Biochemistry of Platelet Desialylation

项目摘要/摘要 在了解血小板清除是如何调节的方面存在着根本的差距。直到我们获得更多 详细了解这一点，我们将缺乏有效的治疗方法来治疗一些低血小板患者 (血小板减少症)有危及生命的出血风险。血小板清除的一个重要触发因素 身体是去唾液酸，指的是从糖蛋白上去除糖分子唾液酸。 血小板的表面。血小板清除在免疫中加速清除血小板中的作用 血小板减少症(ITP)和输注储存在低温下的血小板后引起的疾病。 尽管脱水的临床重要性，但裂解血小板唾液酸的酶的特性 酸及其糖蛋白底物和产品的范围仍然未知，阻碍了开发 针对ITP和其他血小板减少性疾病的靶向治疗。中心假设是人类 神经氨酸酶1可分解糖蛋白(GP)IbαO-聚糖以及其他血小板表面糖蛋白， 从而加速了ITP中和冷藏后的血小板清除。 这一假设将通过以下具体目标来检验：1)确定哪些GPIBα多糖经历 去死神。采用液-质联用(LC/MS)技术对提取的GPIBα进行分析 分离后的血小板在体外通过4℃孵育或与ITP血清孵育而被触发 含有抗GPIBα自身抗体的患者。2)确定哪种人类神经氨酸酶不能分解多糖 与清除血小板有关的物质。一组强效和选择性小分子的作用 神经氨酸酶同工酶抑制剂将在体外血小板抑制实验中进行测试。活体中的一半 用这些止血抑制剂治疗的血小板的寿命将被测量。3)鉴定血小板神经氨酸酶 底物。将采用一种已建立的膜糖蛋白富集化策略来实现LC/MS 需要分析的血小板与对照组的蛋白质组学分析。结果将是描绘出关键的血小板 催化脱氢反应的神经氨酸酶及其糖蛋白底物和产物。这将是 首次提供了血小板脱除过程的生物化学特征，并将为 开发更有效的治疗血小板减少症的方法。 职业发展计划包括1)作为实验室成员接受化学糖生物学培训 在斯坦福大学的Carolyn Bertozzi教授和2)在血小板生物学、小鼠血小板输注方面的培训 在斯坦福大学和斯坦福大学国际专家的指导下进行的实验和免疫性血小板减少症 在其他地方，包括卡琳·霍夫迈斯特博士、大卫·库特和劳伦斯·梁。这将使霍伦霍斯特博士 为了在化学生物学的界面上建立一个独特的利基市场，作为一个独立的医生-科学家研究员， 输血医学和非恶性血液学。

PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how platelet clearance is regulated. Until we gain a more detailed understanding of this, we will lack effective therapies for some patients with low platelet counts (thrombocytopenia) who are at risk for life-threatening bleeding. One important trigger for platelet clearance from the body is desialylation, which refers to the removal of the sugar molecule sialic acid from glycoproteins on the surface of the platelet. Platelet desialylation plays a role in accelerated clearance of platelets in immune thrombocytopenia (ITP) and following transfusion of platelets that have been stored at cold temperatures. Despite the clinical importance of desialylation, both the identity of the enzyme that cleaves platelet sialic acid and its scope of glycoprotein substrates and products remain unknown, hampering efforts to develop targeted therapies for ITP and other thrombocytopenic disorders. The central hypothesis is that human neuraminidase 1 desialylates glycoprotein (GP) Ibα O-glycans as well as other platelet surface glycoproteins, thereby accelerating platelet clearance in ITP and after cold storage. This hypothesis will be tested via the following specific aims: 1) Determine which GpIbα glycans undergo desialylation. Liquid chromatography/mass spectrometry (LC/MS) will be used to analyze GpIbα purified from platelets after desialylation is triggered in vitro either by incubation at 4°C or by incubation with sera from ITP patients containing anti-GpIbα autoantibodies. 2) Determine which human neuraminidase desialylates glycans that are relevant for platelet clearance. The effect of a panel of potent and selective small molecule neuraminidase isoenzyme inhibitors will be tested in in vitro platelet desialylation experiments. The in vivo half life of platelets treated with these desialylation inhibitors will be measured. 3) Identify platelet neuraminidase substrates. An established strategy for enrichment of membrane glycoproteins will be employed to allow LC/MS proteomics analysis of desialylated vs control platelets. The outcome will be a delineation of the key platelet neuraminidase enzyme which catalyzes desialylation and its glycoprotein substrates and products. This will provide the first biochemical characterization of the process of platelet desialylation and will pave the way for development of more effective therapies for thrombocytopenia. The career development plan includes 1) training in chemical glycobiology as a member of the laboratory of Prof. Carolyn Bertozzi at Stanford University and 2) training in platelet biology, mouse platelet transfusion experiments, and immune thrombocytopenia under the mentorship of international experts at Stanford and elsewhere, including Drs. Karin Hoffmeister, David Kuter, and Lawrence Leung. This will position Dr. Hollenhorst to establish a unique niche as an independent physician-scientist investigator at the interface of chemical biology, transfusion medicine, and non-malignant hematology.