RGT, a synthetic peptide corresponding to the integrin (cid:1) 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin (cid:2) IIb (cid:1) 3 with Src kinase

Mutational analysis has established that the cytoplasmic tail of the integrin (cid:1) 3 subunit bindsc-Src(termedasSrcinthisstudy)and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin (cid:1) 3 C-terminal sequence dose-depen-dentlyinhibitedstableplateletadhesionand spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent sec-ondwaveofplateletaggregationinducedby adenosinediphosphate,ristocetin,orthrom-bin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, theRGTpeptideinduceddissociationofSrc from integrin (cid:1) 3 and dose-dependently in-hibitedthepurifiedrecombinant (cid:1) 3cytoplas-mic domain binding to Src-SH3. In addition, phosphorylation of the (cid:1) 3 cytoplasmic tyrosines, Y 747 and Y 759 , was inhibited by myr-RGT. These data indicate an important role for (cid:1) 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disrup-tion of the association of Src with (cid:1) 3 and selective blockade of integrin (cid:2) IIb (cid:1) 3 out-side-in signaling by myr-RGT suggest a potential new antithrombotic strategy. showed that the tyrosine phosphorylation of (cid:1) that inhibits Src-dependent (cid:1) 3 phosphorylation, by disrupting the interaction between (cid:1) 3 Src the effects of the Src- (cid:1) 3 dissociation, as the inhibitory effect on platelet aggregation.

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