Carbohydrate-mediated platelet clearance

描述(申请人提供)：这些研究的目标是确定肝细胞摄取血小板以加速其从循环中清除的途径(S)。直到最近，影响血小板存活的唯一机制是抗体介导的血小板清除、凝血反应对血小板的消耗以及大量出血造成的损失。为了解决一个实际问题，如何冷藏供输血的血小板，导致我们定义了一种基于碳水化合物的血小板清除机制，在这种机制中，Kupffer细胞上的AlphaM-beta 2受体识别短期(&lt；4h)储存的血小板GPIba上聚集的BetaGlcNAc部分。我们现在已经做出了惊人的观察，缺乏唾液酸的小鼠血小板(冷冻48小时)或来自唾液酸转移酶STSGallV基因缺失的小鼠的血小板被从肝脏中移除，主要是由肝细胞，而不是巨噬细胞。由于暴露的半乳糖残基随着血小板的循环而增加，并且经过凝集素结合实验揭示的长期储存，我们假设唾液酸通常覆盖半乳糖残基并允许血小板存活。我们进一步假设，增加半乳糖暴露诱导肝细胞上的去唾液酸糖蛋白受体(ASGPR或HL-1/2)识别。可显著提高清除能力的血小板修饰将被用来确定唾液酸在血小板存活中的作用：1)ST3GallV缺失的血小板；2)酶解去溶的血小板；以及3)冷冻48小时的野生型血小板。目的1将实时记录和表征肝细胞对长期储存的血小板、ST3GallV缺失的血小板和神经氨酸酶处理的血小板的摄取，量化它们在小鼠体内的清除率，确定巨噬细胞清除的贡献(目标1A)，并确定ASGP受体(S)在起始摄取中的作用(目标1B)。我们将确定半乳糖化和唾液酸化的组合是否会改善长期储存的血小板和ST3GallV-/-血小板的存活率(Aim 1C)。我们还将研究血小板在循环过程中是否会失去唾液酸，这一过程可能有助于血小板的老化和清除(目标1D)，以及抗凝是否可以提高存活率(目标1E)。因此，这些实验将建立一种新的衰老血小板清除机制。虽然活体实验应该证明HL-1/2在体内清除冷冻小鼠血小板的功能，但其他方法也是必要的，以确定人类血小板也会遭受同样的命运。因此，在目标2中，我们建议利用HepG2细胞的体外内吞作用作为一个简单的读数(相对于输血研究)(目标2A)来定义宿主血小板糖蛋白的性质，该蛋白携带由HL-1/2(肝细胞)(目标2B)或MGL1(巨噬细胞)受体(目标2D)识别的半乳糖部分。我们将进一步确定，在体外，血小板再溶解是否能阻止所需血小板的摄取(目标2C)。

DESCRIPTION (provided by applicant): The goal of these studies is to determine the pathway(s) by which hepatocytes ingest platelets to accelerate their clearance from the circulation. Until recently, the only well-established mechanisms affecting platelet survival were antibody-mediated platelet clearance, consumption of platelets by coagulation reactions and loss due to massive bleeding. An effort to address a practical problem, how to refrigerate platelets for transfusion, led us to define an unsuspected carbohydrate-based platelet clearance mechanism where the alphaM-beta 2 receptor on Kupffer cells recognized clustered betaGlcNAc moieties on GPIba of short-term (< 4h) stored platelets. We have now made the striking observation that mouse platelets lacking sialic acid (refrigerated for 48h or platelets from sialyl-transferase STSGallV null mice are removed in the liver, principally by hepatocytes, not macrophages. Because exposed galactose residues increase on the surface as platelets circulate, and after long-term storage as revealed by lectin binding experiments, we postulate that sialic acid normally covers the galactose residues and permits platelet survival. We further hypothesize that increases in galactose exposure induce recognition by the Asialoglycoprotein receptor (ASGPR or HL- 1/2) on hepatocytes. Platelet modifications that markedly enhance clearance will be used to determine the role of sialic acid in platelet survival: 1) ST3GallV null platelets; 2) enzymatically desialylated platelets; and 3) wild type platelets refrigerated for > 48h. Aim 1 will document and characterize the uptake of long-term stored platelets, ST3GallV null platelets and neuraminidase-treated platelets by hepatocytes in real time, quantify their clearance rates in mice, determine the contribution of macrophage clearance (Aim 1A), and define the role of the ASGP receptor(s) in initiating ingestion (Aim 1B). We will determine whether a combination of galactosylation and sialylation will improve long-term stored platelet and ST3GallV-/- platelet survival (Aim 1C). We will also investigate if platelets lose sialic acid while circulating, a process that could contribute to platelet aging and clearance (Aim 1D), and whether resialylation can improve survival (Aim 1E). These experiments will, therefore, establish a new clearance mechanism for senile platelets. While the intravital experiments should demonstrate HL-1/2 function in the clearance of chilled murine platelets in vivo, other approaches are necessary to establish that human platelets can suffer the same fate. In Aim 2, therefore, we propose to utilize the in vitro endocytosis of desialylated platelets by HepG2 cells as a simple readout (relative to transfusion studies) (Aim 2A) to define the nature of the host platelet glycoproteins carrying the galactose moieties recognized by HL-1/2 (hepatocytes) (Aim 2B) or MGL1 (macrophages) receptors (Aim 2D). We will further determine if platelet resialylation prevents the ingestion of desialylated platelets in vitro (Aim 2C).