Therapeutic potential of N-acetylcysteine as an antiplatelet agent in patients with type-2 diabetes

Abstract

Background: Platelet hyperaggregability is a pro-thrombotic feature of type-2 diabetes, associated with low levels of the antioxidant glutathione (GSH). Clinical delivery of N-acetylcysteine (NAC), a biosynthetic precursor of GSH, may help redress a GSH shortfall in platelets, thereby reducing thrombotic risk in type-2 diabetes patients. We investigated the effect of NAC in vitro, at concentrations attainable with tolerable oral dosing, on platelet GSH concentrations and aggregation propensity in blood from patients with type-2 diabetes.

Methods: Blood samples (n = 13) were incubated (2 h, 37°C) with NAC (10-100 micromolar) in vitro. Platelet aggregation in response to thrombin and ADP (whole blood aggregometry) was assessed, together with platelet GSH concentration (reduced and oxidized), antioxidant status, reactive oxygen species (ROS) generation, and plasma NOx (a surrogate measure of platelet-derived nitric oxide; NO).

Results: At therapeutically relevant concentrations (10-100 micromolar), NAC increased intraplatelet GSH levels, enhanced the antioxidant effects of platelets, and reduced ROS generation in blood from type-2 diabetes patients. Critically, NAC inhibited thrombin- and ADP-induced platelet aggregation in vitro. Plasma NOx was enhanced by 30 micromolar NAC.

Conclusions: Our results suggest that NAC reduces thrombotic propensity in type-2 diabetes patients by increasing platelet antioxidant status as a result of elevated GSH synthesis, thereby lowering platelet-derived ROS. This may increase bioavailability of protective NO in a narrow therapeutic range. Therefore, NAC might represent an alternative or additional therapy to aspirin that could reduce thrombotic risk in type-2 diabetes.

Figure 1

Therapeutic potential of in vitro additions of NAC to reduce platelet aggregability in whole blood from patients with type-2 diabetes. Inhibition of (A) thrombin- (0.125-1.0 U/ml) and (B) ADP (10 μmol/l)-induced platelet aggregation, by NAC (10-100 μmol/l; 2 h, 37°C), (n = 13 for both). Black circles = vehicle control; white triangles = 10 μmol/l NAC; black squares = 30 μmol/l NAC; white diamonds = 100 μmol/l NAC. Data are expressed as mean ± sem; statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc correction for multiple testing (treatment vs control). **P < 0.01, ***P < 0.001.

Figure 2

NAC impact on (A) total and (B) oxidised glutathione in platelet extracts (n = 11). Data are expressed as mean ± sem; statistical analysis was performed by one-way ANOVA with Dunnett's post hoc test vs control. *P < 0.05; ***P < 0.001.

Figure 3

Effect of in vitro additions of NAC on antioxidant capacity and platelet-derived ROS in platelet samples from patients with type-2 diabetes. (A) Mean data for the effect (n = 12); data are expressed as mean ± sem; statistical analysis was performed by one-way ANOVA with Dunnett's post hoc test vs control. **P < 0.01; ***P < 0.001. (B) Typical 3-line EPR spectra obtained at the 4 h reading for Tyrode's buffer alone and in washed platelets with or without treatment with NAC (10-100 μmol/l; 2 h; 37°C - NAC washed out of supernatant before addition of spin-trap). (C) ROS detection in washed platelets. Luminescence detection of ROS in both the basal state (black bars) and following thrombin-stimulation (white bars) of washed platelets (n = 12). Data are expressed as mean ± sem; statistical analysis was performed by one-way ANOVA with Dunnett's post hoc test vs control for each group (basal and thrombin-activated samples were analysed separately). **P < 0.01; ***P < 0.001 (thrombin-activated vs control): ^#P < 0.05; ^###P < 0.001 (basal vs control). (D) L-NAME sensitive nitrite detection in collagen activated washed platelets (n = 10). Data are expressed as mean ± sem; statistical analysis was performed by one-way ANOVA with Dunnett's post hoc test vs control. **P < 0.01.