Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis
Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis
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DOI：
10.1186/s12964-016-0139-9
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发表时间：
2016-08-11
影响因子：
8.4
通讯作者：
Walter, Ulrich
中科院分区：
文献类型：
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作者：
Gambaryan, Stepan;Subramanian, Hariharan;Walter, Ulrich
背景：红细胞 (RBC) 和血小板之间的直接相互作用早已为人所知。贫血患者的出血时间延长，与血小板计数无关，可以通过输注红细胞来纠正，这表明红细胞在止血和血小板活化中发挥着重要作用。然而，在过去几年中，提出了红细胞衍生的一氧化氮（NO）抑制血小板的相反机制。我们研究的目的是确定红细胞是否可以产生 NO 并激活血小板中的可溶性鸟苷酸环化酶 (sGC)。 方法：为了测试红细胞是否可以在不同条件下（全血、缺氧或什至负载 NO）激活 sGC，我们使用了我们完善且高度敏感的血小板中 NO 依赖性 sGC 激活和纯化 sGC 激活模型。通过流式细胞术和蛋白质印迹检测血管舒张刺激磷酸蛋白（VASPS239）的磷酸化来监测 sGC 的激活。酌情使用方差分析，然后进行 Bonferroni 检验和 Student t 检验。结果：我们表明，在全血中，RBC 可以阻止 NO 介导的 ADP 抑制和 TRAP6 诱导的血小板活化。同样，红细胞与血小板共孵育会强烈抑制 NO 诱导的 sGC 激活。在缺氧条件下，红细胞与 NO 供体一起孵育会导致 Hb-NO 形成，从而抑制血小板中 sGC 的激活。同样，红细胞抑制纯化 sGC 的活化，即使是在红细胞介导的亚硝酸盐生成 NO 的最佳条件下也是如此。结论：我们所有的实验都表明，红细胞充当强 NO 清除剂，并防止 NO 介导的对活化血小板的抑制。在所有测试条件下，红细胞都无法激活血小板或纯化的 sGC。
Background: Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets.Methods: To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni's test and Student's t-test were used as appropriate.Results: We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite.Conclusions: All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.