RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase.
Mutational analysis has established that the cytoplasmic tail of the integrin beta 3 subunit binds c-Src (termed as Src in this study) 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 beta 3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. 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, the RGT peptide induced dissociation of Src from integrin beta 3 and dose-dependently inhibited the purified recombinant beta 3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the beta 3 cytoplasmic tyrosines, Y(747) and Y(759), was inhibited by myr-RGT. These data indicate an important role for beta 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with beta 3 and selective blockade of integrin alpha IIb beta 3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.
Su
Xiaoyu
X
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Mi
Jianqing
J
Yan
Jinsong
J
Flevaris
Panagiotis
P
Lu
Yuanjing
Y
Liu
Hongchen
H
Ruan
Zheng
Z
Wang
Xuefeng
X
Kieffer
Nelly
N
Chen
Saijuan
S
Du
Xiaoping
X
Xi
Xiaodong
X
D003018:Clot Retraction / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D039661:Integrin beta3; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D019061:src-Family Kinases / Q000378:metabolism*
D039661:Integrin beta3; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019061:src-Family Kinases