The platelet receptor for von Willebrand factor (VWF), the glycoprotein Ib-IX complex (GPIb-IX) plays an important role in platelet adhesion, particularly under high shear rate conditions such as in stenotic arteries. GPIb-IX not only mediates the physical adherence of platelets to the site of vascular injury but also initiates signal transduction, leading to activation of the ligand binding function of the platelet integrin alpha-IIb-beta3. Under pathological conditions, binding of GPIb- IX to circulating ultra-large VWF multimers induces microthrombosis and thrombotic thrombocytopenic purpura. Although GPIb-IX has been traditionally believed to be constitutively active in binding VWF, we have obtained evidence supporting the hypothesis that VWF binding function of GPIb-IX is regulated by intracellular signals. GPIb- IX consists of several subunits, GPIb-alpha, GPIb-beta GPIX and GPV. The cytoplasmic domain of GPIb-alpha binds to filamin (actin-binding protein) that links GPIb-IX to the membrane skeleton. We have found that the cytoplasmic domain of GPIb-alpha binds to a phosphoserine-dependent signaling molecule, 14-3-3. Deletion of the 14-3-3 binding site in the C-terminal domain of GPIbalpha or blocking 14-3-3 binding to wild type GPIb-IX with a novel peptide-based inhibitor significantly reduced VWF binding function of GPIb-IX, and affects the association between GPIb-IX and the membrane skeleton. Thus, we hypothesize that 14-3-3 plays a major role in regulating the ligand binding function of GPIb-IX, and in regulating the association between GPIb-IX and the membrane skeleton. An extension to this hypothesis is that the inhibitor of the 14-3-3 interaction with GPIb-IX inhibits platelet adhesion and thrombosis, is thus useful in treating or preventing thrombotic diseases. Furthermore, we have shown that GPIb-IX-mediated integrin activation involves a novel signaling mechanism that requires the cGMP-dependent protein kinase, p38 mitogen-activated protein kinase, extracellular stimuli-responsive kinase (ERK) pathways. Our studies suggest a hypothetic link between GPIb-IX and cGMP pathway via Src and phosphoinositide 3 kinase. To investigate these hypotheses, we propose the following specific aims: (1) To study the mechanisms that regulates the ligand binding function of GPIb-IX and the role of 14-3-3. (2) To investigate the role of 14-3-3 in regulating the GPIb-IX-associated membrane skeleton. (3) To investigate anti-thrombotic effects of the inhibitors of 14-3-3-GPIb-IX interaction. (4) To study the signaling pathways of GPIb-IX-mediated platelet activation.
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