AMP. Eur J Pharmacol 194:63-70. at ASPET Journals on September 27, 2021 Gross S, Tilly P, Hentsch D, Vonesch JL and Fabre JE (2007) Vascular wall-produced prostaglandin E2 exacerbates arterial thrombosis and atherothrombosis through platelet EP3 receptors. J Exp Med 204:311-320.

Grynkiewicz G, Poenie M and Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450.

Hechler B, Cattaneo M and Gachet C (2005) The P2 receptors in platelet function. Semin

Thromb Hemost 31:150-161.

Heptinstall S, Espinosa DI, Manolopoulos P, Glenn JR, White AE, Johnson A, Dovlatova N,

Fox SC, May JA, Hermann D, Magnusson O, Stefansson K, Hartman D and Gurney M (2008) DG-

041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease. Platelets 19:605-613.

27 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Hoshikawa H, Goto R, Mori T, Mitani T and Mori N (2009) Expression of prostaglandin E2 receptors in oral squamous cell carcinomas and growth inhibitory effects of an EP3 selective antagonist, ONO-AE3-240. Int J Oncol 34:847-852.

Iyu D, Glenn JR, White AE, Johnson AJ, Fox SC and Heptinstall S (2010) The role of prostanoid receptors in mediating the effects of PGE(2) on human platelet function. Platelets

21:329-342.

Kiriyama M, Ushikubi F, Kobayashi T, Hirata M, Sugimoto Y and Narumiya S (1997) Downloaded from

Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 122:217-224. jpet.aspetjournals.org Kotani M, Tanaka I, Ogawa Y, Usui T, Mori K, Ichikawa A, Narumiya S, Yoshimi T and

Nakao K (1995) Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger at ASPET Journals on September 27, 2021 systems and tissue-specific distributions. Mol Pharmacol 48:869-879.

Kuriyama S, Kashiwagi H, Yuhki KI, Kojima F, Yamada T, Fujino T, Hara A, Takayama K,

Maruyama T, Yoshida A, Narumiya S and Ushikubi F (2010) Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation. Thromb

Haemost 104 [Epub ahead of print].

Li YJ, Wang XQ, Sato T, Kanaji N, Nakanishi M, Kim M, Michalski J, Nelson AJ, Sun JH,

Farid M, Basma H, Patil A, Toews ML, Liu X and Rennard SI (2010) Prostaglandin E2 Inhibits

Human Lung Fibroblast Chemotaxis through Disparate Actions on Different E-Prostanoid

Receptors. Am J Respir Cell Mol Biol. [Epub ahead of print].

Ma H, Hara A, Xiao CY, Okada Y, Takahata O, Nakaya K, Sugimoto Y, Ichikawa A,

Narumiya S and Ushikubi F (2001) Increased bleeding tendency and decreased susceptibility to thromboembolism in mice lacking the prostaglandin E receptor subtype EP(3). Circulation

104:1176-1180.

28 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Markosyan N and Duffy DM (2009) Prostaglandin E2 acts via multiple receptors to regulate plasminogen-dependent proteolysis in the primate periovulatory follicle. Endocrinology 150:435-

444.

Matthews JS and Jones RL (1993) Potentiation of aggregation and inhibition of adenylate cyclase in human platelets by prostaglandin E analogues. Br J Pharmacol 108:363-369.

Narumiya S, Sugimoto Y and Ushikubi F (1999) Prostanoid receptors: structures, properties,

and functions. Physiol Rev 79:1193-1226. Downloaded from

Patrignani P, Filabozzi P and Patrono C (1982) Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest 69:1366-1372. jpet.aspetjournals.org Patrono C and Rocca B (2009) Nonsteroidal antiinflammatory drugs: past, present and future. Pharmacol Res 59:285-289.

Patrono C, Rocca B (2010) The future of antiplatelet therapy in cardiovascular disease. Annu at ASPET Journals on September 27, 2021 Rev Med 61:49-61.

Paul BZ, Ashby B and Sheth SB (1998) Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells. Br J Haematol

102:1204-1211.

Regan JW, Bailey TJ, Pepperl DJ, Pierce KL, Bogardus AM, Donello JE, Fairbairn CE,

Kedzie KM, Woodward DF and Gil DW (1994) Cloning of a novel human with characteristics of the pharmacologically defined EP2 subtype. Mol Pharmacol 46:213-220.

Rocca B, Secchiero P, Ciabattoni G, Ranelletti FO, Catani L, Guidotti L, Melloni E,

Maggiano N, Zauli G and Patrono C (2002) Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets. Proc Natl Acad Sci U S A 99:7634-

7639.

Rocca B (2006) Targeting PGE2 receptor subtypes rather than : a ‘bridge over troubled water’? Molecular Interventions 6: 68-73.

29 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Sage SO, Reast R and Rink TJ (1990) ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store. Biochem J

265:675-680.

Salzman EW, Kensler PC and Levine L (1972) Cyclic 3',5'-adenosine monophosphate in human blood platelets. IV. Regulatory role of cyclic amp in platelet function. Ann N Y Acad Sci

201:61-71.

Schmid A, Thierauch KH, Schleuning WD and Dinter H (1995) Splice variants of the Downloaded from human EP3 receptor for prostaglandin E2. Eur J Biochem 228:23-30.

Shio H and Ramwell P (1972) Effect of prostaglandin E 2 and aspirin on the secondary jpet.aspetjournals.org aggregation of human platelets. Nat New Biol 236:45-46.

Singh J, Zeller W, Zhou N, Hategan G, Mishra RK, Polozov A, Yu P, Onua E, Zhang J,

Ramirez JL, Sigthorsson G, Thorsteinnsdottir M, Kiselyov AS, Zembower DE, Andresson T and at ASPET Journals on September 27, 2021 Gurney ME (2010) Structure-activity relationship studies leading to the identification of (2E)-3-[l-

[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-lH-indol-7-yl]-N-[(4,5-dichloro-2- thienyl)sulfonyl]-2-propenamide (DG-041), a potent and selective prostanoid EP3 receptor antagonist, as a novel antiplatelet agent that does not prolong bleeding. J Med Chem 53:18-36.

Tsuboi K, Sugimoto Y and Ichikawa A (2002) Prostanoid receptor subtypes. Prostaglandins

Other Lipid Mediat 68-69:535-556.

Tynan SS, Andersen NH, Wills MT, Harker LA and Hanson SR (1984) On the multiplicity of platelet prostaglandin receptors. II. The use of N-0164 for distinguishing the loci of action for

PGI2, PGD2, PGE2 and hydantoin analogs. Prostaglandins 27:683-696.

Vezza R, Roberti R, Nenci GG and Gresele P (1993) Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C. Blood 82:2704-2713.

30 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Footnote

This work was supported by the European Commission FP6 funding [# LSHMCT-2004-005033] and by the funding D1 of the Catholic University School of Medicine of Rome [# 70200368]. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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Legends for figures

Figure 1. Effect of PGE2 pre-incubation on ADP-induced platelet aggregation.

Panels A: Platelets were pre-incubated with vehicle or increasing concentrations of PGE2 indicated in the figure, and then aggregated with 8μM ADP resulting in a biphasic aggregation,

PGE2 enhanced the slope of the second wave of aggregation trace. The dose-response curve of

PGE2 concentrations versus percentage increase of the slopes of the secondary aggregation is shown Downloaded from in panel B; each point represents means±SD (n=7). Panels C and D: at 2 μM ADP a fully reversible aggregation was generated which was stabilized by pre-incubation with increasing concentrations of jpet.aspetjournals.org

PGE2 (panel C). The dose-response fitting of the PGE2 concentrations inhibiting reversible aggregation, measured as light transmittance units 4 minutes after agonist addition, is shown in panel D; each point represents means±SD (n=5). In panels A and C asterisks indicate the addition of at ASPET Journals on September 27, 2021 ADP.

Figure 2. Effect of PGE2 or sulprostone pre-incubation on reverting aspirin inhibition.

Panel A: Platelets were pre-incubated with vehicle, 50 μM aspirin alone, 200 nM PGE2 alone or in combination with aspirin, and then aggregated with 8μM ADP, which resulted in a biphasic aggregation in vehicle-treated samples. The figure shows that PGE2 increased the slope of the second wave of aggregation when used alone and reverted, in part, the inhibitory effect of aspirin. Panel B: platelets were pre-incubated with vehicle, aspirin alone, or 200nM PGE2 in combination with aspirin, and then aggregated with 5 μM ADP. ADP generated a stable primary aggregation wave in vehicle-treated samples, while aspirin caused a reversible aggregation returning to baseline. Panel C: PGE2 partly blunted the effect of aspirin, by stabilizing reversible aggregation (experiment performed in duplicate). In each panel asterisks indicate the addition of the agonist. Panel D: platelets were treated with vehicle, 50 μM aspirin alone or with 50nM sulprostone

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and then aggregated with 4 μM ADP. Sulprostone rescued in part the inhibitory effect exerted by aspirin.

Figure 3. Effect of μM PGE2 pre-incubation on ADP-induced aggregation and of nM PGE2 pre-incubation on collagen-induced aggregation.

Panels A-C: Platelets were pre-incubated with or without vehicle (A), or different Downloaded from concentrations of PGE2 in the high nM and μM range (B and C), and then aggregated with 10 μM

ADP. The panels shows that within this range of concentrations PGE2 dose-dependently inhibited aggregation. Panel D: dose-response curve of PGE2 concentrations versus percentage decrease of jpet.aspetjournals.org the slopes. Panel E: at 1.25 μg/ml collagen, increasing concentrations of PGE2 increased Tmax and slightly shortened the lag time. In each panel asterisks indicate the addition of the agonist.

at ASPET Journals on September 27, 2021

Figure 4. Effects of 11d-16dm PGE2 pre-incubation on ADP- and collagen-induced platelet aggregation.

Panels A and B: PRP was pre-incubated with vehicle or 20 to 200 nM 11d-16dm PGE2 and then aggregated with 8 μM (A) or 4 μM (B) ADP. 11d-16dm PGE2 dose-dependently potentiated the secondary phase of aggregation. Panel C: The dose-response curve of 11d-16dm PGE2 concentrations versus % increase of the slopes of the secondary wave of aggregation in response to

8μM ADP is shown; each point expresses Mean±SD (n=4). Panel D: 2.5 μg/ml collagen-induced aggregation after incubation with vehicle or different concentrations of 11d-16dm PGE2. The main effect was a dose-dependent shortening of the lag interval and abolishment of the shape change. In each panel asterisks indicate the addition of the agonist.

Figure 5. Effects of butaprost pre-incubation on ADP- and collagen-induced platelet aggregation.

33 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Panels A and B: butaprost dose-dependently inhibited the secondary wave of 8 μM ADP- induced aggregation (A) and the correspondent dose-response inhibition of the slopes of the secondary waves is shown in panel B. Panel C: butaprost decreased Tmax and enhanced dis- aggregation at lower (4 μM) ADP concentrations. Panel D: effect of butaprost on collagen-induced aggregation; the response to collagen with a pre-incubation with 200 nM 11d-16dm PGE2 is also shown in this panel. In each panel, asterisks indicate the addition of the agonist.

Downloaded from

Figure 6. Expression of EP2, EP3 and EP4 on peripheral platelets.

Panels A-F: double immunostaining of washed platelets reacted with antibodies against EP2 jpet.aspetjournals.org (A), EP3 (C) or EP4 (E) together with CD61 (B, D, F). Immunoreactions were revealed by FITC- conjugated (A,C,E) or by TRITC-conjugate (B,D,F) secondary Ab. Specimens were observed and digitalized by a fluorescence Zeiss Axioskop equipped with an intensified charge-coupled device at ASPET Journals on September 27, 2021 camera service Phothometrics. Original magnification: 1000X.

Figure 7. Expression of EP2, EP3 and EP4 on bone marrow megakaryocytes.

Panels A-D: bone marrow biopsies were reacted whit anti EP2 (A), anti- EP3 (B) or anti-

EP4 (C) Abs and revealed with DAB chromogen. A negative control (D) was obtained with normal goat serum and omitting the primary antibodies. A reinforcement of the positivity in the periphery of the cytoplasm is visible in EP3-stained megakaryocytes (panel B), as compared to EP2 and EP4- stained samples. Specimens were observed and digitalized by a fluorescence Zeiss Axioskop equipped with an intensified charge-coupled device camera service Phothometrics. Original magnification: 1000X.

Figure 8. Intraplatelet calcium movements in washed platelets.

34 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Panel A shows the traces of fluorescence intensity from FURA-2 loaded washed platelets as a function of time (sec) in response to vehicle, or 11d-16dm PGE2 alone used at 10 nM, 100 nM,

500 nM, 1000 nM, as indicated in the plot. In the inset, the correspondent early phases of intra- platelet calcium movements are shown. Panel B shows a different experiment where washed platelets were stimulated with vehicle, 2.5 μM ADP, or 100 nM 11d-16dm PGE2 alone or by the combination of the two, as indicated in the plot. In the inset, the correspondent early phases of intra-

platelet calcium movements are shown. In each panel the continuous curves were drawn according Downloaded from to equation 2 with the best fit parameter values listed in Table 1.

jpet.aspetjournals.org

Figure 9. Effects of different PGE2 analogs on VASP-P expression.

Panel A: VASP-P expression in samples treated with PGE1 alone or associated with PGE2 or

11d-16dm PGE2, isotypic control of vehicle/PGE1-treated samples is also shown. Panel B: butaprost at ASPET Journals on September 27, 2021 dose-dependently increased VASP-P, isotypic control, butaprost 10 nM, 100 nM and 1μM. Panel C: butaprost pre-incubation in part counteracted the reduction of VASP-P induced in samples treated with the combination of PGE1 and ADP: the plot shows flow cytometry histograms of samples treated with PGE1/ADP + vehicle, PGE1/ADP + 100 nM butaprost, PGE1/ADP + 1μM butaprost.

Figure 10. Effects of different PGE2 analogs on microaggregates or P-selectin expression in whole blood platelets.

Panels A and C show the different FSC-A/SSC-A characteristics of vehicle-treated (A) and

100 nM 11d-16dm PGE2-treated (C) samples, indicating the correspondent % over the total CD61- positive population. Panels B and D represent the CD61 positivity of the ‘P2’ gate drawn in the correspondent FCS/SSC scatter plots. MFI values for CD61 are indicated in each panel. Panel E: membrane P-selectin expression in non-permeabilized platelets treated with vehicle, 100 nM and

250 nM 11d-16dm PGE2.

35 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. JPET #174821

Tables

Table 1. Different parameters of intraplatelet Ca2+ in the presence of ADP with or without

11d-16dm PGE2.

Agonist Lag Time (sec) Vmax (nM Ca2+/sec)

ADP 2.5 µM 2.5±0.3 5.8 ± 1.2 nM/sec Downloaded from 11d-16dm PGE2 100 nM 7±0.4 0.6± 0.1 nM/sec

11d-16dm PGE2 500 nM 5±0.4 1.23± 0.2 nM/sec

11d-16dm PGE2 1000 nM 4.8±0.5 2.3± 0.3 nM/sec jpet.aspetjournals.org

11d-16dm PGE 100 nM 2 2.1±0.3 65± 15 nM/sec + ADP 2.5 µM Values are Means and Standard Deviations (n=4 determinations). at ASPET Journals on September 27, 2021

36 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on November 8, 2010 as DOI: 10.1124/jpet.110.174821 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021