Evaluation of targeting c-Src by the RGT-containing peptide as a novel antithrombotic strategy

Journal of Hematology & Oncology

Xiaodong Xi³  Zhangbiao Long¹  Ping Liu¹  Wenda Xi²  Xiaofeng Shi¹  Jiansong Huang¹ 

[1] 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;Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Second Ruijin Road, Shanghai 200025, China;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

关键词: c-Src;    Integrin αIIbβ3;    Drug design;    Blood platelets;    Antithrombotic agent;   

Others  :  1217379
DOI  :  10.1186/s13045-015-0159-8

 received in 2015-03-30, accepted in 2015-05-22,  发布年份 2015

【 摘 要 】

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 Y⁴¹⁶ and Y⁵²⁷, 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.

【 授权许可】

   
2015 Huang et al.

【 图 表 】

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