Abstract
BackgroundInteraction 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.MethodsThe 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.ResultsThe 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0159-8) contains supplementary material, which is available to authorized users.
Highlights
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
By using reduction-sensitive peptides, the present study aimed to address whether the RGT peptide depends on membrane anchorage to influence Srcregulated signaling and whether it alters the kinase activity of c-Src
A thorough reduction of myr-AC ~ CRGT peptide is achieved in platelet cytoplasm It has been established that myristoylated RGT peptide selectively inhibited integrin αIIbβ3-mediated outside-in signaling [15]
Summary
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. 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. Arterial thrombosis developing on the atherosclerotic lesions causes heart attack and stroke that are currently the most common cause of death. Ligand binding to integrin αIIbβ triggers outside-in signaling that promotes platelet cytoskeletal reorganization leading to the firm association of platelets with the vessel wall or with each other. Talin and kindlin bind to the β3 NxxY motifs and play central roles in inside-out signaling [4], and the association of c-Src with the β3 tail, the RGT residues in particular [5, 6], is obligatory for outside-in signaling [5]. The conventional antagonists prevent ligand binding to αIIbβ receptor and inhibit the platelet functions regulated by both inside-out and outside-in signals
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