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

描述（由申请人提供）：血管性血液病因子（VWF）的血小板受体，糖蛋白Ib-IX复合物（GPIb-IX）在血小板粘附中起重要作用，特别是在高剪切速率条件下，如狭窄动脉。GPIb-IX不仅介导血小板对血管损伤部位的物理粘附，还启动信号转导，导致血小板整合素α - iib - β 3的配体结合功能被激活。病理条件下，GPIb- IX与循环超大型VWF多聚体结合可诱导微血栓形成和血栓性血小板减少性紫癜。虽然传统上认为GPIb-IX在结合VWF中具有组成性活性，但我们已经获得证据支持GPIb-IX的VWF结合功能受细胞内信号调节的假设。GPIb- IX由GPIb-alpha、GPIb-beta、GPIX和GPV几个亚基组成。gpib - α的细胞质结构域与将GPIb-IX连接到膜骨架的丝蛋白（肌动蛋白结合蛋白）结合。我们发现gpib - α的细胞质结构域与磷酸化丝氨酸依赖的信号分子14-3-3结合。缺失GPIbalpha c端14-3-3结合位点或用新型肽基抑制剂阻断14-3-3与野生型GPIb-IX的结合可显著降低GPIb-IX的VWF结合功能，并影响GPIb-IX与膜骨架的关联。因此，我们假设14-3-3在调节GPIb-IX的配体结合功能以及调节GPIb-IX与膜骨架的关联中起主要作用。这一假设的延伸是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，我们已经证明gpib - ix介导的整合素激活涉及一种新的信号机制，该机制需要cgmp依赖性蛋白激酶、p38丝裂原活化蛋白激酶、细胞外刺激反应激酶（ERK）途径。我们的研究表明GPIb-IX通过Src和磷酸肌肽3激酶与cGMP途径之间存在假设的联系。为了验证这些假设，我们提出以下具体目标：(1)研究GPIb-IX配体结合功能的调控机制及14-3-3的作用。(2)探讨14-3-3调控gpib - ix相关膜骨架的作用。(3)探讨14-3-3-GPIb-IX相互作用抑制剂的抗血栓作用。(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.