Transition metal-exchanged zeolite-A adsorbs and stores nitric oxide in relatively high capacity (up to 1 mmol of NO/g of zeolite). The stored NO is released on contact with an aqueous environment under biologically relevant conditions of temperature and pH. The release of the NO can be tuned by altering the chemical composition of the zeolite, by controlling the amount of water contacting the zeolite, and by blending the zeolite with different polymers. The high capacity of zeolite for NO makes it extremely attractive for use in biological and medical applications, and our experiments indicate that the NO released from Co-exchanged zeolite-A inhibits platelet aggregation and adhesion of human platelets in vitro.

1 Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist-induced increase in cytosolic Ca 2 þ concentration through both cGMP-dependent and independent pathways. However, the NO-related (NO x ) species responsible for cGMP-independent signalling in platelets is unclear. We tested the hypothesis that extracellular NO, but not NO þ or peroxynitrite, generated in the extracellular compartment is responsible for cGMP-independent inhibition of platelet activation via inhibition of Ca 2 þ signalling. 2 Concentration-response curves for diethylamine diazeniumdiolate (DEA/NO; a spontaneous NO generator), S-nitroso-N-valerylpenicillamine (SNVP; an S-nitrosothiol) and 3-morpholinosydnonomine (SIN-1; a peroxynitrite generator) were generated in platelet-rich plasma (PRP) and washed platelets (WP) in the presence and absence of a supramaximal concentration of the soluble guanylate cyclase inhibitor, ODQ (20 mM). All three NO x donors displayed cGMP-independent inhibition of platelet aggregation in PRP, but only DEA/NO exhibited cGMP-independent inhibition of aggregation in WP. 3 Analysis of NO generation using an isolated NO-electrode revealed that cGMP-independent effects coincided with the generation of substantial levels of extracellular NO (440 nM) from the NO x donors. 4 Reconstitution of WP with plasma factors indicated that the copper-containing plasma protein, caeruloplasmin (CP), catalysed the release of NO from SNVP, while Cu/Zn superoxide dismutase (SOD) unmasked NO generated from SIN-1. The increased generation of extracellular NO correlated with a switch to cGMP-independent effects with both NO x donors. 5 Analysis of Fura-2 loaded WP revealed that only DEA/NO inhibited Ca 2 þ signalling in platelets via a cGMP-independent mechanism. However, preincubation of SNVP and SIN-1 with CP and SOD, respectively, induced cGMP-independent inhibition of intraplatelet Ca 2 þ trafficking by the NO x donors. 6 Taken together, our data suggest that extracellular NO (440 nM) is required for cGMPindependent inhibition of platelet activation. Plasma constituents may play an important pharmacological role in activating cGMP-independent signalling by S-nitrosothiols or peroxynitrite generators.

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 M) 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 M), cysteinyl-glycine (10 M), or cysteine (10 M). 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. Nitric oxide (NO)1 is a crucial free radical messenger with potent antiplatelet activity (1-5). NO synthesized in vascular endothelial cells and platelets is recognized to be a key mediator that protects against both atherogenesis and thrombosis (6). In platelets, NO primarily acts to stimulate soluble guanylate cyclase, ultimately resulting in a cyclic guanosine monophosphate (cGMP) and G kinase-mediated reduction in calcium mobilization (7,8), although cGMP-independent inhibitory effects have also been identified (9). Under physiological conditions, the half-life of NO is short (ϳ3-10 s) (10, 11), suggesting that NO bioactivity should rapidly dissipate and only impact on cells within close diffusible range of the site of production (12, 13). However, a number of studies suggest that NO can be incorporated into relatively stable endogenous reservoirs that modify its biological activity (14 -19). S-Nitrosothiols rank high among the likely candidates for such a reservoir because of the relative abundance of suitable thiols in the biological environment (20). A physiological role for S-nitrosothiols has been implicated following identification of endogenous S-nitrosothiols at relevant concentrations (14, 21-24), together with plausible pathways that could result in their formation (25-28). In plasma, it has been shown that the vast majority of the Snitrosothiol pool exists in the form of the high molecular weight species S-nitrosoalbumin (14, 29, 30). However, low molecular weight (LMW) thiols such as glutathione are also present in plasma in the low micromolar range and have previously been shown to potentiate the antiplatelet action of S-nitrosoalbumin (31). Given the c...

Background: Serum thyroglobulin (Tg) is useful for monitoring patients with differentiated thyroid cancer (DTC) but is limited by interference from anti-Tg antibodies (TgAb). We determined Tg assay discordance between a radioimmunoassay (RIA) and one of two immunometric assays (IMA) in DTC patients over a 9-year period to gauge assay performance against evidence of recurrent/progressive DTC. Methods: Patients with DTC monitored for >1 year attending local clinics between September 2000 and January 2010 were included. All samples were analysed for Tg using both RIA and IMA. TgAb were measured on all Tg requests made after May 2006. Bias plots comparing RIA against IMA were established to calculate a 2-SD outlier limit. Clinical records were viewed to compare discordant Tg results against clinical evidence of recurrent/progressive DTC. Results: Discordant Tg results were observed in 53/433 patients (12.2%). Four were discordant owing to a higher IMA result, one of which demonstrated recurrence. The remaining 49 patients demonstrated a disproportionately higher RIA result, of which four had recurrent/persistent disease. Twelve patients with a higher RIA result but no evidence of recurrence underwent thyrogen stimulation testing, which was negative in all 12. In many cases, assay discordance appeared more sensitive at indicating interference than direct measurement of TgAb. Conclusions: Interference was evident with both Tg assays, such that neither could be solely relied upon to provide the correct result in the presence of TgAb. The concomitant measurement of Tg by RIA and IMA methods should be considered as an alternative to monitoring TgAb status.

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