GPIbalpha shedding and platelet clearance

 描述（由申请方提供）：本项目的目的是确定糖蛋白（GP）Iba的胞外域脱落在清除室温（RT）或4 ℃下储存的血小板中的作用。尽管血小板用于输血已有50多年的历史，但人血小板只能在室温下储存长达5天，这使得改善血小板储存成为血库的主要挑战。近年来的研究表明，血小板膜受体GP 1b α的胞外区脱落是介导血小板清除的关键步骤。然而，尚无法确定GP 1b α的脱落是否是待清除血小板的无关紧要的指标或实际上是其清除的触发因素。这种模糊性主要是由于在以前的研究中使用金属蛋白酶抑制剂，非特异性地阻止所有受体的胞外域脱落。近年来，人们开发了新型的单克隆抗体（MAbs）来特异性地抑制血小板中人GP 1b α的脱落。对命名为5G 6的原型单克隆抗体的初步研究表明，它对正常血小板功能没有可检测到的影响，并且在仅表达人GP 1b α的转基因hTg小鼠系中不会引起急性血小板减少症。此外，最近在GP 1b α中鉴定出一个质膜机械敏感结构域，极大地促进了我们对GPIb-IX介导的信号传导的理解，并提出了一种将GPIb脱落与去唾液酸化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克隆抗体5G 6及其衍生物来研究GP 1b α脱落在血小板清除中的作用。具体目的1是表征5G 6及其F（ab '）2和Fab片段与血小板的结合，并在分子水平上阐明它们对血小板功能的影响，包括GP 1b α的聚集。具体目标2是测试治疗是否 5G 6或其衍生物在储存过程中可以减轻储存损伤，并改善RT储存的血小板的输血后存活。具体目的3是探索5G 6处理或5G 6与其他储存方法的联合处理对冷储存血小板的输注后存活和止血功能的影响。对于目的2和3，已经开发了两种新的动物模型来监测免疫缺陷小鼠中人白细胞减少的脱血小板衍生的血小板的存活和适当受体小鼠中hTg鼠血小板的存活。对人和鼠血小板研究结果的分析和比较将产生对血小板在室温和4°C储存期间发生的各种变化的独特机制见解。拟定研究的完成将确定GPIb α脱落在RT和冷藏血小板储存和清除中的作用，并提供关于特异性抑制GPIb α脱落作为改善血小板储存用于输血治疗的可行方法的前景的概念验证证据。

 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.