A critical role for 14-3-3ζ protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications

Abstract

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb ␣ and ␤ subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3. Here we show that a novel membrane-permeable inhibitor of 14-3-3-GPIb␣ interaction, MP␣C, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MP␣C also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIb␤ dephosphorylation is diminished by mutagenic disruption of the 14-3-3 binding site in the C-terminal domain of GPIb␣, mimicking MP␣C-induced inhibition, indicating that the inhibitory effect of MP␣C is likely to be caused by disruption of 14-3-3 binding to GPIb␣. These data suggest a novel 14-3-3-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. ( IntroductionPlatelet adhesion plays an important role in hemostasis and thrombosis. Under high shear rate flow conditions such as in arteries and capillaries, platelet adhesion to the subendothelium is dependent on interaction between subendothelial-bound von Willebrand factor (VWF) and its receptor, the platelet glycoprotein Ib-IX-V complex (GPIb-IX-V). 1 VWF-GPIb-IX-V interaction also mediates the formation of platelet microaggregates in patients with abnormally large VWF multimers, 2 resulting in thrombotic thrombocytopenic purpura (TTP). Interaction of VWF with GPIb-IX-V triggers intracellular signaling events such as elevation of intracellular calcium 3 and cyclic guanosine monophosphate (cGMP) levels, 4 activation of phosphoinositide 3-kinase, 5 and activation of multiple protein kinase pathways. 4,[6][7][8] These events lead to the activation of the ligand binding function of the integrin ␣ IIb ␤ 3 , 9-11 which mediates platelet spreading and aggregation.GPIb-IX-V consists of 4 different transmembrane subunits. GPIb, which consists of disulfide-linked GPIb␣ and GPIb␤ subunits, forms a 1:1 complex with GPIX. The GPIb-IX complex (GPIb-IX) is sufficient for ligand binding and signaling function. 11,12 GPIb-IX forms a 2:1 complex with GPV. 12 The N-terminal region of GPIb␣ contains binding sites for VWF and thrombin. Binding of VWF to GPIb-IX is tightly controlled, and normally occurs only at sites of vascular injury or under pathologically high shear stress. Shear stress may induce changes in both VWF and platelets. In vitro, ristocetin and botrocetin are used to induce VWF binding to GPIb-IX by mimicking the effects of the subendothelial matrix and shear stress on VWF and/or GPIb-IX. Increasing evidence indicates that VWF binding to GPIb-IX is regulated by intraplatelet signals such as cyclic adenosine monophosphate (cAMP) levels. [13][14][15] Elevation of intracellular cAMP activates the cAMP-dependent protein kin...

Platelet glycoprotein Ibα supports experimental lung metastasis

“…4). Thus, truncation of GP Ib␣ does impact thrombosis in this ferric-chloride model and presumably relates to alterations in a GPIb␣/14-3-3 signaling pathway, an idea proposed by others (24,25).…”

Transformation, genomic instability and senescence mediated by platelet/megakaryocyte glycoprotein Ibα

“…The cytoplasmic tail of GpIba interacts with proteins such as 14-3-3z and filamin, thus influencing cell signaling and actin cytoskeleton function, respectively (Asazuma et al, 1997;Kanaji et al, 2004;Dai et al, 2005;Ozaki et al, 2005). However, the transcriptional consequences of this are unclear.…”

Brief review: Coronary drug-eluting stents and anesthesia

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