Cardiovascular Research

Cardiovascular Research 1998 40(2):380-388; doi:10.1016/S0008-6363(98)00182-5
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite

Angie S. Brown^a, Maria A. Moro^b, Jean Marc Masse^c, Elizabeth M. Cramer^c, Marek Radomski^d and Victor Darley-Usmar^e,*

^aDepartments of Cardiology and Medicine, King's College Hospital, Denmark Hill, London SE5 9RS, UK
^bDepartmento de Farmacologéa, Facultad de Medicina, Universidad Complutense de Madrid, E-28040 Madrid, Spain
^cUnite INSERM U-91, Hopital Henri Mondor, Creteil, France
^dDepartment of Pharmacology, University of Alberta, Edmonton, Canada
^eDepartment of Pathology, Molecular and Cellular Division, Center for Free Radical Biology, University of Alabama at Birmingham, Volker Hall Room GO38, 1670 University Boulevard, Birmingham, AL 35294-0019, USA

^* Corresponding author.

Received 14 July 1997; accepted 14 April 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Peroxynitrite (ONOO^–) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO^– in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO^– on human platelets, a system in which we have previously shown that ONOO^– has both pro- and anti-aggregatory effects. Methods: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca²⁺ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO^– was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. Results: Peroxynitrite (50–400 µM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca²⁺ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO^– acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma. ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50–100 µM) of ONOO^– inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. Conclusions: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO^– to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca²⁺. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO^– in biological systems. We hypothesize that the conversion of ONOO^– to NO is the critical factor determining the outcome of ONOO^– exposure in vivo.

KEYWORDS Platelets; Nitric oxide; Peroxynitrite; P-selectin; cGMP

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Nitric oxide (NO) generated by constitutive NO synthase isoforms is involved in many physiological processes [1]. When released over long periods, however, NO may be cytostatic and cytotoxic for the cells that release it or for neighbouring cells [2–4]. Superoxide (O₂^–), generated during the respiratory bursts of phagocytes, has also been implicated in tissue damage [5–7]. Nitric oxide reacts rapidly with O₂^– [8], leading to formation of peroxynitrite (ONOO^–), a strong and highly reactive oxidant [9]. Peroxynitrite initiates lipid peroxidation [10]and causes oxidative modification of sulphydryl- and hydroxyl-containing small molecules and proteins [11]. Since the amounts of NO and O₂^– generated are markedly increased during many pathological processes, the rate of ONOO^– formation under these conditions is likely to be substantial [12–21].

Indeed, the balance between NO and O₂^– at the endothelial cell surface has been implicated in the pathophysiology of atherosclerosis and enhanced ONOO^– formation has been demonstrated on reperfusion of ischaemic myocardium [10, 19–21]. In either case, enhanced thrombogenesis may be critical in determining the clinical outcome in a therapeutic setting such as angioplasty. Of particular relevance to cardiovascular disease is the interaction of platelets with ONOO^– at the endothelial cell surface in both the presence and absence of plasma constituents. Several studies have investigated the effects of ONOO^– on platelets, but the results are currently confusing and conflicting: On the one hand, direct exposure of platelets to ONOO^– promotes aggregation and nitration of tyrosine residues in platelet proteins and, on the other, low concentrations of plasma completely inhibit this effect, preventing collagen-induced aggregation [22–26]. These results reflect an important controversy that is developing with respect to the role of ONOO^– in the pathophysiology of human disease and is not restricted to platelets, with similar effects having been reported in other vascular settings [27–30]. In this context, evidence from biochemical studies indicate that ONOO^– can exert both a nitrosative and oxidative stress [28]. The nitrosative stress is of particular interest since this can generate NO donors, based upon modification of polyhydroxylated compounds and thiols [22, 23, 29–32]. These reactions may explain the studies with intact cells or intact organ systems that reveal a biochemical response that is essentially identical to NO and suggests that ONOO^– could exert a cytoprotective function [27, 33]. Isolated platelets are an ideal system in which to dissect these important and topical issues.

We have previously shown that ONOO^– has pro-aggregatory actions on platelets, however, we failed to show that this was associated with increased release of P-selectin, when measured by ELISA in the bathing solution of washed platelets; other aspects of the mechanism were not clarified. Other studies have also questioned a mechanism through which ONOO^– can be converted to NO donors as the explanation for the inhibition of platelet aggregation, suggesting that S-nitrosation of thiols, independent of the guanylate cyclase–cGMP pathway, may be important [25]. Our previous studies using oxy-haemoglobin as a scavenger of NO could not determine whether the guanylate cyclase signal transduction pathway was involved or not and only imply a NO-dependent effect.

We have now further examined the dual actions of ONOO^– on human washed platelets by detecting and measuring P-selectin expression in platelets using more sensitive techniques. This adhesive glycoprotein is present in the -granules of resting platelets and only becomes expressed on the plasma membrane following platelet activation. In addition, we have investigated the possibility that the inhibitory actions of ONOO^– on platelet activation are modulated through activation of the soluble guanylate cyclase (sGC) by comparing the effects of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (ODQ) and oxyhaemoglobin. We have also investigated the mechanisms that underlie ONOO^–-induced platelet activating effects: It has been suggested that changes in Ca²⁺ could explain the pro-aggregatory effects of ONOO^– but this has not been addressed [25]. In the present study the effect of ONOO^– on intraplatelet Ca⁺ concentrations has been determined. In addition, ONOO^– reacts rapidly with thiols through an oxidative reaction [11], to assess the significance of this process on exposure of platelets to ONOO^–, depletion of extracellular thiols has been determined.

Our data indicate that an early event in the interaction of platelets with thiols is the conversion to either NO donors or directly to NO that leads to the activation of soluble guanylate cyclase and the subsequent inhibition of platelet aggregation. This occurs at lower concentrations than the oxidative reactions, the increase in intraplatelet Ca²⁺ and the increase in P-selectin expression.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Washed platelets and platelet aggregometry
Blood was collected from healthy volunteers who had taken no medication within the last seven days, into 3.15% sodium citrate (1:9, v/v) containing 2 µg/ml prostacyclin (Wellcome). Washed platelet suspensions (WPs, 2.0–2.5x10⁸ platelets/ml) were prepared as previously described [34]. Platelet poor plasma (PPP) was obtained by centrifugation of platelet rich plasma at 720 g for 10 min. Aggregation of washed platelets was studied in a platelet ionised calcium aggregometer (Chronolog). In all of the experiments described, WPs were incubated in the aggregometer and stirred at 900 rpm at 37°C.

2.2 Flow cytometry
Expression of the platelet glycoprotein P-selectin was measured following 1 min incubations of ONOO^– (50–400 µM) or decomposed ONOO^– (400 µM) with platelets. In a further series of experiments, the effects of ONOO^– (added 1 min prior to collagen) on collagen (3 µg/ml Hormon-Chemie, Munich, Germany)-induced stimulation of P-selectin were studied in the presence or absence of homologous PPP (1%, v/v). In addition, ONOO^–-induced P-selectin expression was measured in the presence of oxy-haemoglobin (HbO₂; 10 µM, preincubated for 5 min with WPs) or ODQ (2 µM, preincubated for 20 min with WPs) [35, 36]. Following the above experimental protocols, platelets were incubated with a saturating concentration of fluorescein isothiocyanate (FITC)-conjugated P-selectin/GMP140/CD62 monoclonal antibody (Immunotech) for 5 min at room temperature (final dilution, 1:50, v/v). An isotype-matched FITC-conjugated antibody raised against Aspergillus niger glucose oxidase (Dako, UK) was used as a negative control.

Subsequently, ice cold Tyrode's solution (GIBCO) was added and the platelets were immediately analysed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) connected to a Consort 32 computer system. Data were recorded and analysed using the LYSYS II software programme (Becton Dickinson). The instrument was set for the measurements of forward and side scatter, and for the green fluorescence of each cell. The fluidics were set at a low flow rate, to prevent platelet activation and to optimise resolution. A forward scatter acquisition gate was set to exclude background interference and debris so that only platelets, recognised by their characteristic forward and side scattering properties, were recorded. In each sample, data from 10,000 platelets were recorded in list mode. Following data acquisition, all samples were analysed together. A two-parameter display of the green fluorescence vs. forward scatter (a parameter related to cell size) was used to set the analysis gate. The green fluorescence acquisition gate was set above the level of non-specific fluorescence (derived from the analysis of cells stained with the irrelevant antibody) so that the percentage of specifically stained cells could be calculated. The FACScan was calibrated daily with fluorescent microbead standards (Becton Dickinson).

2.3 Platelet fixation and immunogold staining
Following incubation with ONOO^– (400 µM) or decomposed ONOO^– (400 µM), platelets were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer and washed three times with the same buffer. Platelets were post-fixed in glycol methacrylate (GMA) as described by Leduc and Bernhard [37]. Immunocytochemical procedures were performed on thin sections by the method of De May [38]. The antihuman P-selectin antibody (1:100 dilution) was a gift from M. Burt (Melbourne, Australia). Goat antirabbit immunoglobulin fractions coupled to 5-nm colloidal gold particles (GARG5) were purchased from Janssen Pharmaceutica (Beerse, Belgium, used at a 1:10, v/v, dilution).

2.4 Measurement of platelet thiols
The changes in platelet thiol content were measured colorimetrically using dithiobis nitrobenzoate (DTNB). Platelets (3.2x10⁶/ml) were mixed with 1 mM DTNB, incubated for 10 min at room temperature and then centrifuged at 1000 rpm before measurement of the change in absorbance at 412 nm in the supernatant. Peroxynitrite was added and incubated with the platelets for 10 min before measurement of platelet thiols. Absorbance was measured at 412 nm using an extinction coefficient of 13,600 M^–1 cm^–1 [39].

2.5 Synthesis of ONOO^–
Peroxynitrite was synthesized by the reaction of acidified NaNO₂ (1.8 M) with H₂O₂ (2.1 M) in a quenched flow reactor [40, 41]. The reaction was quenched with 4.2 M NaOH and any unreacted H₂O₂ was removed using solid MnO₂. Peroxynitrite was stored at –70°C before use. The concentration was determined prior to each experiment by measuring the absorbance at 302 nm (₃₀₂=1670 M^–1 cm^–1) [41]. Stock solutions containing ONOO^– at concentrations less than 220 mM were discarded. The pH of platelet suspensions was adjusted with 5 mM phosphate buffer (pH 7.4) to avoid alkalinization with the ONOO^– solutions. The experiments were also performed with decomposed ONOO^–, which was prepared by delaying the addition of NaOH to the NaNO₂/H₂O₂ mixture for 3 min or by allowing the ONOO^– to decompose at neutral pH in phosphate buffered saline. Potential contamination of the ONOO^– preparation with H₂O₂ was determined polarographically. Briefly, ONOO^– was decomposed and added to an oxygen electrode to give a final concentration of 30 mM in buffer (200 mM sodium phosphate, pH 7.4) containing 100 U/ml of catalase. No oxygen evolution was detected, indicating that H₂O₂ levels were below 10 µM. This would result in the addition of less than 20 nM H₂O₂ to platelets for a ONOO^– concentration equivalent to 50 µM. Nitrite contamination was determined spectrophotometrically and found to be approximately equivalent, in molar terms, to the amount of authentic ONOO^– prior to decomposition. Human oxy-haemoglobin was prepared by the method of Paterson et al. [42].

2.6 Measurement of intracellular calcium
Washed platelets were incubated for 30 min at 37°C with 2 µM Fura-2AM. Platelets were then washed and resuspended in 5 mM Hepes-buffered (pH 7.4) Ca²⁺-free Tyrodes solution at 2.5x10⁸ platelets/ml. For Ca²⁺ measurements, 0.5 ml of the platelet suspension was placed in a siliconized aggregometer cuvette inside the fluorometer turret by means of an adapter. Fluorescence was measured using a Shimadzu RF 5001-PC spectrofluorometer (Shimadzu, Kyoto, Japan). Excitation and emission wavelengths were set at 340 and 490 nm, respectively. CaCl₂ (1 mM) was added to the suspension before each experiment and platelets were stirred continuously using a teflon-coated stirring bar. In some experiments S-nitroso-DL-penicillamine (SNAP, 3 µM) was incubated with the platelets for 30 s prior to the addition of ONOO^–, and in other experiments, thrombin (0.03 U/ml) was added 30 s after the addition of ONOO^–. After the addition of thrombin, the stirrer was switched off to avoid aggregation. Maximal fluorescence was measured, after 200 mM digitonin (Sigma) was added to the solution and minimal fluorescence was measured after 1 mM MnCl₂ (Sigma) was added. Intracellular Ca²⁺ concentrations were calculated by the method of Grynkiewicz et al. [43].

2.7 Statistics
Results are expressed as the means±SEM of at least three different experiments. They were compared using analysis of variance and Duncan's multiple range test summary (Pharmacologic calculation system, version 4.0). P<0.05 was considered to be statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effects of ONOO^– on P-selectin expression in resting and collagen-activated platelets
Incubation of washed platelets with ONOO^– (50–400 µM) caused a concentration-dependent increase in the proportion of platelets expressing P-selectin (Table 1). The ONOO^– preparation used in these studies contained a number of contaminants that could synergize or otherwise interact to elicit biological responses. For example, it has recently been shown in a series of elegant studies that NO in combination with H₂O₂ can promote platelet aggregation [44]and it has been known for some time that nitrite (NO₂^–) can, under some circumstances, be converted to NO. Since both NO₂^– and H₂O₂ are potential contaminants of ONOO^– preparations, we have performed controls throughout these studies to test for their contribution and that of any other components that might cause effects independent of ONOO^–. We achieved this through allowing the ONOO^– to spontaneously isomerize at neutral pH in a reaction which leaves all the decomposition products intact but with no ONOO^– after 1–2 min. This sample we term ‘decomposed ONOO^–’ and it is evident from Fig. 1 that decomposed ONOO^– has no effect on the process of platelet activation and we therefore ascribe the observed responses (Fig. 1bFig. 1d) to ONOO^– itself. Evidence of platelet activation, with pseudopodia formation and swelling of the surface connected canalicular system (SCCS), was also evident from standard transmission electron microscopy (Fig. 1b), confirming our previous results [22]. As a sensitive index of the initial signalling events leading to adhesion, or its inhibition, the expression of P-selectin was determined. The experiments using electron microscopy coupled with immunogold staining for P-selectin or flow cytometry showed P-selectin expression (Table 1), both in the SCCS and on the platelet membrane (Fig. 1d). Decomposed ONOO^– (400 µM) had no effect on P-selectin expression (Fig. 1c, Table 1).

View this table: [in this window] [in a new window]   Table 1 Effect of ONOO– on platelet P-selectin expression

 

View larger version (138K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Electron micrographs of platelets treated with ONOO–. (a) Washed platelets stirred in an aggregometer in Tyrode's solution in the presence of decomposed ONOO– (400 µM) acted as a control, M:x14,542. (b) After treatment with ONOO– (400 µM), the platelets displayed signs of activation with numerous pseudopodia (p) and evidence of degranulation, with distension of the surface connected canalicular systems (SCCS) (arrows), M:x14,542. (c) In control platelets treated with decomposed ONOO– (400 µM), the immunogold labelled P-selectin is found on the limiting membrane of the -granule (A). The SCCS and plasma membrane are not labelled, M:x52,700. (d) After the addition of ONOO– (400 µM), immunogold labelled P-selectin is seen on the plasma membrane (arrows) and pseudopodia (P) as well as on the luminal side of the dilated SCCS (S), M:x52,700.

 
In contrast, when the expression of P-selectin was induced by collagen (3 µg/ml), the presence of ONOO^– (50–400 µM), but not decomposed ONOO^–, inhibited this expression (Fig. 2). The inhibitory effect of ONOO^– (50–400 µM) on collagen-induced P-selectin expression was further enhanced in the presence of 1% homologous PPP (Fig. 2). Moreover, the presence of 1% PPP prevented the expression of P-selectin induced by ONOO^– (400 µM) itself (data not shown). These data suggest that, concomitant with a pro-aggregatory stimulus, ONOO^– produces a mediator that is capable of inhibiting P-selectin expression.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of ONOO– on collagen-induced P-selectin expression in the presence and absence of 1% plasma. Peroxynitrite (50–400 µM) reduced the expression of P-selectin on WP activated with collagen (3 µg/ml) (solid line). PPP (1%) had no effect on collagen (3 µg/ml)-induced P-selectin expression. However, incubation with PPP in addition to collagen and ONOO– (50–400 µM) reduced the expression of P-selectin (dotted line) to a greater extent than that seen in the absence of PPP. The results are expressed as the mean±SEM of four–six experiments. The significance (P=<0.05) is indicated in all figures by *.

 
To test the involvement of endogenous NO in these inhibitory effects, WPs were preincubated with either the sGC inhibitor ODQ (2 µM) or with the NO scavenger HbO₂ (10 µM), prior to treatment with ONOO^– (50–400 µM). In the presence of ODQ (2 µM), the increase in ONOO^–-induced P-selectin expression was enhanced some two–three-fold and achieved essentially maximal activation (Fig. 3a). The concentration of ODQ was selected on the basis of previous studies as being a level that resulted in essentially maximal inhibition of guanylate cyclase [35, 36]. Moreover, the same concentration of ODQ reversed the inhibition of collagen-induced P-selectin expression by ONOO^– (Fig. 3b), suggesting that, in addition to its activating effects, ODQ unmasks cGMP-dependent ONOO^–-inhibitory effects. The inhibitory effect of the reaction product of PPP and ONOO^– on collagen-induced P-selectin expression was sensitive to either HbO₂ or ODQ, again signifying the importance of the ONOO^–-dependent activation of the NO-soluble guanylate cyclase pathway (Fig. 3c). If ONOO^– results in the formation of NO or a NO donor, this should result in a decreased sensitivity to collagen-induced aggregation. This anticipated result is shown in Fig. 4, together with its reversal by oxy-haemoglobin.

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of ODQ and oxy-haemoglobin on the actions of ONOO– on WP. (a) The increase in P-selectin expression seen with ONOO– (50–400 µM) was augmented by pre-incubation of the WP with ODQ (2 µM) (dotted line). (b) Pre-incubation of WP with ODQ (2 µM) (dotted line) reversed the inhibition of P-selectin expression seen with ONOO– (50–400 µM) on WP activated with collagen (3 µg/ml) (solid line). (c) Collagen (3 µg/ml), platelet poor plasma (PPP, 1%) and ONOO– (400 µM) were added to WP. The inhibition of collagen-induced aggregation seen with ONOO– and PPP together was reversed by pre-incubating the WP with oxy-haemoglobin (oxyHb; 10 µM) or ODQ (2 µM). The results are expressed as the mean±SEM of four experiments.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of oxy-haemoglobin on ONOO–-induced inhibition of collagen-dependent platelet aggregation. The maximal aggregatory dose–response curve produced with increasing concentrations of collagen (0.1–10 µg/ml) was shifted to the right following the addition of ONOO– (100 µM). The inhibitory effect of ONOO– was reversed by pre-incubating the platelets with oxy-haemoglobin (oxyHb; 5 µM). The results are expressed as the mean±SEM of four experiments. *P<0.05 vs. ONOO–.

 
3.2 Effect of ONOO^– on platelet membrane protein thiols and intracellular calcium
As an index of the pro-oxidant reactions of ONOO^–, the oxidation of thiols on the platelet membrane was determined. Peroxynitrite (100–3000 µM) caused a concentration-dependent depletion of platelet membrane thiols but only at concentrations higher than those associated with the release of NO or a NO donor (Fig. 5a). As a further indication of the mechanism of the pro-aggregatory effects of ONOO^–, WPs were pre-loaded with the fluorescent Ca²⁺-sensitive probe FURA-2, washed and then treated with ONOO^– while changes in fluorescence were monitored. Peroxynitrite (200–400 µM) caused a concentration-dependent increase in intracellular calcium levels, which became significant only at concentrations of 300 µM or above (Fig. 5b).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of ONOO– on platelet membrane thiols and intraplatelet Ca2+. (a) The concentration of platelet thiols measured colorimetrically following incubation of WP with increasing concentrations of ONOO– (0–3 mM) is shown. Data are expressed as the percentage (±SEM) decrease in platelet membrane thiols for experiments performed in triplicate for three independent preparations of platelets in which the control level of reactive membrane thiols ranged from 16–39 nmol SH/106 platelets. (b) The concentration of intraplatelet Ca2+ was measured spectrofluorometrically after incubation of WPs with 2 µM Fura-2 AM following their incubation with increasing concentrations of ONOO– (100–400 µM), decomposed ONOO– (400 µM) or thrombin (0.03 U/ml).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Using electron microscopy with immunogold staining and flow cytometry, we have demonstrated that ONOO^– causes the expression of P-selectin on the surface of human washed platelets. In our previous studies using transmission electron microscopy and enzyme immunoassay for the soluble P-selectin, we failed to detect the ONOO^–-induced platelet release reaction or P-selectin release [22]. However, the use of electron microscopy at higher power with immunogold staining increases the sensitivity of the method for detecting membrane-bound P-selectin. Using this technique, we have shown that there is some degranulation of the -granules, with expression of P-selectin in the surface-connected canalicular system and on the platelet membrane. These observations were further supported by flow cytometry.

The mechanism of this signalling may involve Ca²⁺ mobilisation from the extracellular compartment, since the pro-aggregatory effect of ONOO^– was inhibited by EGTA, a Ca²⁺ chelating agent [22]. It is likely that this mechanism represents pro-oxidant damage to ion transport proteins or to the membrane itself, since only the highest concentrations of ONOO^– led to detectable increases in intraplatelet Ca²⁺. This conclusion is supported by the data obtained on depletion of sulphydryl groups in the platelet membrane, which again showed significant changes only at the higher ONOO^– concentrations. These concentrations of ONOO^– have been shown to reverse NO-induced inhibition of platelet aggregation [22]. It is likely that these effects would only occur on chronic exposure to ONOO^–.

At the lower concentrations of ONOO^–, NO-dependent inhibition of the pro-aggregatory effects of collagen was demonstrated. For example, ONOO^– caused inhibition of P-selectin expression on platelets stimulated by collagen. Moreover, the presence of plasma abolished the activating effects of ONOO^– on unstimulated platelets and further enhanced its inhibition of collagen-induced P-selectin expression. An interpretation of this data is that ONOO^– is converted to NO donors and, in support of this, we and others have found that ONOO^– reacts with plasma constituents and polyhydroxylated molecules to yield compounds that donate NO [22, 23, 29–32]. However, this has recently been disputed in favour of a mechanism in which S-nitrosation is the mechanism of platelet inhibition and this is independent of activation of the soluble guanylate cyclase [25].

The low reactivity of thiols in the platelets (Fig. 5) under conditions where a NO-dependent inhibition of aggregation was evident does not support this view. Furthermore, the soluble guanylate cyclase inhibitor, ODQ, also reversed the effects of ONOO^– on collagen-induced P-selectin expression. The interpretation of experiments with oxy-haemoglobin is confounded by potential direct interactions with ONOO^–. However, this would seem unlikely given the high concentration of ONOO^– and platelets and the relatively low concentration of oxy-haemoglobin (10 µM) and, in this case, the data are consistent with NO formation from ONOO^–.

The experiments with washed platelets suggest that the conversion of ONOO^– to NO is a mechanism that is intrinsic to the platelet. This conclusion is supported by our observation that, even in the absence of plasma, the incubation of platelets with ODQ significantly augmented the expression of P-selectin caused by ONOO^–, indicating that NO is also generated from the interaction between platelets and ONOO^–. The ODQ- and oxy-haemoglobin-inhibitable responses in platelets increased as a function of exposure to ONOO^– (Figs. 3 and 4), indicating that NO formation was dependent on a reaction of the oxidant. The chemical identity of the NO donor(s) generated during the interactions between ONOO^–, plasma and platelets is likely to involve the formation of either S-nitrosothiols or organic nitrites [19, 22, 23]. We have previously found that some thiols in proteins such as albumin and in the tripeptide antioxidant glutathione [22], as well as glucose and glycerol [23, 31], are capable of reacting with ONOO^– to generate NO donors. The candidate molecules on the surface of the platelet that mediate these reactions remain uncertain. At the present time, analytical techniques for the detection of NO donors in the nM concentration range are not available, although the biological effects can clearly be demonstrated in the studies shown herein. The present study therefore suggests that both extracellular and intracellular mechanisms may function to detoxify ONOO^– by converting it to NO donor(s).

The limitations of working with ONOO^– are that chronic exposure has to be modelled by bolus addition of what would appear to be unrealistically high concentrations. However, the short half-life of this oxidant results in brief periods of exposure to µM concentrations, with nominal concentrations in the mM range. For example, 100 µM ONOO^– is equivalent to a chronic exposure of 0.034 µM/h. This concentration could be readily formed at sites of inflammation, where rates of NO and O₂^– production are considerably increased. Peroxynitrite has been implicated in the pathophysiology of atherosclerosis. Indeed, nitrated tyrosine, an indirect index of ONOO^– generation, has been found in the atherosclerotic plaque and particularly in macrophages [20]. Since histological sections show that platelet thrombi tend to occur in areas laden with macrophages, it is possible that, in these circumstances, the continuous production of both NO and O₂^– and, hence, ONOO^–, from these phagocytes may lead to a significant depletion of both extracellular and intracellular thiols. In such circumstances, it is possible that the protective pathway we have described may become saturated, leading to further platelet activation and potentiation of the atherosclerotic process [21].

In summary, ONOO^– mediates both nitrosative and oxidative stress. The nitrosative reactions result in the formation of NO donors that can be converted in cells to NO and so exert cytoprotective effects [33, 45, 46]. Our studies illustrate that both classes of reaction can proceed simultaneously and we hypothesize that the net outcome of ONOO^– exposure will be determined by the availability and efficiency of the pathways leading to conversion to NO compared to the competing oxidative reactions.

Time for primary review 38 days.

	Acknowledgements

 
ASB was a British Heart Foundation Junior Research Fellow, MAM was supported by the Human Capital and Mobility program from the European Community. We would also like to thank Derek Smith for his technical advice.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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