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

项目摘要 本实验旨在继续研究血小板膜糖蛋白（GP）Ib-IX-V复合物 physiology.最初确定为血小板传感器的流动剪切应力通过其相互作用与冯 Willebrand因子（VWF），GPIb-IX-V在止血和血栓形成中起关键作用。它也被挖掘为 在炎症、血小板生成和清除中的额外作用。这种多亚基受体的功能障碍 复杂可导致严重的出血素质，并有助于许多心血管疾病。期间 在当前的资助期内，我们首次确定了一个机械感觉域（MSD）， GPIbα亚基的质膜区。MSD在张力介导的牵拉下展开， 接合VWF或抗GPIb α抗体的A1结构域。此外，MSD的解折叠诱导信号传导， 通过GPIb-IX，导致血小板清除加速和血小板减少症。我们对MSD的研究 开创了GPIb-IX-V的机械感知和激活机制的新范式。 抗GPIb α抗体的亲和力和结合表位是GPIb-IX活化的关键。而是 结合抗体产生的张力是展开MSD和激活GPIb-IX所需的。 第二，抗体介导的血小板交联可能是可溶性配体产生血小板聚集的共同特征。 第三，血浆VWF或抗GPIb α抗体对GPIb-IX的激活 导致受体去唾液酸化和血小板表面β-半乳糖残基暴露， 在最近的研究中显示介导血小板清除。这些发现，沿着我们的初步 在粘附条件下的观察表明，存在迄今尚未确定的元素 调节MSD的机械动力学和功能。继续调查 GPIb-IX-V的结构-功能，我们建议在这里识别和表征激活和 在三个具体目标中采用多学科方法调节MSD。目标1：定义 激活GPIb-IX所需的生理配体VWF的结构和机械元件。目的 2是鉴定和表征MSD中的残基和新的调节元件，这些残基和新的调节元件对MSD的功能至关重要。 动态和功能。目的3：探讨GPV对GPIb-IX活性的调控机制。 他们的互动。完成拟议的研究将大大推进基本的机械传感 以及GPIb-IX-V复合物和血小板的机械调节机制。它还将建立一个坚实的 理解GPIb-IX-V在血小板清除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.