Aflac Cancer and Blood Disorders Center, Departments of Pediatrics, Children's Healthcare of Atlanta, Emory University School of Medicine Atlanta, Atlanta, Georgia, USA.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Thromb Haemost. 2022 Jun;20(6):1451-1463. doi: 10.1111/jth.15702. Epub 2022 Mar 29.
Glycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild-type mice. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 inhibits GPIbα-dependent platelet signaling and activation. Further, RAM.1 also inhibits anti-GPIbα antibody-mediated filopodia formation.
To investigate whether RAM.1 can ameliorate trigger sequence exposure-mediated platelet clearance.
Spontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody.
Transfected Chinese hamster ovary cells stably expressing the same chimeric IL4R-Ibα protein complex as in IL4R-IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R-IbαTg platelets.
These results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb-IX-mediated thrombocytopenia.
糖蛋白（GP）Ibα在调节血小板清除中起着关键作用。最近，我们鉴定了 GPIbα中的机械感受器结构域（MSD），并提供了证据表明，GPIbα MSD 的展开会导致其中的触发序列暴露，并随后引发 GPIb-IX 信号，从而加速血小板清除。在一种常用的转基因小鼠模型中，IL4R-IbαTg 中，触发序列被持续暴露，存在组成型 GPIb-IX 介导的细胞信号。它们的血小板清除速度也明显快于野生型小鼠。之前开发了一种抗 GPIbβ抗体 RAM.1。RAM.1 抑制 GPIbα依赖性血小板信号转导和激活。此外，RAM.1 还抑制抗 GPIbα抗体介导的微丝形成。
研究 RAM.1 是否可以改善触发序列暴露介导的血小板清除。
通过共聚焦显微镜测量自发形成的微丝。通过流式细胞术测量其他血小板信号事件。通过 Alexa 488 标记的抗小鼠 GPIX 抗体追踪内源性血小板寿命。
稳定转染表达与 IL4R-IbαTg 小鼠相同嵌合 IL4R-Ibα 蛋白复合物的中国仓鼠卵巢细胞也持续表现出微丝，并且这种微丝可以通过 RAM.1 处理来消除。此外，输注缺乏其 Fc 部分的重组 RAM.1 衍生物可显著延长 IL4R-IbαTg 血小板的内源性寿命。
这些结果提供了关键证据，支持触发序列暴露与加速血小板清除之间的因果关系，并表明可以开发 RAM.1 衍生物来治疗 GPIb-IX 介导的血小板减少症。
