Regular ArticlePlatelet resistance to the antiaggregating effect of N-acetyl-l-cysteine in obese, insulin-resistant subjects
Introduction
N-acetyl-l-cysteine (NAC) exhibits both extra- and intracellular thiol properties and increases cellular synthesis of reduced glutathione [1], [2], [3], thus enhancing the antioxidant defences [4]. NAC inhibits human platelet function by exerting direct antiaggregating effects [5] and by enhancing the antiaggregating action of the endogenous nitric oxide (NO) precursor l-arginine [6] and of NO donors, such as glyceryl trinitrate (GTN) [7], [8], [9].
We recently demonstrated that NAC decreases human platelet aggregation and increases intracellular levels of 3′,5′-cyclic guanosine monophosphate (cGMP) [5] by interplaying with endogenous NO, which is constitutively synthesized by platelets [10] and inhibits aggregation by activating the soluble guanylate cyclase and increasing the cGMP synthesis [11]: actually, the antiaggregating effects of NAC were prevented by both the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA) and the NO-sensitive guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ) [5]. Our observation is in line with the demonstration that NO binding to SH group induces the formation of an S-nitroso-thiol, which activates soluble guanylate cyclase in a more efficient way [4], [7], [12], [13], [14].
In previous studies, we evidenced that platelets from obese subjects exhibit a reduced sensitivity to the antiaggregating effect of insulin [15], which increases endogenous NO synthesis [16], [17] and of GTN [18], which releases NO after enzymatic conversion [19], [20], [21]. The simultaneous resistance to both endogenous and exogenous NO suggests a defect at the level of guanylate cyclase activation, even though the possibility of a reduced NO availability by reactive oxygen species cannot be excluded, since obesity is characterized by an increased oxidative stress [22], [23], [24]. In this case, the defect of the NO/cGMP pathway could be offset by antioxidative substances, such as NAC—which increases NO action within the cells [2], [4]—and superoxide dismutase (SOD), which inactivates extracellular superoxide anion [25], [26], thus protecting from inactivation the NO released extracellularly [25] and increasing its action on the surrounding platelets. An antioxidative effect could be exerted also by amifostine, an organic thiophosphate prodrug acting as an antiaggregating agent through a scavenger action of reactive oxygen species [27], [28].
To test this hypothesis, we carried out in obese subjects experiments designed to evaluate the platelet sensitivity to the effects of NAC, amifostine, and SOD.
In particular, we investigated
- (i)
the direct effects of NAC on platelet aggregation to adenosine-5-diphosphate (ADP) and collagen and on cGMP synthesis to clarify the availability of endogenous NO
- (ii)
the interplay between NAC, SOD, or amifostine and the NO donors GTN and sodium nitroprusside (SNP), to clarify the availability of exogenous NO.
Section snippets
Subjects
The study was carried out on 20 healthy volunteers (10 men, 10 women, aged 38.3±2.1 years) and 20 age-matched obese subjects (10 men, 10 women, aged 36.9±2.7 years). Obesity was defined as the presence of a body mass index (BMI) >27 kg/m2. The study was approved by the Ethics Committee of our department, and all subjects gave an informed consent before investigation. None of them was taking any drug which could influence platelet function in the previous 4 weeks and was long-term smoker.
Results
Baseline demographic and clinical characteristics of the investigated subjects are summarized in Table 1. The two groups were significantly different as far as BMI (p<0.0001), waist circumference (p<0.0001), fasting insulin (p<0.0001), fasting C-peptide (p<0.0001), HOMA index (p<0.0001), systolic blood pressure (p<0.03), triglycerides (p<0.0001), and HDL cholesterol (p<0.0001) are concerned.
Discussion
In the present study, we used different approaches to determine the sensitivity to NAC of platelets from healthy controls and obese subjects, i.e., (i) the evaluation of its antiaggregating action, to clarify the interplay with endogenous NO, and (ii) the interplay with NO donors, to clarify the effect on exogenous NO.
As far as the first topic is concerned, we evidenced relevant differences in obese and healthy subjects. In healthy controls, we confirmed our previous results [5], evidencing
Acknowledgements
This study was supported by a grant from the Italian Diabetes Society (Sezione Piemonte e Valle d'Aosta) and by a grant from Ministero Italiano dell'Istruzione e dell'Università (MIUR) to G.A.
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