Response by Liu and Hu to Letter Regarding Article, "Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase"
Response by Liu and Hu to Letter Regarding Article, "Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase"
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Liu 和 Hu 对有关文章“III 类 PI3K 通过 NADPH 氧化酶的 PI(3)P 导向功能积极调节血小板活化和血栓形成”的信件的回复
DOI：
10.1161/atvbaha.118.310712
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Hu Hu
中科院分区：
文献类型：
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作者：
Liu Yangyang;Hu Hu
我们欢迎这封信对我们最近的研究发表评论，该研究提出了一个问题，即与Vps34(空泡蛋白分类34)缺陷相关的缺陷活性氧(ROS)的产生和血小板激活受损是否如我们的研究所表明的那样是NADPH氧化酶(NOX)的真正功能。1我们的结论是：(1)Vps_(34)通过NO_x发挥功能，其ROS的产生是基于如下观察：(1)Vps_(34)缺乏损害了NO_(X2)的组装；(2)Vps_(34)缺乏减少了ROS的产生；(3)DPI(二苯基碘)抑制了Vps_(34)−/−与野生型血小板的聚集差异，补充H_2O_2可挽救Vps_(34)−/−的聚集缺陷。在使用Vps34抑制剂3-ma的人血小板中也得到了类似的观察结果。这些数据强烈地表明，NOX2是Vps34在血小板中的主要靶点，尽管我们确实同意，包括缺乏NOX2和其他NOX亚单位的小鼠将进一步澄清这一点。我们理解，这个问题主要源于之前关于NOX对血小板激活的重要性的报道之间的争议。因此，尽管最近对NOX1和NOX2缺陷小鼠的研究已经证明，这两种异构体对ROS的生成和血小板激活具有选择性地重要，2先前的报告表明：(1)缺乏NOX亚基的小鼠血小板，如NOX2或p47Phox，显示出正常的ROS生成和对凝血酶和胶原蛋白3，4的聚集反应；(2)缺乏NOX2的人血小板(来自慢性肉芽肿疾病)也表现出正常的聚集反应。5、6然而，值得注意的是，在高浓度激动剂作用下，NOX1和NOX2基因敲除对ROS生成和血小板聚集的抑制作用减弱。2与血管紧张素转换酶34通过一氧化氮合酶发挥作用的结果一致，血管紧张素转换酶34缺乏也可在低剂量激动剂刺激下(凝血酶0.025.0 5U/m L或胶原0.5μg/m L)对ROS的产生和血小板聚集产生明显的抑制作用，但随着激动剂剂量的增加(凝血酶0.0 5U/m L或胶原，1μg/m L)抑制作用减弱。然而，高浓度的激动剂(胶原，30μg/mL3；胶原，4μg/mL5；胶原，2μg/mL6；和C反应蛋白[胶原相关肽]，1μg/mL4)是由相反的研究(包括慢性肉芽肿病患者研究5，6)检验的条件，证明一氧化氮在血小板激活中的作用不显著。然而，我们的研究1和Delaney et al2进行的研究都表明，低剂量激动剂(凝血酶，0.018 U/m l和0.025 U/m L；胶原，0.5μg/m l；或C p，0.5μg/m l)仍然能在血小板缺乏时激活显著的ROS生成，这表明存在其他不依赖于这些蛋白的ROS生成机制。NOX家族由7个催化同系物组成，其中7、8个除NOX1和NOX2外，还在血小板中检测到NOX4。2最近，在慢性肉芽肿性疾病患者的血小板中发现了NOX5的表达，尽管它可能不存在于啮齿动物的血小板中。8不幸的是，除了NOX1和NOX2外，目前还不清楚NOX亚型(如NOX4)在血小板激活中的单独作用以及它们之间可能的相互补偿机制。因为我们的研究确实包含了…
We welcome the letter commenting on our recent study, which raised a question on whether the defective reactive oxygen species (ROS) generation and the impaired platelet activation associated with VPS34 (vacuolar protein sorting 34) deficiency are bona fide function of NADPH oxidase (NOX), as suggested by our study. 1 Our conclusion that platelet VPS34 functions through NOX (likely the isoform NOX2) and the generation of ROS thereof is based on the observations as follows:(1) VPS34 deficiency impairs NOX2 assembly;(2) VPS34 deficiency reduces the production of ROS;(3) NOX inhibition by DPI (diphenyleneiodonium) abolishes the aggregation difference between VPS34−/− and wild-type platelets, and supplementation of H2O2 rescues the aggregation defect of VPS34−/− platelets. Similar observation is obtained in human platelets by using VPS34 inhibitor 3-ma. These data strongly suggest that NOX2 is the main target of VPS34 in platelets although we do agree that including mice deficient in NOX2 and other NOX subunits would further clarify this point. We understand that the question mainly stems from the controversy between the previous reports on the importance of NOX for platelet activation. Hence, although the recent study using NOX1 and NOX2 deficient mice has proven that these 2 isoforms are selectively important for ROS generation and platelet activation, 2 there are previous reports showing that (1) murine platelets lacking NOX subunits, such as NOX2 or p47Phox, display normal ROS generation and aggregation responses to thrombin and collagen3, 4; and (2) human platelets deficient of NOX2 (from chronic granulomatous disease) also exhibit normal aggregation responses. 5, 6 However, it is notable that the inhibitory effects of NOX1 and NOX2 knockout on ROS generation and platelet aggregation diminish at high concentrations of agonists. 2 Consistent with the findings that VPS34 functions through NOX, VPS34 deficiency also causes an obvious inhibition of ROS production and platelet aggregation with low dose agonists stimulation (thrombin, 0.025 U/mL or collagen, 0.5 μg/mL), but the inhibition diminishes on the raise of the agonist dosage (thrombin, 0.05 U/mL or collagen, 1 μg/mL). Yet high concentrations of agonists (collagen, 30 μg/mL3; collagen, 4 μg/mL5; collagen, 2 μg/mL6; and CRP [collagen-related peptide], 1 μg/mL4) is the condition tested by the opposing studies (including the chronic granulomatous disease patient study5, 6) to evidence the insignificant role of NOX in platelet activation. Thus, comparison of the data obtained by different previous reports does not rule out the importance of NOX in platelet activation but rather suggests that the reliance of platelet activation on the VPS34-NOX axis is stimulation intensity-dependent.Nevertheless, both our study1 and the study conducted by Delaney et al2 demonstrate that low dose agonists (thrombin, 0.018 U/mL and 0.025 U/mL; collagen, 0.5 μg/mL; or CRP, 0.5 μg/mL) still activate significant ROS generation in platelets deficiency of NOX1, NOX2, or VPS34, suggesting the existence of other mechanisms of ROS generation independent of these proteins. The NOX family consists of 7 catalytic homologues, 7, 8 in addition to NOX1 and NOX2, NOX4 is also detected in platelets. 2 More recently, NOX5 is found expressed in platelets from patients with chronic granulomatous disease9 although it may not present in rodent platelets. 8 Unfortunately, except for NOX1 and NOX2, neither the individual role of NOX isoforms (eg, NOX4) in platelet activation nor the possible mutual compensational mechanism among them is currently understood. Because our study did …