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，我们定义了一对受体爆发受体对 从循环中去除洗涤后的血小板2-4H。我们提出冷却导致 gpib/v/ix受体复合物（VWFR），以簇状的冰冷血小板的表面簇生，带来裸露的β-n-乙酰基 GP1B-Alpha亚基的N连接聚糖上的葡萄糖胺（β-GLCNAC）残基一起 通过肝脏中的吞噬细胞上的α-Beta2受体的凝集素结构域导致它们的识别。覆盖范围 通过半乳糖（半乳糖基化）在GP1B-Alpha链上裸露的β-GLCNAC的损失 在这些条件下，血小板小鼠的循环。但是，血小板存储在等离子体中 在感冒中48小时引起进一步的变化，导致流通损失未被救出 半乳糖基化。我们现在假设血浆中长期冷却（等于或大于48h）的血小板导致 VWFR的“超聚集”。这增加了簇中半乳糖残基的密度 半乳糖基化的血小板使它们达到临界密度，现在导致清除 使用其ASIALOGLYCOPOR蛋白受体（ASGPR），肝细胞和/或巨噬细胞识别为识别 暴露的半乳糖。 GP1B-Alpha与Filamin A（FLNA）的基础肌动蛋白骨骼有关。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。 AIM 1将确定GP1B-Alpha是否参与了在血浆中储存48h的血小板以及是否参与 长期变化是由于血浆成分与血小板的结合所致。如果GP1B-Alpha是核心 变化，我们将研究FLNA和凝胶素以及基础肌动蛋白连接的作用 VWFR聚类。纯种液体的纯素数量，使用由造血驱动的矿石 已经建立了使用条件flNA的LOXP等位基因的小鼠中的特定启动子GATA1。我们 将确定缺乏FLNA或凝胶素的血小板在野生型（WT）小鼠中循环。我们假设 FLNA无效血小板将被清除。因此，这些结果将达到机制和重要性， 血小板生存和体内功能中的聚类（与Project 1-Wagner博士合作）。如果 缺乏FLNA循环的血小板，我们将确定它们是否无法将VWF受体聚集在寒冷中 在短期存储缓冲液或血浆中长期存储后。我们还将调查是否 长时间的冷血小板存储分离了GP1B-Alpha-FLNA/B复合物。 AIM 2将确定：（1）如果 溶解酶有助于冷藏（> 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-Sialic酸的受体蛋白； （3）确定哪些器官/细胞清除 半乳糖基化和/或siallated血小板（与项目3合作-Von Andrian博士）； （4） 评估修饰和冷藏血小板的体内功能（与项目1合作-Dr. 瓦格纳）; （5）在小鼠中建立人性化的适应性免疫系统，这将使我们能够评估 血小板修饰对体内冷冻的人血小板生存和功能的影响（在 与项目1和3 -DRS合作。瓦格纳和冯·安德里安）。该领域的研究已经 由于缺乏评估人血小板循环和功能的良好动物模型的限制 输血后。 AIM 3（如有必要 在血库条件下，在血浆中冷藏（> 48小时）以供去除并识别 介导去除的吞噬受体。这项工作的总体目标是确定新的 可以调节的目标以防止冷诱导的血小板清除率并开发方法 阻止他们。