Mechanism of cold platelet clearance

冷血小板清除机制

• 批准号: 7340221
• 负责人: John H Hartwig
• 金额: $ 48.42万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-22 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340221
• 关键词: platelets; receptor

During the previous 5 years of this project, we have defined one receptor-counter receptor pair that removes washed platelets, chilled for 2-4h, from the circulation. We proposed that chilling causes the GPIb/V/IX receptor complex (vWfR) to cluster on the surface of chilled platelets bringing exposed beta-N-acetyl glucosamine (beta-GlcNAc) residues on N-linked glycans of the GP1b-alpha subunit together, which leads to their recognition by the lectin domain of alphaM-beta2 receptors on phagocytes in the liver. Coverage of exposed beta-GlcNAc on the GP1b-alpha chain by galactose (galactosylation) rescues the loss of circulation in mice of platelets chilled under these conditions. However, storage of platelets in plasma in the cold for 48 h induces further changes that lead to a loss of circulation not rescued by galactosylation. We now postulate that long-term chilling (equal to or greater than 48h) of platelets in plasma leads to a "hyperclustering" of the vWFR. This increases the density of galactose residues in clusters on galactosylated platelets such that they reach a critical density that now results in clearance by hepatocytes and/or macrophages using their Asialoglycoprotein receptor (ASGPR), which recognizes exposed galactose. GP1b-alpha is linked to the underlying actin skeleton by filamin A (FLNa). We previously showed that chilling induces platelets to remodel their cytoskeleton and assemble actin. Aim 1 will determine if GP1b-alpha is involved in the removal of platelets stored in plasma for 48h and if the long-term changes are due to the binding of plasma components to platelets. If GP1b-alpha is central to the changes, we will investigate the role of FLNa and gelsolin as well as the underlying actin-connection in vWfR clustering. A pure population of FLNa-null platelets using Ore driven by the hematopoetic specific promoter GATA1 in mice using our conditional loxP allele of FLNa has been established. We will determine if platelets lacking FLNa or gelsolin circulate in wild type (WT) mice. We postulate that FLNa null platelets will be cleared. These results will therefore get at the mechanism, and importance, of clustering in platelet survival and in vivo function (in collaboration with Project 1- Dr. Wagner). If platelets lacking FLNa circulate, we will determine if they fail to cluster the vWf receptor in the cold following short-term storage in buffer or long-term storage in plasma. We will also investigate if prolonged cold platelet storage dissociates the GP1b-alpha-FLNa/b complex. Aim 2 will determine: (1) if sialylation facilitates the survival of refrigerated (> 48 hrs) galactosylated platelets; (2) identify platelet acceptor proteins for UDP-galactose and CMP-sialic acid; (3) determine which organ/cells clear galactosylated and/or sialylated platelets (in collaboration with Project 3 - Dr. von Andrian); (4) evaluate in vivo function of modified and refrigerated platelets (in collaboration with Project 1 - Dr. Wagner); and (5) establish a humanized adaptive immune system in mice that will allow us to assess the effects of platelet modification on refrigerated human platelet survival and function in vivo (in collaboration with Projects 1 and 3 - Drs. Wagner and von Andrian). Studies in this area have been limited by a lack of good animal models that evaluate both human platelet circulation and function following transfusion. Aim 3, if necessary, will identify GP1b-alpha independent changes in murine platelets refrigerated (> 48 hrs) in plasma under blood bank conditions that target them for removal and identify the phagocytic receptor that mediates the removal. The overall goal of this work is to identify new targets that can be modulated to prevent cold-induced platelet clearance and develop methodology to block them.

在该项目的前 5 年中，我们定义了一个受体-反受体对 从循环中去除洗涤后的血小板，冷冻 2-4 小时。我们提出，寒冷会导致 GPIb/V/IX 受体复合物 (vWfR) 聚集在冷冻血小板表面，带来暴露的 β-N-乙酰基 GP1b-α 亚基的 N 连接聚糖上的葡萄糖胺 (β-GlcNAc) 残基结合在一起， 导致它们被肝脏吞噬细胞上的 αM-β2 受体的凝集素结构域识别。覆盖范围 通过半乳糖（半乳糖基化）在 GP1b-α 链上暴露的 β-GlcNAc 可挽救 在这些条件下冷冻的血小板在小鼠体内的循环。然而，血浆中血小板的储存 在寒冷中 48 小时会引起进一步的变化，导致循环丧失，但无法通过以下方法挽救 半乳糖基化。我们现在假设血浆中的血小板长期冷冻（等于或大于 48 小时）会导致 vWFR 的“超聚类”。这增加了簇中半乳糖残基的密度 半乳糖基化血小板，使其达到临界密度，现在可通过 肝细胞和/或巨噬细胞使用其去唾液酸糖蛋白受体 (ASGPR)，该受体可识别 暴露的半乳糖。 GP1b-alpha 通过细丝蛋白 A (FLNa) 连接至底层肌动蛋白骨架。我们 先前表明，寒冷会诱导血小板重塑其细胞骨架并组装肌动蛋白。 目标 1 将确定 GP1b-alpha 是否参与血浆中储存 48 小时的血小板的去除，以及是否 长期变化是由于血浆成分与血小板的结合造成的。如果 GP1b-alpha 是 变化，我们将研究 FLNa 和凝溶胶蛋白的作用以及潜在的肌动蛋白连接 vWfR 聚类。使用由造血驱动的矿石的 FLNa-null 血小板的纯群体 使用我们的 FLNa 条件 loxP 等位基因在小鼠中建立了特异性启动子 GATA1。我们 将确定缺乏 FLNa 或凝溶胶蛋白的血小板是否在野生型 (WT) 小鼠中循环。我们假设 FLNa 无效血小板将被清除。因此，这些结果将揭示其机制和重要性， 血小板存活和体内功能的聚类分析（与项目 1-Wagner 博士合作）。如果 缺乏 FLNa 的血小板循环，我们将确定它们是否无法在寒冷中聚集 vWf 受体 在缓冲液中短期储存或在血浆中长期储存后。我们还将调查是否 长时间冷藏血小板会解离 GP1b-α-FLNa/b 复合物。目标 2 将确定： (1) 如果 唾液酸化促进冷藏（> 48小时）半乳糖基化血小板的存活； (2) 识别血小板 UDP-半乳糖和CMP-唾液酸的受体蛋白； (3) 确定哪个器官/细胞透明 半乳糖基化和/或唾液酸化血小板（与项目 3 - von Andrian 博士合作）； (4) 评估改良和冷藏血小板的体内功能（与项目 1 - 博士合作） 瓦格纳）; (5) 在小鼠中建立人源化适应性免疫系统，使我们能够评估 血小板修饰对冷藏人血小板存活和体内功能的影响（在 与项目 1 和 3 的合作 - 博士。瓦格纳和冯·安德里安）。这方面的研究已 由于缺乏评估人类血小板循环和功能的良好动物模型而受到限制 输血后。如有必要，目标 3 将识别小鼠血小板中 GP1b-α 独立的变化 在血库条件下冷藏（> 48 小时）血浆，以去除和识别血浆 介导清除的吞噬细胞受体。这项工作的总体目标是确定新的 可以调节的目标以防止寒冷诱导的血小板清除并开发方法 阻止他们。