Dnic Capacity Of Inhibiting Platelet Aggregation

Investigations in the 1980s demonstrated that DNICs with various anion ligands (thio-sulfate, cysteine or phosphate) are capable of preventing platelet aggregation induced by diverse agents (ADP, collagen, thrombin or epinephrine) [18,19]. This activity was suggested to be mediated by NO release from the complexes. According to the earlier proposed mechanisms [51], the activation of guanylate cyclase by NO radicals in platelets leads to the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) attenuating the formation of diacylglyceride, the activator of protein kinase C, which is a factor that directly suppresses the ability of platelets to aggregate.

Comparative studies on human platelet aggregation in the presence of SNP and DNICs added 3 min before the inductor of the process (ADP) showed a much higher inhibiting activity of the latter (Fig. 17).

The effect correlated with the extensive accumulation of cGMP in platelets [19]. It cannot completely be excluded that this effect was due to the toxic action of the DNICs on platelets. However, the experiments with addition of phorbol 12-myristate 13-acetate 4-O-methyl ether (MPMA) did not support this hypothesis. Due to the capability for activation of protein kinase C, MPMA stimulates platelet aggregation even in the presence of cGMP [52]. It was demonstrated that MPMA efficiently induced platelet aggregation despite the addition of DNICs (Fig. 18). Moreover, MPMA stimulated the platelet aggregation when added after DNICs at the time when the process induced by ADP was blocked by DNIC addition

Interestingly, DNIC administration could induce the process of platelet disaggregation in human plasma. The effect was more notable than that characteristic of SNP (Fig. 19).

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Fig. 17. Effect of DNIC with thiosulfate and nitroprusside on platelet aggregation induced by ADP (10 2 M). Curve 1, control preparation, curves 2-4, in the presence of 7 x 10-5, 3.5 x 10-4 or 7 x 10-4 M DNIC (non-dashed numbers) or nitroprusside (dashed numbers), respectively. The agents were added before the inductor administration. Platelet plasma preparations are prepared from the blood of two donors (a) and (b) [18].

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Fig. 17. Effect of DNIC with thiosulfate and nitroprusside on platelet aggregation induced by ADP (10 2 M). Curve 1, control preparation, curves 2-4, in the presence of 7 x 10-5, 3.5 x 10-4 or 7 x 10-4 M DNIC (non-dashed numbers) or nitroprusside (dashed numbers), respectively. The agents were added before the inductor administration. Platelet plasma preparations are prepared from the blood of two donors (a) and (b) [18].

Fig. 18. Effect of phorbolic ether on platelet aggregation in the presence of DNIC with thiosulfate. Curves 1 and 3 demonstrate platelet aggregation induced by phorbolic ether (10-5 M) or ADP (10-2 M), respectively. DNIC with thiosulfate (3.5 x 10-4 M) was added 3 min before the addition of phorbolic ether or ADP (curves 2, 4, respectively). Consequently, phorbolic ether (10-5 M) was added at the time point shown by the arrow (curve 4). Platelet plasma preparations are prepared from blood of two donors [(a) and (c), curves 1, 2 and 3, 4, respectively] [18].

Fig. 18. Effect of phorbolic ether on platelet aggregation in the presence of DNIC with thiosulfate. Curves 1 and 3 demonstrate platelet aggregation induced by phorbolic ether (10-5 M) or ADP (10-2 M), respectively. DNIC with thiosulfate (3.5 x 10-4 M) was added 3 min before the addition of phorbolic ether or ADP (curves 2, 4, respectively). Consequently, phorbolic ether (10-5 M) was added at the time point shown by the arrow (curve 4). Platelet plasma preparations are prepared from blood of two donors [(a) and (c), curves 1, 2 and 3, 4, respectively] [18].

Fig. 19. Disaggregating effect of nitroprusside (curve 1) or DNIC with thiosulfate (curve 2) added to platelet plasma [donor (d)] at the concentration of 3.5 x 10-4 M after pre-aggregation of platelets induced by ADP (10-2 M) [18].

Fig. 19. Disaggregating effect of nitroprusside (curve 1) or DNIC with thiosulfate (curve 2) added to platelet plasma [donor (d)] at the concentration of 3.5 x 10-4 M after pre-aggregation of platelets induced by ADP (10-2 M) [18].

EPR assay of human blood plasma containing platelets revealed the formation of protein-bound DNIC in the plasma after the addition of low-molecular DNIC with thiosulfate. Evidently, these complexes function as NO donors inhibiting the process of platelet aggregation. No accumulation of protein-bound DNICs was detected immediately in platelet cells [19].

The capability of Roussin's Black Salt, another representative of iron-sulfur-nitrosyl compounds to inhibit platelet aggregation was demonstrated in the 1990s by British researchers [53]. Inhibition of platelet aggregation by this compound was eliminated by the NO scavenger Hb. Addition of a selective inhibitor of cGMP phosphodiesterase (M&B22948) enhanced the effect of Roussen's Black Salt [53]. Therefore, the data indicated that

Roussen's Black Salt affected the process of platelet aggregation as an NO donor that activated guanylate cyclase.

In conclusion, the data presented in this chapter indicate a high biological potential of DNICs with various anion ligands. Without any doubt, they can be considered as the promising basis for the development of medicines with a wide range of therapeutic effects. Moreover, the investigations on biological activities of synthetic DNICs described in this chapter can give insights into the mechanisms of endogenous DNIC function, which forms in living systems generating NO by enzymatic or non-enzymatic pathways.

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