Carbohydrate-Mediated Platelet Clearance

描述(申请人提供)：人体每天产生和取出1011个血小板，以维持正常的稳态血小板计数，在血小板破坏的条件下，可以大大提高产生水平。我们提供了第一个证据，表明血小板的存活与表面多糖密切相关，并表明具有受损的Siaa2-3Galb1-4GlcNAc(LacNAc)结构的血小板可被Kupffer细胞和肝细胞从肝脏中移除。在这里，我们将继续研究这一途径，并将剖析靶向肝细胞或Kupffer细胞的调控方式。目的1确定肝脏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，并将剖析每种受体对O-和N-连接的糖链识别的贡献。我们将检测缺乏肝脏去唾液酸糖蛋白受体2的小鼠的血小板糖蛋白 (Asgr2)，巨噬细胞半乳糖凝集素(MGL)，或两者兼而有之，以定义AsgR和MGL的含糖血小板配体，因为这些血小板携带由于清除力降低而增加的去解离蛋白水平。此外，利用唾液酸基转移酶3(ST3)Gal-I或Gal-IV缺陷的小鼠通常唾液酸化N-连接或O-连接的多糖，将破译期望分析的O-和N-连接的多糖作为这些半乳糖受体的配体的相对作用。对所需目标的表征将揭示哪些目标在循环血液中通常是被修饰的。此数据 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-连接的糖链的渴望是否能促进TACE释放GPIba。我们的工作进一步表明，在血小板、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Asgr1/2的表达。目标2将阐述O-和N-连接的去唾液酸在肝细胞摄取血小板中的作用，并确定其功能后果，重点是它们如何影响肝细胞的动态平衡和肝损伤后的重塑。此外，我们将开始研究肝细胞糖链介导的血小板摄取受损是如何导致骨髓异常的。我们将仔细测定Asgr2-/-、ST3GalIV-/-和WT骨髓(BMS)中造血干细胞(HSCs)、成骨细胞和基质细胞的比例。在缺乏Asgr1/2的情况下，全身信号改变调节骨髓内环境平衡的假说将得到证实，我们将开始识别这些系统性因素。

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.