RGT, a Sythetic Peptide, Selectively Inhibits Integrin αIIbβ3 Outside-In Signaling in Human Platelets through Disassociating SRC Kinase from β3 Subunit.
Authors: Su Xiaoyu , Mi Jianqing , Lu Yuanjing , Liu Hongchen , Chen Saijuan , Xi Xiaodong 
Published: October 2019

Integrin αIIbβ3 plays pivotal roles in platelet activation by interacting with ligands and by mediating bidirectional signal transduction as well. The molecular mechanisms underlying the signal transduction pathways by which integrin αIIbβ3 regulates platelet activation have not yet been completely understood. Previous truncational analyses with CHO cell model have established that the C-terminal RGT sequence of integrin β3 subunit is critical to outside-in signaling but not inside-out signaling. To examine whether this mechanism functions in intact human platelets, a synthetic cell-permeable peptide corresponding to C-terminal RGT sequence of integrin β3 was employed in this work in an attempt to compete with native integrin β3 subunit for downstream signaling molecules. Myristoylated RGT peptide (myr-RGT) dose-dependently inhibited adhesion and spreading of normal human platelets on immobilized fibrinogen and prevented fibrin clot retraction. Also, myr-RGT selectively inhibited the second wave of platelet aggregation induced by ADP, ristocetin or thrombin with unaffected first wave of aggregation, while it had no inhibitory effect on ADP-induced soluble fibrinogen binding. These results suggest that the integrin outside-in signaling pathway is impaired by the treatment of RGT peptide and that the inside-out pathway remained undisrupted. Thus we showed for the first time in intact platelets that the outside-in signaling could be selectively regulated by synthetic peptide with sequence corresponding to the last three amino acids of integrin β3 cytoplasmic tail. To further define the molecular basis of the roles of RGT peptide in signal transduction we examined the effect of RGT peptide on tyrosine phosphorylation of integrin β3 cytoplasmic domain. We found that when platelets were stimulated by thrombin the phosphorylation of both cytoplasmic tyrosine residues (Y747 and Y759) of integrin β3 was substantially inhibited by the presence of myr-RGT peptide. This inhibition could be attributed to the findings in immunoprecipitation experiments whereby RGT peptide attenuated the interaction of integrin β3 with Src kinase. On the contrary, the RGT peptide was unable to interfere talin binding to β3 subunit. These results support the conclusion that, in intact human platelets, the RGT peptide selectively blocked outside-in signaling by disrupting the constitutive interaction of Src kinase with integrin β3 and hence causing a decreased phosphorylation level of tyrosine residues at integrin β3 cytoplasmic domain. Thus, in platelets, the interaction of the RGT sequence in integrin β3 cytoplasmic tail with Src kinase is necessary and sufficient to transduce outside-in signals while the disassociation of Src from integrin β3 has no regulatory effect on talin binding to β3 cytoplasmic domain. The application of RGT peptide and derivatives in the practice of platelet studies enables us to regulate outside-in signaling without affecting inside-out pathway and to speculate the better understanding of the molecular basis of mechanisms for integrin signal transdution. These results also underline the potential use of RGT peptide as a new antithrombotic strategy.