N-acetylcysteine Amide AD4/NACA and Thioredoxin Mimetic Peptides Inhibit Platelet Aggregation and Protect against Oxidative Stress

Abstract

In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4/NACA), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bioavailability compared to NAC, and the thioredoxin-mimetic (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB₂ and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM peptides, or NAC, when tested at the highest concentration (0.6 mM). The effects of AD4, TXM-peptides, and NAC were also tested on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the greatest increase in CT. Furthermore, two representative compounds, TXM-CB3 and NAC, showed an increase in the anti-oxidant free sulfhydryl groups of plasma detected via Ellman's method, suggesting a contribution of plasma factors to the antiaggregating effects. Our results suggest that these small-molecular-weight redox peptides might become useful for the prevention and/or treatment of oxidative stress conditions associated with platelet activation.

Keywords: 12-HETE; N-acetylcysteine; N-acetylcysteine amide; TxB2; aggregation; clotting time; platelet; thioredoxin-mimetic peptides.

Figure 1

Inhibition of platelet aggregation by N-acetylcysteine amide (AD4), thioredoxin mimetics (TXM-CB3, TXM-CB13, TXM-CB30), and N-acetylcysteine (NAC). PRP was incubated with the vehicle or the different compounds for 30 min at 37 °C with constant stirring. Subsequently, platelets were stimulated with collagen (0.5 µg/mL). Concentration-dependent inhibition of platelet aggregation induced by collagen (0.5 µg/mL) after pretreatment with (A) AD4, n = 3; (B) TXM-CB3, n = 4; (C) TXM-CB13, n = 4; (D) TXM-CB30, n = 4; (E) NAC, n = 7. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. collagen-stimulated PRP for ANOVA and Dunnett’s post hoc test.

Figure 2

Inhibition of TxB₂ and 12-HETE synthesis by N-acetylcysteine amide (AD4), thioredoxin-mimetics (TXM-CB3, TXM-CB13, TXM-CB30), and N-acetylcysteine (NAC). PRP was incubated with the vehicle or the different compounds for 30 min at 37 °C with constant stirring. Subsequently, platelets were stimulated with collagen (0.5 µg/mL). At the end of aggregation, platelet aggregates were pelleted and the levels of (A) TxB₂ and (B) 12-HETE were simultaneously measured. n = 3. * p < 0.05, ** p < 0.01, vs. collagen-stimulated PRP for ANOVA and Dunnett’s post hoc test.

Figure 3

N-acetylcysteine amide (AD4), thioredoxin-mimetics (TXM-CB3, TXM-CB13, TXM-CB30), and N-acetylcysteine (NAC) prolonged the closure time (CT) in PFA-200 analysis. Whole blood was incubated for 30 min at 37 °C and perfused in a (A) coll/epi or (B) coll/ADP cartrige of the PFA-200 system, and the CT was recorded. n = 4. * p < 0.05, **** p < 0.0001, vs. vehicle-incubated blood for ANOVA and Dunnett’s post hoc test.

Figure 4

N-acetylcysteine amide (AD4), TXM-CB3, and NAC did not inhibit aggregation of washed platelets. Washed platelets were preincubated with the vehicle or AD4, or TXM-CB3, or NAC for 30 min at 37 °C with constant stirring, and subsequently stimulated with collagen (8 µg/mL). Aggregation was monitored for 6 min. n = 7.

Figure 5

Quantitation of sulfhydryl groups. PRP was preincubated with the (A) vehicle, (B) thioredoxin mimetics (TXM-CB3), or (C) N-acetylcysteine (NAC), for 30 min at 37 °C with constant stirring. Platelets were stimulated with collagen (0.5 µg/mL). After 6 min, plasma was separated via centrifugation and free sulfhydryl groups were quantified using Ellman’s reagent. n = 5. * p < 0.05; *** p < 0.001; **** p < 0.0001 vs. collagen-stimulated PRP.

Figure 6

Total antioxidant activity. PRP was preincubated with vehicle, TXM-CB3, or NAC for 30 min at 37 °C with constant stirring. Platelets were stimulated with 0.5 µg/mL collagen. After 6 min plasma was separated via centrifugation and antioxidant activity was detected via DCF fluorescence. Data are expressed as fluorescence units of DCF. n = 5. * p < 0.05 vs. unstimulated PRP (70 min).