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

通过 GPIb-IX 和 14-3-3 的血小板激活信号传导

• 批准号: 6988819
• 负责人: Xiaoping Du
• 金额: $ 34.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6988819
• 关键词: CHO cells; biological signal transduction; blood coagulation disorders; cell adhesion; clinical research; glycoproteins; human tissue; integrins; intermolecular interaction; laboratory mouse; laboratory rabbit; membrane proteins; phosphatidylinositol 3 kinase; phosphoproteins; platelet activation; protein binding; protein kinase A; protein kinase C; protein protein interaction; thrombosis; von Willebrand' s disease

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.

描述（由申请人提供）：血管性血友病因子（VWF）的血小板受体，糖蛋白Ib-IX复合物（GPIb-IX）在血小板粘附中发挥重要作用，特别是在高剪切率条件下，例如在狭窄动脉中。 GPIb-IX 不仅介导血小板与血管损伤部位的物理粘附，而且还启动信号转导，从而激活血小板整合素 α-IIb-β3 的配体结合功能。在病理条件下，GPIb-IX 与循环超大 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。尽管传统上认为 GPIb-IX 在结合 VWF 方面具有组成型活性，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 由几个亚基组成：GPIb-α、GPIb-β GPIX 和 GPV。 GPIb-α 的胞质结构域与细丝蛋白（肌动蛋白结合蛋白）结合，将 GPIb-IX 与膜骨架连接起来。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。删除 GPIbalpha C 端结构域中的 14-3-3 结合位点或用新型肽抑制剂阻断 14-3-3 与野生型 GPIb-IX 的结合，可显着降低 GPIb-IX 的 VWF 结合功能，并影响 GPIb-IX 与膜骨架之间的关联。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与膜骨架之间的结合中起主要作用。该假说的延伸是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，我们还发现，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 丝裂原激活蛋白激酶、细胞外刺激反应激酶 (ERK) 途径。我们的研究表明 GPIb-IX 和 cGMP 途径之间通过 Src 和磷酸肌醇 3 激酶假设存在联系。为了研究这些假设，我们提出以下具体目标：（1）研究调节GPIb-IX配体结合功能的机制和14-3-3的作用。 (2)探讨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作用。 (4)研究GPIb-IX介导的血小板活化的信号通路。