The F₂-Isoprostane 8-Epiprostaglandin F₂ Increases Platelet Adhesion and Reduces the Antiadhesive and Antiaggregatory Effects of NO

From the Institutes of Clinica Medica (P.M., M.D., A.L.), Chimica e Microscopia Clinica (G.A., S.G., R.T., V.Z., C.L.), and Immunologia e Malattie Infettive (R.O.), University of Verona, Verona, Italy.

Correspondence to Dr P. Minuz, Clinica Medica, Policlinico di Borgo Roma, Via delle Menegone, 37134 Verona, Italy.

	Abstract

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Abstract—F₂-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical–catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F₂ (8-epi-PGF₂) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF₂ (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3±0.2% to 5.5±0.2%, EC₅₀ of 48 nmol/L; fibrinogen from 3.3±0.3% to 6.4±0.2%, EC₅₀ of 35 nmol/L; mean±SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF₂ in resting platelets (from 64.8±2.1% to 83.9±1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF₂ (from 48.5±3.1% to 63.1±2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4±4.6%; with 8-epi-PGF₂, 30.8±6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5±4.6%; with 8-epi-PGF₂, 30.7±5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF₂. In conclusion, F₂-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F₂. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.

Key Words: F₂-isoprostanes • platelet adhesion • nitric oxide

	Introduction

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F₂-isoprostanes are prostaglandin (PG) isomers synthesized in vivo, mostly independently of the activity of cyclooxygenase, through the free radical–catalyzed peroxidation of arachidonic acid in biological membranes.^{1 2} The major interest for studying these compounds comes from the possibility of quantifying in vitro and in vivo the oxidative damage and lipid peroxidation by measuring their rate of production. Increased cellular production of F₂-isoprostanes has been described,^{3 4} and there is evidence of increased synthesis of 8-epi-PGF₂ during cell-mediated and copper-mediated lipoprotein oxidation.^{5 6} Increased urinary excretion or plasma concentrations of 8-epi-PGF₂ have been observed in subjects who smoke cigarettes, in elderly subjects, and in patients suffering from diabetes and familial hypercholesterolemia.^{7 8 9 10} One of the main products of F₂-isoprostane synthesis in vivo, 8-epi-PGF₂, has also been characterized for its biological activities and found to be a potent constrictor of the renal and pulmonary vasculature and a broncoconstrictor,^{11 12} sharing its activities with those of previously described prostanoids. Indeed, the effects of 8-epi-PGF₂ on smooth muscle cells appear to be mediated by the activation of a receptor that is closely related to that of thromboxane A₂.¹³ Limited effects of 8-epi-PGF₂ on platelet function were described in the first reports. Shape change but not irreversible platelet aggregation was induced by high concentrations of 8-epi-PGF₂.^{14 15} Recently, 8-epi-PGF₂ has been observed to potentiate the aggregation induced by subthreshold doses of platelet agonists and increases in platelet [Ca²⁺]_i.¹⁶ All of these effects appear to be mediated by the activation of a receptor that does not induce irreversible aggregation. Therefore, 8-epi-PGF₂ may be active as a partial platelet activator, and this might be relevant in clinical conditions in which increased platelet activation, increased oxidative stress, and increased F₂-isoprostane biosynthesis occur.^{17 18 19}

It has been postulated that lipid oxidation is a key event in the development of atherosclerotic lesions and its thrombotic complications, and 8-epi-PGF₂ might be one of the mediators operating in the complex network of biological messages evoked by oxidative injury.²⁰ However, none of the direct effects on platelet function that have now been described occurred at concentrations of 8-epi-PGF₂ comparable to those observed in plasma.¹⁶

The current study was aimed at further investigating the activity of 8-epi-PGF₂ on platelets by focusing on the early and critical events of platelet activation: platelet adhesion and the expression of adhesion molecules on cell membranes.²¹ We also tried to better define the potential biological role of 8-epi-PGF₂ by using a simulation of the antiaggregatory environment in which circulating platelets are located. We therefore studied the effects of 8-epi-PGF₂ in the presence of endothelial cells releasing NO and in the presence of NO donors. The hypothesis was that 8-epi-PGF₂ might affect platelet-endothelium interaction, since reduced biological activity of endothelium-derived NO has been described in the same clinical conditions characterized by increased lipid oxidation.^{22 23 24}

	Methods

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Platelet Preparation
Venous blood samples (40 mL) were collected from healthy adult volunteers who had not been taking any drugs during the previous 3 weeks and who had given their informed, written permission; an acid-citrate-dextrose mixture (14 mmol/L sodium citrate, 11.8 mmol/L citric acid, and 18 mmol/L dextrose; Merck) was used as an anticoagulant. Washed platelets were obtained from platelet-rich plasma by further centrifugation (700g for 15 minutes), which had been prepared according to Hallam et al²⁵ by low-speed centrifugation (200g for 10 minutes at room temperature) of 10-mL blood samples. Platelets were then suspended in 2 to 3 mL of 145 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES (Sigma Chemical Co), 0.5 mmol/L Na₂HPO₄, and 6 mmol/L glucose, pH 7.4, at 37°C (HEPES buffer) and counted by using an automatic analyzer (model T-890, Coulter Co). A final concentration of 2x10⁸ platelets/mL was obtained by dilution. The cells were kept at room temperature and used for the tests within 2 hours.

Preparation of Endothelial Cells
Cultured human endothelial cells (from human umbilical cord veins; American Type Culture Collection) were used. Cells were grown as a monolayer in standard culture F-12 medium (Sigma Chemical Co) containing FBS (Biochrom Kg, Seromed; 10:100, vol/vol), 100 mg/L heparin (Sigma Chemical Co), and 30 to 50 mg/L endothelial cell growth supplement from bovine neural tissue (Sigma Chemical Co). When the cells reached confluence, 100 µmol/L acetylsalicylic acid (ASA, Bayer) was added to prevent the synthesis of prostanoids; cells were then detached from the flask with 0.5 g/L trypsin (Sigma Chemical Co) in HEPES buffer containing 0.2 g/L EDTA (Carlo Erba). Digestion was stopped by the addition of FBS. Endothelial cells were collected by centrifugation (70g to 80g for 10 minutes at room temperature) and suspended in HEPES buffer containing 100 µmol/L ASA. Cell number was determined by using a Neubauer improved hemocytometer chamber. Concentrations of endothelial cells between 5x10⁴ and 7.5x10⁵/mL were tested within 3 hours for their inhibitory effects on platelet aggregation. N^G-Monomethyl-L-arginine (300 µmol/L, Calbiochem) was used to confirm that the antiaggregatory effects were dependent on NO release, and radioimmunoassay measurement of 6-keto-PGF₁ in the supernatant was used to exclude the presence of the antiaggregatory prostacyclin.^{26 27} The reduction in prostacyclin release (measured by radioimmunoassay as the concentration of 6-keto-PGF₁ in the medium) caused by 100 µmol/L ASA or 10 µmol/L indomethacin exceeded 90% in thrombin-stimulated endothelial cells.

Assay of Platelet Adhesion
Platelet adhesion was assayed, as previously described,²⁸ under static conditions in culture microplates (Flow Laboratories) precoated by means of overnight incubation at 4°C with 0.2 mg/mL human fibrinogen (Sigma Chemical Co) and human plasma diluted 1:1 in Dulbecco's PBS (Gibco Ltd). Plasma used for the adhesion experiments was obtained from the same pool of 10 healthy subjects, as previously described.²⁸ Immediately before use, microplates were washed twice with 0.9% NaCl in an automatic plate washer (Easy Washer 2, SLT Labs Instruments).

Washed platelets were suspended in HEPES buffer containing 0.2% human albumin (Behring Institute) and supplemented with 1 mmol/L CaCl₂ and 1 mmol/L MgSO₄. This platelet suspension was incubated for 10 minutes with or without scalar doses of platelet agonists in a humidified thermostatic chamber (37°C, 5% CO₂). At the end of the incubation period, plates were transferred to an automatic washer and subjected at room temperature to 2 washing cycles with Dulbecco's PBS. Each well contained 2.5x10⁶ platelets in a final volume of 75 µL/well. Platelet adhesion was measured by assaying, as an index of the number of adhering cells, the activity of platelet acid phosphatase.²⁸ The percentage of adherent cells was calculated on the basis of a standard curve obtained with a defined number of platelets from the same subject.

To explore the effects of 8-epi-PGF₂ on platelet adhesion, the assay was carried out by incubating platelets for 40 minutes in the absence or presence of 1 to 1000 nmol/L 8-epi-PGF₂ (Cayman Chemical Co) and in the presence of 8-epi-PGF₂ and thrombin (0.025 U/mL, Calbiochem). Low concentrations of thrombin were used to obtain a limited increase in platelet adhesion and therefore to allow sensitive evaluation of the effects of 8-epi-PGF₂, which had been dissolved in ethanol (1 mg/mL) and then diluted in the assay buffer. The final concentration of ethanol was always <0.3 µL/mL of the total volume.

To investigate the contribution of the endogenous generation of thromboxane A₂ to the activity of 8-epi-PGF₂, experiments were also performed after the addition to platelet suspensions of 50 µmol/L ASA, which almost completely inhibited platelet thromboxane A₂ production. The effects of 8-epi-PGF₂ on platelet adhesion were compared with those of the thromboxane A₂ analogue 15(S)-hydroxy-11,9-(epoxymethano)prostadienoic acid (U46619, Cayman Chemical Co) and those of PGF₂ (Cayman Chemical Co). The effects of 8-epi-PGF₂ on platelet adhesion were also tested in the presence of the NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR-3, Calbiochem).²⁹ These experiments were performed by adding NOR-3 (from 1 µmol/L to 5 µmol/L) to platelet suspensions in the absence or presence of 8-epi-PGF₂ (1 to 1000 nmol/L).

To investigate any possible competition between 8-epi-PGF₂ and U46619 on platelet adhesion, these compounds were added simultaneously to the platelet suspension and tested at a wide range of concentrations (1 to 1000 nmol/L). Possible desensitization of the thromboxane receptor by 8-epi-PGF₂ was investigated by using washed platelets that had been incubated for 30 minutes with 8-epi-PGF₂ or U46619 (0.1 to 1 µmol/L) and then adding U46619 (1 to 10 µmol/L) or 8-epi-PGF₂ (0.01 to 1 µmol/L) when the platelets were seeded in plasma-coated microwells. All of these experiments were performed in the presence of 50 µmol/L ASA to minimize the possible contribution of endogenous thromboxane A₂ generation.

To evaluate whether the activity of 8-epi-PGF₂ was mediated by receptor activation, the thromboxane A₂/PG endoperoxide receptor inhibitor [1r-[1(Z),2ß,3ß,5]]-(±)-7–5-[(1,1'-biphenyl)-4-yl]methoxyl]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid hydrochloride (10 nmol/L, GR32191, Glaxo Group Research) was added to the platelet suspensions 10 minutes before the addition of the studied agonist in the presence of ASA.³⁰ Only 10 nmol/L GR32191 was used, because at higher concentrations it induces increases in platelet adhesion and [Ca²⁺]_i (data not shown).

To investigate whether activation of the fibrinogen receptor was necessary to induce platelet adhesion in the presence of 8-epi-PGF₂ and U46619, we used a monoclonal antibody able to bind the activated fibrinogen receptor IIb/IIIa (PAC1, Becton Dickinson). Washed platelets were incubated with 0.9 µg of PAC1 per 2.5x10⁶ platelets for 10 to 20 minutes to block the receptor.³¹ The adhesion of resting platelets to fibrinogen was reduced by 29.4±4.1% (n=5, P<0.001) when the binding of fibrinogen to its platelet receptor was prevented by using the specific antibody PAC1. Platelet adhesion was then assayed by using fibrinogen-coated microwells in the presence of 100 nmol/L 8-epi-PGF₂ or 1000 nmol/L U46619 in the presence of ASA.

Expression of Glycoprotein (GP) IIb/IIIa (CD41/IIb) and P-Selectin (CD62/GMP-140)
To explore the effects of 8-epi-PGF₂ on the surface expression of GP IIb/IIIa and GMP-140, 1 mL of platelet suspension (2x10⁵ cells/mL) was incubated for 40 minutes at 37°C in the presence or absence of scalar doses of 8-epi-PGF₂. After incubation, 100-µL aliquots of the platelet suspensions were taken from each test tube and immediately diluted. Ten microliters of saturating concentrations of FITC-labeled anti-human P-selectin (clone CLB-thromb/6, Immunotech) and phycoerythrin-labeled anti-human GP IIb/IIIa (clone P2, Immunotech) antibodies were added. The antibody used for the detection of GP IIb/IIIa specifically recognizes the heterodimeric complex, which is the receptor of fibrinogen.³² All experiments included an isotype-matched, nonspecific, conjugated mouse IgG1 FITC/IgG1 phycoerythrin (Immunotech) as a negative control. These platelet suspensions were incubated for 20 minutes at 4°C and the reaction was stopped by addition of 1 mL HEPES buffer at 4°C. Samples were analyzed in duplicate by flow cytometry (FACScan, Becton Dickinson) using dual-color fluorescence. The platelet population was identified on the basis of size and granularity.³³ To explore the possibility that 8-epi-PGF₂ might amplify the expression of GP IIb/IIIa and GMP-140 in the presence of platelet agonists, low doses of thrombin were added to platelet suspensions. All of these experiments were carried out both with and without 50 µmol/L ASA.

To further investigate the effects of 8-epi-PGF₂ on the fibrinogen receptor, experiments were performed using an antibody that specifically binds the active form of the GP IIb/IIIa complex (PAC1-FITC, Becton Dickinson).³⁴ To this purpose, 100 µL of platelet suspension (5x10⁵ cells/mL) was incubated for 2, 10, or 30 minutes at 37°C in the presence or absence of scalar doses of 8-epi-PGF₂. After incubation, 5 µL of a saturating concentration of PAC1 (100 µg/mL) was added, and the test tubes were incubated for an additional 25 minutes at room temperature. The reaction was stopped by addition of 1 mL HEPES buffer at 4°C, and the sample was analyzed. Confirmatory experiments were also performed using different antibodies to detect GMP-140 and GP IIIa (Becton Dickinson).

Platelet Aggregation
The rate of platelet aggregation was monitored for 3 minutes after the addition of the agonist by using a 4-channel aggregometer (Aggrecorder II, PA-3220; Daiichi); the rate was measured as the change in percentage of transmitted light according to Born.³⁵ Suspensions of washed platelets (0.5 mL, 2x10⁸ cells/mL) were maintained in the aggregometer at 37°C for 1 minute in HEPES buffer in the presence of 1 mmol/L CaCl₂ and 1 mmol/L MgSO₄, with continuous stirring at 1000 rpm. They were then stimulated with the agonists. The effects of 8-epi-PGF₂ (10 to 1000 nmol/L) on platelet aggregation were tested in the presence of thrombin at a concentration (0.03 U/mL) able to induce 70% to 80% of the maximum aggregation and of the NO donor NOR-3 (1 to 10 µmol/L). These tests were also carried out in the presence of 10 nmol/L GR32191 and 50 µmol/L ASA. Some experiments were repeated without the addition of ASA. NOR-3 (1 mmol/L) was dissolved in DMSO (Sigma Chemical Co) and then suitably diluted. The final volume of DMSO was <0.1% of the total volume.

The effects of 8-epi-PGF₂ on the antiaggregatory activity of endothelial cells, mediated through the release of NO, were also determined. Submaximal doses of thrombin (0.02 to 0.03 U/mL) were used in these experiments to optimize the antiaggregatory effect of endothelial cells. In fact, endothelial cells, after stimulation with thrombin, release NO, which inhibits platelet activation.^{26 27} The concentration of endothelial cells necessary to achieve a residual platelet aggregation of 0% to 30% ranged between 2x10⁵ and 7.5x10⁵/mL. In these experiments 1 µmol/L 8-epi-PGF₂ was added to the platelet–endothelial cell preparation immediately before thrombin was added.

Platelet [Ca²⁺]_i
[Ca²⁺]_i was measured in thrombin-stimulated platelets with the fluorescence indicator fura 2, according to the method described by Pollock et al.³⁶ After loading platelet-rich plasma with 2 µmol/L fura 2-AM (Calbiochem) for 30 minutes at 32°C plus 15 minutes at room temperature, platelets were collected by centrifugation and suspended (2 to 4x10⁷ cells/mL) in HEPES buffer. The external calcium and magnesium concentrations were restored to 1 mmol/L. Fluorescence measurements were carried out at 37°C by using an F-2000 fluorescence spectrophotometer (Hitachi) with magnetic stirring. The fluorescence signal was monitored by using an excitation wavelength of 340 nm and an emission wavelength of 500 nm. In some experiments [Ca²⁺]_i fluorescence was monitored at excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm.

The effects of 8-epi-PGF₂ on resting platelets were investigated by using the same conditions previously described by Kinsella et al.³⁷ Platelet-rich plasma was incubated with 3 µmol/L fura 2-AM in the dark at 37°C for 45 minutes; platelets were collected after centrifugation (2.5 to 5x10⁷ cells/mL). Experiments were carried out to study desensitization to the effects of U46619 on [Ca²⁺]_i by 8-epi-PGF₂. The fluorescence signal was monitored by using excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm. Experiments were performed to investigate the effects of 8-epi-PGF₂ (1 to 1000 nmol/L) on platelet [Ca²⁺]_i increases induced by thrombin (0.02 to 0.03 U/mL) and ADP (5 µmol/L), both in the presence and absence of 50 µmol/L ASA. The role of 1 µmol/L 8-epi-PGF₂ on the release of calcium from intracellular stores was investigated by comparing [Ca²⁺]_i measured in resting platelets and in thrombin (0.02 to 0.03 U/mL)-stimulated platelets in the presence of 5 mmol/L EGTA.

To evaluate the activity of 8-epi-PGF₂ on thrombin-induced [Ca²⁺]_i increases in the presence of NO, platelet [Ca²⁺]_i was measured after the addition of NOR-3 (1 to 10 µmol/L) to the platelet suspension 10 minutes before 8-epi-PGF₂ (10 to 1000 nmol/L) was added. These experiments were carried out both with and without the addition of ASA to platelet suspensions.

Statistical Analysis
Data are expressed as mean±SEM. One-way ANOVA was used when multiple variables were compared and the data had been obtained from the same platelet preparation; 2-sided Dunnett's test was used for post hoc analysis (computer programs from SPSS Italia). Student's t test for paired data was used when the experiments compared 2 variables and a single platelet preparation had been used. EC₅₀ was calculated by using a computer program (GraphPAD InPlot). P values <0.05 were considered significant.

	Results

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Effects of 8-Epi-PGF₂ on Platelet Adhesion and Adhesion GP Expression
The effects of 8-epi-PGF₂ on platelet adhesion were tested in vitro under static conditions. We observed that the percentage of platelets adhering both to fibrinogen and to plasma-coated microwells was increased by 8-epi-PGF₂ at concentrations ranging from 1 nmol/L to 1 µmol/L. This increase was dose dependent (Figure 1A). Calculated EC₅₀s for this proadhesive effect were 4.8±0.5 and 3.5±0.5x10^-8 mol/L, respectively, when plasma and fibrinogen were used as adhering surfaces. When the experiments were performed in the presence of thrombin (0.03 U/mL), 8-epi-PGF₂ dose-dependently amplified the response of platelets to this agonist (Figure 1B). Calculated EC₅₀s were 4.8±0.3x10^-8 mol/L when 8-epi-PGF₂ was tested in the presence of thrombin on plasma-coated microwells and 2.1±0.5x10^-8 mol/L when tested on fibrinogen-coated microwells. The effects of 8-epi-PGF₂ were additive to those of thrombin, since the increase in platelet adhesion was not statistically different when each dose of 8-epi-PGF₂ was tested in resting versus stimulated platelets (except for a slightly higher increase with 1 µmol/L 8-epi-PGF₂ plus thrombin).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of scalar doses of 8-epi-PGF2 on platelet adhesion of resting (A), thrombin-stimulated (B), and NO-inhibited (C) platelets. Microwells coated with human plasma or fibrinogen were used as proadhesive surfaces. n=8 for A and B; n=5 for C. Values are mean±SEM. *P<0.001, +P<0.01 vs 0 mol/L 8-epi-PGF2.

The effects of 8-epi-PGF₂ on platelet adhesion were also tested in the presence of the NO donor NOR-3. The antiadhesive effect of NO was antagonized by 8-epi-PGF₂, and a dose-dependent increase in platelet adhesion was observed (Figure 1C). These experiments were repeated after the addition of 50 µmol/L ASA to the platelet suspension to inhibit cyclooxygenase-dependent thromboxane synthesis. Under these conditions the effects of 8-epi-PGF₂ were similar to those observed without ASA (Figure 2A). When platelets were incubated with 10 nmol/L of the thromboxane receptor antagonist GR32191, the effects of 8-epi-PGF₂ on platelet adhesion were significantly reduced (Figure 2A).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effects of scalar doses of 8-epi-PGF2 on resting (A) and thrombin-stimulated (B) ASA-treated platelets (n=5). Effects of 8-epi-PGF2 on platelet adhesion of ASA-treated platelets in the presence of 10 nmol/L of the thromboxane receptor inhibitor GR32191 are also shown (A). Values are mean±SEM. *P<0.001 vs 0 mol/L 8-epi-PGF2.

Although both 8-epi-PGF₂ and U46619 increased platelet adhesion, PGF₂ had no effects (Figure 3). The thromboxane mimetic U46619 had no effects on platelet adhesion at concentrations <100 nmol/L. A dose-dependent increase in platelet adhesion was observed when U46619 was tested at higher concentrations (100 to 1000 nmol/L, Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of scalar doses of 8-epi-PGF2 (n=8), U46619 (n=5), and PGF2 (n=6) on in vitro platelet adhesion to microwells coated with human plasma. Values are mean±SEM. *P<0.001 vs 0 mmol/L.

Platelet adhesion to plasma was not further increased when 8-epi-PGF₂ and U46619 were added to ASA-treated-platelet suspensions at the same time (Table 1). The cumulative effects of 1 µmol/L 8-epi-PGF₂ plus 100 nmol/L U46619 were lower than those observed with 1 µmol/L 8-epi-PGF₂ alone (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effects of 8-Epi-PGF2 on Platelet Adhesion in the Presence of the Thromboxane Mimetic U46619

The proadhesive activity of 1 to 10 µmol/L U46619 to human plasma was not affected by preincubation with 1 µmol/L 8-epi-PGF₂ (U46619, 15.4±1.4%; 8-epi-PGF₂ plus U46619, 16.6±0.9%; n=6, P=NS), while preincubation of platelets with 1 µmol/L U46619 resulted in a marked reduction in the activity of 1 to 10 µmol/L U46619 (U46619, 16.3±1.0%; U46619 plus U46619, 10.4±0.4%; n=4, P<0.01). A reduction in the proadhesive activity of 8-epi-PGF₂ was observed only when 1 µmol/L 8-epi-PGF₂ was added to platelets preincubated with 100 nmol/L U46619 (8-epi-PGF₂, 5±0.1%; 8-epi-PGF₂ plus U46619, 1.7%±0.06; n=3, P<0.01). With higher concentrations of these compounds, no additive effects of 8-epi-PGF₂ to the activity of U46619 were observed. The increase in platelet adhesion to fibrinogen induced by 0.1 µmol/L 8-epi-PGF₂ and 1 µmol/L U46619 was not altered by inhibition of GP IIb/IIIa by PAC1 (difference in platelet adhesion with and without PAC1 for 8-epi-PGF₂,-0.6±0.5%; for U46619, -1.2±0.7%; n=3, P=NS).

The effects of 8-epi-PGF₂ on the expression of adhesion molecules were tested by using fluorescence-labeled monoclonal antibodies and flow cytometry. Increased immunodetection of the GP IIb/IIIa complex, but not of the secretory GP GMP-140, was observed in resting platelets after prolonged incubation with 8-epi-PGF₂. This effect was dose dependent (Figure 4A). The effects of 8-epi-PGF₂ were additive to those of thrombin, since the increase in GP IIb/IIIa expression was not statistically different when each dose of 8-epi-PGF₂ was tested in resting versus stimulated platelets (except for a slightly lower increase with 1 µmol/L 8-epi-PGF₂ plus thrombin). A dose-dependent increase in the expression of both GP IIb/IIIa and GMP-140 on platelet surfaces was observed when 8-epi-PGF₂ was added to thrombin. The increase in GP IIb/IIIa expression was not different from that observed when 8-epi-PGF₂ was tested on resting platelets (Figure 4B). All of these experiments were repeated in the presence of ASA to investigate the role of intraplatelet-generated thromboxane A₂ on the activity of 8-epi-PGF₂. No reduction in the activity of 8-epi-PGF₂ was observed after the addition of ASA (Figure 5). No evident increase in the expression of the active form of the fibrinogen receptor was observed after using the specific antibody PAC-1, after both short and prolonged incubation with 8-epi-PGF₂ (<3% of positive cells in all experiments, n=9).

View larger version (23K): [in this window] [in a new window]   Figure 4. Flow cytometry measurement of surface expression of GP IIb/IIIa (CD41) and GMP-140 (CD62) GPs on resting (A) and thrombin-stimulated (B) platelets in the presence of scalar doses of 8-epi-PGF2 (n=5). Values are mean±SEM. *P<0.05, **P<0.005, and ***P<0.001 vs 0 mol/L 8-epi-PGF2.

View larger version (24K): [in this window] [in a new window]   Figure 5. Surface expression of GPs GP IIb/IIIa (CD41) and GMP-140 (CD62) on ASA-treated resting (A) and thrombin-stimulated (B) platelets in the presence of scalar doses of 8-epi-PGF2 (n=5). Values are mean±SEM. *P<0.05, **P<0.01, and ***P<0.001 vs 0 mol/L 8-epi-PGF2.

Effects of 8-Epi-PGF₂ on Platelet [Ca²⁺]_i
The effects of 8-epi-PGF₂ on free [Ca²⁺]_i were tested by using fluorescent probes. Relatively low concentrations of platelets were utilized (2.5x10⁷/mL). Under these conditions, 1 µmol/L 8-epi-PGF₂ increased [Ca²⁺]_i (28.0±4.7 nmol/L; n=3, P<0.05). Preincubation of platelets with 1 µmol/L 8-epi-PGF₂ strongly reduced the calcium increase in response to 1 µmol/L U46619 (U46619, 418.5±77.6 nmol/L; 8-epi-PGF₂ plus U46619, 111.1±21.1 nmol/L; n=4, P<0.05; Figure 6). When thrombin was added to platelet suspensions together with 1 to 1000 nmol/L 8-epi-PGF₂, an increase in [Ca²⁺]_i was observed (Figure 7). An enhanced [Ca²⁺]_i increase by 8-epi-PGF₂ (10 to 1000 nmol/L) also occurred when 5 µmol/L ADP was used as the platelet agonist (Table 2).

View larger version (17K): [in this window] [in a new window]   Figure 6. Typical traces showing effects of the thromboxane analogue U46619 on free [Ca2+]i (A). Addition of 1 µmol/L 8-epi-PGF2 to the platelet suspension caused a marked reduction in [Ca2+]i increase (B).

View larger version (44K): [in this window] [in a new window]   Figure 7. Effects of scalar doses of 8-epi-PGF2on thrombin-induced [Ca2+]i increase in ASA-treated platelets. For each concentration of 8-epi-PGF2, 11 experiments were performed in duplicate. Values are mean±SEM. *P<0.02, ***P<0.001 vs thrombin plus ASA (first of each pair of bars).

View this table: [in this window] [in a new window]   Table 2. Effects of 8-Epi-PGF2 on [Ca2+]i in ADP-Stimulated Platelets Without ASA

The effects of 10 nmol/L and 1 µmol/L 8-epi-PGF₂ on platelet [Ca²⁺]_i were also investigated in the presence of the NO donor NOR-3. The inhibitory effect of NOR-3 on platelet [Ca²⁺]_i in thrombin-stimulated platelets was antagonized by 8-epi-PGF₂ (Figure 8). An increase in [Ca²⁺]_i was also observed in the presence of EGTA (resting platelets, 8.9±1.9 nmol/L; n=7, P<0.01; thrombin-stimulated platelets, 63.0±25.2 nmol/L; n=7, P<0.05).

View larger version (33K): [in this window] [in a new window]   Figure 8. Effects of 8-epi-PGF2 (10 nmol/L and 1 µmol/L) on [Ca2+]i in the presence of the NO donor NOR-3 (percentage of inhibition), with (A) and without (B) ASA. For A, n=9; for B, n=5. Values are mean±SEM.

Effects of 8-Epi-PGF₂ on Platelet Aggregation
Platelet aggregation was not induced by 1 µmol/L 8-epi-PGF₂ alone (0% variation in optical density in all experiments; n=6). The aggregation induced by submaximal doses of thrombin was slightly increased in the presence of 1 µmol/L 8-epi-PGF₂ (thrombin, 67.0±2.1%; thrombin plus 8-epi-PGF₂, 70.1±1.8%; n=32, P<0.05).

The effects of 8-epi-PGF₂ on platelet aggregation were studied by using a model of platelet–endothelial cell interaction. ASA-treated platelets and endothelial cells were used. When thrombin was added to this cell mixture in the aggregometer, we observed that thrombin-induced platelet aggregation was inhibited by the NO released by endothelial cells. This inhibitory effect was significantly reduced in the presence of 1 µmol/L 8-epi-PGF₂. Similarly, the NO donor NOR-3 reduced the aggregatory activity of thrombin in ASA-treated platelets, and the addition of 1 µmol/L 8-epi-PGF₂ blunted the antiaggregatory effects of NOR-3 (Figure 9). This effect was also observed in the absence of ASA (thrombin plus NOR-3, 4.3±1.9%; thrombin plus NOR-3 plus 1 µmol/L 8-epi-PGF₂, 31.0±8.3%; n=5, P<0.01). The thromboxane receptor antagonist GR32191 at 10 nmol/L reduced the effects of 8-epi-PGF₂ on the antiaggregatory activity of NOR-3 (Figure 9).

View larger version (39K): [in this window] [in a new window]   Figure 9. Effects of 1 µmol/L 8-epi-PGF2 on antiaggregatory activity of endothelial cells mediated by NO release (n=15). Effects of 1 µmol/L 8-epi-PGF2 on the antiaggregatory activity of the NO donor NOR-3 (n=13) and NOR-3 plus 10 nmol/L GR32191 (n=6) in ASA-treated platelets are also shown. Numbers of endothelial cells and NOR-3 concentration were determined on the basis of their inhibitory activity of thrombin-induced platelet aggregation. Values are mean±SEM. *P<0.05, **P<0.001 vs first of each pair of bars.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The recent identification of PG isomers as products of lipid peroxidation allows investigators to hypothesize that their biological activity could be responsible for alterations in cell function. This leads to the fascinating speculation that the effects of oxidative damage might be partly due to the activation of specific receptors and be amplified by intracellular signaling systems. The results of the current study indicate that 8-epi-PGF₂ activates platelets by inducing platelet adhesion and reducing the inhibitory activity of NO.

Experiments were performed to test the adhesion of platelets to proadhesive surfaces and the expression of adhesion receptors on platelet surfaces in the presence of 8-epi-PGF₂. Platelet adhesion is a complex process induced by platelet agonists and the specific binding of ligands to their membrane receptors.^{21 38} Receptor-induced adhesion can be potentiated by the presence of agonists, such as thrombin, which not only directly activate platelets but also strengthen the receptor-ligand binding by increasing both the affinity of the receptors for their ligands and the expression of the receptors on platelet surfaces. This has been described in detail for the fibrinogen receptor: the expression of the heterodimeric GP IIb/IIIa on the plasma membrane can be increased by 30% by a variety of platelet agonists, which can also induce the conformational modifications that increase its affinity for fibrinogen.³⁹

This early step in platelet activation, which is observed when the vascular endothelium is damaged or dysfunctional, can be experimentally reproduced by allowing platelets to come in contact with proadhesive surfaces. Under static conditions very few platelets adhere in vitro to human plasma or fibrinogen, but as much as a 10-fold increase in platelet adhesion is observed when relatively low concentrations of thrombin are added to the medium.²⁸

The increase in platelet adhesion induced by 8-epi-PGF₂ occurs at concentrations much lower than those previously described that induce shape change or platelet aggregation.^{14 15 16} The effects of 8-epi-PGF₂ (and the thromboxane mimetic U46619) are specific, since they have not been observed with PGF₂ and can be prevented by the thromboxane receptor antagonist GR32191⁴⁰ but not by ASA. This finding indicates that endogenous generation of thromboxane A₂ is not involved in the proadhesive activity of 8-epi-PGF₂ and that platelet adhesion in vitro does not necessarily involve platelet cyclooxygenase activity.⁴¹

Although our results might suggest occupancy of the thromboxane A₂ receptor by 8-epi-PGF₂, this compound is not simply a thromboxane mimetic: it proved to be more active as a proadhesive agent than the thromboxane analogue U46619, which did not induce platelet adhesion at concentrations <100 nmol/L. Nevertheless, the effects of U46619 were cumulative with those of thrombin but not with those of 8-epi-PGF₂, indicating that a common activator pathway (either a receptor site or a signaling system) could exist for 8-epi-PGF₂ and thromboxane A₂.¹⁴ We also observed that 8-epi-PGF₂ desensitized platelets to the increases in [Ca²⁺]_i induced by U46619 but not to the proadhesive activity of this thromboxane analogue, as already described for its proaggregatory activity,¹⁶ whereas the proadhesive activity of 8-epi-PGF₂ can be partially reduced by U46619. These results indicate that the effects of 8-epi-PGF₂ on platelet [Ca²⁺]_i are mediated by the activation of thromboxane A₂ receptors. 8-Epi-PGF₂, at least at high concentrations, may also interact with the thromboxane A₂ receptor site that mediates the effects of U46619 on platelet adhesion.

Taken together, the available data suggest that 8-epi-PGF₂ has strong affinity for a receptor closely related to the thromboxane receptor, which mediates its effects on platelet adhesion and is also an agonist/antagonist of the receptor site that mediates the increase in calcium induced by U46619. The proadhesive activity of 8-epi-PGF₂ is not dependent on activation of membrane adhesion molecules by their ligands (such as the binding of GP IIb/IIIa to fibrinogen). In fact, we observed that increases in platelet adhesion to fibrinogen also occurred after inactivation of the fibrinogen receptor.

However, nanomolar concentrations of 8-epi-PGF₂ not only increased platelet adhesion but also caused a direct, though weak, increase in the immunodetection of GP IIb/IIIa, but not of P-selectin. Using a different antibody, which specifically binds the active form of the GP IIb/IIIa complex, we observed no increase in binding, indicating that 8-epi-PGF₂ did not induce those conformational changes responsible for its increased affinity to fibrinogen and further platelet activation. When thrombin was added, both the expression of the integrins GP IIb/IIIa and P-selectin increased, and a dose-dependent additive effect by 8-epi-PGF₂ was observed. The GP P-selectin is secreted from the -granules only after platelet activation by agonists.⁴² This result indicates that 8-epi-PGF₂ does not induce platelet secretion unless an additional platelet agonist is present.

Thrombin induces platelet activation through the phospholipase C pathway and the recently described tyrosine phosphorylation processes, leading to platelet adhesion, platelet secretion, and finally irreversible aggregation.^{43 44} The thromboxane analogue U46619 has been observed to induce the phosphorylation of Ras protein via activation of protein kinase C, and this activity may be relevant to platelet activation.⁴⁵ 8-Epi-PGF₂ has been shown to stimulate inositol triphosphate generation and to increase free [Ca²⁺] in platelets.¹⁶ These 2 events might represent the inside-out signal for both the expression of adhesion receptors and the increase in platelet adhesion. 8-Epi-PGF₂ does not induce platelet secretion and platelet aggregation; therefore, these 2 signals are adequate to cause the phenotypic appearance of increased expression of the fibrinogen receptor and increased platelet adhesion to immobilized substrates, whereas complete platelet activation may require the engagement of different signaling mechanisms.⁴⁶

To further investigate the activatory effects of 8-epi-PGF₂ we tested its antagonism toward the antiadhesive and antiaggregatory effects of NO. The antiplatelet activity of NO is recognized as one of the major mechanisms responsible for the antithrombotic properties of the vascular endothelium,⁴⁷ and antiadhesive effects of NO have been described in a variety of experimental models.^{48 49} We tested the effects of 8-epi-PGF₂ on platelet adhesion and platelet aggregation by using experimental models that we found useful for investigating the antiplatelet activity of NO.^{26 50 51} Although platelet adhesion was increased by nanomolar concentrations of 8-epi-PGF₂, platelet aggregation was increased by micromolar concentrations. These antagonistic effects were similar when NO donors and NO derived from endothelial cells were used. NOR-3 spontaneously releases NO in aqueous solution, and the endothelial cells that we used maintained their antiaggregatory effects solely through the release of NO, this effect being prevented by the NO synthase inhibitor N^G-monomethyl-L-arginine.²⁶ The mechanism by which NO inhibits platelet activation is thought to be the increase in intraplatelet cGMP and the activation of G kinases.⁵² Although it might depend on inhibition of an agonist-induced cytoskeleton reorganization, a cGMP-dependent decrease in free [Ca²⁺]_i is a crucial event for the antiplatelet activity of NO.^{53 54 55}

It has been described that 8-epi-PGF₂ increases platelet [Ca²⁺].^{16 36} We observed this effect by using a relatively low cell number in platelet preparations (the same used to test platelet adhesion). The activity of 8-epi-PGF₂ was more evident in the presence of platelet agonists under our experimental conditions. This increase in [Ca²⁺]_i is mainly dependent on the release of calcium from intracellular stores, because it was only partially reduced in the presence of EGTA and is most likely dependent on phospholipase C pathway activation.^{13 16 36} We tested the effects of NO and 8-epi-PGF₂ on free [Ca²⁺]_i in thrombin-stimulated platelets. Under these conditions NO reduces platelet [Ca²⁺]_i, and 8-epi-PGF₂ antagonizes this effect. On the basis of the available data, the observed antagonism of 8-epi-PGF₂ toward the antiadhesive activity of NO may be explained, at least partly, by their opposite effects on intraplatelet[Ca²⁺]_i.

In conclusion, our results indicate that nanomolar 8-epi-PGF₂ concentrations increase platelet adhesiveness and the expression of the fibrinogen receptor. 8-Epi-PGF₂ also reduces the activity of NO through its effects on platelet function. However, the presence of a platelet agonist such as thrombin or ADP is required to induce platelet secretion and aggregation. The signaling system by which 8-epi-PGF₂ is able to induce this initial platelet activation is somewhat different from that engaged by thromboxane A₂, although the effects are prevented by thromboxane receptor antagonists. It appears that 8-epi-PGF₂ does not operate through the system that mediates platelet secretion and aggregation induced by thromboxane A₂. The receptor activated by 8-epi-PGF₂ might be functionally similar to that irreversibly inactivated by GR32191, which mediates platelet shape changes and increases in [Ca²⁺]_i^{56 57} and may not be a single receptor. Finally, platelet activation induced by 8-epi-PGF₂ is not affected by ASA; this finding indicates that the prothrombotic activity of F₂-isoprostanes in vivo may be completely ASA-insensitive.

Although our experimental setting may not entirely represent the physiological conditions in which platelet-endothelium and platelet-subendothelium interactions take place, the results of the current study suggest that 8-epi-PGF₂ may alter the equilibrium between prothrombotic and antithrombotic factors directly activating platelets, also in the presence of a functional endothelium, able to release NO.

It has been suggested that 8-epi-PGF₂ may not be a circulating prothrombotic factor. However, the concentrations that were observed to induce platelet adhesion may be obtained in vivo, particularly where cellular release of isoprostanes⁵⁸ or high concentrations of oxidized lipids^{5 6} occur. This may contribute to local platelet activation by 8-epi-PGF₂ and possibly, other isoeicosanoids. The observed effects of 8-epi-PGF₂ on platelet function may help explain the association between increased oxidative stress, altered NO activity, and increased risk of thromboembolic events observed in clinical conditions such as cigarette smoking, diabetes, and atherosclerosis.^{59 60 61}

	Acknowledgments

 
This study was supported by grants from the University of Verona (fondi di ricerca 60%) to C.L. We acknowledge Glaxo Wellcome for kindly providing us GR32191.

Received July 31, 1997; accepted February 24, 1998.

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