Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Cardiology, Huashan Hospital, Fudan University, Shanghai, China.
J Thromb Haemost. 2017 Aug;15(8):1679-1688. doi: 10.1111/jth.13755. Epub 2017 Jul 14.
Essentials The mechanisms of extracellular signal-regulated kinase 5 (ERK5) in GPIb-IX signaling are unclear. Function of ERK5 in GPIb-IX was tested using aggregation, western blotting, and mass spectrometry. The protein interacting with ERK5 in human platelets was identified as casein kinase II (CKII). ERK5 associates with CKII to regulate the activation of the PI3K/Akt pathway in GPIb-IX signaling.
Background The platelet glycoprotein (GP) Ib-IX complex plays essential roles in thrombosis and hemostasis. The mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 have been shown to be important in the GPIb-IX-mediated signaling leading to integrin activation. However, the roles of the MAPK extracellular signal-regulated kinase 5 (ERK5) in GPIb-IX-mediated platelet activation are unknown. Objective To reveal the function and mechanisms of ERK5 in GPIb-IX-mediated platelet activation. Methods The functions of ERK5 in GPIb-IX-mediated human platelet activation were assessed using botrocetin/VWF, ristocetin/VWF, or platelet adhesion to von Willebrand factor (VWF) under shear stress in the presence of a specific inhibitor of ERK5. ERK5-associated proteins were pulled down from Chinese hamster ovary (CHO) cells transfected with HA-tagged-ERK5, identified by mass spectrometry, and confirmed in human platelets. Roles of ERK5-associated proteins in GPIb-IX-mediated platelet activation were clarified using specific inhibitors. Results The phosphorylation levels of ERK5 were significantly enhanced in human platelets stimulated with botrocetin/VWF or ristocetin/VWF. The ERK5 inhibitor XMD8-92 suppressed the second wave of human platelet aggregation induced by botrocetin/VWF or ristocetin/VWF and inhibited human platelet adhesion on immobilized VWF under shear stress. Casein kinase II (CKII) was identified as an ERK5-associated protein in human platelets. The CKII inhibitor TBB, similar to the ERK5 inhibitor XMD8-92, specifically restrained PTEN phosphorylation, therefore suppressing Akt phosphorylation in human platelets treated with botrocetin/VWF. Conclusion ERK5 associates with CKII to play essential roles in GPIb-IX-mediated platelet activation via the PTEN/PI3K/Akt pathway.
要点 细胞外信号调节激酶 5（ERK5）在 GPIb-IX 信号转导中的机制尚不清楚。使用聚集、western blot 和质谱法测试了 ERK5 在 GPIb-IX 中的功能。在人血小板中与 ERK5 相互作用的蛋白质被鉴定为酪蛋白激酶 II（CKII）。ERK5 与 CKII 结合，调节 GPIb-IX 信号转导中 PI3K/Akt 通路的激活。
背景 血小板糖蛋白（GP）Ib-IX 复合物在血栓形成和止血中起重要作用。已显示丝裂原激活的蛋白激酶（MAPKs）ERK1/2 和 p38 在 GPIb-IX 介导的信号转导中对整合素激活很重要。然而，ERK5 在 GPIb-IX 介导的血小板激活中的作用尚不清楚。目的 揭示 ERK5 在 GPIb-IX 介导的血小板激活中的功能和机制。
方法 使用特异性 ERK5 抑制剂，在存在玻连蛋白/VWF、瑞斯托菌素/VWF 或血小板在切应力下黏附于血管性血友病因子（VWF）的情况下，评估 ERK5 在 GPIb-IX 介导的人血小板激活中的功能。从转染 HA 标记的 ERK5 的中国仓鼠卵巢（CHO）细胞中下拉 ERK5 相关蛋白，通过质谱鉴定，并在人血小板中确认。使用特异性抑制剂阐明 ERK5 相关蛋白在 GPIb-IX 介导的血小板激活中的作用。
结果 玻连蛋白/VWF 或瑞斯托菌素/VWF 刺激的人血小板中 ERK5 的磷酸化水平显著增强。ERK5 抑制剂 XMD8-92 抑制了玻连蛋白/VWF 或瑞斯托菌素/VWF 诱导的人血小板第二波聚集，并抑制了剪切应力下固定化 VWF 上的人血小板黏附。酪蛋白激酶 II（CKII）被鉴定为人血小板中的 ERK5 相关蛋白。CKII 抑制剂 TBB 与 ERK5 抑制剂 XMD8-92 相似，特异性抑制 PTEN 磷酸化，从而抑制玻连蛋白/VWF 处理的人血小板中 Akt 的磷酸化。
结论 ERK5 与 CKII 结合，通过 PTEN/PI3K/Akt 通路在 GPIb-IX 介导的血小板激活中发挥重要作用。
