Structure and function of platelet glycoprotein Ib-IX-V complex

项目总结 这项建议是为了继续我们对血小板必需的糖蛋白(GP)Ib-IX-V复合体的研究 生理学。通过与von的相互作用，最初被确定为流动剪应力的血小板传感器 Willebrand因子(VWF)、GPIB-IX-V在止血和血栓形成中起重要作用。它还被开发为 在炎症、血小板生成和清除中的其他作用。这种多亚单位受体的功能障碍 复合体可导致严重的出血素质，并导致许多心血管疾病。在.期间 在目前的资金期限内，我们首次确定了一个机械感应域 GPIBα亚基的膜旁区域。MSD在张力调节的拉力作用下展开 结合VWF或抗GPIBα抗体的A1结构域。此外，MSD的展开诱导了信号传递 通过GPIB-IX，导致加速的血小板清除和血小板减少。我们对MSD的研究有 开创了GPIB-IX-V机械传感和激活机制的新范式。首先，两者都不是 抗GPIB-α抗体的亲和力或结合表位是GPIB-IX激活的关键。相反，一个 结合抗体产生的张力是打开MSD和激活GPIB-IX所需的。 其次，抗体介导的血小板交联可能是可溶性配体产生的共同特征。 对GPIB-IX施加压力并将其激活。第三，血浆vWF或抗GPIBα抗体对GPIB-IX的激活 导致受体缺失和暴露在血小板表面的β-半乳糖残基，这具有 最近的研究表明，它可以调节血小板的清除。这些发现，以及我们初步的 在附着条件下的观察表明，存在迄今未确定的元素 调节MSD的机械动力学和功能。以继续我们对 GPIB-IX-V的结构和功能，我们建议在这里鉴定和表征激活和 在三个具体目标中用多学科方法调整MSD。目标1是定义 激活GPIB-IX所需的生理配体VWF的结构和机械元件。目标 2是确定和表征MSD中对其关键的残基和新的调节元件 动力和功能。目的3探讨GPV对GPIB-IX活性的机械调节。 他们的互动。拟议研究的完成将极大地推进基本的机械传感 以及GPIB-IX-V复合体和血小板的机械调节机制。它还将建立一个坚实的 了解GPIB-IX-V在清除血小板中的不同功能作用的结构基础 止血和血栓形成。

PROJECT SUMMARY This proposal is to continue our studies of glycoprotein (GP)Ib-IX-V complex that is essential to platelet physiology. Initially identified as a platelet sensor for flow shear stress through its interaction with von Willebrand factor (VWF), GPIb-IX-V plays a critical role in hemostasis and thrombosis. It is also tapped for additional roles in inflammation, platelet genesis and clearance. Malfunction of this multi-subunit receptor complex can lead to severe bleeding diathesis and contribute to many cardiovascular diseases. During the current funding period we have identified, for the first time, a mechanosensory domain (MSD) in the juxtamembrane region of the GPIbα subunit. The MSD unfolds upon tension-mediated pulling on an engaged A1 domain of VWF or anti-GPIbα antibody. Furthermore, unfolding of the MSD induces signaling through GPIb-IX, resulting in accelerated platelet clearance and thrombocytopenia. Our study on MSD has initiated a new paradigm for the mechanosensing and activation mechanism of GPIb-IX-V. First, neither the affinity nor the binding epitope of an anti-GPIbα antibody is the key to GPIb-IX activation. Instead, a tensile force generated by the bound antibody is what is needed to unfold the MSD and activate GPIb-IX. Second, antibody-mediated platelet crosslinking may be a common feature for soluble ligands to generate tension on GPIb-IX and to activate it. Third, activation of GPIb-IX by plasma VWF or anti-GPIbα antibodies results in receptor desialylation and exposure of β-galactose residues on the platelet surface, which have been shown in recent studies to mediate platelet clearance. These findings, along with our preliminary observations under adherent conditions, suggest the existence of hitherto unidentified elements modulating the mechanical dynamics and function of the MSD. To continue our investigation of the structure-function of GPIb-IX-V, we propose here to identify and characterize the elements activating and modulating the MSD with multidisciplinary approaches in three Specific Aims. Aim 1 is to define the structural and mechanical elements of physiological ligand VWF that are required to activate GPIb-IX. Aim 2 is to identify and characterize residues and novel modulatory elements in the MSD that are critical to its dynamics and function. Aim 3 is to explore the mechanical regulation of GPIb-IX activity by GPV through their interaction. Completion of the proposed study will critically advance the fundamental mechanosensing and mechanoregulatory mechanism of the GPIb-IX-V complex and the platelet. It will also establish a solid structural foundation for understanding the diverse functional roles of GPIb-IX-V in platelet clearance, hemostasis, and thrombosis.