Mechanotransduction of platelet receptors GPIb and GPIIb-IIIa

项目概要/摘要 血小板可以说是止血和血栓形成中最重要的细胞参与者。许多治疗 策略靶向血小板分子以抑制它们在血栓形成中的功能。特别是两层膜的粘附 受体，糖蛋白（GP）Ib-IX和GPIIb-IIIa（整联蛋白IIb-3），协同介导血小板粘附， 高剪切流动下的血栓形成，因此是抑制狭窄动脉中血栓形成的理想靶点 以及微脉管系统中。此外，目前临床上用于治疗血栓形成的所有抗血小板药物都具有 不良出血副作用，因为止血所需的血小板栓形成也受到抑制。 在过去一段时间的资助下，我们和其他人证明了GPIbα和GPIIb β 3都是 能够接受机械提示的机械感受器。我们发现GPIb机械信号导致 整联蛋白由内而外信号传导，其从具有弯曲构象的非活性状态激活β IIb β 3， 具有低亲和力和短键寿命的封闭头片段（BC），配体具有 延伸的构象，但具有中等亲和力和中等键寿命的封闭头片段（EC 对于配体，这与可溶性激动剂刺激的血小板中常见的完全活性状态的β IIb β 3不同 其具有延伸的构象和开放的头部（EO），具有高亲和力和长的键合寿命， 配体。值得注意的是，β IIb β 3的中间状态介导了“生物力学血栓”的形成， 短暂和不稳定的血小板聚集体，类似于体内血栓形成的动态外层 在高剪切血流下，这与外层下面的血小板聚集体不同， 受释放的可溶性激动剂刺激，并且其中E1 IIb β 3被进一步激活至完全活性的EO状态。进一步 从中间状态到完全活性状态的活化也可以通过由外向内的机械活化来诱导。 信号传导，其也诱导血小板的进一步活化。这些数据突出了力诱导的作用 GPIb-IX和GPIIb-3的、通过GPIb-IX和GPIIb-3的以及GPIb-IX和GPIIb-3之间的信号传导-机械信号传导，以及它们在血栓中的独特重要性 阵我们的总体假设是GPIbα和GPIIb β 3机械信号的平衡调节， 它们与可溶性血小板激动剂诱导的生物化学信号的协同作用，有望控制 血栓形成，无止血不稳定。具体目的是阐明：1）14-3-3 α和细丝蛋白的作用 A对GPIbα配体结合、去折叠和机械信号传导的影响，2）GPIb-IX机械传感的机制。 诱导的细胞内信号通路导致整合素活化; 3）G β 13、talin 1和Rap 1的作用 对GPIIb-IIIa结合、构象和机械信号传导的影响。这些研究将表征配体结合 以及与止血和血栓形成相关的两种主要血小板机械受体GPIb和GPIIb-IIIa的信号传导。 研究结果将提供对血管生理学和病理学的深入了解，并有助于制定治疗策略 以治疗血栓形成，并具有最小的不良出血副作用。

Project Summary/Abstract Platelets are arguably the most important cellular participant in hemostasis and thrombosis. Many therapeutic strategies target platelet molecules to inhibit their functions in thrombosis. In particular, two membrane adhesion receptors, glycoprotein (GP) Ib-IX and GPIIb-IIIa (integrin IIb3), cooperatively mediate platelet adhesion and thrombus formation under high shear flow, and thus are ideal targets for inhibiting thrombosis in stenotic arteries as well as in microvasculature. Also, all anti-platelet drugs currently being used to treat thrombosis clinically have adverse hemorrhagic side effects, because the platelet plug formation required to stop bleeding is also inhibited. Under the support of this grant in its past period, we and others showed that both GPIbα and IIb3 are mechanoreceptors capable of receiving mechanical cues. We found that GPIb mechano-signaling leads to integrin inside-out signaling, which activates IIb3 from the inactive state having a bent conformation and a closed headpiece (BC) with a low affinity and short bond lifetime for ligand to an intermediate state having an extended conformation but a closed headpiece (EC) with an intermediate affinity and intermediate bond lifetime for ligand, which is distinct from the fully active state of IIb3 often seen in platelets stimulated by soluble agonists that has an extended conformation and an open headpiece (EO) with a high affinity and long bond lifetime for ligand. Significantly, the intermediate state of IIb3 mediates the formation of “biomechanical thrombi”, consisting of transient and unstable platelet aggregates resembling the dynamic outer layer of an in vivo thrombus formed under high shear blood flow, which is distinct from platelet aggregates underneath the outer layer that are further stimulated by released soluble agonists and where IIb3 are further activated to the fully active EO state. Further activation from the intermediate state to the fully active state can also be induced by IIb3 outside-in mechano- signaling, which also induces further activation of platelets. These data highlight the role of force-induced signaling – mechano-signaling – of, by, and between GPIb-IX and IIb3, and their unique importance in thrombus formation. Our overarching hypothesis is that a balanced regulation of mechano-signals of GPIbα and IIb3, and their synergy with biochemical signals induced by soluble platelet agonists, holds the promise to control thrombosis without unsetting hemostasis. The specific aims are to elucidate 1) the roles of 14-3-3ζ and filamin A on GPIbα ligand-binding, unfolding and mechano-signaling, 2) the mechanism of GPIb-IX mechanosensing- induced intracellular signaling pathway leading to integrin activation, and 3) the roles of G13, talin1 and Rap1 on GPIIb-IIIa binding, conformation and mechano-signaling. These studies will characterize the ligand-binding and signaling of of two major platelet mechanoreceptors, GPIb and GPIIb-IIIa, as related to hemostasis and thrombosis. The results will provide insights into vascular physiology and pathology and help develop therapeutic strategies to treat thrombus formation with minimal adverse bleeding side effects.