Mechanotransduction of platelet receptors GPIb and GPIIb-IIIa

项目摘要/摘要 可以说，血小板是止血和血栓形成过程中最重要的细胞参与者。许多治疗方法 策略针对血小板分子，以抑制其在血栓形成中的功能。具体地说，两种膜粘合 受体，糖蛋白(GP)Ib-IX和GPIIb-IIIa(整合素IIb3)，协同介导血小板黏附和 在高剪切率下形成血栓，因此是抑制狭窄动脉血栓形成的理想靶点。 在微血管中也是如此。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有 不利的出血副作用，因为止血所需的血小板堵塞的形成也被抑制。 在过去一段时间这笔赠款的支持下，我们和其他人表明，GPIBα和IIb3都是 机械感受器能够接收机械信号的机械感受器。我们发现，GPIB机械信号会导致 整合素内向外信号，从具有弯曲构象的非激活状态激活IIb3 具有低亲和力和短键寿命的封闭式头饰(BC)，用于配体到具有 延长构象，但具有中间亲和力和中间键寿命的封闭头(EC) 对于配体，这不同于在可溶性激动剂刺激的血小板中常见的IIb3的完全活性状态 它具有延长的构象和具有高亲和力和长键合寿命的开放式头饰(EO) 莱兰德。值得注意的是，IIb3的中间状态参与了“生物力学血栓”的形成，包括 暂时性和不稳定的血小板聚集体，类似于体内形成的血栓的动态外层 在高切变血流下，这与外层下的血小板聚集物不同，后者进一步 受到释放的可溶性激动剂的刺激，其中IIb3进一步被激活到完全活跃的EO状态。进一步 IIb3由外向内机制也可诱导从中间态到完全活化态的激活。 信号，这也诱导了血小板的进一步激活。这些数据突出了力诱导的作用 GPIB-IX和IIb3之间的信号-机械信号-机制信号及其在血栓中的独特重要性 队形。我们的总体假设是，GPIBα和IIb3的机械信号的平衡调节，以及 它们与可溶性血小板激动剂诱导的生化信号的协同作用，有望控制 血栓形成而不止血。具体目的是阐明1)14-3-3ζ和丝氨酸的作用 关于GPIB-α配体的结合、解折叠和机械信号传递，2)GPIB-IX机械传感机制-- 诱导细胞内信号通路导致整合素活化；3)G13、talin1和Rap1的作用 关于GPIIb-IIIa的结合、构象和机械信号转导。这些研究将描述配体结合的特征。 以及与止血和血栓形成相关的两个主要的血小板机械受体GPIb和GPIIb-IIIa的信号转导。 这些结果将为血管生理学和病理学提供洞察力，并有助于制定治疗策略 治疗血栓形成，不良出血副作用最小。

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.