In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets

Abstract

Nitric oxide (NO) is an important physiological signaling molecule. However, when produced in excessive amounts, NO can also have toxic effects. The aim of this study is to investigate the effects of exogenous- and endogenous-derived NO on oxidative modifications of proteins and apoptosis in activated platelets. Washed platelets were incubated with l-arginine or nitroso-glutathione (GSNO) in the presence of adenosine diphosphate (ADP). After incubation, caspase-3 activity, phosphatidylserine (PS) externalization and the potential of mitochondrial membrane as markers of apoptosis were measured. In addition, the alterations in protein carbonylation (PCO) and nitrotyrosine (NT) formation as markers of protein oxidation were examined. Platelet activation with ADP (20 µM) significantly increased PCO and NT levels and apoptotic events. After incubation with l-arginine, platelet NO production increased significantly. This l-arginine-induced increase caused decreases in formerly increased PCO and NT levels associated with ADP-induced platelet activation. Stimulation of NO production with l-arginine protected platelets from apoptosis. GSNO caused an increase in protein NT levels. Despite this change, GSNO was effective in inhibition of P-selectin expression, platelet aggregation, protein carbonylation and apoptosis. The results suggest that l-arginine and GSNO-mediated NO leads to the inhibition of key apoptotic processes including caspase-3 activation, PS exposure and low mitochondrial membrane potential in washed platelets. The inhibitory effect of platelet clearance of l-arginine and GSNO may be a novel useful therapeutic property in clinical application.

Introduction

Apoptosis, or programmed cell death, is a main physiologic mechanism that regulates clearance of cells. Over the last decade, it has been discovered that apoptosis occurs not only in nucleated cells but also in anucleated cells such as platelets. ¹ Platelets show many of the signs of the apoptotic mechanism such as depolarisation of the mitochondrial membrane potential (ΔΨm), microparticle formation, activation of caspase-3 and exposure of phosphatidylserine (PS) on the platelet surface. ^2,3 Apoptosis is frequently accompanied by the generation of reactive oxygen and nitrogen species (RONS). ⁴ However, a specific role of RONS in apoptotic cell death has not been established yet. Platelets can also generate RONS that may play an important role in platelet functions. But, increased RONS were reported to provoke oxidative reactions in platelets. ⁵

Nitric oxide (NO) is an important messenger molecule involved in many biological processes. NO is synthesized from l-arginine and molecular oxygen in a reaction catalyzed by one of the three isoenzymes of NO synthases (NOS) such as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) in many cell types. ⁶ Platelets produce NO in smaller amounts than the endothelial cells do. eNOS and iNOS isoforms have been described in platelets, but eNOS is the predominant form. ⁷ The iNOS produces large quantities of NO, while the constitutive isoforms (nNOS and eNOS) produce lower levels. ⁸ The functional role of NO can vary by cell type and the involved isoenzyme. Endothelial cell-derived NO prevents adhesion of platelets to the vessel wall ⁹ and inhibits platelet aggregation. ¹⁰ The platelet-derived NO also limits the activation of neighboring platelets to avoid excessive recruitment of these cells into thrombi. ¹¹

An important feature of NO is that its properties and cellular targets at higher concentrations are profoundly different, particularly under conditions of oxidative stress, where it rapidly reacts with superoxide to form peroxynitrite which is an RONS. ¹² Peroxynitrite is a potent oxidant. Peroxynitrite not only decreases NO biovailability but also induces oxidation and/or nitration of cellular components such as DNA, lipids, proteins and low-molecular-weight biomolecules. When peroxynitrite is generated in excess, it may damage cells by oxidizing and nitrating cellular components. Nitration of some amino acid residues, particularly tyrosine in proteins by peroxynitrite can result in the alteration of protein function or structure and inhibition of enzyme activities. Nitrotyrosine (NT) has been used extensively as a marker of peroxynitrite formation. Thiols also are the preferential targets of peroxynitrite. ¹³ Exposure to NO or activation of iNOS has been reported to inhibit ¹⁴ or stimulate ¹⁵ apoptosis in several cell types.

It has been observed that incubation of platelets with peroxynitrite nitrated the platelet proteins and impaired platelet functions. ¹⁶ But it is still unclear whether l-arginine or nitroso-glutathione (GSNO)-derived NO modifies platelet proteins or induces platelet apoptosis. In a previuos study, we have shown that endogenous NO suppressed oxidized LDL-mediated platelet activation. ¹⁷ In this study, we aimed to investigate the role of l-arginine and GSNO-mediated NO signaling in the development of protein oxidative modifications and apoptosis in human platelets. The effects of l-arginine and GSNO on platelet aggregation and P-selectin expression as platelet activation marker were also investigated.

Material and methods

Blood collection

Blood was collected from healthy donors (12 subjects) with a mean age of 22 years. All donors were nonsmokers; none of them have been taking aspirin or other drugs affecting platelet function for at least 7 days prior to blood collection and none of them had a medical history suggesting haemostatic disorders.

Preperation of washed platelets and platelet-rich plasma (PRP)

Blood collected from healthy volunteers was anticoagulated with 1/9 volume of acid citratedextrose (ACD, 2.5% trisodium citrate, 2.0% d-glucose, 1.5% citric acid) and centrifuged at 150g for 10 min to separate PRP. PRP was centrifuged at 800g for 10 min. The platelet pellets were washed in modified Tyrode’s Ca²⁺/Mg²⁺ free buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH₂PO₄, 12 mM NaHCO₃, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5.6 mM glucose, pH: 7.4) and centrifuged at 800g for 10 min. The isolated platelets contained no detectable erythrocytes and less than 1 leukocyte per 5000 platelets.

For aggregation experiments, fresh blood was anticoagulated with 1/9 volume of 3.8% trisodium citrate and centrifuged at 150g for 10 min at room temperature to get PRP.

Incubations

For protein modification and apoptosis experiments, washed platelets were suspended in the modified Tyrode’s buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH₂PO₄, 12 mM NaHCO₃, 5 mM HEPES ve 5.6 mM glucose, 1 mM MgCl₂ and 1 mM CaCl₂, pH: 7.4). Adenosine diphosphate (ADP) (20 µM) was used to trigger oxidative stress and apoptosis in the platelets. Platelet suspensions were incubated with l-arginine (1 mM) or GSNO (100 µM) for 30 min at 37°C in the presence of ADP. For the detection of NO levels, protein oxidation markers and caspase-3, ADP, ADP+L-arginine or ADP+GSNO-treated platelets (5 × 10⁸ platelets/mL) were sonicated and lysates were used for further investigation.

Mitochondrial membrane potential (ΔΨm), PS exposure and P-selectin levels were analyzed by flow cytometry as mentioned below.

NO assay

The NO levels in platelets were measured using the method of Chen et al. ¹⁸ with minor modifications, indicated as nitrite plus nitrate (NOx) which is commonly used to monitor NO concentration. 1 mM l-arginine or GSNO (100 µM) were added to the washed platelets. Lysates of platelet were deproteinized with 75 mM ZnSO₄ and incubated with 1.44 mM NADPH and 20 mU nitrate reductase for 15 min at 37°C for the reduction of nitrate to nitrite. After centrifugation for 15 min at 2500g, the supernatant was used for assessment of the nitrite levels. Samples were allowed to react with the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% H₃PO₄). NO concentration was estimated from a standard curve with sodium nitrite as the standard and results were given as nmol NOx/10⁸ platelets.

Platelet aggregation

To assess the effects of exogenous l-arginine or GSNO on platelets, platelet aggregation in PRP was studied in duplicate. PRP contained no detectable erythrocytes and less than 1 leukocyte per 5000 platelets. ADP (10 µM) was used as agonist. Platelet aggregation was monitored by measurement of the changes in light transmission in a lumiaggregometer (Chronolog, Inc., Havertown, Pennsylvania, USA) at 37°C with stirring. Platelets in 0.5 mL PRP were stimulated with agonist and change in light transmission was measured. In some experiments, l-arginine or GSNO was added to PRP and incubated for 10 min at 37°C without stirring before ADP stimulation. The change in light transmission was recorded for 5 min. The maximum aggregation (%) was determined as the peak light transmission after the addition of ADP.

Assay of oxidative/nitrosative stress parameters

Protein carbonyl (PCO) and NT formation were measured as indices of oxidative/nitrosative stress. Carbonyl groups (aldehydes and ketones) are produced on protein side chains (especially of proline, arginine, lysine and threonine) when they are oxidized. These moieties are chemically stable. ¹³ The contents of PCO were assayed according to the method of Levine et al. ¹⁹ with minor modifications. Supernatants were incubated with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl for 1 h at room temperature. Protein hydrazone derivates were precipitated with TCA 20% and then the precipitates were washed three times with ethanol:ethyl-acetate (1:1). The final pellet was resuspended in 6 M guanidine hydrocholoride and incubated for 15 min at 37°C. The absorbance was measured at 360 nm, using a molar extinction coefficient of 2.2 × 10⁴ M⁻¹ cm⁻¹. The results were expressed as nmol PCO/mg protein.

NT modification of proteins is a well-established marker of protein damage by oxidative stress. 3-nitrotyrosine is a product of protein tyrosine nitration resulting from oxidative damage to proteins by peroxynitrite. ¹³ The levels of NT in platelet proteins were measured by Nitrotyrosine Chemiluminescence Detection Assay Kit following the protocol supplied by the manufacturer (Millipore, Cat No: 17-376). Briefly, 96-well plates were coated with 5 μg/mL nitrated BSA overnight at 4°C, and blocked with blocking buffer for 1 h; 50 μL of sample or standard and 50 μL of 2X anti-NT were added to each well, then the plates were incubated at 37°C for 60 min; 100 μl per well of 1X anti-rabbit immunoglobulin G (IgG), horse radish peroxidase (HRP) conjugate was added and incubated at 37°C for 60 min. The freshly prepared LumiGLO^® Chemiluminescent Substrate (75 μL) was added to wells and incubated at room temperature for 10 min. The plates were washed between each step. The amount of NT was calculated with the standard curve and expressed as µg nitrated BSA equivalents per mg protein.

Caspase-3 activity assay

Caspase activity was measured using a caspase-3 colorimetric assay kit (R&D Systems BF3100) following the manufacturer’s protocol. This method uses a colorimetric assay to monitor cleavage of an acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate, which resembles the caspase-3 cleavage site. First, platelets were lysed in the lysis buffer at room temperature for 15 min. The platelet lysates were then centrifuged at 2000g for 10 min at 4°C to precipitate cellular debris. Cell lysates were incubated with substrate solution Ac-DEVD-pNA for 1 h at 37°C. In order to measure specific caspase-3 activity, platelets were also treated with a caspase-3-specific inhibitor, N-Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO, Sigma A0835). The absorbance was read at 405 nm using a micro reader. The caspase-3 activity was calculated from the cleavage of the specific colorimetric substrate (Ac-DEVD-pNA). The difference between the amount of pNA produced in the absence of inhibitor and in the presence of inhibitor is a measure of specific caspase-3 activity. The results were expressed as nmol pNA/mg protein.

P-Selectin expression assay

For determination of platelet activation, P-selectin expression was monitered as described previously. ¹⁷ Platelets were treated with ADP, ADP + l-arginine or ADP + GSNO as described above, and the reaction was stopped after 30 min by adding an equal volume of 2% paraformaldehyde. Aliquots of the fixed platelets were incubated for 10 min with 1 μg/mL fluorescein isothiocyanate (FITC)-labeled anti-CD62P (clone AC1.2 MoAb; Becton Dickinson, San Jose, California, USA) and examined by flow cytometry.

PS exposure assay

For PS exposure experiments, platelet suspensions of 100 μL aliquots were placed in siliconized glass cuvettes at 37°C. ADP, l-arginine or GSNO was added and, after an initial mixing, incubated for 30 min at 37°C. The platelet suspensions were transferred to a water bath of approximately 22°C temperature and, after 1 min, 50 μL was combined with FITC-labeled annexin V (annexinV-FITC; 1 μg/mL; PharMingen, San Diego, California, USA) and phycoerythrin (PE)-labeled anti-CD41 (GPIIb) MoAb (150 ng/mL; Coulter/ Immunotech, Miami, Florida, USA) and incubated for 10 min at approximately 22°C. Samples were diluted fivefold with platelet buffer with 2 mM CaCl₂ for immediate analysis by flow cytometry.

ΔΨm assay

Changes in ΔΨm were measured according to the kit procedure (MitoPT JC-1 #911) using the J-aggregate-forming lipophilic cationic fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide (JC- 1). Briefly, JC-1 was dissolved in 500 μL dimethyl sulfoxide (DMSO) for preparing the stock solution and then diluted in platelet buffer on the day of staining. First, washed platelets (5 × 10⁸ /ml) were incubated with agents as described above at 37°C for 30 min. Then, 5 μL JC-1 was added to tubes and incubated for 20 min in the dark at 37°C. Platelet buffer (1.5 ml) was added and centrifuged at 1000g for 5 min and the supernatant was discharged. Buffer was added and centrifuged at 1000g and the supernatant was removed. Buffer (1 ml) was added and samples were analyzed immediately by flow cytometry.

For flow cytometric analyses, all the samples which were prepared as described above were analyzed on a Beckman Coulter, EPICS XL-MCL flow cytometer or a Becton-Dickinson, FACScan. The flow cytometer was equipped with a 488 nm argon ion laser. The platelet population was identified by forward scatter for cell size and by side scatter for cell granularity. Alignment of the instrument was checked daily by calibration beads. An electronic bitmap was placed around the platelet population and CD41a-FITC was used to form a gate. The results were expressed as the percentage of antibody-positive platelets. Nonspecific and background fluorescence was determined by the use of FITC-conjugated IgG. Fifty thousand platelets were counted in each tube.

Protein assay

Protein content was analyzed according to Bradford’s method ²⁰ using bovine serum albumine as the standard and expressed as milligram protein per milliliter platelet suspension.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was carried out using the Instat statistical package (GraphPad Software, San Diego, California, USA). The statistical difference between groups was determined by the Student's t test. A p value less than 0.05 was considered statistically significant.

Results

NO production

Washed platelets in the presence of ADP were incubated with or without l-arginine for 30 min at 37°C and NOx levels were measured. NOx levels in resting platelets (baseline) were significantly lower than those in the ADP-induced platelets (p < 0.001). Incubation of platelets with l-arginine caused significant increases in NOx levels compared with the ADP group (p < 0.01) (Figure 1).

Figure 1.

Mean values of NOx levels in platelets (⁺⁺⁺ p < 0.001 compared to baseline, **p < 0.01 compared to ADP). NOx: nitrite plus nitrate; ADP: adenosine diphosphate.

Oxidative/nitrosative stress parameters in platelet protein

We measured the levels of PCO and NT as markers of protein modification. Figure 2(a) shows the levels of PCO after ADP and ADP+l-arginine or ADP+GSNO treatments. After activation with ADP, there was a significant increase in the PCO levels as compared with the baseline group (p < 0.05). Addition of l-arginine or GSNO in ADP-induced platelets caused significant reduction in PCO levels (p < 0.05).

Figure 2.

Mean values of PCO (a) and NT (b) levels in platelets (⁺ p > 0.05 compared to baseline, *p < 0.05, **p < 0.01 compared to ADP). PCO: protein carbonylation; NT: nitrotyrosine; ADP: adenosine diphosphate; GSNO: nitroso-glutathione.

NT levels of platelet proteins are shown in Figure 2(b). After activation with ADP, there was a significant increase in the NT levels as compared with the baseline group (p < 0.05). Addition of l-arginine in ADP-induced platelets caused significant reductions in NT levels (p < 0.05). On the other hand, NT levels increased after GSNO as compared with the ADP group (p < 0.01).

Platelet aggregation and P-selectin expression

Platelet aggregation responses after l-arginine or GSNO are shown in Figure 3(a). After l-arginine or GSNO, there was a significant reduction in the aggregation (%) as compared with the ADP group (p < 0.001). Figure 3(b) shows the aggregation curves after l-arginine or GSNO.

Figure 3.

(a) Platelet aggregation responses (%) and (b) a sample of aggregation curve (***p < 0.001 compared to ADP group). ADP: adenosine diphosphate; GSNO: nitroso-glutathione.

Figure 4(a-d) shows the expression of platelet P-selectin as platelet activation marker in all groups. After ADP, there was a significant difference in the expression of P-selectin on the platelet surface as compared with the baseline group (p < 0.001). The expression of platelet P-selectin significantly reduced after l-arginine and GSNO treatment (p < 0.05 and p < 0.01, respectively, Figure 4(b)).

Figure 4.

Histogram samples of baseline, ADP, ADP+l-arginine and ADP+GSNO (a-d, respectively) of the P-selectin measurement. (e) Mean values of P-selectin in platelets (⁺⁺⁺ p < 0.001 compared to baseline group, *p < 0.05 and **p < 0.01, compared to the ADP group). ADP: adenosine diphosphate; GSNO: nitroso-glutathione.

Apoptotic events

In order to investigate whether NO can induce platelet apoptosis, platelets were incubated with l-arginine and GSNO. Caspase-3 activation, PS exposure and ΔΨm were used for characterizing cytoplasmic, plasma membrane and mitochondrial manifestations of platelet apoptosis, respectively.

Figure 5(a-d) shows flow cytometer histograms of annexin-V binding in platelets.

Figure 5.

PS exposure of platelets was determined using annexin-V. (a-d) Flow cytometric histogram samples of baseline, ADP, ADP+l-arginine and ADP+GSNO groups, respectively. (e) Mean values of platelet PS exposure. (f) Platelet caspase-3 levels (⁺⁺⁺ p < 0.001 compared to the baseline group, **p < 0.01, *p < 0.05, compared to the ADP group). ADP: adenosine diphosphate; GSNO: nitroso-glutathione; PS: phosphatidylserine.

After ADP stimulation of platelets, there was a significant increase in PS exposure as compared with the baseline group (p < 0.001). Treatment with l-arginine or GSNO reduced platelet PS exposure as compared with the ADP group (p < 0.01; Figure 5(e)).

Caspase-3 is the major effector caspase involved in apoptotic pathways. ¹ Caspase-3 levels are shown in Figure 5(f). Activation with ADP caused a significant increase in platelet caspase-3 activity (p < .001). After l-arginine and GSNO treatment, caspase-3 levels in platelets significantly decreased as compared with the ADP group (p < 0.05 and p < 0.01, respectively).

To investigate whether the activation of caspase-3 and PS exposure are apoptotic events we have tested using ΔΨm. Cell-permeable lipophilic cationic dye JC-1 was used for the determination of ΔΨm. If the ΔΨm is maintained, this dye accumulates in the mitochondria and shows a shift in fluorescence from green (monomeric form, FL1) to red (aggregate form FL2). Mitochondria have a high ΔΨm, breakdown of ΔΨm is characteristic of apoptosis. ² In this study, changes of ΔΨm in platelets were monitored by red fluorescence (FL2) in JC-1-stained platelets. Besides, the results were also expressed as dividing the arithmetic mean fluorescence intensity (MFI) of FL2 by the MFI of FL1 (FL2/FL1 ratio).

Figure 6(a-d) shows dot graphs of experimental groups after platelets were stained with JC-1. As shown in Figure 6(E), after platelet activation with ADP, ΔΨm decreased as compared with the baseline group (p < 0.001). l-arginine and GSNO significantly increased ΔΨm as compared with the ADP-activated group (p < 0.05 and p < 0.01, respectively). While ADP decreased FL2/FL1 ratio as compared with the baseline group (p < 0.001), l-arginine and GSNO significantly increased FL2/FL1 ratio as compared with the ADP-activated group (p < 0.001; Figure 6(F))

Figure 6.

Effects of l-arginine and GSNO on the mitochondrial membrane potential (ΔΨm) in ADP-activated platelets. The dot graph samples of baseline, ADP, ADP+l-arginine or ADP+GSNO (a-d, respectively). Mean values of ΔΨm (e), FL2/FL1 ratio (f) in platelets (⁺⁺⁺ p < 0.001, compared to the baseline group, ***p < 0.001, **p < 0.01, *p < 0.05, compared to the ADP group). ADP: adenosine diphosphate; GSNO: nitroso-glutathione.

Discussion

In this study the role of NO and protein modifications in mechanisms underlying platelet apoptosis which has a crucial importance in thrombosis and hemostasis were investigated. Stimulation of platelets by ADP caused an increase in platelet NO production as observed in previous studies. ¹⁸ The intracellular uptake of l-arginine is one of the important mechanisms regulating endogenous NO synthesis. ²¹ In our study, l-arginine (1 mM) addition in the presence of ADP also increased NO production when compared with the ADP group.

The stimulation of the platelets with agonists leads to increases in levels of reactive oxygen species such as superoxide and H₂O₂; thus, increases the oxidative markers. ⁵ ADP also causes superoxide production. ²² NO reacts with superoxide at a high rate to form peroxynitrite, which can induce nitration of proteins. ¹⁶ The stimulation of platelets results in the activation of several key tyrosine kinases and phosphatases, leading to the phosphorylation of several key cellular proteins. ²³ Peroxynitrite-induced nitration of proteins has been shown to reduce their phosphorylation when exposed to specific protein kinases, suggesting that protein nitration may interfere with protein phosphorylation signaling pathways. ¹⁶ Exogenous peroxynitrite can activate or inhibit platelets depending on its concentration. ¹³ In our study, ADP increased carbonylated and tyrosyl nitrated proteins, when compared with resting platelets. During collagen-induced activation of platelets, spontaneous nitration of platelet proteins have been reported. ²⁴ NO is required for nitration of proteins. It was shown that when the platelets are activated with agonists, the expression of iNOS and particularly eNOS increase. In our study, due to the short incubation period the increase in nitrated protein levels could have resulted from the increased activation of the present active iNOS form or eNOS rather than the increase in protein expression of iNOS. ²⁵ In parallel to our findings, small amounts of nitrated proteins were found in resting platelets, while the nitrated protein levels were found to increase as a result of collagen stimulation. ^23,24

As the importance of NO has been understood, some several NO releasing agents have gained extensive medical use. S-nitrosothiols are not being used clinically at present, but there are a large number of animal and clinical studies demonstrating their beneficial features. Some of these agents show tissue selectivity; such as S-nitrosoglutathione which is selective for arteries over veins, thus, has a distinct hemodynamic profile of action when compared with the classical organic nitrates. ¹² GSNO has been shown to decrease the prevalence of cerebral embolism after carotid endarterectomy in patients already receiving aspirin and heparin. Additionally, GSNO reduces platelet adhesion in bypass grafts. ¹² Therefore, we used GSNO as the exogenous NO donor in our study. NO acts upon being released from GSNO. The enzymatic activity necessary for this relase is also present in the platelets besides many other cells. ^26,27

We observed that l-arginine blocked the ADP-induced oxidative changes in the platelets. Despite increasing NO production in the platelets, l-arginine caused slight reductions in NT levels which is indicative of peroxynitrite production. Besides, l-arginine inhibited ADP-induced platelet aggregation and the increase in P-selectin expression. It has been known that NO is an inhibitor of platelet activation due to its inhibition of increasing intracellular cyclic guanosine monophosphate (GMP).7 At same time, l-arginine has antioxidant effects as a scavenger for superoxide anion. ²⁸ This effect of l-arginine may also play a role in its inhibition of platelet activation and oxidative stress.

GSNO caused significant increases in nitrated protein levels, while decreasing the oxidative changes. GSH found in the structure of GSNO may play role in its antioxidative effect. ¹² It has been shown that GSNO might lead to NT formation in the vessel wall. ²⁹ We observed that 100 µM GSNO significantly decreased platelet aggregation and activation. These findings are parallel with those of previous studies. ^30,31

NO’s effect on cell apoptosis varies depending on NO concentration and type of the cell involved. ³² Apoptosis is frequently accompanied by increasing oxidative stress. ⁴ The role of increased oxidative stress in NO-induced apoptosis is indicated by the suppression of NO-induced apoptosis by antioxidants. ³³ The recent studies verified that platelet apoptosis is induced by physiological agonists or calcium ionophores or platelet storage. ¹ In this report, we observed that platelet stimulation with 20 µM ADP resulted in caspase-3 activation, PS externalization and loss of mitochondrial potential. Despite ADP is a weak platelet agonist, it was shown that it caused increases in some platelet apoptotic markers at the concentration we used in our study. ³⁴ l-arginine and GSNO caused suppression of ADP-mediated caspase-3 activation and PS exposure in the platelets. Mitochondria are both major sources and targets for RONS. Peroxynitrite can cause mitochondrial damage including depolarization with alterations in the permeability transition, and thus, results in cytochrome c release and caspase activation. ³⁵ It was shown that peroxynitrite caused dose-dependent increases in apoptotic markers in the platelets including the change in mitochondrial potential; however, nitration of tyrosine was found to be partially associated with apoptosis. ³⁶ In our study, NO derived from l-arginine and GSNO inhibited the ADP-induced decrease in mitochondrial potential. These findings indicated that oxidative stress inhibitory effect of l-arginine may have a role in its apoptotic effect on activated platelets. It is reported that NO inactivates caspases by nitrating them. ³⁷ Despite decreasing the protein NT levels with l-arginine and increasing the protein NT levels with GSNO, both of them have shown antiapoptotic effects on platelets. This effect of GSNO different from l-arginine may possibily be due to its effect on the protein nitration as well as its suppressive effects on the oxidative events.

At a study conducted on rats, it was shown that oral administration of l-arginine following ischemia–reperfusion resulted in decreased apoptosis and necrosis. ³⁸ In an in vitro study, l-arginine administration was shown to block apoptosis in rat mesangial culture cells ³⁹ and neutrophils. ⁴⁰ In the literature, studies demonstrating both an apoptosis inhibitory effect ⁴¹ and an apoptosis promoting effect ⁴² of GSNO exist. These disparities may have been derived from differing administration times, concentrations and other conditions. Besides, studies on platelets may yield different results depending on the use of PRP or washed platelets. We used washed platelets in our study in order not to be affected by antioxidant and oxidant molecules and the NO metabolites in the plasma.

It can be concluded that both endogenous NO and exogenous NO provided from GSNO protect the platelets from apoptosis. The protein nitration in platelets is NO- and NO-production-dependent but is independent of increased oxidative stress. This in vitro study can be a basis for novel in vivo studies that would investigate the use of l-arginine and GSNO in antithrombotic treatment.