Wang Su Bin, Jang Ji Yong, Chae Yun Hee, Min Ji Hyun, Baek Jin Young, Kim Myunghee, Park Yunjeong, Hwang Gwi Seo, Ryu Jae-Sang, Chang Tong-Shin
Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
Lab of Cell Differentiation Research, College of Oriental Medicine, Gachon University, Seongnam 461-701, Republic of Korea.
Free Radic Biol Med. 2015 Jun;83:41-53. doi: 10.1016/j.freeradbiomed.2015.01.018. Epub 2015 Jan 31.
Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.
胶原蛋白刺激产生的活性氧（ROS）作为第二信使，可引发各种血小板激活事件。在产生活性氧的酶中，NADPH氧化酶（NOX）在血小板激活中起重要作用。因此，NOX已被认为是抗血小板药物开发的新靶点。尽管山奈酚已被鉴定为一种NOX抑制剂，但山奈酚对血小板激活的影响及其潜在机制从未被研究过。在此，我们研究了山奈酚对胶原蛋白刺激的血小板中NOX激活、ROS依赖的信号通路和功能反应的影响。山奈酚以浓度依赖的方式显著抑制了胶原蛋白刺激产生的超氧阴离子生成。更重要的是，山奈酚直接结合NOX的主要调节亚基p47(phox)，并显著抑制胶原蛋白诱导的p47(phox)磷酸化和NOX激活。与NOX的抑制作用一致，山奈酚有效保护了含SH2结构域的蛋白酪氨酸磷酸酶-2（SHP-2）的ROS依赖性失活。随后，胶原蛋白受体信号通路关键成分（Syk、Vav1、Btk和PLCγ2）的特异性酪氨酸磷酸化被山奈酚抑制。山奈酚还减弱了下游反应，包括胞质钙升高、P-选择素表面暴露和整合素-αIIbβ3激活。最终，山奈酚在体外抑制了胶原蛋白诱导的血小板聚集和黏附，并延长了小鼠颈动脉的体内血栓形成反应。这项研究表明，山奈酚通过抑制NOX衍生的ROS产生以及随后SHP-2的氧化失活来损害胶原蛋白诱导的血小板激活。这种作用表明山奈酚在预防和治疗血栓血管疾病方面具有治疗潜力。
