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Originally published In Press as doi:10.1074/jbc.M208608200 on September 23, 2002

J. Biol. Chem., Vol. 277, Issue 49, 46858-46863, December 6, 2002
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Novel Role for Low Molecular Weight Plasma Thiols in Nitric Oxide-mediated Control of Platelet Function*

Michael S. CraneDagger , Richard Ollosson§, Kevin P. Moore§, Adriano G. Rossi, and Ian L. MegsonDagger ||

From the Dagger  Centre for Cardiovascular Science and the  Centre for Inflammation Research, University of Edinburgh, Edinburgh EH8 9XD and the § Centre for Hepatology, Royal Free and University College, London NW3 2PF, United Kingdom

Received for publication, August 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) is a powerful antiplatelet agent, but its notoriously short biological half-life limits its potential to prevent the activation of circulating platelets. Here we used diethylamine diazeniumdiolate (DEA/NO) as an NO generator to determine whether the antiplatelet effects of NO are prolonged by the formation of a durable, plasma-borne S-nitrosothiol reservoir. Preincubation of both platelet rich plasma (PRP) and washed platelets (WP) with DEA/NO (2 µM) for 1 min inhibited collagen-induced platelet aggregation by 82 ± 5 and 91 ± 2%, respectively. After 30 min preincubation with DEA/NO, NO was no longer detectable in either preparation, but aggregation remained markedly inhibited (72 ± 7%) in PRP. In contrast, the inhibitory effect in WP was almost completely lost at this time (5 ± 3%) but was partially restored (39 ± 10%) in WP containing human serum albumin (1%) and fully restored by co-incubation with albumin and the low molecular weight (LMW) thiols, glutathione, (5 µM), cysteinyl-glycine (10 µM), or cysteine (10 µM). This NO-mediated effect was not seen with LMW thiols in the absence of albumin and was associated with S-nitrosothiol formation. Our results demonstrate that LMW thiols play an important role in both the formation and activation of an S-nitrosoalbumin reservoir that significantly prolongs the duration of action of NO.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO)1 is a crucial free radical messenger with potent antiplatelet activity (1-5). NO synthesized in vascular endothelial cells and platelets is recognized to be a key mediator that protects against both atherogenesis and thrombosis (6). In platelets, NO primarily acts to stimulate soluble guanylate cyclase, ultimately resulting in a cyclic guanosine monophosphate (cGMP) and G kinase-mediated reduction in calcium mobilization (7, 8), although cGMP-independent inhibitory effects have also been identified (9). Under physiological conditions, the half-life of NO is short (~3-10 s) (10, 11), suggesting that NO bioactivity should rapidly dissipate and only impact on cells within close diffusible range of the site of production (12, 13). However, a number of studies suggest that NO can be incorporated into relatively stable endogenous reservoirs that modify its biological activity (14-19). S-Nitrosothiols rank high among the likely candidates for such a reservoir because of the relative abundance of suitable thiols in the biological environment (20). A physiological role for S-nitrosothiols has been implicated following identification of endogenous S-nitrosothiols at relevant concentrations (14, 21-24), together with plausible pathways that could result in their formation (25-28). In plasma, it has been shown that the vast majority of the S-nitrosothiol pool exists in the form of the high molecular weight species S-nitrosoalbumin (14, 29, 30). However, low molecular weight (LMW) thiols such as glutathione are also present in plasma in the low micromolar range and have previously been shown to potentiate the antiplatelet action of S-nitrosoalbumin (31). Given the close proximity of platelets to the vascular endothelium and the unique sensitivity of platelets toward S-nitrosothiol-mediated inhibition, it is important to dissect the role of plasma-borne thiols in the modification of NO activity in platelets.

Here, we tested the hypothesis that the activity of a short-acting NO donor drug, diethylamine diazeniumdiolate (DEA/NO), is prolonged in the presence of plasma albumin through formation and subsequent activation of an S-nitrosoalbumin NO reservoir. Furthermore, we explored the hypothesis that low molecular weight thiols have a unique role in both the formation and activation of an S-nitrosoalbumin reservoir, potentiating NO-mediated inhibition of platelet aggregation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate sodium salt (DEA/NO; Alexis Biochemicals, Lausen, Switzerland) was dissolved in 0.01 M NaOH and stored at -20 °C. DEA/NO was diluted in phosphate-buffered saline (pH 7.4) immediately before use. Bovine met-hemoglobin was reduced to the ferro (Fe2+) form by sodium dithionite as previously described (32). Spectrophotometric analysis indicated that ferrohemoglobin existed primarily in the oxygenated form. Collagen reagent was purchased from Labmedics (Stockport, UK). All other chemicals were purchased from Sigma.

Platelet Preparation-- Venous blood was drawn from the antecubital fossa of healthy volunteers (age 20-40 years) into citrated tubes (0.38% final concentration). Volunteers had not taken any medication known to affect platelet aggregation within the last 10 days. Platelet-rich plasma (PRP) and platelet poor plasma (PPP) were prepared as previously described (34). Washed platelets (WP) were prepared by centrifugation of PRP (1200 × g; 10 min) in the presence of PGI2 (300 ng/ml), and the platelet pellet resuspended in an equal volume of modified HEPES-tyrode buffer containing (in mM): 137 NaCl, 2.7 KCl, 1.05 MgSO4, 0.4 NaH2PO4, 1.8 CaCl2, 12.5 NaHCO3, 5.6 glucose, 10 HEPES, and 10.9 trisodium citrate. Following a secondary centrifugation (1200 × g; 10 min) in the presence of 300 ng/ml PGI2; platelets were resuspended in an equal volume of PGI2-free HEPES-tyrode. Platelet count was determined using a Coulter Ac.T 8 Hematology Analyzer (Coulter Electronics, Luton, UK) and standardized to 250 × 109 liter-1 via dilution with PPP (PRP) or HEPES-tyrode (WP).

NO Electrode Measurements-- Samples (2 ml) of PRP and WP were prewarmed to 37 °C before addition of DEA/NO (2 µM). NO concentration was measured for 30 min by an isolated NO electrode (World Precision Instruments, Stevenage, UK). The electrode was calibrated using DEA/NO (0.1-3.2 µM) in phosphate buffer (pH 4.0); DEA/NO spontaneously decomposes at pH <=  5 (33). In a different series of experiments, WP were reconstituted with 0.46 µM hemoglobin derived from red blood cell (RBC) lysate, prior to addition of DEA/NO (2 µM) and recording for 30 min.

Hemoglobin Measurements-- Plasma hemoglobin was quantified using an assay (Sigma Diagnostics) based on the hemoglobin catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine by hydrogen peroxide and colorimetric determination at 600 nm as described (34, 35).

Aggregometry-- Aggregometry studies were performed via turbidometric analysis using a two-channel platelet aggregometer (Chronolog Ca560, Labmedics, Stockport, UK). Signals were processed by a MacLab/4e analogue-digital converter (AD Instruments, Sussex, UK) and displayed through Chart software (AD Instruments, Sussex, UK). Aliquots (0.5 ml) of PRP and WP were equilibrated at 37 °C before the addition of 2 µM DEA/NO (~IC90 for DEA/NO in platelets (9)). Platelet aggregation was then induced via the addition of collagen (2.5 µg/ml) 1-30 min later. Aggregation was monitored for 5 min, and the maximum response recorded. In a different series of experiments, WP were reconstituted with the LMW thiols glutathione (GSH; 5 µM), cysteinyl-glycine (Cys-gly; 10 µM), and cysteine (Cys; 10 µM) to approximate plasma concentrations (36). Thiol-reconstituted WP was also incubated in the absence and presence of 1% human serum albumin (HSA); higher concentrations of HSA that approximate plasma levels (4%) were found to have nonspecific effects in platelets, even after extensive dialysis. Platelets were incubated with DEA/NO (2 µM) before stimulation with collagen (2.5 µg/ml) 30 min later. In further experiments, WP reconstituted with GSH (5 µM) ± HSA (1%) were preincubated with donor RBC lysate to produce a final hemoglobin concentration of 0.46 µM. DEA/NO (2 µM) was added to WP for 30 min prior to the addition of collagen (2.5 µg/ml) 30 min later. In control experiments, DEA/NO (2 µM) was added to WP 25 min before the addition of oxy-hemoglobin (10 µM). Platelets were then stimulated with collagen (2.5 µg/ml) 5 min later, and aggregation measured.

Thiol Measurements-- The reduced thiol content of plasma and HSA (1%)-reconstituted tyrodes ± the LMW thiols GSH (5 µM), Cys-gly (10 µM), or cysteine (10 µM) was quantified via reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and colorimetric determination at 412 nm, as previously described (37).

S-Nitrosothiol Detection-- Samples of PRP and WP were equilibrated in the aggregometer for 15 min. To establish baseline S-nitrosothiol levels, 0.5-ml aliquots of PRP or WP were transferred to vials containing N-ethylmaleimide (NEM) and EDTA (final concentration 5 mM and 2 mM, respectively). Samples were centrifuged (1800 × g; 5 min), and the supernatant aspirated. Acidified sulfanilamide (2.5% dissolved in 1 M HCl) was added to the supernatant, and the mixture stored at -70 °C prior to S-nitrosothiol detection. To determine S-nitrosothiol formation after bolus NO injection, WP and PRP samples were prewarmed as before and 2 µM DEA/NO added. Aliquots (0.5 ml) of DEA/NO-treated WP or PRP were aspirated 1-30 min later and added to NEM/EDTA to stop the reaction. Samples were centrifuged (1800 × g; 5 min), and the supernatant and pellet treated with acidified sulfanilamide and stored at -70 °C. S-Nitrosothiols were quantified by copper/iodide-induced cleavage of the S-NO bond and subsequent measurement by chemiluminescence as described (21).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hemoglobin Measurements-- Hemoglobin (Hb) concentration in PRP was 0.46 ± 0.18 µM (n = 5) and did not differ significantly (p > 0.05) from the hemoglobin concentration determined in PPP (0.39 ± 0.01 µM).

NO Electrode Studies-- Addition of 2 µM DEA/NO to WP resulted in a rapid increase in NO concentration, which reached a maximum of 3.2 ± 0.18 µM (mean ± S.E.) NO before it declined to basal levels within 20-25 min (Fig. 1; n = 6). Administration of 2 µM DEA/NO to PRP showed that DEA/NO-derived NO was partially quenched in plasma, reaching a maximum extracellular concentration of 0.53 ± 0.11 µM. Addition of 2 µM DEA/NO to WP reconstituted with 0.46 µM hemoglobin derived from donor RBC lysate produced a profile matching that observed in PRP with a maximum extracellular NO concentration of 0.59 ± 0.05 µM (n = 6). Reconstitution of WP with 1% HSA ± GSH (5 µM) produced a NO trace similar to that observed with WP (results not shown).


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  		Fig. 1.   Generation of NO by DEA/NO in WP, PRP, and Hb reconstituted WP. Platelets were equilibrated at 37 °C before the addition of 2 µM DEA/NO. Recording was stopped after a 30-min incubation of DEA/NO. Data are expressed as the mean of six experiments.

Effect of DEA/NO on Inhibition of Platelet Aggregation in PRP and WP-- Bolus administration of DEA/NO (2 µM) to PRP resulted in sustained inhibition of collagen-induced platelet aggregation that was maintained for at least 30 min (Fig. 2A; n = 8). In WP, however, inhibition of collagen-induced platelet aggregation by DEA/NO (2 µM) was attenuated at 20 min and abolished after 30 min (Fig. 2A; n = 8). The difference between inhibition of aggregation in PRP and WP was significant (p < 0.001). Representative traces from each time point in both PRP and WP are included (Fig. 2B).


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  		Fig. 2.   Inhibition of platelet aggregation by DEA/NO in WP and PRP. WP or PRP were equilibrated to 37 °C before the addition of DEA/NO (2 µM). Platelet aggregation was then stimulated via the addition of collagen (2.5 µg/ml) 1-30 min later. Data are expressed as mean ± S.E. of eight experiments. (*, p < 0.05; ***, p < 0.001; A). Representative traces obtained from PRP and WP are also included (B).

Effect of Thiols on DEA/NO-mediated Inhibition of Platelet Aggregation in WP-- Reconstitution of WP with the LMW thiols GSH (5 µM), Cys-gly (10 µM), and Cys (10 µM) did not alter the inhibition of platelet aggregation by DEA/NO after 30 min (n = 8; p > 0.05). However, reconstitution of WP with 1% HSA resulted in a modest restoration of the inhibitory effect of DEA/NO after 30 min (Fig. 3; p < 0.001). Co-incubation of WP with 1% HSA and either GSH, Cys-gly, or Cys fully restored the inhibitory effect of DEA/NO after 30 min (Fig. 3; p < 0.001). Inhibition of platelet aggregation by DEA/NO at 30 min in the presence of HSA and GSH was partially quenched by preincubation of 0.46 µM RBC-derived hemoglobin in WP (p < 0.01), although inhibition was still significantly enhanced when compared with WP alone (Fig. 4; n = 8).


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  		Fig. 3.   Effect of LMW thiols and HSA on inhibition of platelet aggregation by DEA/NO. LMW thiols GSH (5 µM), Cys-gly (10 µM), and Cys (10 µM) ± HSA (1%) were preincubated in WP before the addition of 2 µM DEA/NO. Platelet aggregation was then stimulated via the addition of collagen (2.5 µg/ml) 30 min later. PRP data are also included for a comparison. Data are expressed as mean ± S.E. of eight experiments. (***, p < 0.001).


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  		Fig. 4.   Effect of Hb on DEA/NO mediated inhibition of platelet aggregation. WP ± RBC-derived Hb (0.46 µM) were incubated with GSH (5 µM) ± HSA (1%) before the addition of DEA/NO (2 µM). Aggregation was then induced by the addition of collagen (2.5 µg/ml) 30 min later. Data are expressed as mean ± S.E. of eight experiments. (**, p < 0.001).

Effect of Hemoglobin on Prolonged Inhibition of Platelet Aggregation-- Prolonged inhibition of platelet aggregation in WP reconstituted with HSA alone or with HSA and any of the LMW thiols was abolished by addition of the NO scavenger oxy-Hb (10 µM; p < 0.001, n = 8).

Thiol Measurements-- The concentration of reduced thiol in plasma was 0.32 ± 0.01 mM (n = 5). In HEPES-tyrode buffer containing 1% HSA, thiol concentration was 0.11 ± 0.01 mM (n = 5) and did not differ significantly from 1% HSA containing GSH (5 µM; 0.12 ± 0.01 mM), Cys-gly (10 µM; 0.10 ± 0.01 mM), or Cys (10 µM; 0.11 ± 0.01 mM).

S-Nitrosothiol Detection-- Incubation of DEA/NO in PRP caused a rapid increase in S-nitrosothiol production which reached a maximum of 73.5 ± 15.4 nM after 10 min and diminished gradually over the 30-min incubation period (Fig. 5; n = 6). Addition of DEA/NO to WP + 1% HSA resulted in a slower increase in S-nitrosothiol concentration, which reached a level close to that observed in PRP after 30 min (46.0 ± 8.8 nM). The presence of 5 µM GSH increased the formation of S-nitrosothiol ~2-fold after a 30 min-incubation of DEA/NO (104.5 ± 18.7 nM). There was no significant difference in S-nitrosothiol formation in WP + 1% HSA compared with 1% HSA alone (p > 0.05).


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  		Fig. 5.   S-nitrosothiol formation in PRP and reconstituted WP after treatment with DEA/NO. DEA/NO (2 µM) was incubated in PRP or reconstituted WP before the addition of NEM/EDTA 1-30 min later to stop the reaction. Samples were then centrifuged, and the supernatant treated with acidified sulfanilamide (2.5% in 1 M HCl) before S-nitrosothiol detection. Data are expressed as mean ± S.E. of six experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results clearly demonstrate that the biological activity of DEA/NO, a short-acting NO-donor drug with a half-life of ~2 min at physiological temperature and pH, is significantly prolonged in PRP compared with WP, where activity closely mirrored NO concentration. Importantly, the prolonged inhibition of aggregation observed in PRP is mediated by NO, despite the clear decay of DEA/NO-derived NO to undetectable levels within the 30-min incubation period. Reconstitution of WP with HSA caused a partial restoration of DEA/NO-mediated inhibition after 30 min, but when combined with the LMW thiols GSH, Cys-gly, or Cys, the inhibitory action was fully restored to that seen in PRP. Furthermore, the degree of inhibition of aggregation was associated with S-nitrosothiol formation in PRP and reconstituted platelets, indicating a crucial role for both protein and LMW thiols in prolonging the biological activity of NO.

NO was clearly detected in both WP and PRP treated with DEA/NO, a compound known to generate two molar equivalents of NO upon hydrolysis. Importantly, while there was a clear divergence in the concentration of NO detected in PRP and WP, DEA/NO-derived NO declined to undetectable levels after a 20-min incubation period in both PRP and WP. There was a delay in the appearance of NO in PRP after bolus injection of DEA/NO, suggesting that plasma has some NO scavenging ability. Analysis of Hb concentration revealed that PRP contained 0.46 µM Hb, with a potential capacity to scavenge ~1.5-2 µM NO, assuming that all four heme groups are available for reaction with NO. Given that the delay in appearance of extracellular NO in PRP is ~2 min, during which time ~2 µM NO is released, our data indicate that Hb-mediated scavenging is responsible for the discrepancy between extracellular NO in PRP and WP. The concentration of Hb in PRP equated with that in PPP, indicating that the vast majority of Hb was cell-free. The physiological relevance of these findings is, as yet, unclear because blood sampling and platelet isolation is likely to cause significant hemolysis. However, increased scavenging of NO by cell-free Hb might have important implications in conditions where hemolysis is increased in vivo, such as in subarachnoid hemorrhage (38).

The ability of plasma components to prolong the antiplatelet effects of bolus DEA/NO is profound. While inhibition of platelet aggregation was sustained in PRP, substitution of plasma with HEPES-tyrode buffer resulted in a marked reduction in the duration of the inhibitory effect. Predictably, there was a close correlation between extracellular NO concentration and inhibition of platelet aggregation in WP, indicating that the degree of inhibition is closely defined by the extracellular NO concentration. In PRP, however, inhibition of aggregation was maintained, despite the progressive loss of extracellular NO from the system. The NO scavenger, oxy-Hb, abolished the sustained inhibitory effect in PRP, confirming that the effect was entirely NO-mediated. Given that human plasma is an abundant source of reduced thiol (20) and that the concentration of S-nitrosothiols in human plasma is relatively high (30-120 nM) (21, 24, 30), we hypothesized that thiols may have a role in the prolongation of NO bioactivity observed here. In human plasma, the single free cysteine residue present on serum albumin (Cys-34) accounts for the majority of reduced thiol. However, LMW thiols are present in human plasma in the low micromolar range (36), and S-nitrosothiols have previously been shown to undergo thiol-nitrosothiol exchange in vivo (15). Therefore, WP was reconstituted with albumin and LMW thiols to dissect thiol function on the antiplatelet activity of NO. Our results clearly indicate that incubation of the LMW thiols GSH, Cys-gly, and Cys did not alter the duration of antiplatelet action of DEA/NO, but reconstitution with 1% HSA significantly prolonged inhibition of aggregation. Crucially, while DEA/NO-mediated aggregation was only partially restored with HSA, co-incubation of HSA with each of the LMW thiols completely restored the inhibitory action of DEA/NO at 30 min despite a negligible increase in the thiol pool. Furthermore, hemoglobin completely reversed this inhibition, indicating that the LMW thiol/HSA effect is entirely NO-mediated.

The correlation observed between S-nitrosothiol formation and inhibition of platelet aggregation strongly indicates that the role of thiols in prolongation of NO-mediated inhibition of platelet aggregation is through provision of a substrate for S-nitrosation. Interestingly, our results indicate that there is a clear difference in the rate by which S-nitrosothiols are generated in PRP compared with thiol-reconstituted solutions. In PRP, S-nitrosothiol formation was very fast compared with that observed in reconstituted WP, with significant amounts being formed (~60 nM) after 1 min of incubation with DEA/NO. Conversely, 1 min of incubation of DEA/NO in HSA-reconstituted WP resulted in very low level S-nitrosothiol formation (<10 nM), which gradually increased to a maximum concentration of 50.5 ± 6.7 nM after 20-30 min. Despite rather different kinetics of formation of S-nitrosothiols in PRP and HSA-reconstituted WP, by 30 min, total S-nitrosothiol concentration is the same (~50 nM). However, inhibition of platelet aggregation is markedly different in PRP and HSA reconstituted WP after a 30-min incubation of DEA/NO. Previous data indicating that LMW thiols such as GSH can increase the antiplatelet action of S-nitrosoalbumin (31) are supported by our results. We suggest that S-nitrosoalbumin formed in reconstituted WP is an inefficient NO donor and requires the presence of low molecular weight thiols such as those found in PRP to efficiently control physiological function as has previously been proposed (14, 15, 17, 31). However, data obtained here emphasize an additional role for GSH and other LMW thiols in the formation of S-nitrosothiols. Co-incubation of GSH with HSA-reconstituted WP resulted in an increase in S-nitrosothiol concentration by ~2-fold. Furthermore, this increase was accompanied by a large augmentation of DEA/NO-mediated inhibition of platelet aggregation.

The mechanism for formation of S-nitrosothiols in vivo is a source of considerable debate; NO itself is a weak nitrosating agent, but higher oxides of NO such as N2O3 are potent nitrosating species (39). The rate-limiting step in the formation of N2O3 is the reaction of NO with molecular oxygen, which is third order (k ~ 4 × 106 M-2 s-1) (40, 41). Although originally thought too slow to account for endogenous levels of S-nitrosothiols, the reaction between NO and O2 can be catalyzed by ceruloplasmin (42), a copper-containing protein abundant in plasma. Moreover, accelerated formation of S-nitrosothiols has been observed in the presence of biological membranes (39, 43) and in the hydrophobic core of proteins such as albumin (44), which act as "NO sinks" to concentrate nitrosating species. We recognize that the pharmacological levels of NO used here are sufficiently high to facilitate significant formation of N2O3 that might subsequently nitrosate thiols. However, our results with GSH and HSA confirm previous findings that the ability of albumin to catalyze S-nitrosothiol formation is greatly increased in the presence of low molecular weight thiols (44). A modest increase (~5%) of thiol pool through addition of GSH to HSA-treated WP failed to significantly affect total thiol concentration, while causing a disproportionate increase in S-nitrosothiol formation (~2-fold). Our data demonstrate that the presence of platelets did not significantly alter S-nitrosothiol production, suggesting that plasma membrane-mediated acceleration does not play a part in this system. Given that we observed more rapid production of S-nitrosothiols in plasma than in reconstituted WP, we suggest that S-nitrosothiol formation catalyzed by plasma components like ceruloplasmin may be a key factor in the difference observed. Alternatively, the full complement of thiols in plasma may be required to provide an efficient pathway for the incorporation of NO into S-nitrosothiols. Our results indicate that cell-free Hb at plasma concentrations has a net scavenging effect, implying that cell-free Hb functions to remove NO rather than to conserve NO bioactivity through the formation of additional S-nitrosated species. We recognize that many pathways for S-nitrosothiol formation exist (26, 28), and thus may play a significant role in this system.

It is noteworthy that low serum GSH levels are an independent predictor of coronary heart disease (45) and that thiol supplementation in humans has been shown to cause an increase in both endothelium-dependent and -independent relaxation (46-48), especially in subjects at risk from coronary artery disease (46). Furthermore, a number of potential mechanisms for the cardioprotective role of thiols have been identified, including scavenging of oxygen-derived free radical species (48) and direct stimulation of NO synthase itself (49). Our results imply that the bioavailability of LMW thiols may have a significant impact on the ability of plasma to form S-nitrosothiols and, therefore, prolong the antiplatelet action of endothelium-derived NO (Fig 6). Moreover, in light of evidence that S-nitrosoglutathione is relatively platelet-selective (50), we suggest that the ability of GSH and other LMW thiols to assist in S-nitrosothiol formation and delivery may be of crucial importance in the maintenance of hemostasis and might be compromised in coronary artery disease.


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  		Fig. 6.   Summary of proposed mechanism. DEA/NO hydrolyzes in aqueous solution to generate NO. NO diffuses into the platelet where it activates various cellular processes leading to inhibition of platelet aggregation (path a). Alternatively, DEA/NO-derived NO reacts with molecular oxygen to form nitrosating species such as N2O3, which react with the sulfhydryl group on HSA to form relatively stable SNO-HSA. SNO-HSA inhibits aggregation via generation of NO at the platelet membrane surface (path b). In the presence of LMW thiols, N2O3 preferentially reacts with LMW thiols to form LMW S-nitrosothiols (RSNO). LMW S-nitrosothiols transnitrosate with HSA to form the S-nitrosoalbumin reservoir. Bioactive NO can be delivered to the platelet via a reverse of the previous process, leading to prolonged inhibition of aggregation (path c).

Our results have important implications with respect to the potential for NO donor-mediated antithrombotic therapy. Formation of a durable plasma reservoir of NO that is slowly liberated through the action of LMW thiols suggests that prolonged antiplatelet activity might be afforded by delivery of short acting NO donor drugs that were previously considered too labile for this purpose.

    FOOTNOTES

* This work was funded by British Heart Foundation FS/2001060 (to M. S. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Centre for Cardiovascular Science, Hugh Robson Bldg., University of Edinburgh, Edinburgh EH8 9XD, UK. Tel.: 44-131-651-1193; Fax: 44-131-650-6527; E-mail: ian.megson@ed.ac.uk.

Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.M208608200

    ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; cGMP, cyclic guanosine monophosphate; DEA/NO, diethylamine diazeniumdiolate; LMW, low molecular weight; PRP, platelet-rich plasma; PPP, platelet-poor plasma; WP, washed platelets; RBC, red blood cell; HSA, human serum albumin; NEM, N-ethylmaleimide; Hb, hemoglobin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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