Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, UK.
J Thromb Haemost. 2017 Aug;15(8):1668-1678. doi: 10.1111/jth.13738. Epub 2017 Jul 18.
Essentials Platelet shape change requires cytoskeletal rearrangement via myosin-mediated actin contraction. We investigated whether nitric oxide (NO) affected thrombin-induced platelet shape change. NO inhibits shape change, RhoA/ROCK signalling and myosin light chain (MLC) phosphorylation. NO promotes MLC phosphatase activity, thus prevents MLC phosphorylation and shape change.
Background Platelet shape change, spreading and thrombus stability require activation of the actin cytoskeleton contractile machinery. The mechanisms controlling actin assembly to prevent unwanted platelet activation are unclear. Objectives We examined the effects of nitric oxide on the signaling pathways regulating platelet actin-myosin activation. Results S-nitrosoglutathione (GSNO) inhibited thrombin-induced platelet shape change and myosin phosphorylation of the myosin light chain (MLC). Because thrombin stimulates phospho-MLC through the RhoA/ ROCK dependent inhibition of MLC phosphatase (MLCP) we examined the effects of NO on this pathway. Thrombin caused the GTP loading and activation of RhoA, leading to the ROCK-mediated phosphorylation of MLCP on threonine 853 (thr ), which is known to inhibit phosphatase activity. Treatment of platelets with GSNO blocked ROCK-mediated increases in phosphoMLCP-thr induced by thrombin. This effect was mimicked by the direct activator of protein kinase G, 8-pCPT-PET-cGMP, and blocked by the inhibition of guanylyl cyclase, but not inhibitors of protein kinase A. Further exploration of the mechanism demonstrated that GSNO stimulated the association of RhoA with protein kinase G (PKG) and the inhibitory phosphorylation (serine188) of RhoA in a cGMP-dependent manner. Consistent with these observations, in vitro experiments revealed that recombinant PKG caused direct phosphorylation of RhoA. The inhibition of RhoA by GSNO prevented ROCK-mediated phosphorylation and inhibition of MLCP activity. Conclusions These data suggest novel crosstalk between the NO-cGMP-PKG and RhoA/ROCK signaling pathways to control platelet actin remodeling.
血小板形状变化需要通过肌球蛋白介导的肌动蛋白收缩来进行细胞骨架重排。我们研究了一氧化氮 (NO) 是否影响凝血酶诱导的血小板形状变化。NO 抑制形状变化、RhoA/ROCK 信号和肌球蛋白轻链 (MLC) 磷酸化。NO 促进 MLC 磷酸酶活性，从而防止 MLC 磷酸化和形状变化。
背景血小板形状变化、伸展和血栓稳定性需要激活肌动球蛋白细胞骨架收缩机制。控制肌动蛋白组装以防止血小板不必要激活的机制尚不清楚。目的我们研究了一氧化氮对调节血小板肌动蛋白-肌球蛋白激活的信号通路的影响。结果 S-亚硝基谷胱甘肽 (GSNO) 抑制凝血酶诱导的血小板形状变化和肌球蛋白轻链 (MLC) 的磷酸化。由于凝血酶通过 RhoA/ROCK 依赖性抑制肌球蛋白轻链磷酸酶 (MLCP) 刺激磷酸化-MLC，我们研究了 NO 对该途径的影响。凝血酶导致 RhoA 的 GTP 加载和激活，导致 ROCK 介导的 MLCP 在 threonine 853 (thr) 上的磷酸化，这已知抑制磷酸酶活性。GSNO 处理血小板可阻止凝血酶诱导的磷酸化-MLCP-thr 的 ROCK 介导增加。此作用可被蛋白激酶 G 的直接激活剂 8-pCPT-PET-cGMP 模拟，并被鸟苷酸环化酶抑制，但不受蛋白激酶 A 抑制剂的抑制。对机制的进一步探索表明，GSNO 以 cGMP 依赖性方式刺激 RhoA 与蛋白激酶 G (PKG) 的结合以及 RhoA 的抑制性磷酸化（丝氨酸 188）。与这些观察结果一致，体外实验表明重组 PKG 导致 RhoA 的直接磷酸化。GSNO 抑制 RhoA 可防止 ROCK 介导的磷酸化和 MLCP 活性抑制。结论这些数据表明，NO-cGMP-PKG 和 RhoA/ROCK 信号通路之间存在新的串扰，以控制血小板肌动蛋白重塑。
