Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Inhibition of platelet P2Y12 and alpha2A receptor signaling by cGMP-dependent protein kinase

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.