ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways
ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways
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DOI：
10.1111/j.1538-7836.2012.04734.x
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发表时间：
2012-06-01
影响因子：
10.4
通讯作者：
Andrews, R. K.
中科院分区：
文献类型：
--
作者：
Arthur, J. F.;Qiao, J.;Andrews, R. K.
背景资料：血小板特异性胶原蛋白受体GPVI/FcR γ的连接导致快速、瞬时的二硫键依赖性同源二聚化，并产生由NADPH氧化酶产生的细胞内活性氧（ROS），通过TRAF 4与GPVI连接。目的：本研究的目的是评估GPVI/FcR γ或血小板上第二种含免疫受体酪氨酸激活基序（ITAM）的受体Fc γ RIIa参与后早期信号传导事件在ROS生成中的作用。方法和结果：使用基于H2 DCF-DA的流式细胞术测定来测量细胞内ROS，我们表明用GPVI激动剂、胶原相关肽（CRP）或惊厥素（Cvx）或Fc γ RIIa激动剂14 A2处理血小板增加了血小板内ROS;其他血小板激动剂如ADP和TRAP则没有。来自14名健康供体的富血小板血浆中的基础ROS显示出很小的个体间变异性。CRP、Cvx或14 A2在2 min内诱导ROS的初始爆发，随后在15-20 min后额外的ROS达到平台。阻断ITAM依赖性信号传导的Syk抑制剂BAY 613606对初始ROS爆发没有影响，但完全抑制第二阶段。结论：总之，这些结果首次表明GPVI或Fc γ RIIa下游的ROS生成由两个不同的阶段组成：初始Syk非依赖性爆发，随后是额外的Syk依赖性生成。
Background: Ligation of the platelet-specific collagen receptor, GPVI/FcR gamma, causes rapid, transient disulfide-dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. Objectives: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcR gamma or a second immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor on platelets, Fc gamma RIIa. Methods and Results: Using an H2DCF-DA-based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen-related peptide (CRP) or convulxin (Cvx), or the Fc gamma RIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet-rich plasma from 14 healthy donors displayed little inter-individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15-20 min. The Syk inhibitor BAY613606, which blocks ITAM-dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. Conclusions: Together, these results show for the first time that ROS generation downstream of GPVI or Fc gamma RIIa consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation.