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 p47phox, a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47phox 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.
|Number of pages||13|
|Journal||Free Radical Biology and Medicine|
|State||Published - 1 Jun 2015|
Bibliographical noteFunding Information:
This study was supported by a National Research Foundation grant ( M10642040002-07N4204-00210 ) funded by the Korean Ministry of Science, ICT , and Future Planning, a grant from the Health Technology R&D Project (Grant HI13C1046 ) funded by the Korean Ministry of Health & Welfare, and the Brain Korea 21 Plus Program (to J.Y. Jang and S.B. Wang) funded by the Korean Ministry of Education.
© 2015 Elsevier Inc.
- Free radicals
- NADPH oxidase
- Oxidative inactivation
- Reactive oxygen species
- Redox regulation
- SH2 domain-containing protein tyrosine