Carbohydrate-mediated platelet clearance

碳水化合物介导的血小板清除

• 批准号: 7837517
• 负责人: Karin Maria Hoffmeister
• 金额: $ 27.49万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7837517
• 关键词: Address; Affect; Age; Aging; Antibodies; Asialoglycoprotein Receptor; Binding; Biological Assay; Blocking Antibodies; Blood Circulation; Blood Platelets; Carbohydrates; Cells; Chills; Coagulation Process; Consumption; Data; Development; Eating; Employee Strikes; Endocytosis; Galactose; Goals; Hemorrhage; Hepatocyte; Human; In Vitro; Ingestion; Knockout Mice; Kupffer Cells; Lectin; Ligands; Liver; Mediating; Metabolic Clearance Rate; Modification; Mus; Nature; Neuraminidase; Pathway interactions; Platelet Glycoproteins; Platelet Transfusion; Process; Relative (related person); Role; Sialic Acids; Site; Surface; System; Time; Transfusion; base; improved; in vivo; inhibitor/antagonist; macrophage; platelet typing; prevent; receptor; research study; sialylation; sugar; uptake

The goal of these studies is to determine the pathway(s) by which hepatocytes ingest platelets to acceler- ate their clearance from the circulation. Until recently, the only well-established mechanisms affecting platelet survival were antibody-mediated platelet clearance, consumption of platelets by coagulation reac- tions and loss due to massive bleeding. An effort to address a practical problem, how to refrigerate platelets for transfusion, led us to define an unsuspected carbohydrate-based platelet clearance mechanism where the ccM|}2 receptor on Kupffer cells recognized clustered pGlcNAc moieties on GPIba of short-term (< 4h) stored platelets. We have now made the striking observation that mouse platelets lacking sialic acid (re- frigerated for 48h or platelets from sialyl-tranferase STSGallV null mice) are removed in the liver, principally by hepatocytes, not macrophages. Because exposed galactose residues increase on the surface as platelets circulate, and after long-term storage as revealed by lectin binding experiments, we postulate that sialic acid normally covers the galactose residues and permits platelet survival. We further hypothesize that increases in galactose exposure induce recognition by the Asialoglycoprotein receptor (ASGPR or HL- 1/2) on hepatocytes. Platelet modifications that markedly enhance clearance will be used to determine the role of sialic acid in platelet survival: 1) ST3GallV null platelets; 2) enzymatically desialylated platelets; and 3) wild type platelets refrigerated for > 48h. Aim 1 will document and characterize the uptake of long-term stored platelets, STSGallV null platelets and neuraminidase-treated platelets by hepatocytes in real time, quantify their clearance rates in mice, determine the contribution of macrophage clearance (Aim 1A), and define the role of the ASGP receptor(s) in initiating ingestion (Aim 1B). We will determine whether a com- bination of galactosylation and sialylation will improve long-term stored platelet and STSGallV-/- platelet survival (Aim 1C). We will also investigate if platelets lose sialic acid while circulating, a process that could contribute to platelet aging and clearance (Aim 1D), and whether resialylation can improve survival (Aim 1E). These experiments will, therefore, establish a new clearance mechanism for senile platelets. While the intravital experiments should demonstrate HL-1/2 function in the clearance of chilled murine platelets in vivo, other approaches are necessary to establish that human platelets can suffer the same fate. In Aim 2, therefore, we propose to utilize the in vitro endocytosis of desialylated platelets by HepG2 cells as a simple readout (relative to transfusion studies) (Aim 2A) to define the nature of the host platelet glycoproteins car- rying the galactose moieties recognized by HL-1/2 (hepatocytes) (Aim 2B) or MGL1 (macrophages) re- ceptors (Aim 2D). We will further determine if platelet resialylation prevents the ingestion of desialylated platelets in vitro (Aim 2C).

这些研究的目的是确定肝细胞摄取血小板以加速的途径 吃掉了流通中的间隙。直到最近，唯一完善的影响机制 血小板存活是抗体介导的血小板清除，凝血反应消耗血小板。 因大量失血而造成的损失和损失。努力解决一个实际问题：如何冷藏血小板 对于输血，使我们定义了一种意想不到的基于碳水化合物的血小板清除机制，其中 Kupffer 细胞上的 ccM|}2 受体短期（< 4 小时）识别 GPIba 上聚集的 pGlcNAc 部分 储存的血小板。我们现在做出了惊人的观察，即小鼠血小板缺乏唾液酸（重新 冷藏 48 小时或从唾液酸转移酶 STSGallV 缺失小鼠中取出血小板，主要是在肝脏中去除 由肝细胞而非巨噬细胞产生。因为暴露的半乳糖残留物在表面上增加 血小板循环，并且经过凝集素结合实验揭示的长期储存后，我们假设 唾液酸通常覆盖半乳糖残基并允许血小板存活。我们进一步假设 半乳糖暴露增加会诱导去唾液酸糖蛋白受体（ASGPR 或 HL- 1/2) 在肝细胞上。显着增强清除率的血小板修饰将用于确定 唾液酸在血小板存活中的作用：1) ST3GallV 无效血小板； 2) 酶促去唾液酸化血小板；和 3) 野生型血小板冷藏> 48小时。目标 1 将记录并描述长期的吸收情况 肝细胞实时保存的血小板、STSGallV 无效血小板和神经氨酸酶处理的血小板， 量化小鼠体内的清除率，确定巨噬细胞清除率的贡献（目标 1A），以及 定义 ASGP 受体在启动摄入中的作用（目标 1B）。我们将确定是否有一个公司 半乳糖基化和唾液酸化的结合将改善长期储存的血小板和 STSGallV-/- 血小板 生存（目标 1C）。我们还将研究血小板在循环时是否会失去唾液酸，这一过程可能会导致 有助于血小板老化和清除（目标 1D），以及再唾液酸化是否可以提高生存率（目标 1E）。因此，这些实验将为老年血小板建立新的清除机制。虽然 活体实验应证明 HL-1/2 在清除冷冻小鼠血小板中的功能 在体内，需要其他方法来证实人类血小板也能遭受同样的命运。在目标 2 中， 因此，我们建议利用 HepG2 细胞对去唾液酸化血小板的体外内吞作为简单的方法 读出（相对于输血研究）（目标 2A）来定义宿主血小板糖蛋白的性质 重新检测 HL-1/2（肝细胞）（目标 2B）或 MGL1（巨噬细胞）识别的半乳糖部分 感受器（Aim 2D）。我们将进一步确定血小板再唾液酸化是否可以防止去唾液酸化的摄入 体外血小板（目标 2C）。