title: Evaluation of targeting c-Src by the RGT-containing peptide as a novel antithrombotic strategy.
abstract: BACKGROUND
  Interaction of integrin β3 with c-Src plays critical roles in cellular signaling which is heavily implicated in platelet adhesion and aggregation, as well as in tumor cell proliferation and metastasis or in osteoclastic bone resorption. Selectively blocking integrin αIIbβ3 outside-in signaling in platelets has been a focus of attention because of its effective antithrombotic potential together with a sufficient hemostatic capacity. The myristoylated RGT peptide has been shown to achieve this blockade by targeting the association of c-Src with the integrin β3 tail, but the lack of key information regarding the mechanisms of action prevents this strategy from being further developed into practical antithrombotics. Therefore, in-depth knowledge of the precise mechanisms for RGT peptide in regulating platelet function is needed to establish the basis for a potential antithrombotic therapy by targeting c-Src.
  
  
  METHODS
  The reduction-sensitive peptides were applied to rule out the membrane anchorage after cytoplasmic delivery. The c-Src activity was assayed at living cell or at protein levels to assess the direct effect of RGT targeting on c-Src. Thrombus formation under flow in the presence of cytoplasmic RGT peptide was observed by perfusing whole blood through the collagen-coated micro-chamber.
  
  
  RESULTS
  The RGT peptide did not depend on the membrane anchorage to inhibit outside-in signaling in platelets. The myr-AC ~ CRGT peptide readily blocked agonist-induced c-Src activation by disrupting the Src/β3 association and inhibited the RhoA activation and collagen-induced platelet aggregation in addition to the typical outside-in signaling events. The myr-AC ~ CRGT had no direct effect on the kinase activity of c-Src in living cells as evidenced by its inability to dissociate Csk from c-Src or to alter the phosphorylation level of c-Src Y(416) and Y(527), consistent results were also from in vitro kinase assays. Under flow conditions, the myr-AC~ CRGT peptide caused an inhibition of platelet thrombus formation predominantly at high shear rates.
  
  
  CONCLUSIONS
  These findings provide novel insights into the molecular mechanisms by which the RGT peptide regulates integrin signaling and platelet function and reinforce the potential of the RGT peptide-induced disruption of Src/β3 association as a druggable target that would finally enable in vivo and clinical studies using the structure-based small molecular mimetics.
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. hjiansong1234@126.com.
authors.forename: Jiansong
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. shixiaofeng1977@163.com.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Shi
authors.affiliation: Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. xwendax@live.com.
authors.forename: Wenda
authors.initials: W
authors.lastname: Xi
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. ping8705@163.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. longzhangbiao@163.com.
authors.forename: Zhangbiao
authors.initials: Z
authors.lastname: Long
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. xixiaodong@shsmu.edu.cn.|Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai, 200025, China. xixiaodong@shsmu.edu.cn.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xi
mesh_terms: D001792:Blood Platelets / Q000378:metabolism*; D015195:Drug Design; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D039661:Integrin beta3; D016392:Proto-Oncogene Proteins pp60(c-src)