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 effect with vasorelaxation, to determine the effects 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 micro g ml(-1)) and adenosine diphosphate (ADP; 2 micro M) in platelet rich plasma. It was (i). more effective 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 micro M) caused only a small shift (approximately half a log unit) in the concentration-response 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 micro M), 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 micro M 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 ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Figure 1

Mean concentration-response curves to (a) MAHMA NONOate, (b) GSNO (S-nitrosoglutathione), (c) sodium nitroprusside and (d) glyceryl trinitrate on rat platelets aggregated sub-maximally with collagen (2–5 μg ml⁻¹) or ADP (2 μM) and on rat main pulmonary arteries pre-contracted with phenylephrine (0.1 μM). Responses in platelets are expressed as percentage of inhibition of the aggregation response obtained in the absence of NO donor drug. Relaxation responses in pulmonary artery are expressed as percentage of reversal of the phenylephrine-induced contraction. Points are mean values with s.e.mean shown by vertical bars except when smaller than the size of the symbols. (n=4–6).

Figure 2

Mean concentration-response curves to (a,b) MAHMA NONOate and (c,d) GSNO (S-nitrosoglutathione) on rat platelets aggregated sub-maximally with collagen (2–5 μg ml⁻¹) or ADP (2 μM) in the absence (control) or presence of ODQ (10 μM). Responses to the NO donor drugs are expressed as percentage of inhibition of the aggregation response obtained in the absence of NO donor drug. Points are mean values with s.e.mean shown by vertical bars except when smaller than the size of the symbols. (n=5–6). Note that the control concentration-response curves are the same as those shown in Figure 1. *0.05>P>0.01, **0.01>P>0.001, ***P<0.001 when compared with corresponding concentration-response curve in the absence of ODQ (two-way ANOVA).

Figure 3

Mean inhibition of responses to MAHMA NONOate (3 μM) by ODQ (1, 3, 10 and 30 μM) on rat platelets aggregated sub-maximally with ADP (2 μM). Responses are expressed as percentage of inhibition of the control response to MAHMA NONOate. Points are mean values with s.e.mean shown by vertical lines. (n=5–6).

Figure 4

Mean concentration-response curves to (a) YC-1, a soluble guanylate cyclase activator, and (b) 8-pCPT-cGMP, a cGMP analogue, on rat platelets aggregated sub-maximally with collagen (5 μg ml⁻¹; n=3–4) or ADP (2 μM; n=4) and on rat main pulmonary arteries pre-contracted with phenylephrine (0.1 μM; n=4). Responses in platelets are expressed as percentage of inhibition of the aggregation response obtained in the absence of NO donor drug. Relaxation responses in pulmonary artery are expressed as percentage of reversal of the phenylephrine-induced contraction. Points are mean values with s.e.mean shown by vertical bars except when smaller than the size of the symbols.

Figure 5

Mean inhibitory responses to (a,b) MAHMA NONOate (1 and 3 μM) and (c,d) GSNO (S-nitrosoglutathione; 10 and 100 μM) on rat platelets aggregated sub-maximally with (a,c) collagen (2–5 μg ml⁻¹) or (b,d) ADP (2 μM) in the absence (control) or presence of thapsigargin (200 nM). ODQ (10 μM) present throughout. Responses to the NO donor drugs are expressed as percentage of inhibition of the aggregation response obtained in the absence of NO donor drug. Points are mean values with s.e.mean shown by vertical lines. (n=4–8). *0.05>P>0.01, **0.01>P>0.001 when compared with corresponding control values in the absence of thapsigargin (paired t-test).