Biochemistry of Platelet Desialylation

血小板脱唾液酸化的生物化学

• 批准号: 10833844
• 负责人: Marie Hollenhorst
• 金额: $ 16.74万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10833844
• 关键词: Acceleration; 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; Idiopathic Thrombocytopenic Purpura; Immune Sera; 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; Polysaccharides; Positioning Attribute; Process; Proteins; Proteomics; Recombinants; Research Personnel; Resistance; Risk; Scientist; Sialic Acids; Stimulus; Surface; Techniques; Testing; Thrombocytopenia; Time; Training; Transfusion; 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; sialylation; 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 了解如何调节血小板清除率存在根本的差距。直到我们获得更多 对此的详细了解，我们将缺乏一些血小板计数低的患者的有效疗法 （血小板减少症）有威胁生命的出血风险。从 人体是脱酰化的，它是指从糖蛋白上去除糖分子唾液酸 血小板的表面。血小板脱酰溶质在免疫中的血小板的加速清除率中起作用 血小板减少症（ITP），然后在冷温下储存的血小板输血后。 尽管脱酰化的临床重要性，但裂解血小板唾液的酶的身份 酸及其糖蛋白底物和产品的范围仍然未知，阻碍了发展的努力 ITP和其他血小板减少性疾病的靶向疗法。中心假设是人类 神经氨酸酶1脱酰基糖糖蛋白（GP）IBαO-聚糖以及其他血小板表面糖蛋白， 从而加速ITP和冷藏后的血小板间隙。 该假设将通过以下特定目的进行检验：1）确定哪种GPIBα聚糖经历 脱酰基。液相色谱/质谱法（LC/MS）将用于分析从中纯化的GPIBα 在4°C下孵育或与ITP的血清一起孵育，在体外触发脱酰基后血小板。 含有抗GPIBα自身抗体的患者。 2）确定哪种人神经氨酸酶脱酰基化聚糖 与血小板间隙有关。一个有效和选择性小分子的效果 神经氨酸酶同工酶抑制剂将在体外血小板目的地实验中进行测试。体内半 将测量用这些脱酰化抑制剂处理的血小板的寿命。 3）识别血小板神经氨酸酶 基材。将聘请富含膜糖蛋白的既定策略，以允许LC/MS 脱伊拉德与对照血小板的蛋白质组学分析。结果将是钥匙血小板的描述 神经氨酸酶酶催化脱酰化及其糖蛋白底物和产物。这会 提供血小板目的地过程的第一个生化特征，并为 开发对血小板减少症的更有效疗法。 职业发展计划包括1）化学糖生物学培训作为实验室成员 斯坦福大学Carolyn Bertozzi教授和2）血小板生物学培训，鼠标血小板输血 在斯坦福大学和国际专家的心态下，实验和免疫血小板减少症 在其他地方，包括Drs。 Karin Hoffmeister，David Kuter和Lawrence Leung。这将定位Hollenhorst博士 为了在化学生物学界面建立独特的生态位作为独立的身体科学研究者， 输血医学和非恶性血液学。

项目成果

Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation.

• DOI: 10.1016/j.jtha.2023.01.009
• 发表时间: 2023-04
• 期刊: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
• 影响因子: 10.4
• 作者: Hollenhorst, Marie A.;Tiemeyer, Katherine H.;Mahoney, Keira E.;Aoki, Kazuhiro;Ishihara, Mayumi;Lowery, Sarah C.;Rangel-Angarita, Valentina;Bertozzi, Carolyn R.;Malaker, Stacy A.
• 通讯作者: Malaker, Stacy A.