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入血小板加速的途径。 从循环中消除了它们的清除。直到最近，影响的唯一建立的机制 血小板的存活率是抗体介导的血小板清除率，通过凝结浓度消耗血小板 由于大量出血而导致的损失。努力解决实际问题，如何冷藏血小板 为了输血，使我们定义了一种未使用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受体在短期的GPIBA上识别簇的PGLCNAC部分（<4H） 存储的血小板。现在，我们已经对缺乏唾液酸的小鼠血小板进行了惊人的观察（重新 从肝脏中移除了48h或siAllyl-tranferase stsgallv null小鼠的48h或血小板），主要是在肝脏中去除 通过肝细胞，而不是巨噬细胞。因为暴露的半乳糖残基在表面上增加了 血小板循环，在长期储存之后，如凝集素结合实验所揭示的，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 半乳糖暴露的增加会诱导亚乳糖蛋白受体（ASGPR或HL--）识别 1/2）在肝细胞上。明显增强间隙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）酶上的脱夹血小板；和 3）野生型血小板以> 48h的速度冷藏。 AIM 1将记录并表征长期的吸收 实时通过肝细胞储存的血小板，Stsgallv null血小板和神经氨酸酶处理的血小板， 量化其在小鼠中的清除率，确定巨噬细胞清除率（AIM 1A）的贡献，然后 定义ASGP受体在开始摄入（AIM 1B）中的作用。我们将确定是否com- 半乳糖基化和溶解的结合将改善长期储存的血小板和stsgallv - / - 血小板 生存（AIM 1C）。我们还将调查血小板是否在循环时失去唾液酸，这一过程可能 有助于血小板的老化和清除（AIM 1D），以及重苷是否可以提高生存率（AIM 1E）。因此，这些实验将为老年血小板建立新的清除机制。而 插入性实验应证明HL-1/2的功能在清除冷冻鼠血小板中 体内，其他方法对于确定人血小板可能会遭受同样的命运。在AIM 2中， 因此，我们建议利用HEPG2细胞对desiapated血小板的体外内吞作用作为简单的 读数（相对于输血研究）（AIM 2A）定义宿主血小板糖蛋白的性质 通过HL-1/2（肝细胞）（AIM 2B）或MGL1（巨噬细胞）重新识别的半乳糖部分 CEPTORS（AIM 2D）。我们将进一步确定血小板重苷是否阻止了脱水的摄入 体外血小板（AIM 2C）。