Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

Abstract

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1- {N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory eect with vasorelaxation, to determine the eects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and to investigate the possible role of activation of sarco-endoplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 ± 10 mg ml 71) and adenosine diphosphate (ADP; 2 mM) in platelet rich plasma. It was (i) more eective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mM) caused only a small shift (approximately half a log unit) in the concentrationresponse curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; 1 ± 100 mM), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 ± 1 mM), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 mM present) were abolished by thapsigargin (200 nM). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQresistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

A potential role for extracellular nitric oxide generation in cGMP‐independent inhibition of human platelet aggregation: biochemical and pharmacological considerations

“…To date, numerous potential targets for cGMP-independent inhibition have been established. There is convincing evidence that NO can activate the platelet sarcoendoplasmic reticulum Ca 2 þ ATPase (SERCA; Trepakova et al, 1999;Homer & Wanstall, 2002). Our results suggest that the cGMP-independent effects of NO do indeed impact on Ca 2 þ trafficking (Figure 5), in agreement with earlier findings.…”

“…Plasma, nitric oxide, cGMP and platelet function investigate NO-mediated cGMP-independent antiplatelet effects (Tsikas et al, 1999;Homer & Wanstall, 2002); however, a 100 : 1 excess of the NO donor SNAP can result in a partial reversal of ODQ-mediated inhibition of sGC (Moro et al, 1996). In our experiments, a theoretical maximum of 20 mM NO will be released by 10 mM DEA/NO, which is equivalent to the concentration of ODQ used here, and unlikely to be sufficiently high to overcome ODQ-mediated inhibition.…”

“…Recently, several cGMP independent signalling mechanisms have been identified (Gordge et al, 1998;Trepakova et al, 1999;Tsikas et al, 1999;Ahern et al, 2002;Homer & Wanstall, 2002;Thyagarajan et al, 2002;White et al, 2002). In platelets, NO accelerates sarco-endoplasmic reticulum Ca 2 þ ATPase (SERCA)-dependent refilling of internal Ca 2 þ stores (Trepakova et al, 1999;Homer & Wanstall, 2002), and the unstable S-nitrosothiol, S-nitrosocysteine, inhibits agonistinduced TxA 2 synthesis in human platelets (Tsikas et al, 1999).…”

“…In platelets, NO accelerates sarco-endoplasmic reticulum Ca 2 þ ATPase (SERCA)-dependent refilling of internal Ca 2 þ stores (Trepakova et al, 1999;Homer & Wanstall, 2002), and the unstable S-nitrosothiol, S-nitrosocysteine, inhibits agonistinduced TxA 2 synthesis in human platelets (Tsikas et al, 1999). Furthermore, the importance of cGMP-independent mechanisms is underpinned by the recent discovery that G-kinase has an excitatory role in platelet activation (Li et al, 2003).…”

Ginkgolide C Inhibits Platelet Aggregation in cAMP- and cGMP-Dependent Manner by Activating MMP-9

“…20) Thus, we measured the cAMP and cGMP production by GC in resting and collagen-stimulated platelets. As the results, GC acts as a strong intracellular inducer of cAMP/cGMP, endogenous negative regulators of platelet aggregation, [26][27][28] in the presence of collagen (Figs. 4A, C).…”