Carbohydrate-mediated platelet clearance

这些研究的目的是确定肝细胞摄取血小板以加速血小板生成的途径。 把他们的通行证都取消了直到最近，唯一行之有效的机制， 血小板存活率分别为抗体介导的血小板清除率，凝血反应消耗血小板， 由于大量出血造成的损失。努力解决一个实际问题，如何使血小板 使我们确定了一种意外的基于碳水化合物的血小板清除机制， 的ccM| Kupffer细胞上的β 2受体识别短期（<4 h）GPIba上的成簇的pGlcNAc部分。 储存血小板。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（re-sal-acid） 在肝脏中去除血小板（主要是来自唾液酸转移酶STSGallV缺失小鼠的血小板）， 而不是巨噬细胞。因为暴露的半乳糖残基在表面上增加， 血小板循环，并且在凝集素结合实验揭示的长期储存后，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去唾液酸糖蛋白受体（ASGPR或HL- 1/2）。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)野生型血小板冷藏> 48小时。目标1将记录和描述长期 通过肝细胞真实的实时储存血小板、STSGallV无效血小板和神经氨酸酶处理的血小板， 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），以及 定义ASGP受体在启动摄取中的作用（目的1B）。我们将决定是否一个COM- 半乳糖基化和唾液酸化的结合将改善长期储存的血小板和STSGallV-/-血小板 存活率（目标1C）。我们还将研究血小板在循环过程中是否会丢失唾液酸，这一过程可能会导致 有助于血小板老化和清除（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。而 活体实验应证明HL-1/2在冷冻小鼠血小板的清除中的功能， 在体内，其他方法是必要的，以建立人血小板可以遭受相同的命运。在目标2中， 因此，我们建议利用体外HepG 2细胞对去唾液酸化血小板的内吞作用作为一种简单的 读数（相对于输血研究）（目的2A），以确定宿主血小板糖蛋白的性质， 使HL-1/2（肝细胞）（Aim 2B）或MGL 1（巨噬细胞）识别的半乳糖部分重新结合， 感受器（Aim 2D）。我们将进一步确定血小板再唾液酸化是否能阻止去唾液酸化的 体外血小板（Aim 2C）。

The goal of these studies is to determine the pathway(s) by which hepatocytes ingest platelets to acceler- ate 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 reac- tions 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 ccM|}2 receptor on Kupffer cells recognized clustered pGlcNAc moieties on GPIba of short-term (< 4h) stored platelets. We have now made the striking observation that mouse platelets lacking sialic acid (re- frigerated for 48h or platelets from sialyl-tranferase 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, STSGallV 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 com- bination of galactosylation and sialylation will improve long-term stored platelet and STSGallV-/- 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 car- rying the galactose moieties recognized by HL-1/2 (hepatocytes) (Aim 2B) or MGL1 (macrophages) re- ceptors (Aim 2D). We will further determine if platelet resialylation prevents the ingestion of desialylated platelets in vitro (Aim 2C).