Abstract
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 microm) 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 microm), cysteinyl-glycine (10 microm), or cysteine (10 microm). 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.
Highlights
Nitric oxide (NO)1 is a crucial free radical messenger with potent antiplatelet activity [1,2,3,4,5]
Addition of 2 M DEA/NO to washed platelets (WP) reconstituted with 0.46 M hemoglobin derived from donor red blood cell (RBC) lysate produced a profile matching that observed in platelet rich plasma (PRP) with a maximum extracellular NO concentration of 0.59 Ϯ 0.05 M (n ϭ 6)
Our results clearly demonstrate that the biological activity of DEA/NO, a short-acting NO-donor drug with a half-life of ϳ2 min at physiological temperature and pH, is significantly prolonged in PRP compared with WP, where activity closely mirrored NO concentration
Summary
Nitric oxide (NO)1 is a crucial free radical messenger with potent antiplatelet activity [1,2,3,4,5]. 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. 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 M RBC-derived hemoglobin in WP (p Ͻ 0.01), inhibition was still significantly enhanced when compared with WP alone (Fig. 4; n ϭ 8).
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