Molecular Mechanism Of Platelet Activation Through 14-3-3ζ-c-Src-integrin β3 Complex

Platelets are anucleated cells generated from the nucleated precursor cells known as megakaryocytes in the bone marrow.Platelets play key roles in hemostasis,the basic function of which is rapidly binding to damaged blood vessels,aggregates to form thrombi,and preventing excessive bleeding.However,activated platelets also aggregate at the site of atherosclerotic plaque rupture or endothelial cell erosion,stimulating thrombus formation and promoting atherothrombotic disease.Antiplatelet aggregation drugs can effectively inhibit the abnormal activation of platelets and prevent thrombosis.However,most of the already marketed antiplatelet drugs are accompanied by severe bleeding and other side effects.The research on antiplatelet drugs without significant bleeding risk has always been a popular issue in hematological research.Integrin αⅡbβ3 mediates bi-directional signaling and plays crucial roles in platelet activation,hemostasis,and thrombosis.Several adaptor molecules such as 14-3-3ζ and c-Src bind to cytoplasmic tails of integrins β3 and facilitates bi-directional signaling.We have shown how several adaptor molecules bind to cytoplasmic tails of integrinsβ3 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 antithrombotic drugs.14-3-3ζ-c-Src interaction is mediated by thePIRLGLALNFSVFYYE-fragment(PE16)on the 14-3-3ζ and SH2-domain on c-Src,whereas the 14-3-3ζ-integrin-β3 interaction is mediated by theESKVFYLKMKGDYYRYL-fragment(EL17)on the 14-3-3ζ and-KEATSTFfragment(KF7)on the β3-integrin cytoplasmic tail.The EL17-motif inhibitor,or KF7 peptide,interferes with the formation of the 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 characterized a previously unidentified 14-3-3ζ-c-Src-integrin-β3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.We screened the laboratory compound library according to the previous screening methods and found that a variety of isoflavone(3’,4’,7’-trihydroxyisoflavone,genistein and daidzein)compounds can significantly bind to 14-3-3ζ protein.Isoflavones can inhibit agonist-induced platelet aggregation.The 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 a 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 an 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 Gplba deficient mice.The interaction of these soy isoflavones 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 an inhibitory effect on thrombosis formation in the perfusion chamber,especially under high shear force(1800 s-1).These soy isoflavones interacted with 14-3-3ζ and inhibited both GP1B-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 GP1B-Ⅸ and αⅡbβ3-mediated platelet activation is involved in soy isoflavone-mediated platelet inhibition.In this paper,we show for the first time that 14-3-3ζ is a new adaptor between cSrc and integrin β3,which formed the 14-3-3ζ-c-Src-integrin-β3 complex during platelet activation,and prove that 14-3-3ζ can facilitate the interaction between c-Src and integrin β3 during platelet activation.They interferes with the formation of the 143-3ζ-c-Src-integrin-β3 complex and selectively inhibits β3 outside-in signaling without affecting the integrin-ligand interaction,which suppresses thrombosis without causing the significant bleeding.Based on the new theory,we filtered new antithrombotic drugs,which have a great effect on inhibiting thrombosis without significant bleeding.The new theory provides a potential idea for the development of antithrombotic therapy.