Platelet activation signaling via GPIb-IX and 14-3-3

描述（由申请方提供）：血管性血友病因子（VWF）的血小板受体，即糖蛋白Ib-IX复合物（GPIb-IX）在血小板粘附中起重要作用，特别是在高剪切率条件下，如狭窄动脉中。GPIb-IX不仅介导血小板对血管损伤部位的物理粘附，而且还启动信号转导，导致血小板整联蛋白α-IIb-β 3的配体结合功能的活化。在病理条件下，GPIb-IX与循环中的超大VWF多聚体的结合诱导微血栓形成和血栓性血小板减少性紫癜。虽然GPIb-IX传统上被认为是组成型活性结合VWF，我们已经获得的证据支持的假设，GPIb-IX的VWF结合功能的细胞内信号调节。GPIb-IX由几个亚基GPIb-alpha、GPIb-beta GPIX和GPV组成。GPIb-α的胞质结构域结合到将GPIb-IX连接到膜骨架的细丝蛋白（肌动蛋白结合蛋白）。我们发现GPIb-α的胞质结构域与磷酸丝氨酸依赖性信号分子14-3-3结合。在GPIb α的C-末端结构域中缺失14-3-3结合位点或用新的基于肽的抑制剂阻断14 - 3-3与野生型GPIb-IX的结合显著降低GPIb-IX的VWF结合功能，并且影响GPIb-IX与膜骨架之间的缔合。因此，我们推测14-3-3在调节GPIb-IX的配体结合功能以及调节GPIb-IX与膜骨架之间的结合中起主要作用。该假设的延伸是14-3-3与GPIb-IX相互作用的抑制剂抑制血小板粘附和血栓形成，因此可用于治疗或预防血栓性疾病。此外，我们已经表明，GPIb-IX介导的整合素激活涉及一种新的信号传导机制，需要cGMP依赖性蛋白激酶，p38丝裂原活化蛋白激酶，细胞外刺激应答激酶（ERK）途径。我们的研究表明GPIb-IX和cGMP途径之间通过Src和磷酸肌醇3激酶的假设联系。为了研究这些假说，我们提出了以下具体目标：（1）研究GPIb-IX的配体结合功能的调节机制和14-3-3的作用。(2)研究14-3-3在调节GPIb-IX相关膜骨架中的作用。(3)研究14-3-3-GPIb-IX相互作用抑制剂的抗血栓形成作用。(4)研究GPIb-IX介导血小板活化的信号通路。

DESCRIPTION (provided by applicant): 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.