Carbohydrate-mediated platelet clearance

描述（由申请人提供）：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，唯一确定的影响血小板存活的机制是抗体介导的血小板清除、凝血反应消耗血小板和大出血导致的血小板损失。为了解决一个实际问题，即如何冷藏血小板供输血，我们定义了一种意想不到的基于碳水化合物的血小板清除机制，其中Kupffer细胞上的α - β 2受体识别短期（< 4h）储存血小板GPIba上的聚集性betaGlcNAc片段。我们现在已经取得了惊人的观察结果，缺乏唾液酸的小鼠血小板（冷藏48小时）或唾液转移酶STSGallV缺失小鼠的血小板在肝脏中被移除，主要是由肝细胞，而不是巨噬细胞。由于暴露的半乳糖残基在血小板循环时在表面增加，并且经凝集素结合实验显示，经过长期储存，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，半乳糖暴露的增加诱导了肝细胞上的亚洲糖蛋白受体（ASGPR或HL- 1/2）的识别。血小板修饰显著增强清除将用于确定唾液酸在血小板存活中的作用：1)ST3GallV无效血小板；2)酶解脱盐血小板；3)野生型血小板冷冻48小时。Aim 1将记录和描述肝细胞对长期储存的血小板、ST3GallV无效血小板和神经氨酸酶处理的血小板的实时摄取，量化它们在小鼠中的清除率，确定巨噬细胞清除的贡献（Aim 1A），并确定ASGP受体在启动摄取中的作用（Aim 1B）。我们将确定半乳糖基化和唾液酰化联合治疗是否会改善长期储存的血小板和st3galv -/-血小板的生存（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老化和清除（Aim 1D），以及重磷酸化是否可以提高生存率（Aim 1E）。因此，这些实验将建立一种新的老年性血小板清除机制。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中的功能，但还需要其他方法来确定人类血小板也可能遭受同样的命运。因此，在Aim 2中，我们建议利用HepG2细胞体外内吞脱氮血小板作为简单的读出（相对于输血研究）（Aim 2A）来定义宿主血小板糖蛋白的性质，这些糖蛋白携带被HL-1/2（肝细胞）（Aim 2B）或MGL1（巨噬细胞）受体（Aim 2D）识别的半乳糖片段。我们将进一步确定血小板重乙酰化是否能在体外阻止去乙酰化血小板的摄入（Aim 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).