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

描述(申请人提供)：von Willebrand因子的血小板受体(VWF)，糖蛋白Ib-IX复合体(GPIB-IX)在血小板粘连中发挥重要作用，特别是在高剪切率条件下，如狭窄的动脉。GPIB-IX不仅介导了血小板与血管损伤部位的物理黏附，还启动了信号转导，从而激活了血小板整合素α-IIb-β3的配体结合功能。在病理条件下，GPIB-IX与循环中的超大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在与VWF的结合中具有结构性活性，但我们已经获得了证据，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，即GPIB-α、GPIB-β、Gpix和GPV。GPIB-α的细胞质结构域与丝蛋白(肌动蛋白结合蛋白)结合，后者将GPIB-IX与膜骨架连接起来。我们发现，GPIB-α的细胞质结构域与依赖于磷酸丝氨酸的信号分子14-3-3结合。GPIbalpha C-末端结构域14-3-3结合位点的缺失或用一种新型多肽抑制剂阻断14-3-3与野生型GPIb-IX的结合显著降低了GPIb-IX的VWF结合功能，并影响了GPIb-IX与膜骨架的结合。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与膜骨架的结合中起主要作用。这一假设的延伸是14-3-3与GPIB-IX相互作用的抑制剂可以抑制血小板黏附和血栓形成，因此在治疗或预防血栓性疾病方面是有用的。此外，我们还证明了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依赖的蛋白激酶、p38丝裂原活化蛋白激酶、细胞外刺激反应激酶(ERK)通路。我们的研究表明，GPIB-IX和cGMP途径之间可能通过Src和磷脂酰肌醇3激酶联系在一起。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)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转导途径。

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.