Mechanism of cold platelet clearance

冷血小板清除机制

• 批准号: 7904079
• 负责人: John H Hartwig
• 金额: $ 51.16万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904079
• 关键词: Actins; Address; Alleles; Animal Model; Area; Asialoglycoprotein Receptor; Binding; Biology; Blood Banks; Blood Circulation; Blood Platelets; Buffers; Cell Adhesion Molecules; Chills; Clear Cell; Clinical; Collaborations; Complex; Cryopreservation; Cytidine Monophosphate N-Acetylneuraminic Acid; Cytoskeleton; Defect; Disease; Dissociation; Excision; GATA1 gene; Galactose; Gelsolin; Glucosamine; Goals; Hepatic; Hepatocyte; Human; Human Volunteers; Immune system; Interleukin 2 Receptor; Label; Lead; Lectin; Life; Link; Liver; Mediating; Methodology; Modification; Mus; Normal Statistical Distribution; Organ; Pathway interactions; Patients; Phagocytes; Plasma; Plasma Proteins; Platelet Glycoproteins; Platelet Transfusion; Polysaccharides; Population; Process; Proteins; Receptor Inhibition; Research; Role; Sepsis; Shapes; Skeleton; Surface; System; Technology; Temperature; Testing; Time; Transfusion; Uridine Diphosphate Galactose; Wild Type Mouse; Work; cold temperature; density; design; filamin; in vivo; macrophage; prevent; promoter; receptor; research study; sialylation; sugar; von Willebrand Factor; von Willebrand factor 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，我们已经定义了一个受体-反受体对， 从循环中取出冷却2- 4小时的洗涤血小板。我们认为，冷冻会导致 GPIb/V/IX受体复合物（vWfR）聚集在冷冻血小板表面， GP 1b-alpha亚基的N-连接聚糖上的葡糖胺（β-GlcNAc）残基， 导致它们被肝脏中吞噬细胞上的α M-β 2受体的凝集素结构域识别。覆盖 通过半乳糖（半乳糖基化）在GP 1b-α链上暴露的β-GlcNAc的缺失挽救了 在这些条件下冷冻的血小板在小鼠中的循环。然而，血小板在血浆中的储存 在寒冷的48小时诱导进一步的变化，导致循环损失，而不是拯救 半乳糖基化我们现在假设，血浆中血小板的长期冷冻（等于或大于48小时）导致血小板减少。 vWFR的“超聚类”。这增加了半乳糖残基在聚簇中的密度， 半乳糖基化的血小板，使得它们达到临界密度，现在导致被 肝细胞和/或巨噬细胞使用其去唾液酸糖蛋白受体（ASGPR），其识别 暴露的半乳糖GP 1b-α通过细丝蛋白A（FLNa）与底层肌动蛋白骨架连接。我们 先前的研究表明，低温诱导血小板重塑其细胞骨架并组装肌动蛋白。 目的1将确定GP 1b-α是否参与清除储存在血浆中48小时的血小板， 长期变化是由于血浆成分与血小板的结合。如果GP 1b-α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。使用由造血系统驱动的Ore的FLNA无效血小板的纯群体 已经建立了使用我们的FLNa的条件loxP等位基因的小鼠中的特异性启动子GATA 1。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。我们推测 FLNa无效血小板将被清除。因此，这些结果将得到的机制，和重要性， 血小板存活和体内功能的聚集（与项目1-瓦格纳博士合作）。如果 缺乏FLNa的血小板循环，我们将确定它们是否不能在寒冷中聚集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， 血小板长期低温储存使GP 1 b-α-FLNa/B复合物解离。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）鉴定血小板 UDP-半乳糖和CMP-唾液酸的受体蛋白;（3）确定哪些器官/细胞清除 半乳糖化和/或唾液酸化血小板（与项目3 -von Andrian博士合作）;（4） 评价修饰和冷藏血小板的体内功能（与项目1 -Dr. 瓦格纳）;和（5）在小鼠中建立人源化适应性免疫系统，这将使我们能够评估 血小板修饰对冷冻人血小板存活和功能的影响 与项目1和3 -瓦格纳博士和冯安德里安博士合作）。这方面的研究已经 由于缺乏评价人类血小板循环和功能的良好动物模型， 输血后。目的3，如果需要，将确定小鼠血小板中GP 1b-α的非依赖性变化 在血库条件下冷藏（> 48小时）在血浆中，以进行清除和鉴定 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 可以调节以防止冷诱导血小板清除的靶点，并开发方法， 阻止他们。