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Research ArticleCellular and Molecular

N-Stearoylethanolamine Inhibits Integrin-Mediated Activation, Aggregation, and Adhesion of Human Platelets

Iehor A. Hudz, Volodymyr O. Chernyshenko, Ludmila O. Kasatkina, Lesia P. Urvant, Vitaliy M. Klimashevskyi, Oksana S. Tkachenko, Halyna V. Kosiakova, Nadiia M. Hula and Tetyana M. Platonova
Journal of Pharmacology and Experimental Therapeutics October 2022, 383 (1) 2-10; DOI: https://doi.org/10.1124/jpet.122.001084

Abstract

N-stearoylethanolamine (NSE), a lipid mediator that belongs to the N-acylethanolamine (NAE) family, has anti-inflammatory, antioxidant, and membranoprotective actions. In contrast to other NAEs, NSE does not interact with cannabinoid receptors. The exact mechanism of its action remains unclear. The aim of this study is to evaluate the action of NSE on activation, aggregation, and adhesion of platelets that were chosen as a model of cellular response. Aggregation of platelets was measured to analyze the action of NSE (10−6–10−10 M) on platelet reactivity. Changes in granularity and shape of resting platelets and platelets stimulated with ADP in the presence of NSE were monitored by flow cytometry, and platelet deganulation was monitored by spectrofluorimetry. In vivo studies were performed using obese insulin-resistant rats. Binding of fibrinogen to the GPIIb/IIIa receptor was estimated using indirect ELISA and a scanning electron microscopy (SEM). It was found that NSE inhibits the activation and aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. These effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory effect and allows us to prioritize studies of NSE as a potent anti-thrombotic agent.

SIGNIFICANCE STATEMENT N-stearoylethanolamine (NSE) was shown to possess inhibitory action on platelet activation, adhesion, and aggregation. The mechanism of inhibition possibly involves integrin receptors. This finding complements the known anti-inflammatory effects of NSE.

Introduction

N-acyletanolamines (NAEs) are low-molecular-weight derivatives of saturated and unsaturated fatty acids, involved in the homeostatic response to cell damage. Their action on cells may be realized via both receptor-independent and receptor-dependent mechanisms (Mato et al., 2009). The action of NAEs is directed at the neutralization of pathologic damage in many physiologic processes, and explains why the level of NAEs, normally in the picomolar range in (human) plasma, increases substantially during pathologic or inflammatory processes (Kilaru et al., 2011). The concentration of NАEs in human plasma under certain pathologic conditions can reach 2.70±3.37 ng/ml (versus 0.83±0.47 ng/ml in healthy controls) (Hauer et al., 2013).

N-stearoylethanolamine (NSE) is an NAE, with potent anti-inflammatory properties (Gorid’ko et al., 1999; Voitychuk et al., 2012). NSE is able to modulate voltage-gated sodium and calcium channels and, as a consequence, impacts the membrane potential of cardiac myocytes. Previous studies have shown that NSE has membranotropic action and could influence the cholesterol level and balance between saturated and unsaturated fatty acids (Gorid’ko et al., 1999). The modification of membrane lipid composition by NAEs led to the inhibition of veratridine-activated ion channels (Di Marzo et al., 1996) and decreased the production of 11-oxy steroids by the adrenal cortex in rats (Mikosha et al., 1998). NSE was also shown to prevent Fe2+-dependent oxidation of lipids induced by free radicals in cardiac and liver mitochondria (Parinardi and Schmid, 1988; Gulaya et al., 1998). In contrast with other NAEs, NSE does not interact with cannabinoid receptors (Movahed et al., 2005), and its action may be realized via rearranging membrane lipids and modulation of proinflammatory signals (Onopchenko et al., 2018). However, the exact mechanisms of the action of NSE remain unclear.

The aim of our study is to evaluate NSE action on platelet activation, aggregation, and adhesion. These processes in particular involve integrin signaling and platelet degranulation and are highly dependent on membrane lipid rearrangement and cholesterol-enriched domains. Platelets were chosen to study the mechanisms of NSE action on living cells because they are small anuclear cells that can be activated through diverse pathways and their responses to an inducer can be easily measured. By studying the activation, adhesion, and aggregation of platelets using different approaches and comparing the results, we aimed to detect the peculiarities of NSE action separately on different branches of platelet signaling pathways. Taking into account that selective targeting of integrin outside-in signaling mechanisms provides potent inhibition of thrombosis, while maintaining blood coagulation in animal models (Estevez et al., 2015), we also studied the action of NSE on platelet reactivity in vivo.

Materials and Methods

Materials

Chemicals

Both thrombin (50 NIH/ml) and platelet activating factor (PAF) were purchased from Sigma (St. Louis, MO). ADP, collagen, epinephrine, and ristocetin were purchased from Tekhnologia-standard (Barnaul, Russia). Monoclonal antibody (mAb) 2d-2a to human fibrin(ogen) was provided by I.M. Kolesnikova, Palladin Institute of Biochemistry, NAS of Ukraine. Rabbit anti-mouse IgG conjugated with horseradish peroxidase and chromogenic substrate o-phenylenediamine (OPD) was purchased from Sigma. Acridine orange (AO) was purchased from Invitrogen (Molecular Probes, Eugene, OR).

NSE and NOE Preparation and Characterization

NSE was synthesized at the Department of Lipids Biochemistry, Palladin Institute of Biochemistry (Kyiv, Ukraine) using the condensation of ethanolamine and stearic acid (Gula et al., 2008).

Purity of the NSE preparation was confirmed by gas chromatography (HRGC 5300, Carlo Erba Instruments, Val De Reuil, France) using packed column Chromosorb W 100-125 (Supelco, Montclair, NJ) with phase 10% Silar 5 CP. The temperature of the injector was −250°C and the temperature of the detector was −270°C.

The sample of NSE for gas chromatography was prepared as follows: 0.1 mg of NSE, 1 ml of benzene, and 1 ml of 3 М HCl in methanol were mixed in a glass ampoule. The ampoule was sealed and heated on a water bath at 100°C for 1.5 hours. The ampoule was then cooled and opened. The solution was removed and put into a sample tube and mixed with 5 ml of hexane. The upper fraction of the solution was collected and transferred into a glass flask. The glass flask with the NSE sample was then dried up using a rotary evaporator at 35°C. The dried sample was dissolved into 0.2 ml benzene. The solution was separated by a thin-layer chromatography plate using benzene. The methyl ester of stearic acid was used as the marker probe. The zone, which consisted of methyl ester NSE, was extracted into benzene.

N-oleoylethanolamine (NOE) was synthesized and characterized in a manner similar to that described above for NSE, except oleic acid was used instead of the stearic acid.

Dried NSE and NOE powders were suspended in distilled water under sonication at a concentration of 10−3 M. Serial dilutions of NSE (10−5 – 10−9 M) were prepared in dimethyl sulfoxide (DMSO) ex tempore. The final concentration of DMSO in the studied samples was 1% for all experiments. An equal volume of DMSO was added to the control probes.

Methods

Platelet-Rich Plasma and Blood Plasma Preparation

The venous blood of healthy male volunteers (n = 4) who had not taken any medication for seven days prior to blood sampling was collected into a 3.8% sodium citrate solution (9 parts blood to 1 part sodium citrate). Platlet-rich plasma (PRP) was prepared by centrifuging blood at 160 g for 20 minutes at 25°C. Washed platelets were obtained by spinning down the platelets at 300 g followed by resuspension of the platelet pellet in 0.14 M NaCl, 0.01 M KH2PO4, pH 7.4 (PBS) (Korolova et al., 2014).

Flow Cytometry

Platelet shape and cytoplasmic granularity were monitored on a Beckman Coulter Epics XL Flow Cytometer (Backman Coulter, Ramsey, MN) (Adan et al., 2017). NSE (10−8 M) was added to 1 ml of PRP, and the samples were incubated for 60 minutes at 25°С. Platelets were analyzed in comparison with thrombin-stimulated samples. Forward scattering (FS) and side light scattering were monitored to detect any changes of platelet granularity and shape.

Platelet Aggregation

Platelet aggregation measurements were based on changes in the turbidity of human PRP (Cattaneo et al., 2013). Aggregation was registered for 10 minutes using the Aggregometer Solar AP2110 (Solar, Minsk, Belarus). The initial rate and final level of aggregation were estimated at 37°C. In a typical experiment, 250 μl of PRP was activated by the following platelet agonists: ADP (2.5 and 12.5 μM), collagen (2 mg/ml), epinephrine (0.6 μg/ml), ristocetin (0.2 and 0.4 mg/ml), thrombin (0.125 NIH/ml), or PAF (50 ng/ml).

Spectrofluorimetric Analysis of Platelet Degranulation

Platelet degranulation was registered with pH-sensitive fluorescent AO dye that accumulates in platelet acidic compartments and is released after activation. PRP was preincubated with or without NSE (10−8 M) at 37°C for 10 minutes and fluorescence measurements were started after the AO application. Changes in fluorescence intensity were measured as described previously (Kasatkina, 2016) at excitation and emission wavelengths of 490 and 530 nm, respectively (slit bands 5 nm each). The ADP (0.3 and 2 µM), epinephrine (0.2 μg/ml), collagen (0.1 and 0.5 mg/ml), or ristocetin (0.4 mg/ml) were applied at the steady-state level of AO fluorescence. Fluorescence intensities were normalized to similar values in the absence of platelets (Ft/F0). Degranulation was estimated at plateau level as a percent of released AO (from total accumulated dye).

Evaluation of Fibrinogen Binding to Platelets

The modified ELISA technique was used to evaluate the binding of fibrinogen to activated platelets in the presence or absence of NSE (10−7 M). Washed platelets were re-suspended in PBS and added to wells of a 96-well plate and kept for 1 hour at 37°C. The 96-well plate was washed three times with PBS containing 0.05% Tween-20 (TPBS). Then, NSE (10−7 M) and fibrinogen (3 mg/ml) in PBS were added to the wells and incubated for 1 hour at 37°C. The plate was washed three times with TPBS. A solution of mAb 2d-2a in PBS was added to the wells, the plate was incubated for 1 hour at 37°Cs and washed with TPBS. The rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:1000) was added as a secondary antibody and incubated for 1 hour at 37°C. The plate was then washed three times with TPBS and 0.4 mg/ml OPD, and 0.06% H2O2 was added. The reaction was terminated by the addition of 4 N H2SO4. The 2,3-diaminophenazine formed after OPD cleavage by horseradish peroxidase was determined at 492 nm using the Thermo Multiskan EX multiplate reader (Thermo Electron; West Palm Beach, FL).

For scanning electron microscopy (SEM) measurements of platelet bindings to fibrinogen, plastic discs used for cell culture dishes were incubated with fibrinogen solution (2 mg/ml) for 1 hour at room temperature. Discs with adsorbed fibrinogen were washed gently and placed in the wells of 24-well plate. Then, 0.25 ml of PRP was added to the wells and incubated at 37°C for 1 hour incubation; a 3% glutaraldehyde solution buffered with 0.05 M Tris-HCl buffer, pH 7.4, was then poured onto the wells, and the plate was incubated at room temperature for 1 hour. After 1 hour, the buffered glutaraldehyde solution was changed to a series of ethanol/water dilutions (with increasing ethanol concentrations) and the plate was incubated at each dilution for 30 to 60 minutes. As the final solution, 100% ethanol was added to the wells and the plate was left to dry. The dried samples were taken from the wells and placed onto stubs for coating with palladium by ion sputtering analysis via SEM.

Animal Model

Male Wistar rats (170 ± 4 g, n = 40) were purchased from the Vivarium of the Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine. Only male rats were used in the study, using the same logic as only male volunteers were involved as blood plasma donors for direct platelet studies. However, no sex-specific differences were expected.

Insulin resistance in rats (n = 30) was induced by feeding a prolonged high-fat diet for six months. The amount of lipids in the diet was increased by adding pork lard, in addition to the standard pellet chow, so the fat content was 58%, proteins at 23%, and carbohydrates at 10%. The lard contained a high level of palmitic and stearic acids, 24% and 28% of total fatty acids (FAs), respectively. The FA composition was at a ratio of 55% saturated (SFA) to 45% unsaturated FA. Control rats (n = 10) during the experiment were on normal pellet diet (4% fat; 23% protein; 65% carbohydrates) with a SFA/unsaturated FAs ratio of 38%/62%, respectively. The cholesterol content was 0.57 mg/g of lard.

The lard was provided to the rats every day of the experiment, in the same amount to each rat. All animals were keen to obtain their daily portions, eagerly took the lard from hands of laboratory assistants, and ate it immediately. According to rats’ preferences, we provided lard to them thinly sliced and slightly chilled.

The animals were kept in standard cages (five animals in each cage) under controlled conditions (22°C ± 2, 12-hour/12-hour light/dark cycle), with unlimited access to drinking water and rodent chow (Animal Feed Manufacturer “Agrovita,” Ukraine). Throughout the experiments, the rats gained weight gradually. On the 24th week, the average weight of the high-fat diet rats was 410–430 g (due to visceral fat), in comparison with the weight gain of the control rats at 330–350 g.

Six months after the high-fat diet period, we conducted an oral glucose tolerance test according to (Collier et al., 1985). The rats with impaired glucose tolerance (the level of blood glucose within 150 minutes after an oral glucose administration of 6 ± 1.05 mmol/l) were selected and divided randomly into two groups: IR (n = 9) and IR + NSE (n = 10) with equal distribution of tolerance data in each test group. Control rats were further subdivided into control (n = 10) and NSE (n = 7) groups. The size of the NSE group (control rats + NSE) was reasonably reduced according to previous data reported where there was no physiologic action of NSE in the case of healthy control animals (Onopchenko et al., 2018).

Animals in the NSE and IR + NSE groups were orally given the water suspension of NSE for two weeks at a dose of 50 mg/body weight kg−1.

This particular dose of NSE was chosen on the basis of our previous experimental data, as an optimal reacting dose for the biologic effect investigations. The exact concentration of NSE calculated during the experiment on dog coronary artery occlusion was reported earlier (Epps et al., 1980).

Samples of rat blood were collected by heart puncture. Pentobarbital (Nembutal) anesthesia (dosage of 50 mg/kg of body weight) was used intraperitoneally according to ethical standards and principles (as specified below). A 3.8% sodium citrate solution was added to the blood samples immediately after collection. PRP was prepared by centrifugation at 1000 rpm for 30 minutes.

Animals were decapitated immediately after blood collection, still under anesthesia. This study was carried out with the approval of animal care and use committee of the Palladin Institute of Biochemistry, NAS of Ukraine (Protocol #08/09-2015).

Statistical Analysis

Statistical analysis of the data was performed using Microsoft Excel (Microsoft Corp., Redmond, WA). All assays were performed in a series of three replicates and the data were fitted with standard errors using Statistica 7 (Dell Technologies, Round Rock, Texas). Results are presented as mean ± standard deviation. The difference between the two significantly different groups (control and studied group) was analyzed by one-way ANOVA. Statistical data analysis was performed on the groups of insulin-resistant animals with or without administration of NSE using the Wilcoxon-Mann-Whitney test. Differences were considered significant for P < 0.05.

Results

NSE Specifically Inhibits Platelet Aggregation

To evaluate the concentrations of NSE that could affect platelets we used the standard test with ADP, the most common inducer of platelet aggregation in clinical tests (Born and Cross, 1963). The concentration range was selected based on previous observations of the biologic action of NSE (Cattaneo et al., 2013). We studied the ADP-induced aggregation of platelets in the presence of NSE at concentrations of 10−5–10−10 M and found it inhibited platelet aggregation in a dose-dependent manner. However, the correlation was not linear and the effective inhibition of platelet aggregation was observed when 10−7 and 10−8 M concentrations of NSE were applied (Fig. 1). Lower or higher doses did not exhibit statistically significant effects on platelet aggregation induced by ADP. These results correspond to previous findings that showed maximal antiviral effects of NSE in the 10−7–10−8 M range of concentrations (Gulaya et al., 1998). Thus these concentrations were selected for all further experiments.

Fig. 1.
Fig. 1.

The action of NSE (gray line) and NOE (black line) on the rate of ADP-induced platelet aggregation in PRP. Final concentrations of NSE or NOE ranged from 10−5 to 10−9 M. Platelet aggregation was induced by ADP (12 μM). Results are presented as mean ± standard deviation, n = 5. NOE, N-oleoylethanolamine; NSE, N-stearoylethanolamine; PRP, platelet-rich plasma.

To confirm that the effect was specific, we performed similar experiments using NOE, which is analogous to NSE. However, no anti-aggregatory action of NOE was observed (Fig. 1).

NSE Does Not Stimulate Resting Platelets

To verify any possible effect of NSE on human platelets, resting platelets in PRP were analyzed by flow cytometry in the presence or absence of NSE (10−8 M). Non-activated PRP was incubated with NSE for 60 minutes. No changes in platelet granularity or shape were observed (Fig. 2). The count of platelets with normal shape and granularity in control probes (Fig. 2A) and in samples with NSE (Fig. 2B) did not differ significantly (61.5 ± 3% vs. 60.0 ± 3%, respectively). Thus we assumed that NSE did not activate platelets.

Fig. 2.
Fig. 2.

Flow cytometry of resting human platelets in PRP incubated with (A) 10−8 M NSE or (B) an equal volume of solvent (DMSO) in a control probe. Forward scattering (FS) and side light scattering (SS) of platelets were monitored to detect any changes of platelet granularity and shape. The analysis was based on 20 000 particles after preincubation with NSE for 60 minutes. Then, 2.5-µM ADP was applied for platelet stimulation, n = 5. NSE, N-stearoylethanolamine; PRP, platelet-rich plasma.

NSE Attenuates Ristocetin-Induced Platelet Activation

To verify if NSE affects the platelet activation step we analyzed the action of NSE on platelet responses to agonists acting via G-protein-coupled receptors, i.e., ionotropic and metabotropic purinergic receptors, P2X and P2Y, and estimated the secretion during ristocetin- and collagen-induced interactions of the platelet membrane glycoprotein Ib (GPIb) and the von Willebrand factor (vWF). We used a range of inducers including “weak” inducers: ADP [agonist for P2Y1 and P2Y12 receptors (Zhou and Schmaier, 2005)], epinephrine [binds to α2-adrenergic receptor (Gachet, 2000)], ristocetin [binds with vWF and then adheres to GPIb-IX-V complex (Spalding et al., 1998)], and “strong” inducers: thrombin [activates protease-activated receptors (Koutts et al., 1976)], collagen, and PAF [binds to the PAF-receptor (Kahn et al., 1999)].

The application of NSE (10−8 and 10−7 M) or an equivalent volume of DMSO did not affect the proton gradient of platelet secretory granules. In the next set of experiments, after 6 minutes of preincubation with NSE (10−7 M), platelets were stimulated with ADP (0.3 μM), ristocetin (0.4 mg/ml), epinephrine (0.6 μg/ml), or collagen (0.5 mg/ml) to detect the release of secretory granule constituents. The degranulation was estimated in percent from total accumulated dye.

ADP at a concentration of 2 μM promoted a fast secretion of granule constituents. The comparative analysis showed that ADP-stimulated platelet degranulation was not affected in the presence of 10−7 M NSE. However, at lower doses of ADP (0.3 μM) NSE inhibited platelet degranulation by 20.8±4% (Fig. 3A). Epinephrine-induced degranulation of platelets was also partially inhibited by 10−7 M NSE (9.5 ± 2.1% versus 11.3 ± 2.4% in control, Fig. 3B).

Fig. 3.
Fig. 3.

The action of NSE on the acidification of platelet secretory granules and the release of granule constituents during agonist-induced activation. Platelets were loaded with pH-sensitive fluorescent dye acridine orange in the presence of an equivalent volume of DMSO (control) or 10−7 M NSE and stimulated with (A) 0.3-μM ADP, (B) 0.6-μg/ml epinephrine, (C) 0.5-mg/ml collagen, or (D) 0.4-mg/ml ristocetin. The concentration of DMSO in the control samples was the same as that used in the preparation of 10−7 M NSE. Fluorescence intensities were normalized to similar values in the absence of platelets (Ft/F0). Represented traces were selected as the most common for independent experiments with the use of samples of PRP from 2 donors (each in triplicate). NSE, N-stearoylethanolamine.

We found that NSE slightly inhibited the degranulation induced by low doses of collagen (0.5 mg/ml), which did not induce platelet aggregation (Fig. 3C).

Another approach was to test the platelet secretion produced by the interaction of vWF and GPIb in the presence of ristocetin. Such stimulation in a mode of outside-in signaling was typically characterized by a lower level of secretion and was substantially attenuated in the presence of NSE (10−7 M) (33.3 ± 3.5% versus 44.8 ± 4.2% in control, Fig. 3D). As demonstrated in Fig. 3D, platelets preincubated with NSE displayed attenuation of both the rate and the final level of ristocetin-induced secretion.

NSE Preferentially Inhibits Ristocetin-Induced Platelet Aggregation

Studies of platelet aggregation induced by different agonists allow specifying the action of inhibitors and provide a detailed pharmacological approach, which significantly increases the likelihood of detecting platelet function (Pacher and Haskó, 2008).

The effects of low doses of ADP and epinephrine on platelet aggregation are in accordance with the data of activation studies. The effect of 10−8 and 10−7 M NSE on ADP-induced aggregation was moderate. When a low concentration of ADP (2 µM) was applied, the rate of the first wave of platelet aggregation decreased from 30±5% (control samples) to 25 ± 6% and 15 ± 6% in the presence of 10−8 and 10−7 M NSE, respectively (Fig. 4A). Such a concentration of ADP is normally assumed to be a weak stimulus for platelet aggregation. NSE also decreased the rate of platelet aggregation induced by epinephrine, from 24 ± 3% (control samples) to 20 ± 6% and 17 ± 6% in the presence of 10−8 and 10−7 M NSE, respectively (Fig. 4B).

Fig. 4.
Fig. 4.

The action of NSE on platelet aggregation in PRP induced by (A) 2-µM ADP, (B) 0.6- μg/ml epinephrine, (C) 3 -mg/ml collagen, (D) 0.4-mg/ml ristocetin, (E) 0.125NIH/ml thrombin. or (F) 50-nM PAF; C – control samples; 10−7, 10−8 –samples with 10−7 and 10−8 M NAEs added. Represented traces were selected as the most common for independent experiments with the use of samples of PRP from 2 donors (each in triplicate). NAE, N-acyletanolamines; NSE, N-stearoylethanolamine; PAF, platelet activating factor; PRP, platelet-rich plasma.

It was observed that 10−8 and 10−7 M NSE did not affect the rate of platelet aggregation induced by collagen (41 ± 8% and 40 ± 6%, respectively, versus 50 ± 6% in control), thrombin (33 ± 5% and 38 ± 6%, respectively, versus 40 ± 6% in control), and PAF (28 ± 5% and 21 ± 6%, respectively, versus 27 ± 4% in control). Typical curves are shown on Fig. 3, C, E, and F.

The most prominent effect of NSE was shown for ristocetin-induced aggregation. NSE at concentrations of 10−8 and 10−7 M decreased the rate of platelet aggregation induced by ristocetin (0.4 mg/ml) from 45 ± 8% in control samples to 31 ± 6% and 10 ± 3%, respectively (Fig. 4D). This effect is in accordance with the prominent inhibition of ristocetin-induced platelet activation and suggests that NSE modulates platelet signaling at the early stage of blood clotting.

Thus we showed the inhibitory effect of NSE on platelet activation and aggregation. This effect was more evident for “weak” inducers and the most prominent impact was observed for the ristocetin-induced process. As the binding of fibrinogen to the glycoprotein GPIIb/IIIa of activated platelets is the crucial step of platelet aggregation, we next studied the fibrinogen binding properties of platelets in the presence of NSE.

NSE Inhibits Fibrinogen Binding to Platelets

Fibrinogen has high affinity for GPIIb/IIIa receptors of activated platelets and much lower affinity for those of resting cells (Tselepis et al., 1999). Moreover, fibrinogen-GPIIb/IIIa interactions not only connect platelets to each other, but also induce outside-in signaling and promote the facilitation of platelet activation (Hantgan et al., 2010). The amount of fibrinogen that was bound to platelets was evaluated using specific anti-fibrinogen mAb. It was found that fibrinogen binding to platelets adhered to the surface was 15% lower in the presence of NSE (Fig. 5).

Fig. 5.
Fig. 5.

(A) Immunoassay of fibrinogen binding to activated platelets in the presence of NSE (from 10−6 to 10−9 M; K – control sample). Platelets were adsorbed to the microtiter wells in the presence of ADP, the amount of fibrinogen bound to platelets was estimated using mAb 2d-2a. Results are presented as mean ± standard deviation, n = 8, *P < 0.05. (B) Scanning electron microscopy (SEM) of platelets attached to plastic coated with fibrinogen in the presence of different concentrations of NSE (from 10−6 to 10−9 M; Control, control sample). mAb, monoclonal antibody; NSE, N-stearoylethanolamine.

Thus this approach allowed us to obtain measurable data. To reconfirm these results we also applied SEM as the traditional method for studying platelet adhesion to the fibrinogen-coated surface. As seen in Fig. 5B, platelets in the presence of 10−7 and 10−8 M of NSE were more spherical and did not expand pseudopodia as did those in the control sample or in samples in the presence of NSE in insufficient concentrations.

NSE Administration Leads to Normalization of Platelet Aggregation Rate in Animal Model

Previously we developed and characterized a model of obesity-induced insulin resistance in rats. Along with changes in blood coagulation parameters, this condition was shown to cause the over-reactivity of platelets (Dziuba et al., 2018). In the current study, we measured the aggregation of platelets from obese IR rats and compared them to those from rats that had been given NSE per os for two weeks until the end of the experiment.

As shown in Fig. 6, we observed a statistically significant decrease in the rate of platelet aggregation in blood plasma of rats treated with NSE. This finding may be due to the known anti-inflammatory action of NSE, as well as by the direct action of NSE on platelets, similar to that observed in vitro.

Fig. 6.
Fig. 6.

Aggregation rate of platelets from obese insulin-resistant (IR) rats after 2 weeks of NSE administration (IR+NSE). Platelets were activated by 12.5 μM of ADP (IR versus IR+NSE, P < 0.05 according to the Mann-Whitney U test), n = 9 (IR), n = 10 (IR+NSE), *P< 0.05. NSE, N-stearoylethanolamine.

Discussion

While the main mechanisms of platelet activation and aggregation induced by different agonists are similar, the intracellular signaling pathways differ significantly. All agonists have their unique receptors on the platelet surface, but the main result of their action is the activation of phospholipases leading to thromboxane A2 generation, granule secretion, and integrin activation (Li et al., 2010; Brantl et al., 2014). A summarized scheme of platelet activation of the agonists we studied is shown in Fig. 7.

Fig. 7.
Fig. 7.

Platelet signaling pathways during activation by the studied agonists. ADP, PAF, and thrombin act via G-protein-coupled receptors and promote the activation of PLA2 and PLCβ. Ristocetin-vWF and collagen bind to GPIb-IX-V and GPVI, respectively, via kinases and adaptor proteins that stimulate PLCγ and increase the production of IP3. IP3 interacts with the IP3receptor on the dense tubular system and stimulates the release of intracellular Ca2+, which is the main second messenger of platelet activation. Small GTP-ase Rap1b is controlled by Ca2+ and the diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI). Downstream effectors of the Rap1, Rap1-GTP-interacting adaptor molecule (RIAM) and Akt are important molecules involved in talin-1 recruitment to the cytoplasmic integrin tail. Binding of talin-1 and kindlin-3 to cytoplasmic domains of β3-integrin triggers a conformational change in the extracellular domains that increase the affinity for ligands, such as fibrinogen and vWF. AA–arachidonic acid; ADP –adenosine diphosphate; COX, cyclooxygenase; DAG, diacylglycerol; GP, glycoprotein; GTP, guanosine triphosphate; IP3, inositol-1,4,5-trisphosphate; P2Y1, P2Y12, purinergic receptors; PAF, platelet activating factor; PAR, protease-activated receptor; PL, phospholipase; vWF,von Willebrand factor. Artwork by Ludmila Kasatkina.

ADP, PAF, and thrombin act via G-protein-coupled receptors and lead to the activation of phospholipase Cβ (PLCβ) (Yang et al., 2002; Offermanns, 2006; Roka-Moya et al., 2014). In our studies NSE did not affect aggregation stimulated by thrombin, PAF, or high doses of ADP. However, NSE displayed inhibitory action on epinephrine-induced platelet aggregation and degranulation that is also dependent on G-protein signaling. In contrast to protease-activated receptor, purinergic (i.e., P2Y1, P2Y12) and PAF receptors, the αA2 receptor of epinephrine induces the signaling pathway that preferentially activates phospholipase A2 (PLA2). In this case, phospholipase Cγ (PLCγ) is activated through a PLA2-dependent mechanism as a result of stimulation by mediators released from platelets after PLA2 action (Banga et al., 1986; Coughlin, 2000).

We observed the prominent inhibitory effect of NSE on ristocetin-induced platelet degranulation. The rate and the final level of ristocetin-induced secretion of secretory granule constituents were attenuated in the presence of 10−7 M NSE. The receptor of ristocetin (in a complex with vWF) is GPIb-IX-V, which is a complex of integrins. This complex generates the signal in a G-protein-independent manner and mediates the activation of PLCγ (Wu et al., 1992). Signal transduction mediated by the GPIb-IX-V receptor is associated with the rearrangement of actin filaments (Rivera et al., 2009).

We did not observe prominent effects of NSE on collagen-induced activation or aggregation of platelets despite the fact that the collagen receptor GPVI is also an integrin and transduces the signal in a G-protein-independent manner (Bryckaert et al., 2015).

Surprisingly, it was reported that collagen and ristocetin can induce PLA2 activation and the subsequent enzymatic generation of free arachidonic acid and the formation of cyclooxygenase (COX) products also activate PLC (Nakano et al., 1989; Börsch-Haubold et al., 1995). Under inflammatory conditions, COX-mediated formation of thromboxanes and prostaglandin provides amplification signals in platelet activation; inhibition of their synthesis by anti-inflammatory agents is suggested as an effective mechanism for preventing platelet-dependent vascular occlusion (FitzGerald, 1991).

NSE also inhibited platelet degranulation induced by low doses of ADP (0.3 μM). The activation of the metabotropic purinergic receptors P2Y1 and P2Y12 leads to phosphorylation of PLCβ, but not PLA2. Low doses of ADP are known to induce two-wave aggregation with the second wave strongly dependent on GPIIb/IIIa activation and outside-in signaling (Kroll et al., 1991). Outside-in signaling after fibrinogen binding to GPIIb/IIIa and their clustering further increases platelet activation (Buensuceso et al., 2003; Hantgan et al., 2010).

The last hypothesis is in accordance with the data on fibrinogen binding to platelets that was distinctly affected by NSE. The decreased binding of fibrinogen to activated and absorbed platelets could be evidence for the impaired GPIIb/IIIa activity in the presence of NSE.

As was previously shown, the localization of the GPIb-IX-V complex within cholesterol-enriched domains is crucial for both platelet adhesion and post-adhesion signaling (Ginsberg et al., 2005). Thus the NSE-mediated impact on cholesterol-enriched membrane microdomains may influence the stage of vWF and GPIb interaction, as well as the activation and clustering of GPIIb/IIIa receptors. This suggestion is supported by the prevailing effects of NSE on integrin-dependent steps of platelet responses.

NSE was shown for the first time to be an inhibitor of aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. We concluded that these effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory action and allows us to prioritize studies of NSE as a potent anti-thrombotic agent.

Ethics Approval and Consent to Participate

The blood donor volunteers signed informed consent prior to blood sampling according to the Helsinki declaration. This study was approved by the Ethics Committee of the Palladin Institute of Biochemistry (23.08.2015, N5).

Animals were kept in accordance with the General Ethical Principles of Experiments on Animals adopted by the First National Congress of Ukraine on Bioethics (2001), which are consistent with the provisions of “The European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” adopted in 1986 at Strasbourg. This study was carried out with the approval of the Animal Care and Use Committee of the Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine (Protocol N1 from 08/09-2015).

Acknowledgments

The authors thank the Cedars-Sinai Medical Center International Research and Innovation in Medicine Program, and the Association for Regional Cooperation in the Fields of Health, Science, and Technology (RECOOP HST Association) for their support of our organization as a participating Cedars-Sinai Medical Center–RECOOP Research Center (CRRC), and for encouraging communication for inflammation studies at the Palladin Institute of Biochemistry (Kyiv).

Authorship Contributions

Participated in research design: Hudz, Chernyshenko, Kasatkina.

Conducted experiments: Hudz, Chernyshenko, Kasatkina, Urvant, Klimashevsky, Tkachenko.

Performed data analysis: Kosiakova, Hula, Platonova.

Contributed new reagents or analytic tools: Urvant, Klimashevsky.

Wrote or contributed to the writing of the manuscript: Hudz, Chernyshenko, Kasatkina.

Footnotes

    • Received January 19, 2022.
    • Accepted July 28, 2022.

  • This research was funded by the National Academy of sciences of Ukraine for Youth Laboratories [Grant 0122U002132].

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • dx.doi.org/10.1124/jpet.122.001084.

Abbreviations

AO
acridine orange
COX
cyclooxygenase
FA
fatty acid
FS
forward scattering
GP
glycoprotein
IR
insulin resistance
IP3
inositol-1,4,5-trisphosphate
mAb
monoclonal antibody
NAE
N-acyletanolamines
NOE
N-oleoylethanolamine
NSE
N-stearoylethanolamine
OPD
o-phenylenediamine
PAF
platelet activating factor
PL
phospholipase
PRP
platelet-rich plasma
SEM
scanning electron microscopy
vWF
von Willebrand factor

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Research ArticleCellular and Molecular

N-Stearoylethanolamine Inhibits Platelet Reactivity

Iehor A. Hudz, Volodymyr O. Chernyshenko, Ludmila O. Kasatkina, Lesia P. Urvant, Vitaliy M. Klimashevskyi, Oksana S. Tkachenko, Halyna V. Kosiakova, Nadiia M. Hula and Tetyana M. Platonova
Journal of Pharmacology and Experimental Therapeutics October 1, 2022, 383 (1) 2-10; DOI: https://doi.org/10.1124/jpet.122.001084
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