Mechanism of cold platelet clearance

在这个项目的前5年中，我们定义了一个受体-计数器受体对， 将冰冻2-4小时的洗涤过的血小板从循环中移除。我们提出，寒冷会导致 GPIB/V/IX受体复合体(VWfR)聚集在冷却的血小板表面，带来暴露的β-N-乙酰 氨基葡萄糖(β-GlcNAc)残基在GP1b-α亚基的N-连接的糖链上，这 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(半乳糖化)在GP1b-阿尔法链上暴露的β-GlcNAc挽救 在这些条件下冷却的小鼠的血小板的循环。然而，血小板在血浆中的储存 在寒冷中停留48小时会引起进一步的变化，导致血液循环丧失，而不是通过 半乳糖化。我们现在假设，血小板在血浆中的长期低温(等于或大于48小时)会导致 VWFR的“超级集群”。这增加了簇中半乳糖残基的密度 半乳糖化的血小板，使它们达到临界密度，现在导致清除 肝细胞和/或巨噬细胞利用其去唾液酸糖蛋白受体(ASGPR)识别 暴露的半乳糖。GP1b-α通过细丝A(Flna)与潜在的肌动蛋白骨架相连。我们 先前的研究表明，低温可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-α是否参与去除储存在血浆中48小时的血小板，以及是否 长期的变化是由于血浆成分与血小板的结合。如果GP1b-α是 ，我们将研究Flna和明胶蛋白的作用以及潜在的肌动蛋白连接 VWfR群集。一种纯FLANA缺失型血小板的血液学研究 利用我们的条件loxP等位基因建立了小鼠的特异性启动子GATA1。我们 将确定缺乏Flna或明胶的血小板是否在野生型(WT)小鼠中循环。我们假定 将清除Fla空的血小板。因此，这些结果将触及机制和重要性， 研究聚集在血小板存活和体内功能中的作用(与项目1-Wagner博士合作)。如果 缺乏Flna的血小板可以循环，我们将确定它们是否在寒冷中无法聚集VWF受体 在缓冲液中短期存储或在等离子体中长期存储之后。我们还将调查是否 长时间的冷保存会使GP1b-α-Flna/b复合体解离。目标2将确定：(1)是否 唾液酸化促进冷藏(48小时)半乳糖化血小板存活；(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 半乳糖和/或唾液酸化血小板(与项目3--冯·安德里安博士合作)；(4) 评估改良和冷冻的血小板的体内功能(与项目1-DR合作)。 以及(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体内血小板存活和功能的影响 与项目1和3的合作--Wagner博士和von Andrian博士)。这方面的研究一直在进行 受限于缺乏良好的动物模型来评估人类血小板的循环和功能 在输血后。如有必要，目标3将确定小鼠血小板中GP1b-α的非依赖性变化 在血库条件下冷藏(&gt；48小时)以清除和识别为目标的血浆 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 可调节以防止冷诱导的血小板清除的靶点和开发方法学 挡住他们。

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.