title: Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.
abstract: 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.
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Su Bin
authors: SB
authors: Wang
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Ji Yong
authors: JY
authors: Jang
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Yun Hee
authors: YH
authors: Chae
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Ji Hyun
authors: JH
authors: Min
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Jin Young
authors: JY
authors: Baek
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Myunghee
authors: M
authors: Kim
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Yunjeong
authors: Y
authors: Park
authors: Lab of Cell Differentiation Research, College of Oriental Medicine, Gachon University, Seongnam 461-701, Republic of Korea.
authors: Gwi Seo
authors: GS
authors: Hwang
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea.
authors: Jae-Sang
authors: JS
authors: Ryu
authors: Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea. Electronic address: changts@ewha.ac.kr.
authors: Tong-Shin
authors: TS
authors: Chang
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D002712:Chlorides / Q000633:toxicity; D003094:Collagen / Q000494:pharmacology*; D005290:Ferric Compounds / Q000633:toxicity; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D044949:Kaempferols / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010084:Oxidation-Reduction; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000072377:Syk Kinase; D013927:Thrombosis / Q000139:chemically induced / Q000188:drug therapy / Q000378:metabolism
chemical_list: D002712:Chlorides; D005290:Ferric Compounds; D047908:Intracellular Signaling Peptides and Proteins; D044949:Kaempferols; D051567:Proto-Oncogene Proteins c-vav; D017382:Reactive Oxygen Species; C496618:VAV1 protein, human; C006552:kaempferol; D003094:Collagen; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D051966:Phospholipase C gamma; C024555:ferric chloride
keywords: Anti-platelet; Free radicals; Kaempferol; NADPH oxidase; Oxidative inactivation; Reactive oxygen species; Redox regulation; SH2 domain-containing protein tyrosine phosphatase-2; p47(phox)