Carbohydrate-Mediated Platelet Clearance

描述（申请人提供）：人体每天产生并清除1011个血小板，以维持正常稳态血小板计数，在血小板破坏的情况下，产生水平可大大增加。我们提供了血小板存活与表面聚糖密切相关的第一个证据，并表明具有受损Siaa 2 - 3Galb 1 -4GlcNAc（LacNAc）结构的血小板在肝脏中被枯否细胞和肝细胞清除。在这里，我们将继续研究这条道路，并将剖析如何调节靶向肝细胞或枯否细胞。目的1提出定义肝和巨噬细胞半乳糖结合受体在终止血小板循环中的不同作用，并将剖析O-和N-连接聚糖识别的贡献。我们将调查肝脏去唾液酸糖蛋白受体2缺陷小鼠的血小板糖蛋白 在一些实施方案中，血小板可以是具有多聚糖（Asgr 2）、巨噬细胞半乳糖凝集素（MGL）或两者的血小板，以限定Asgr和MGL的携带聚糖的血小板配体，因为这些血小板由于清除率降低而携带增加水平的去唾液酸化蛋白。此外，使用通常唾液酸化N-连接或0-连接聚糖的唾液酸转移酶3（ST 3）Gal-I或Gal-IV缺陷的小鼠将破译去唾液酸化的0-和N-连接聚糖作为这些半乳糖受体的配体的相对作用。去唾液酸化靶标的表征将揭示哪些在循环血液中被正常修饰。该数据 将与储存的血液中发现的进行比较。最后，我们将确定O-O-N-连接聚糖的去唾液酸化是否促进TACE释放GPIba。我们的工作进一步表明血小板，肝细胞和骨髓之间存在新的和持续的串扰。肝再生依赖于血小板向肝细胞输送因子和Asgr 1/2的表达。目的2将解决的作用，O-和N-连接的脱唾液酸聚糖在血小板摄取肝细胞和确定功能的后果，重点是它们如何影响肝细胞的稳态以及肝损伤后的重塑。此外，我们将开始研究肝细胞对聚糖介导的血小板摄取受损如何导致骨髓异常。我们将仔细确定Asgr 2-/-、ST 3GalIV-/-和WT骨髓（BM）中造血干细胞（HSC）和成骨细胞龛细胞和基质细胞的比例。在缺乏Asgr 1/2调节BM稳态的情况下系统信号改变的假设将得到巩固，我们将开始识别这些系统因素。

DESCRIPTION (provided by applicant): The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. We provided the first evidence that survival of platelets is intimately tied to surface glycans and have shown that platelets with impaired Siaa2-3Galb1-4GlcNAc (LacNAc) structures are removed in the liver by Kupffer cells and hepatocytes. Here, we continue to investigative this path and will dissect how targeting to hepatocytes or Kupffer cells is regulated. Aim 1 proposes to define the differential roles of hepatic and macrophage galactose- binding receptors in terminating platelet circulation and will dissect the contribution of O- and N-linked glycan recognition by each of these receptors. We will survey platelet glycoproteins from mice deficient for the hepatic asialoglycoprotein receptor2 (Asgr2), the macrophage galactose lectin (MGL), or both to define the glycan- bearing platelet ligands for the Asgr and MGL, as these platelets carry increased levels of desialylated proteins because of diminished clearance. Further, use of mice deficient in sialyltransferase 3 (ST3) Gal-I or Gal-IV that normally sialylate N-linked or O-linked glycans will decipher the relative roles o desialylated O- and N-linked glycans as ligands for these galactose receptors. Characterization of the desialylated targets will reveal which are normally modified in circulating blood. This data will be compared to those found in stored blood. Lastly, we will establish if desialylation of O- o N-linked glycans promotes GPIba release by TACE. Our work further suggests the existence of novel and continuous crosstalk between platelets, liver cells and bone marrow. Liver regeneration depends on the delivery of factors to hepatocytes by platelets and on the expression of the Asgr1/2. Aim 2 will address the role of O- and N-linked asialoglycans in platelet ingestion by hepatocytes and determine the functional consequences focusing on how they influence hepatocyte homeostasis as well as remodeling following liver injury. In addition we will begin to investigate how impaired glycan-mediated platelet uptake by hepatocytes leads to bone marrow abnormalities. We will carefully determine the proportion of hematopoietic stem cells (HSCs) and osteoblastic niche cells and stromal cells in Asgr2-/-, ST3GalIV-/- and WT bone marrows (BMs). The hypothesis that systemic signals altered in the absence of Asgr1/2 regulate BM homeostasis will be solidified, and we will begin to identify these systemic factors.