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.
ABSTRACT 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.
Article: A palmitylated peptide derived from the glycoprotein Ib beta cytoplasmic tail inhibits platelet activation.[show abstract] [hide abstract]
ABSTRACT: The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIb alpha, GPIb beta, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIb beta subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell-permeable palmitylated peptide (Pal-RRLRARARARA) and investigated its effect on platelet function. Pal-RRLRARARARA completely inhibited low dose thrombin- and ristocetin-induced aggregation in washed platelets but only partially inhibited collagen- and U46619-induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal-RRLRARARARA compared with collagen. Activation of the integrin alpha IIb beta 3 in response to thrombin was significantly reduced when platelets were preincubated with Pal-RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal-RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal-RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIb beta in platelet activation.Journal of Thrombosis and Haemostasis 01/2004; 1(12):2643-52. · 5.73 Impact Factor
Article: Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy.[show abstract] [hide abstract]
ABSTRACT: Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outside-in" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the beta3 integrin, thereby amplifying outside-in signaling via alphaIIbbeta3. Blocking the Gas6-R-alphaIIbbeta3 integrin cross-talk might be a novel approach to the reduction of thrombosis.Journal of Clinical Investigation 03/2005; 115(2):237-46. · 15.39 Impact Factor
Article: A critical role for 14-3-3zeta protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications.[show abstract] [hide abstract]
ABSTRACT: The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb alpha and beta subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3zeta. Here we show that a novel membrane-permeable inhibitor of 14-3-3zeta-GPIbalpha interaction, MPalphaC, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPalphaC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbbeta dephosphorylation is diminished by mutagenic disruption of the 14-3-3zeta binding site in the C-terminal domain of GPIbalpha, mimicking MPalphaC-induced inhibition, indicating that the inhibitory effect of MPalphaC is likely to be caused by disruption of 14-3-3zeta binding to GPIbalpha. These data suggest a novel 14-3-3zeta-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis.Blood 10/2005; 106(6):1975-81. · 9.90 Impact Factor