Several adaptor molecules bind to cytoplasmic tails of β-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe anti-thrombotics. Here we show for the first time that 14-3-3ζ-c-Src-integrin-β3 complex is formed during platelet activation. 14-3-3ζ-c-Src interaction is mediated by -pirlglalnfsvfyye- fragment (PE16) on 14-3-3ζ and SH2-domain on c-Src, while 14-3-3ζ-integrin β3 interaction is mediated by -eskvfylkmkgdyyrYL- fragment (EL17) on 14-3-3ζ and -keatstf- fragment (KF7) on β3 integrin cytoplasmic tail. EL17-motif inhibitor or KF7 peptide interferes with the formation of 14-3-3ζ-c-Src-integrin-β3 complex and selectively inhibits β3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterizes a previously unidentified 14-3-3ζ-c-Src-integrin-β3 complex in platelets and provides a novel strategy for the development of safe and effective anti-thrombotic therapies.

It was recently discovered that Ssm Spooky Toxin (SsTx) with 53 residues serves as a key killer factor in red-headed centipede’s venom arsenal, due to its potent blockage of the widely expressed KCNQ channels to simultaneously and efficiently disrupt cardiovascular, respiratory, muscular, and nervous systems, suggesting that SsTx is a basic compound for centipedes’ defense and predation. Here, we show that SsTx also inhibits KV1.3 channel, which would amplify the broad-spectrum disruptive effect of blocking KV7 channels. Interestingly, residue R12 in SsTx extends into the selectivity filter to block KV7.4, however, residue K11 in SsTx replaces this ploy when toxin binds on KV1.3. Both SsTx and its mutant SsTx_R12A inhibit cytokines production in T cells without affecting the level of KV1.3 expression. The results further suggest that SsTx is a key molecule for defense and predation in the centipedes’ venoms and it evolves efficient strategy to disturb multiple physiological targets.

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s−1) and high (1800 s−1) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbβ3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s−1). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbβ3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbβ3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.