Biochemistry of Platelet Desialylation

血小板脱唾液酸化的生物化学

• 批准号: 10402246
• 负责人: Marie Hollenhorst
• 金额: $ 16.74万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2023-01-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402246
• 关键词: Alberta province; Antibodies; Asialoglycoproteins; Autoantibodies; Biochemical; Biochemistry; Biology; Blood Platelets; Carbohydrates; Chemicals; Clinical; Cryopreservation; Development; Development Plans; Disease; Enzymes; Excision; Flow Cytometry; General Hospitals; Glycobiology; Glycoproteins; Half-Life; Hematology; Hemorrhage; Human; Immune; Immune Sera; Immunotherapy; In Vitro; Incubated; International; Isoenzymes; Knowledge; Laboratories; Lead; Lectin; Life; Link; Massachusetts; Measures; Mediating; Membrane Glycoproteins; Mentorship; Molecular; Mucins; Mus; Neuraminidase; Non-Malignant; Outcome; Pathologic; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Physicians; Platelet Count measurement; Platelet Membrane Glycoproteins; Platelet Transfusion; Play; Polysaccharides; Positioning Attribute; Process; Proteins; Proteomics; Recombinants; Research Personnel; Resistance; Risk; Role; Scientist; Sialic Acids; Stimulus; Surface; Techniques; Testing; Thrombocytopenia; Time; Training; Universities; Work; career development; cold temperature; effective therapy; experimental study; glycoproteomics; glycosylation; in vivo; inhibitor; innovation; liquid chromatography mass spectrometry; member; mouse model; mucinase; platelet storage; small molecule; small molecule inhibitor; sugar; targeted treatment; tool; transfusion medicine

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.

项目摘要/摘要 在理解血小板清除是如何调节的方面存在根本性的差距。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，一些患者的血小板计数低 （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是 身体是去唾液酸化，这是指从糖蛋白上去除糖分子唾液酸， 血小板的表面。血小板去唾液酸化在加速血小板清除免疫中起作用 血小板减少症（ITP）和输注储存在低温下的血小板后。 尽管去唾液酸化在临床上很重要，但切割血小板唾液酸的酶的特性 酸及其糖蛋白底物和产物的范围仍然未知，阻碍了开发 ITP和其他血小板减少性疾病的靶向治疗。核心假设是人类 神经氨酸酶1使糖蛋白（GP）Ibα O-聚糖以及其它血小板表面糖蛋白去唾液酸化， 从而加速ITP中和冷藏后的血小板清除。 将通过以下具体目的检验这一假设：1）确定哪些GpIbα聚糖经历 去唾液酸化将使用液相色谱/质谱法（LC/MS）分析从 通过在4 ℃下孵育或通过与来自ITP的血清孵育，在体外触发脱唾液酸化后的血小板 含有抗GpIb α自身抗体的患者。2)确定哪种人神经氨酸酶去唾液酸化聚糖 与血小板清除率相关的一组有效的选择性小分子的作用 将在体外血小板去唾液酸化实验中测试神经氨酸酶同工酶抑制剂。体内半 将测量用这些去唾液酸化抑制剂处理的血小板的寿命。3)鉴定血小板神经氨酸酶 印刷受体.将采用既定的膜糖蛋白富集策略，以允许LC/MS 去唾液酸化血小板与对照血小板的蛋白质组学分析。结果将是描绘出关键的血小板 催化去唾液酸化的神经氨酸酶及其糖蛋白底物和产物。这将 提供了血小板去唾液酸化过程的第一个生化表征，并将为 开发更有效的血小板减少症治疗方法。 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 Carolyn Bertozzi教授在斯坦福大学和2）培训血小板生物学，小鼠血小板输注 在斯坦福大学的国际专家的指导下， 其他地方，包括博士卡琳Hoffmeister，大卫库特，和劳伦斯梁。这将使霍伦霍斯特博士 在化学生物学的界面上建立一个独特的利基，作为一个独立的物理学家-科学家调查员， 输血医学和非恶性血液学。