GPIbalpha shedding and platelet clearance

 描述(申请人提供)：这个项目的目标是确定糖蛋白(GP)Iba的胞外区域脱落在清除储存在室温(RT)或4℃下的血小板中的作用。尽管血小板用于输血已有50多年的历史，但人的血小板只能在RT条件下储存5天，这使得改善血小板的储存成为血库的一大挑战。最近的研究表明，血小板表面丰富而特异表达的膜受体GP1bα的胞外区脱落是介导血小板清除的关键步骤。然而，目前还无法确定GP1bα的脱落是否是需要清除的血小板的一个无关紧要的指标，或者实际上是其清除的触发因素。这种模糊性在很大程度上是因为在以前的研究中使用了金属蛋白酶抑制剂，这些抑制剂非特异性地阻止所有受体的胞外结构域脱落。最近，已开发出新型的单抗，以特异性地抑制人GP1bα在血小板中的脱落。对一种命名为5G6的原型单抗的初步研究表明，它对正常的血小板功能没有可检测到的影响，也不会在只表达人GP1bα的转基因HTG小鼠中引起急性血小板减少。此外，最近在GP1bα中发现了一个膜旁机械敏感结构域，这极大地促进了我们对GPIB-IX介导的信号转导的理解，并提出了一种将GPIba脱落与止血和血小板清除联系起来的新机制。本项目旨在通过利用新型单抗5G6及其衍生物来研究GP1bα脱落在清除血小板中的作用。具体目的1是研究5G6及其F(ab‘)2和Fab片段与血小板的关系，并从分子水平阐明它们对血小板功能的影响，包括GP1bα的聚集性。具体目标2是测试是否治疗 5G6或其衍生物在保存过程中可减轻保存损伤，提高RT保存的血小板输注后存活率。目的3探讨5G6处理或5G6与其他保存方法联合应用对冷藏血小板输注后存活率和止血功能的影响。对于AIMS 2和AIMS 3，已经建立了两个新的动物模型来监测免疫缺陷小鼠中人白细胞减少的采集源血小板和适当受体小鼠中HTG小鼠血小板的存活。对人和小鼠血小板的研究结果的分析和比较将对血小板在RT和4°C储存过程中发生的各种变化产生独特的机械见解。完成拟议的研究将确定GPIba脱落在RT和冷藏血小板的储存和清除中的作用，并提供关于特定抑制GP1bα脱落作为一种可行方法的前景的概念验证证据，以此作为一种可行的方法来改善输血治疗中的血小板储存。

 DESCRIPTION (provided by applicant): The objective of this project is to define the role of ectodomain shedding of glycoprotein (GP)Iba in the clearance of platelets that are stored at room temperature (RT) or 4°C. Although platelets have been used for transfusion for more than 50 years, human platelets can only be stored at RT for up to 5 days, making improving platelet storage a major challenge in blood banking. Recent studies have suggested that ectodomain shedding of GP1bα, a membrane receptor abundantly and exclusively expressed on the surface of platelets, as a key step in mediating platelet clearance. However, it has not been possible to determine whether shedding of GP1bα is an inconsequential indicator for the to-be-cleared platelet or actually the trigger for its clearance. Such ambiguity is largely due to the usage of metalloprotease inhibitors in previous studies that nonspecifically block ectodomain shedding of all receptors. Recently novel monoclonal antibodies (MAbs) have been developed to specifically inhibit shedding of human GP1bα in the platelet. Preliminary studies of a prototypical MAb designated 5G6 showed that it exhibited no detectable effect on normal platelet functions and it did not cause acute thrombocytopenia in a transgenic hTg mouse line expressing only human GP1bα. In addition, recent identification of a juxtamembrane mechano-sensitive domain in GP1bα has greatly advanced our understanding of GPIb- IX-mediated signaling and suggested a novel mechanism linking GPIba shedding to desialylation and platelet clearance. This project seeks to address the role of GP1bα shedding in platelet clearance by utilizing the novel MAb 5G6 and its derivatives. Specific Aim 1 is to characterize the association of 5G6 and its F(ab')2 and Fab fragments with platelets and to elucidate their effects on platelet functions at the molecular level, including the clustering of GP1bα. Specific Aim 2 is to test whether treatment of 5G6 or its derivative during storage can mitigate the storage lesion and improve the post-transfusion survival of RT-stored platelets. Specific Aim 3 is to explore the effects of 5G6 treatment, or the combined treatment of 5G6 with other storage methods, on the post-transfusion survival and hemostatic functions of cold- stored platelets. For Aims 2 and 3, two novel animal models have been developed to monitor the survival of human leukoreduced apheresis-derived platelets in the immunodeficient mice and that of hTg murine platelets in appropriate recipient mice. Analysis and comparison of the results obtained from studies on both human and murine platelets will generate unique mechanistic insights into the various changes the platelet undertake during the storage at RT and 4°C. Completion of the proposed studies will define the role of GPIba shedding in storage and clearance of RT- and cold-stored platelets, and provide proof-of- concept evidence regarding the prospect of specific inhibition of GP1bα shedding as a viable approach to improve platelet storage for the transfusion treatment.