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Circulation. 1995;91:2080-2088

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(Circulation. 1995;91:2080-2088.)
© 1995 American Heart Association, Inc.

Articles

Effects of Aspirin on Platelet-Neutrophil Interactions

Role of Nitric Oxide and Endothelin-1

A. López-Farré, PhD; C. Caramelo, MD; A. Esteban, MD; M. L. Alberola, MD; I. Millás, MS; M. Montón, PhD; S. Casado, MD

From Laboratorio de Nefrologia-Hipertensión, Instituto de Investigaciones Médicas, Fundación Jiménez Díaz, Facultad de Medicina, Universidad Autónoma, Madrid, Spain.

Correspondence to Carlos Caramelo, MD, Instituto de Investigaciones Médicas, Fundación Jiménez Díaz, Facultad de Medicina, Universidad Autónoma de Madrid, Avda Reyes Católicos, 2, 28040 Madrid, Spain.


*    Abstract
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 *Abstract
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Background In recent studies, the hypothesis has been raised that the mechanisms by which aspirin acts as a protective anti-ischemic agent exceed the inhibition of platelet thromboxane A2 synthesis. Recently, new data have been obtained disclosing a platelet-antiaggregating effect by neutrophils, which occurs through a nitric oxide (NO)/cGMP-dependent pathway.

Methods and Results The present study, using platelets and neutrophils from normal subjects, was undertaken to assess the putative effect of aspirin on the neutrophil-mediated, platelet-inactivating effect. Aspirin facilitated the inhibitory effect of neutrophils on platelet activation by thrombin, ADP, or epinephrine. This effect was equally evident in vitro and in blood samples of normal individuals taking aspirin. A significant stimulation of NO-mediated mechanisms in the presence of aspirin was disclosed by different methods, as follows: (1) the increased metabolism of arginine to citrulline, (2) the increase of cGMP in the platelet/neutrophil system, and (3) the inhibitory action of the L-arginine (L-Arg)–competitive analogue L-NMMA, which was reversed by L-Arg. The effect of aspirin appeared to be related to cyclooxygenase inhibition, since it was reproduced by using indomethacin. The vasoconstricting peptide endothelin-1 (ET-1) reversed the effect of aspirin through the endogenous production of platelet-activating factor (PAF) by neutrophils, as judged by the marked inhibitory effect of the PAF antagonist BN-52021.

Conclusions Our results show that a significant part of the effect of aspirin on platelet activation involves a neutrophil-mediated, NO/cGMP-dependent mechanism. The presence of ET-1 counterbalances these effects of neutrophils on platelet activation, therefore acting as an indirect proactivating agent. These results add new elements for interpreting the effects of aspirin on the interactions between blood cells, with special reference to high endothelin states (for example, ischemia/reperfusion processes).


Key Words: aspirin • platelets • endothelin


*    Introduction
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 up arrowAbstract
 *Introduction
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In recent years, relevant changes have occurred in the knowledge of the cellular mechanisms regulating platelet aggregation and adhesion to the endothelial surface.1 2 3 In particular, major aspects of the interactions between platelets and endothelial cells and neutrophils have been clarified.1 2 3 4 These interactions involve not only thrombosis-promoting or thrombosis-inhibiting properties but also several aspects of the regulation of vascular function.5 A new concept has progressively emerged showing thrombosis as a multicellular event in which cell-to-cell interactions between platelets, neutrophils, and endothelium regulate the size of a growing thrombus.4 6 7 8 9 10 11 In brief, there is consistent evidence showing that two vasodilating mediators produced by endothelial cells and neutrophils (nitric oxide [NO] and prostacyclin) have synergistic antiaggregating effects. NO also has platelet-antiadhesive properties that appear to be, similar to its antiaggregating effects, related to the induction of cGMP formation (see Reference 5 for review).

With regard to the interactions between platelets and neutrophils affecting platelet aggregation, the results published to date are less than homogeneous. A study by Del Maschio et al12 has shown that neutrophils may favor platelet aggregation, whereas other communications described an antiaggregating effect of neutrophils.13 14 15

Adding more complexity to the interactions between diverse types of cells at the microcirculatory level, activated platelets release vasoactive mediators with potential implications in the pathogenesis of vascular occlusion or vasospasm. In this regard, activating platelets release the vasoconstricting agent thromboxane A2 and induce the production of endothelin-1 (ET-1) by the vascular endothelium.2 ET-1 increases in ischemic processes, but its actual role in the final outcome of the ischemic phenomenon is still incompletely clarified. ET-1 is apparently devoid of any direct effect on platelets2 16 but has, nonetheless, effects on neutrophils, which may account for the interactions between neutrophils, platelets, and endothelial cells. In this regard, ET-1 is involved in neutrophil Ca2+ release,17 superoxide anion generation,18 cGMP production,19 aggregation,20 and adhesion to the endothelium.21

In human pathology, platelet activation is particularly relevant in myocardial ischemia, and several pharmacological strategies have been devised to prevent intravascular platelet activation. Aspirin remains a keystone of these preventive and damage-limiting strategies.22 23 24 Current knowledge maintains that low doses of aspirin decrease in vivo platelet aggregation by a selective inhibitory effect on thromboxane A2 production by platelets with maintenance of prostacyclin production by the endothelium.22 23 24 25 The role of aspirin, however, must be readdressed in light of the new information concerning platelet and neutrophil interactions.

In several studies, antiaggregating effects of aspirin have been reported that cannot be explained only by the above-mentioned action on thromboxane A2 and prostacyclin production. In this regard, Gaspari et al26 found that aspirin prolonged bleeding time in uremia by a mechanism distinct from platelet cyclooxygenase inhibition. Di Gaetano et al27 suggested that the inhibition of cyclooxygenase by aspirin does not fully explain the antithrombotic effects of the drug. The existence of non–platelet-mediated antithrombotic effects of aspirin was further suggested by Mehta and Mehta.28 It is clear that besides its well-known effects on endothelial cells and platelets, which involve cyclooxygenase inhibition, aspirin affects neutrophil function by mechanisms that are still incompletely understood.27 Especially interesting are the reports showing that in activated neutrophils, aspirin can inhibit the production of superoxide anion29 and antagonize the effects on pulmonary artery contractility.30 Recently, Valles et al31 described that aspirin enhanced the neutrophils' downregulatory effect on thrombin-induced platelet aggregation. These findings, together with the above-mentioned data regarding the cross-talk between platelets and neutrophils by exchanging active metabolites, give additional support to the hypothesis that aspirin may possess a relevant effect on neutrophil/platelet interactions.

Further evidence has been accumulated in recent years about the protective role of aspirin in the control of the complications of myocardial infarction24 30 and in the prevention of coronary bypass closure.32 Of interest, a decreased incidence of non–platelet-mediated arrhythmias accompanying myocardial infarction, which putatively are a neutrophil-related complication, has been found in aspirin-treated individuals.3 Based on the above-mentioned data, the aim of the present investigation was therefore to examine the role of aspirin in the inactivating effects of neutrophils on activated platelets. In addition, in an effort to further understand the effects of aspirin in pathophysiological conditions, we examined the effects of ET-1, a vasoactive mediator that augments in ischemic circumstances, on the platelet-neutrophil interaction.


*    Methods
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Chemicals
Ficoll-Hypaque medium was obtained from Flow Laboratories. Acetylsalicylic acid and L-arginine were purchased from Sigma Chemical Co. NG-monomethyl-L-arginine (L-NMMA) was obtained from Calbiochem and ET-1 from Neosystem Laboratoire. Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes. The cGMP radioimmunoassay kit and L-[3H]-arginine were purchased from Amersham. [3H]-acetate was purchased from New England Nuclear. Thrombin was purchased from Ortho Diagnostic Systems. All other chemicals were from the highest commercially available quality from Sigma. The monoclonal antibody against CD18, TS1/18, was a gift from Prof Francisco Sánchez Madrid.

The words aspirin and acetylsalicylic acid can be used indistinctly, but preference has been given to aspirin.

Neutrophils and Platelet-Rich Plasma Preparation
All the experiments were done using human platelet-rich plasma (PRP) and neutrophils. Rabbit PRP and neutrophils were also used to examine the validity of the findings in cells of a different species. Therefore, unless otherwise indicated, the findings reported in the "Results" section correspond to experiments performed on human cells. Human or rabbit PRP and neutrophils were obtained from the same donors for each experiment. With the exception of the experiments with previous aspirin administration (see below), volunteer donors and animals did not receive drugs for at least 20 days before the experiments. All the experiments performed in the present study were approved by the Clinical Research Committee, and all the subjects gave written informed consent. Blood extractions in the rabbits were done by ear venipuncture, after local anesthesia.

Human neutrophils were isolated from peripheral blood by Ficoll/Hypaque centrifugation as previously described.18 19 20 21 Neutrophils (95% pure, 98% viable by trypan blue exclusion) were resuspended in calcium–physiological saline solution (PSS) containing (in mmol/L): 140 NaCl, 4.6 KCl, 2.0 CaCl2, 1.0 MgCl2, 5.0 D-glucose, and 10.0 HEPES, pH 7.4. A similar procedure was followed for isolation of New Zealand rabbit neutrophils.

PRP was prepared as described.33 In brief, whole blood was obtained in 10% (vol/vol) acid-citrate-dextrose and centrifuged at 800g for 15 minutes. PRP was collected and the platelet number counted by a Coulter counter. Platelet number was adjusted with platelet-poor plasma, obtained from the same individual or animal, to 2.5x108 cells/mL plasma.

An additional study was performed to ascertain the in vivo relevance of the in vitro findings. For this purpose, aspirin (200 mg/d, one oral morning dose) was given to human control subjects (n=6) during 4 days, and blood was processed as indicated above. The results of these experiments were compared with those of baseline experiments done in the same individuals before the administration of aspirin. As an estimate of the circulating levels of aspirin, serum salicylate levels were measured by a Du Pont ACA analyzer at 510-nm wavelength.

Platelet Aggregometry
Platelet activation was registered in a Lumiaggregometer (Aggrecorder, four channels) by the change in light transmission. Previously, a platelet-poor sample was used as control for 100% light transmission. To correct for the possible light absorption induced by the presence of neutrophil suspension, the platelet-poor sample contained a number of neutrophils equal to the platelet-rich sample.

PRP (500 µL) was incubated at 37°C for 10 minutes in the aggregometer with continuous stirring (500 rpm) and was then stimulated with submaximal concentrations of thrombin (0.025 U/mL). Five minutes before, 100 µL of the neutrophil suspension was added to PRP to reach a final amount of 1.25x108 platelets and 1x106 neutrophils (125:1), which approximates the relative concentrations in normal blood. When required, indomethacin, aspirin, or ET-1 was added to the platelet-neutrophil suspension. In all cases, the comparative baseline measurements were done in the presence of the solvent of aspirin and indomethacin, ethanol, in the same dilution used for the incubations with the cyclooxygenase inhibitors (final ethanol concentration in the assay <0.1%). No significant effects were detected in any case with these concentrations of ethanol. In some experiments, PRP was incubated with the specific platelet-activating factor (PAF) antagonist BN-52021 10 minutes before adding ET-1. In other experiments, neutrophils were preincubated with the L-arginine analogue L-NMMA or with a monoclonal antibody against the CD18 antigen of the ß2-integrins, TS1/18, for 45 or 10 minutes, respectively, before they were added to the assay. For standardizing the measurements, only the values of turbidimetry at 3 minutes were used for doing the calculations. This time period corresponds to the maximal or near-maximal value of the first wave of platelet aggregation. This primary wave is representative of platelet activation rather than platelet aggregation and is partially reversible. In this regard, at the relatively high concentrations of thrombin, ADP, or epinephrine used, the first wave was the only relevant process in the platelet activation phenomenon.

Measurement of Intracellular [Ca2+]
Cytosolic [Ca2+] ([Ca2+]i) was measured as previously described.17 Platelets were loaded by incubating PRP, prepared as detailed above, with 2 µmol/L fura-2/AM during 60 minutes at 37°C at room atmosphere. Platelets were pelleted by centrifugation (2500 rpm, 10 minutes), washed twice, and resuspended in platelet-poor plasma obtained from the same individual and adjusted to 2.5x108 cells/mL plasma. Neutrophils (2x106 cells/mL), aspirin (600 µg/mL), or both were added when required, allowing 5 minutes of preincubation in all cases. Measurements were performed with an LS-50 Perkin Elmer Luminescence Spectrometer in a stirring-warmed cuvette (37°C) as previously described.17 19 Experiments were done in the presence of thrombin (0.025 U/mL), ADP (10-6 mol/L), or epinephrine (10-5 mol/L). The results are expressed in nmol/L of [Ca2+]i.

Measurement of cGMP
cGMP concentrations were measured as described previously19 in acetylated samples by means of a radioimmunoassay kit (Amersham). PRP, neutrophils, or both were preincubated for 10 minutes at 37°C with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 2 mmol/L). cGMP was measured in suspensions of platelets, neutrophils, and platelets plus neutrophils, both in the presence and absence of aspirin. cGMP levels were measured 3 minutes after the cells were activated with thrombin (0.025 U/mL) to reproduce the conditions of the platelet aggregation experiments.

Determination of L-[3H]-Citrulline Content
As detailed elsewhere,19 neutrophils were incubated (30 minutes, 37°C) in PSS-Ca2+ containing 10-4 mol/L L-arginine and 1 µCi/mL L-[3H]-arginine. Unincorporated L-[3H]-arginine was washed twice with PSS-Ca2+ buffer. After L-[3H]-arginine labeling, neutrophils were coincubated with PRP in the presence and absence of aspirin (600 µg/mL) or vehicle (ethanol, final concentration <0.1%). Platelets were activated with thrombin (0.025 U/mL) during 3 minutes at 37°C. Further control experiments in the absence of thrombin-stimulated platelets were carried out. After centrifugation, PRP and neutrophils were lysed with cold methanol, and the supernatant was evaporated to dryness under N2 at 37°C. As previously described,19 the extracts were resuspended in 20 mmol/L HEPES/KOH, pH 5.5, and applied to columns of Dowex AG50WX8 (Na+ form), which were subsequently eluted with water (L-citrulline fraction) and 0.5 mol/L NaOH (L-arginine fraction). L-[3H]-citrulline fraction was quantified by liquid scintillation counting.

Measurement of PAF Production
PAF production by the neutrophils was measured as [3H]-acetate incorporation into PAF. [3H]-acetate–labeled neutrophils (25 µCi/5x106 neutrophils) were resuspended in PSS-Ca2+ buffer and incubated at 37°C for 3 minutes in the presence or absence of vehicle (ethanol <0.1), aspirin (600 µg/mL), or 10-7 mol/L ET-1. Incubation was finished by the addition of 3 mL 1 mol/L HCl in methanol. Lipids were extracted from the methanolic phase as described by Bligh and Dyer.34 Samples were dried under N2 and separated by thin-layer chromatography on precoated plates of silica gel 60, using propionic:propanol:water:chloroform (2:2:1:1) as the mobile phase. The silica was scraped off in narrow bands, based on their coimigration with a PAF standard.35 Radioactivity was determined by liquid scintillation counting.

To be certain that the measured material corresponded to [3H]-PAF, selected samples were analyzed by high-performance liquid chromatography using a dual Kontron model 420, as previously described.20 The retention time of [3H]-PAF obtained from neutrophils was identical to a synthetic labeled PAF (C16-C18 mixture, Amersham).

Statistical Methods
Results are expressed as mean±SEM. Unless otherwise stated, each value corresponds to a minimum of six experiments done in triplicate. Comparisons were done by ANOVA or paired and unpaired Student's t test when appropriate. Bonferroni's correction for multiple comparisons was used to determine the level of significance of the P value.


*    Results
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*Results
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Inhibitory Effects of Aspirin on Platelet Activation
In the presence of aspirin, neutrophils significantly inhibited thrombin-induced platelet activation as assessed by aggregometry and measurements of [Ca2+]i.

The data represented in Fig 1Down were taken at 3 minutes after the stimulus, corresponding to maximal platelet activation. However, the turbidimetry curves in the presence or absence of aspirin were already different when measured at 1 minute from the start of the experiment (% light transmission: -aspirin, 26±2, +aspirin, 16±2; P<.01). The effect of aspirin was concentration dependent (Fig 1Down), showing the maximal inhibition with 600 µg/mL. This latter concentration was used for most of the following studies. In the absence of aspirin, no significant effect of neutrophils on platelet activation was detected (Fig 1Down). To examine whether the effect of aspirin was due to its cyclooxygenase-inhibiting action, similar experiments were done in the presence of a different cyclooxygenase inhibitor, namely, indomethacin. A 38±6% inhibitory effect of platelet activation by neutrophils was observed in the presence of 10-6 mol/L indomethacin (n=4, P<.01 with respect to controls). This effect was not different from that obtained with aspirin (P=NS).



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  		Figure 1. Plot shows effects of increasing concentrations of aspirin on thrombin-induced platelet activation in the presence (n=6) or absence (n=6) of neutrophils (PMN). Platelet activation is plotted as % light transmission at 3 minutes after the addition of thrombin. Results are represented as mean±SEM. {star}P<.05 with respect to % light transmission in the absence of neutrophils.

In the absence of neutrophils, aspirin alone (600 µg/mL) did not result in changes of thrombin-induced platelet activation (Fig 1Up). A significant inhibition of thrombin-induced platelet activation was still present when only the neutrophils were previously treated with aspirin (% inhibition, 20±3, P<.05), albeit with lesser intensity than that observed when both platelets and neutrophils were incubated with aspirin.

A concentration-dependent, aspirin-related inhibition of platelet activation by neutrophils was also observed when ADP or epinephrine was used as platelet activator (Fig 2ADown and 2BDown). An effect of aspirin per se was detected on the epinephrine-induced activation curve (Fig 2BDown); also, a small but significant effect of aspirin on ADP-stimulated platelet activation was observed (Fig 2ADown).



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  		Figure 2. Plots show A, effects of increasing concentrations of aspirin on ADP-stimulated (10-6 mol/L) platelet activation in the presence (n=4) or absence (n=4) of neutrophils (PMN). B, Effects of increasing concentrations of aspirin on epinephrine-induced (10-5 mol/L) platelet activation in the presence (n=4) or absence (n=4) of neutrophils. Platelet activation is plotted as % light transmission at 3 minutes after the addition of ADP or epinephrine. *P<.05 with respect to % light transmission in the absence of neutrophils. {star}P<.05 with respect to platelet activation in the absence of aspirin.

The spontaneous platelet activation (% light transmission <5%) was not changed by the incubation of PRP with neutrophils and aspirin. Moreover, no differences were observed in the above-mentioned effects of aspirin when similar aggregometry experiments were done using rabbit PRP and neutrophils (data not shown).

Measurements of [Ca2+]i transients in platelets were performed as a second method to examine platelet activation in the presence or absence of aspirin and neutrophils. As shown in Table 1Down, thrombin-induced [Ca2+]i transients in platelets were not modified by neutrophils or aspirin (600 µg/mL) alone. In the presence of aspirin, neutrophils markedly decreased the thrombin-stimulated [Ca2+]i transients in platelets (Table 1Down). A neutrophil-mediated, aspirin-dependent inhibition of [Ca2+]i transients in platelets was also observed when ADP (10-6 mol/L) or epinephrine (10-5 mol/L) was used as platelet activator (% decrease of [Ca2+]i peak, 62±7 and 68±6, respectively, both P<.01). An inhibitory effect induced by aspirin on ADP- or epinephrine-stimulated [Ca2+]i transients was also observed in the absence of neutrophils, although with lesser intensity than that shown when neutrophils were present (% decrease of [Ca2+]i peak by aspirin in the absence of neutrophils: ADP, 10±3; epinephrine, 18±6; P<.05).


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  		Table 1. Effect of Neutrophils and Aspirin on [Ca2+]i Transients in Thrombin-Stimulated Platelets

Baseline [Ca2+]i in platelets (110±8 nmol/L) was not modified by the incubation of PRP with neutrophils or aspirin.

The inhibitory effect of neutrophils in the presence of aspirin on the [Ca2+]i transients in either thrombin-, ADP-, or epinephrine-stimulated platelets was reverted by the preincubation of neutrophils with the L-arginine–competitive analogue L-NMMA (10-5 mol/L) (% inhibition of the aspirin effect, 88±8, 82±5, and 84±3, respectively, P<.01).

In an effort to address the actual in vivo importance of the above-mentioned findings, the role of neutrophils in platelet activation was examined in normal subjects in samples obtained before and after the oral intake of aspirin. In all cases, each individual was used as his or her own control before and after the intake of aspirin; moreover, in all cases, both platelets and neutrophils were obtained from the same donor. In these experiments, neutrophils obtained before aspirin treatment did not significantly influence platelet activation induced by thrombin (Fig 3Down). However, after oral aspirin administration, a significant inhibition of thrombin-induced platelet activation by neutrophils was observed (Fig 3Down). The serum salicylate level in these individuals was 49.4±0.4 µg/mL, which corresponds to 68.6±0.5 µg/mL of aspirin on a molar basis.



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  		Figure 3. Bar graph shows inhibition of thrombin-induced platelet (PLT) activation by neutrophils (PMN) obtained from donors (n=6) receiving oral aspirin (+ASA, 200 mg/d, 4 days). Neutrophils were also preincubated with the L-arginine–competitive antagonist L-NMMA (10-5 mol/L) during 45 minutes before addition to the assay. Experiments without aspirin were performed in the same individuals (-ASA). Results are represented as mean±SEM. {star}P<.01 with respect to platelet activation induced by thrombin in the absence of neutrophils and aspirin.

In the absence of neutrophils, platelet activation in PRP taken from the human donors before or after oral aspirin administration was similar (Fig 3Up).

Mechanisms of the Effect of Aspirin on Platelet/Neutrophil Interactions: Role of Nitric Oxide Synthesis Inhibition
Based on the data of Salvemini et al,13 the hypothesis was raised that NO would be a relevant mediator of the aspirin-induced effect of neutrophils on platelet activation. Subsequently, to examine the implication of NO in the above-described effects, neutrophils were preincubated with L-NMMA (10-5 mol/L). Incubation of neutrophils alone with L-NMMA blocked the aspirin-sensitive inhibition of platelet activation by neutrophils (% inhibition, 92±6; P<.05). Preincubation of neutrophils with L-NMMA produced a concentration-dependent inhibition of the antiaggregating effect in the presence of aspirin (% inhibition by the threshold concentration 10-8 mol/L, 7±1; P<.05). The inhibitory effect of neutrophils on thrombin-stimulated platelet activation in the presence of lower concentrations of aspirin was also blocked by L-NMMA (data not shown). However, no effect of L-NMMA was detected on thrombin-induced platelet activation in the absence of neutrophils (% light transmission in L-NMMA–preincubated PRP stimulated by thrombin, 78±9; n=3; P=NS). Like in the in vitro studies, L-NMMA also suppressed the neutrophil-induced inhibition of platelet activation in samples obtained from aspirin-treated donors (Fig 3Up).

More data supporting the putative role of NO in the effect of neutrophils on platelet activation were obtained by preincubating neutrophils in the presence of excess L-arginine, the substrate for NO formation. In this regard, the addition of L-arginine (10-4 mol/L) to 10-5 mol/L L-NMMA–preincubated neutrophils restored up to 86±4% of the inhibitory effect of neutrophils in the presence of aspirin. In the absence of aspirin and L-NMMA, L-arginine–loaded neutrophils showed a small but significant inactivating effect (% inhibition, 11±3; n=3; P<.05).

Additional experiments were done to examine the role of oxygen free radicals on the platelet-inactivating effect of neutrophils observed in the presence of aspirin. In this regard, a small but significant effect of the superoxide anion scavenger superoxide dismutase (60 U/mL) was detected on the platelet-inactivating effect of neutrophils in the presence of aspirin (% inhibition of platelet activation by neutrophils in the presence of 300 µg/mL of aspirin, 37±4% with and 28±3% without superoxide dismutase; P<.05). Furthermore, a small effect of superoxide dismutase by itself was detected on platelet activation in the presence of neutrophils incubated without aspirin (% inhibition, 10±2; n=3; P<.05). Of note, aspirin concentrations with submaximal effect were used in these experiments to facilitate the observation of any putative interaction. No effect was detected on either spontaneous activation or thrombin-induced platelet activation by the incubation of platelets alone with superoxide dismutase (data not shown).

Formation of Nitric Oxide by Neutrophils
To determine whether aspirin favored NO generation by neutrophils, we studied the accumulation of L-[3H]-citrulline in L-[3H]-arginine–loaded neutrophils. To reproduce the conditions of the platelet activation experiments, platelets and thrombin were also added during the assay, with a time and concentration profile similar to that used in the experiments referred to above. In the presence of aspirin, the generation of L-[3H]-citrulline on neutrophils increased significantly (Table 2Down), indicating that the aspirin-sensitive inhibition of platelet activation mediated by neutrophils is coupled to an increased NO production. The stimulatory effect of aspirin on NO production did not need the presence of activated platelets, since it occurred also when neutrophils alone were incubated with aspirin (Table 2Down). Thrombin did not change NO production by neutrophils in the absence of platelets (Table 2Down).


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  		Table 2. Measurement of Nitric Oxide Production by Neutrophils Determined as the Generation of L-[3H]-Citrulline From L-[3H]-Arginine

Effect of Aspirin on cGMP Levels
The inhibition of thrombin-stimulated platelet activation mediated by aspirin and neutrophils was accompanied by a potentiated increase of cGMP levels (Fig 4Down). On the other hand, when the cGMP levels were measured in the mixed cellular suspensions (platelets plus neutrophils) in the absence of aspirin, they increased only in an additive manner (Fig 4Down).



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  		Figure 4. Bar graph shows cGMP production by platelets (PLT), neutrophils (PMN), or both in the presence (n=5) or absence (n=5) of aspirin (ASA, 600 µg/mL). Results are represented as mean±SEM. {star}P<.05 with respect to the cGMP assay in the absence of aspirin; *P<.01 with respect to the cGMP assay in the absence of aspirin.

The presence of aspirin did not alter platelet baseline cGMP levels. However, aspirin increased neutrophil cGMP content (Fig 4Up), although in a small proportion.

Effect of Endothelin-1 on Aspirin-Dependent Modulation of Platelet Activation by Neutrophils
An additional set of experiments was performed to examine whether the above-mentioned findings were valid in pathological conditions. In particular and based on recent data18 19 20 21 showing its effects on neutrophil activation, we studied the effect of ET-1, a mediator that increases during ischemia. Experiments were therefore conducted to determine whether the presence of ET-1 could modify the interaction between neutrophils and platelets. Table 3Down summarizes the effects of ET-1 (10-7 mol/L) on the platelet/neutrophil interaction in the presence of aspirin. ET-1 blocked the aspirin-dependent inhibition of thrombin-stimulated platelet activation mediated by neutrophils. Moreover, in the absence of aspirin, the addition of ET-1 to the platelet-neutrophil mixture elicited a small but significant increase in spontaneous platelet activation (% light transmission, 12±2; P<.05 with respect to spontaneous activation without ET-1). This effect of ET-1 appeared to involve mostly the neutrophil side of the interaction, since in the absence of neutrophils, ET-1 did not stimulate platelet activation (% light transmission, 4±1; P=NS) or change thrombin-induced activation (% light transmission, 77±5; P=NS). The concentration of ET-1 was chosen based on our previous studies, in which 10-7 mol/L ET-1 consistently provoked a maximum peak of [Ca2+]i on neutrophils, which correlated with neutrophil activation and neutrophil adhesion to the endothelial surface.17 19 20 21 Simultaneous experiments were done based on the previous evidence showing that ET-1 provokes neutrophil activation20 in order to rule out the possibility that the blocking action of ET-1 on the platelet-antiaggregating effect of neutrophils could be due to the ET-1–induced activation of neutrophils. These additional experiments were performed by preincubating the neutrophils with a monoclonal antibody against the CD18 antigen TS1/18 to block the neutrophil-neutrophil aggregating interactions induced by ET-1, as previously observed in our laboratory (López-Farré et al, unpublished observations). The inhibitory effect of ET-1 was not dependent on the increase of neutrophil activation, since it persisted in TS1/18-preincubated neutrophils (Table 3Down). No effect of the TS1/18 antibody by itself was detected on the platelet activation curve either in the presence or in the absence of neutrophils (P=NS, data not shown).


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  		Table 3. Effect of Endothelin-1 on Neutrophil-Dependent Inhibition of Thrombin-Induced Platelet Activation in the Presence of Aspirin

Thereafter, since ET-1 was devoid of direct actions on platelets, we examined the hypothesis that ET-1 induces the release of a mediator from neutrophils that blocks the aspirin-dependent inhibition of platelet activation. In this regard, we have found previously that ET-1 stimulates PAF release from neutrophils.20 Therefore, we investigated the role of PAF as a possible mediator of the ET-1 effects described above by preincubating the platelets during 10 minutes with the specific PAF antagonist BN-52021 (5x10-5 mol/L) and then adding the neutrophils and ET-1. As shown in Table 3Up, the blockade by ET-1 of the platelet-antiaggregating effect of ET-1 was reverted by BN-52021. No effect of BN-52021 by itself was detected on thrombin-induced platelet activation in the absence of neutrophils (Table 3Up). To further ascertain whether aspirin would interfere with ET-1–induced PAF production by neutrophils, additional experiments to examine PAF production by neutrophils were carried out in the presence or absence of aspirin. These experiments confirmed that ET-1 induced PAF production by neutrophils and disclosed no effect of aspirin on this phenomenon (Table 4Down).


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  		Table 4. Effect of Endothelin-1 on Platelet-Activating Factor Generation by Neutrophils in the Presence or Absence of Aspirin


*    Discussion
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The present results provide new evidence to clarify the antiaggregating effects of aspirin based on its role on platelet/neutrophil interactions. Our findings support the hypothesis that aspirin acts as an anti–platelet-activating agent not only through the traditionally described mechanisms22 23 24 25 but also by favoring the platelet-inactivating effect of neutrophils by means of an NO/cGMP-related pathway.

The existence of platelet inactivation by neutrophils was demonstrated in 1989 by Salvemini et al.13 These authors have also found that this effect was mediated by an NO-dependent, cGMP-mediated mechanism. On the other hand, Del Maschio et al12 described a proactivating effect of neutrophils. In part, the apparent disagreement of the results from these two groups could be attributed, in light of the data obtained in the present study, to the presence13 or absence12 of aspirin in the platelet incubation media and to relevant differences in the experimental conditions, namely, use of washed platelets12 or PRP, prestimulation of the neutrophils with cytochalasin B,13 and aequorin loading.12 The results by Salvemini et al13 are in agreement with ours and with those of other authors, including other communications from Del Maschio's group.12 15 The new information in our experiments includes the finding of the aspirin dependence of the inactivating effect of neutrophils, the relation of aspirin and NO production, and the potential role of ET-1 through a PAF-related pathway.

The effect of aspirin was evident within a wide range of concentrations. Moreover, in the presence of aspirin, neutrophils not only counteracted the effect of thrombin but also those of ADP and epinephrine, therefore suggesting that platelet activation was blocked at a postreceptor level. Importantly, the concentrations of aspirin used are within the in vivo therapeutic range of the drug.23 24 25 36 37 38 In this regard, the present experiments using blood from subjects taking aspirin confirm the in vivo relevance of the described in vitro effects and the similarity of the NO-dependent mechanisms.

Different types of experiments were performed to address the mechanisms involved in the effect of aspirin on neutrophils. First, the identity of the effects of both aspirin and indomethacin strongly suggested that the main effect of aspirin in the neutrophil-mediated platelet inactivation was due to cyclooxygenase inhibition. The inhibition of the effect of neutrophils plus aspirin on platelet aggregation by the L-arginine competitive analogue L-NMMA supported the NO-mediated nature of the inactivating phenomenon. The specificity of this inhibition was established by the reversal of the L-NMMA effect by excess L-arginine. The experiments using separate incubations with aspirin of either neutrophils alone or neutrophils with platelets suggested that the effect of aspirin on platelet activation and L-[3H]-citrulline production occurred preferentially on the neutrophil side of the neutrophil-platelet association. Therefore, this effect appears to involve either the existence of a mediator that would appear in conditions of cyclooxygenase inhibition and activate NO production from neutrophils or the interference by aspirin with an inhibitory effect on NO formation.

Further insight into the characteristics of the aspirin effect was obtained by studying the [Ca2+]i response, which allowed a very sensitive assessment of the early events of platelet activation. In our study, in the presence of aspirin, neutrophils interfered with the [Ca2+]i signaling pathway by an L-NMMA–inhibitable mechanism. Therefore, it appears that NO can inhibit platelet activation through the blockade of calcium-stimulated pathways. The fact that thrombin-mediated as well as ADP- and epinephrine-mediated responses were affected showed that aspirin blocked a postreceptor mechanism involving [Ca2+]i mobilization. An explanation for these results could be found in the increase of cGMP observed when platelets were incubated with aspirin-treated neutrophils, since cGMP is a potent blocker of calcium-related activation in numerous cellular types, including platelets.39

The present experimental model did not allow specific identification of the putative agent(s) responsible for the aspirin effect on NO production. However, as suggested by preliminary observations in our laboratory,40 a role by arachidonic acid or noncyclooxygenase arachidonic acid metabolites is suspected. Arachidonic acid or its metabolites can be expected to increase after cyclooxygenase inhibition by aspirin. Arachidonic acid fulfills several conditions to be considered a possible mediator, since it can activate NO release from endothelial cells41 and has been proposed as a direct modulator of neutrophil function.42 Despite the possible effects of aspirin on the inhibition of superoxide anion production,27 28 which may have consequences on the rate of NO degradation,43 44 our results support the existence of an increased NO formation induced by the presence of aspirin. In this regard, the absence of a relevant effect of superoxide dismutase in the present experimental conditions diminishes the potential importance of a superoxide anion–related mechanism. However, an effect of aspirin on the platelet side of the association can be suspected from the observation that, even though the increase in L-[3H]-citrulline production was already at maximum when neutrophils were incubated with aspirin in the absence of platelets, the platelet antiactivating effect of the neutrophils was more intense when both platelets and neutrophils were simultaneously incubated with aspirin.

Several recent findings indicated that aspirin might contribute to increased NO levels or NO effects. In this sense, an antagonistic effect of aspirin has been observed on the vasoconstrictor properties of the L-arginine–competitive analogue L-NMMA45 and hypoxic vasoconstriction.46 Even closer to our results, aspirin might alter the contractile effects of neutrophils on the pulmonary artery.30 In addition, salicylate, a part of the aspirin molecule, has been shown recently to interfere with leukocyte adhesion,47 a property modulated by NO.48 Finally, as a direct demonstration, our experiments showed that aspirin increased NO in neutrophils.

The results of the experiments using ET-1 illustrate the possible in vivo outcome of the platelet/neutrophil/aspirin interactions in conditions of increased ET-1. The inhibitory effect of ET-1 on the neutrophil-dependent platelet inactivation adds more insight into the possible platelet/neutrophil interactions in high ET-1 states, namely, ischemia/reperfusion or endothelial damage of different causes. As observed by other authors16 and confirmed in the present study, ET-1 appears to be devoid of a direct effect on platelets. Our results, however, showed for the first time that ET-1 may indirectly modulate platelet activation by interfering with the antiaggregating effect of neutrophils. Actually, in the presence of ET-1, the neutrophils appear to promote platelet activation rather than inactivation. Since this effect was not inhibited by the TS1/18 antibody, it can be considered to be independent of the neutrophil-aggregating effect of endothelin.20 A role of P-selectin,47 48 49 which is involved in the platelet-neutrophil physical interaction,49 50 51 is theoretically possible and cannot be ruled out from our results. However, recent data from Rinder et al52 suggested that aspirin does not affect P-selectin surface expression and therefore do not support the hypothesis of a P-selectin–related effect.

The inhibition of the effects of ET-1 on the platelet-neutrophil association by the PAF antagonist BN-52021 strongly suggests that it is due to PAF-induced activation. Moreover, PAF production by neutrophils was enhanced in the presence of ET-1, an effect that was not blocked by aspirin (see Reference 20 and the present study). This effect may account for the observed neutrophil-dependent platelet activation in the presence of ET-1. ET-1 may, therefore, act as an in vivo negative modulator of the platelet-antiaggregating effect of neutrophils through a PAF-mediated mechanism. This pathway may be potentially relevant in pathological conditions associated with increased local levels of ET-1.

The observed effects of aspirin are of potential interest in the setting of ischemia/reperfusion, particularly in the myocardium. In circumstances of coronary occlusion and subsequent reperfusion, there exists a deep perturbation of endothelial function, with decreased formation of vasodilating/antiaggregating mediators, for example, NO and prostacyclin, and blunted endothelium-dependent relaxation.53 54 The role of neutrophils, which adhere to the damaged endothelium, is not completely understood in this setting.55 Of further interest, recent results obtained in our laboratory indicate the existence of neutrophil-related effects of NO on red blood cells, which may be relevant for the regulation of microcirculatory hemorheology and may expand the scope of the possible blood cell interactions beyond those mentioned in the present study.56

Conclusions
The present results raise the possibility that aspirin modifies the way in which neutrophils and platelets interact. These findings may, therefore, add a novel interpretation to the protective effect of aspirin in the control of ischemic damage.


*    Acknowledgments
 
This work was supported by grants PM 92/04 of Dirección General de Investigación Científica y Técnica and 239/93 of Fondo de Investigaciones Sanitarias de la Seguridad Social, Fundación Ramón Areces, and Fundación Renal Iñigo Alvarez de Toledo. Dr López-Farré is a Senior Researcher of Fundación Jiménez Díaz/Boehringer Mannheim. Dr Alberola and Dr Montón are fellows of Fundación Renal and Conchita Rábago, respectively. The authors wish to thank Prof Francisco Sánchez Madrid (Universidad Autónoma, Madrid) for the gift of TS1/18 and Concepción San Martín, Elena Rubio, and Liselotte Gulliksen for editorial assistance.

Received October 12, 1994; accepted November 26, 1994.


*    References
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*References
 

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