N-acetylcysteine attenuates systemic platelet activation and cerebral vessel thrombosis in diabetes

Abstract

Objective: We previously demonstrated that diabetes exacerbates stroke-induced brain injury, and that this correlates with brain methylglyoxal (MG)-to-glutathione (GSH) status. Cerebral injury was reversed by N-acetylcysteine (NAC). Here we tested if the pro-thrombotic phenotype seen in the systemic circulation and brain during diabetes was associated with increased MG-glycation of proteins, and if NAC could reverse this.

Methods: The streptozotocin (STZ)-induced mouse model of type 1 diabetes was used. Thrombus formation in venules and arterioles (pial circulation) was determined by intravital videomicroscopy using the light-dye method. Circulating blood platelet-leukocyte aggregates (PLAs) were analyzed by flow cytometry 1 wk before other measurements. GSH and MG levels in platelets were measured by HPLC. MG-modified proteins, glutathione peroxidase-1 (GPx-1), and superoxide dismutase-1 (SOD1) levels were detected in platelets by western blot at 20 weeks. Proteins involved in coagulation were quantified by ELISA. NAC (2mM) was given in drinking water for 3 weeks before the terminal experiment.

Results: Thrombus development was accelerated by diabetes in a time-dependent manner. % PLAs were significantly elevated by diabetes. Plasma activated plasminogen activator inhibitor type 1 levels were progressively increased with diabetes duration, with tail bleeding time reduced by 20 wks diabetes. Diabetes lowered platelet GSH levels, GPx-1 and SOD-1 expression. This was associated with higher MG levels, and increased MG-adduct formation in platelets. NAC treatment partly or completely reversed the effects of diabetes.

Conclusion: Collectively, these results show that the diabetic blood and brain become progressively more susceptible to platelet activation and thrombosis. NAC, given after the establishment of diabetes, may offer protection against the risk for stroke by altering both systemic and vascular prothrombotic responses via enhancing platelet GSH, and GSH-dependent MG elimination, as well as correcting levels of antioxidants such as SOD1 and GPx-1.

Keywords: Diabetes; Glutathione; Methylglyoxal; N-acetylcysteine; Thrombosis.

Graphical abstract

Fig. 1

Effect of NAC on acceleration of thrombosis during early diabetes: Thrombosis in blood vessels of the mouse brain at 6 wks after injection with vehicle (Veh), or streptozotocin to induce diabetes (STZ 6wk). One STZ 6wk group was treated with N-acetylcysteine in drinking water for 3 wks before observation (STZ 6wk +NAC). Times to thrombus onset and cessation are shown in postcapillary venules (A&B) and arterioles (C&D). * P < 0.01 vs. Veh; # P < 0.05 vs. STZ 6wk.

Fig. 2

Impact of NAC on systemic platelet and coagulation changes induced by early diabetes: Mice were injected with vehicle (Veh) or streptozotocin (STZ) to induce diabetes. A third group of mice was injected with STZ, and 3 weeks later started on NAC drinking water. 5 wks after injections, blood was drawn for flow cytometry to measure (A) the % of leukocytes and leukocyte subsets forming aggregates with platelets, and (B) CD41 (platelet marker) expression on the aggregates. One week later, at 6 wks post-injection, (C-E) blood was drawn to measure circulating plasma levels of PAI-1, tPA and the ratio of tPA/PAI-1, and (F) tail bleed time was determined. * P < 0.05 vs. Veh; # P < 0.05 vs. STZ.

Fig. 3

Outcome of NAC treatment for the acceleration of thrombosis at 5 months diabetes. (A) Thrombosis in blood vessels of the mouse brain at 20 wks after injection with vehicle (Veh), or streptozotocin to induce diabetes (STZ 20wk). One STZ 20wk group was treated with N-acetylcysteine in drinking water for 3 wks before observation (STZ 20wk +NAC). (B) Comparison of thrombosis times between STZ groups 6wk and 20wk after induction of diabetes. Times to thrombus onset and cessation are shown in postcapillary venules and arterioles in both panels. * P < 0.05 vs. Veh; # P < 0.05 vs. STZ 20wk; ^ P < 0.05 vs. STZ 6wk.

Fig. 4

Effect of NAC on systemic platelet activation and coagulation during diabetes: Mice were injected with vehicle (Veh) or streptozotocin (STZ) to induce diabetes. A third group of mice was injected with STZ, and 3 or 17 wks later started on NAC drinking water. At 2 wks of NAC, blood was drawn for flow cytometry to measure (A-B) the % of leukocytes and leukocyte subsets forming aggregates with platelets, and (C-D) CD41 (platelet marker) expression on the aggregates. One week later, at 6 or 20 wks post-injection, (E-I) blood was drawn to measure circulating plasma levels of PAI-1, tPA and the ratio of tPA/PAI-1, and (J-K) tail bleed time was determined. * P < 0.05 vs. Veh; # P < 0.05 vs. STZ; ^ P < 0.05 vs. STZ 6wk.

Fig. 5

Impact of NAC on diabetes-induced alterations in GSH and MG: HPLC was used to measure GSH, MG or GSSG (A-F) and western blot was used to probe for MG-adducts (G) in platelets isolated from mice 6 or 20 wks after injection with vehicle (Veh), or streptozotocin to induce diabetes (STZ). Separate STZ groups were treated with N-acetylcysteine in drinking water for 3 wks before obtaining platelets (STZ +NAC). In (G), a sample blot and the quantification of 24kD and 54kD protein band intensities normalized to β-actin are shown. * P < 0.01 vs. Veh; # P < 0.01 vs. STZ.

Fig. 6

Expression of antioxidant enzymes in platelets from diabetic mice without and with NAC treatment. Western immunoblotting was employed to measure levels of (A) SOD-1 and (B) GPx-1 in platelets isolated from mice 20 wks after injection with vehicle (Veh), or streptozotocin to induce diabetes (STZ 20wk). One STZ 20wk group was treated with N-acetylcysteine in drinking water for 3 wks before obtaining platelets (STZ 20wk +NAC). For both, sample immunoblots are shown at the top, and the bar graphs show the quantification of protein band intensities normalized to β-actin. * P < 0.05 vs. Veh; # P < 0.05 vs. STZ 20wk.

Fig. S1

Examples of flow cytometry analysis for platelet-leukocyte aggregates (PLAs). Left: CD45 (leukocytes), CD41 (platelets) double positive events identified platelet-leukocyte aggregates. Right: This population was further divided into F4/80+ (Pl-Mono aggregates), Ly-6 G+ (Pl-Neut aggregates) or F4/80-negative, Ly-6 G-negative (Pl-Lymph aggregates). Examples are provided from the three experimental groups: mice injected with vehicle (Veh 19 week), mice injected with STZ to induce diabetes (STZ 19 week) and STZ-treated mice given NAC in drinking water for 2 wks before observation (STZ 19 week+NAC 2wk).