Department of Physiological Sciences, Eastern Virginia Medical School, PO Box 1980, Norfolk, Virginia 23501, USA.
Thromb Res. 2011 Jan;127(1):e8-14. doi: 10.1016/j.thromres.2010.10.002. Epub 2010 Nov 5.
In this study the effects of nitric oxide (NO) donors on intracellular free calcium (Ca(2+)) 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 Ca(2+) which suggests that a small portion of the thrombin mediated increase in Ca(2+) was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases Ca(2+) by stimulating store-operated Ca(2+) entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase Ca(2+) however it inhibited the ability of thrombin to increase Ca(2+) 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 Ca(2+) whereas higher concentrations (>200 nM) of SNP inhibited thrombin induced increases in Ca(2+). Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba(2+) 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 Ba(2+) 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.
在这项研究中，研究了一氧化氮（NO）供体对人血小板细胞内游离钙（Ca(2+)）的影响。用亚甲蓝或 ODQ 抑制鸟苷酸环化酶（GC），略微抑制了低浓度凝血酶增加Ca(2+)的能力，这表明凝血酶介导的Ca(2+)增加的一小部分是由于NO的增加，随后 cGMP 增加和 cGMP 依赖性蛋白激酶（cGPK）的激活。凝血酶主要通过刺激储存操作的 Ca(2+)内流（SOCE）来增加Ca(2+)。先前的研究表明，NO 供体 GEA3162 可刺激某些细胞中的 SOCE。在血小板中，GEA3162 没有增加Ca(2+)的作用，但它抑制了凝血酶增加Ca(2+)的能力，而 ODQ 可逆转这种作用。添加低浓度（2.0-20 nM）的 NO 供体硝普钠（SNP）可轻微增强凝血酶增加Ca(2+)的能力，而较高浓度（>200 nM）的 SNP 抑制凝血酶诱导的Ca(2+)增加。SNP 的这两种作用均被 ODQ 逆转，这表明它们均由 cGPK 介导。低浓度（2.0 nM）SNP 刺激 Ba(2+)内流，高浓度（>200 nM）SNP 抑制 Ba(2+)内流，这两种作用均被 ODQ 抑制。先前的研究表明，SOCE 抑制剂 2-氨基乙氧基二苯硼酸盐和己烯雌酚可阻断 Ba(2+)内流。结论是，低水平的 SNP 可刺激血小板中的 SOCE，这一作用可能解释了先前观察到的低浓度 NO 供体引起的聚集和分泌增加。在已知参与 SOCE 的蛋白质（例如基质相互作用分子 1（Stim1）、Stim2 和 Orai1）中，只有 Stim2 具有 cGPK 磷酸化位点。讨论了 Stim2 磷酸化是否调节血小板中的 SOCE。
