Carbohydrate-mediated platelet clearance

描述（由申请方提供）：这些研究的目的是确定肝细胞摄取血小板以加速其从循环中清除的途径。直到最近，影响血小板存活的唯一公认机制是抗体介导的血小板清除、凝血反应引起的血小板消耗和大出血引起的血小板丢失。为了解决一个实际问题，即如何将血小板用于输血，我们定义了一种未被怀疑的基于碳水化合物的血小板清除机制，其中枯否细胞上的α M-β 2受体识别短期（<4小时）储存血小板的GPIba上的成簇β GlcNAc部分。我们现在已经做出了惊人的观察，即缺乏唾液酸的小鼠血小板（冷藏48小时）或来自唾液酸转移酶STSGallV缺失小鼠的血小板在肝脏中主要被肝细胞而不是巨噬细胞去除。由于暴露的半乳糖残基增加的表面血小板循环，并在长期储存后，凝集素结合实验所揭示的，我们假设，唾液酸通常覆盖的半乳糖残基，并允许血小板存活。我们进一步假设，半乳糖暴露的增加诱导肝细胞上的去唾液酸糖蛋白受体（ASGPR或HL- 1/2）的识别。显著增强清除率的血小板修饰将用于确定唾液酸在血小板存活中的作用：1）ST 3GallV无效血小板; 2）酶促去唾液酸化血小板;和3）冷藏> 48小时的野生型血小板。目的1将记录和表征肝细胞对长期储存的血小板、ST 3 GallV无效血小板和神经氨酸酶处理的血小板的真实的摄取，定量其在小鼠中的清除率，确定巨噬细胞清除的贡献（目的1A），并定义ASGP受体在启动摄取中的作用（目的1B）。我们将确定半乳糖基化和唾液酸化的组合是否会改善长期储存的血小板和ST 3GallV-/-血小板存活率（目的1C）。我们还将研究血小板在循环过程中是否会失去唾液酸，这一过程可能导致血小板老化和清除（Aim 1D），以及再唾液酸化是否可以改善生存率（Aim 1 E）。因此，这些实验将建立一种新的老年血小板清除机制。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，但需要其他方法来确定人血小板可能遭受相同的命运。因此，在目标2中，我们建议利用HepG 2细胞对脱唾液酸化血小板的体外内吞作用作为简单的读数（相对于输血研究）（目标2A），以确定携带HL-1/2（肝细胞）（目标2B）或MGL 1（巨噬细胞）受体（目标2D）识别的半乳糖部分的宿主血小板糖蛋白的性质。我们将进一步确定血小板再唾液酸化是否会阻止体外去唾液酸化血小板的摄入（目的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).