Liver-Mediated Clearance of Low Molecular Weight Heparins

肝脏介导的低分子量肝素清除率

基本信息

批准号：
9241420
负责人：
EDWARD N HARRIS
金额：
$ 36.32万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-15 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9241420
关键词：
Affect Affinity Affinity Labels Animal Sources Animals Anticoagulants Binding Binding Sites Biochemical Biological Biological Assay Biological Availability Biological Process Blood Blood Circulation Blood Coagulation Factor Blood coagulation Cell Line Cells Coagulants Coagulation Process Complement Custom Data Degradation Pathway Deletion Mutagenesis Dialysis procedure Effectiveness Endocytosis Endothelium Enzymes Factor Xa Family suidae Foundations Future Goals Half-Life Heparin Heparin Binding Human Human Cell Line Individual Injection of therapeutic agent Kidney Kinetics Knock-out Knockout Mice Knowledge Label Length Libraries Ligands Liver Low-Molecular-Weight Heparin Maps Mass Spectrum Analysis Measures Mediating Metabolic Metabolic Clearance Rate Methodology Methods Modification Molecular Molecular Weight Monitor Monosaccharides Mus Mutagenesis Oligosaccharides Operative Surgical Procedures Patients Physiological Polymers Primates Procedures Production Property Proteins Rattus Recombinants Research Rodent Rodent Model Route Safety Site Source Structure Sulfides System Technology Testing Therapeutic Thrombosis Unspecified or Sulfate Ion Sulfates Urine Vesicle affinity labeling analog base blood filter cofactor experimental study heparin receptor high risk insight mouse model physiologic model prevent public health relevance receptor stable cell line subcutaneous success sugar sulfation trafficking

项目摘要

DESCRIPTION (provided by applicant): Porcine-derived heparins are among the most commonly used anticoagulant drugs in the world and are needed for invasive surgery, dialysis, thrombosis/blood clotting treatments, and numerous other procedures that involve the handling of blood. The effectiveness of heparin is dependent on the half-life in blood (clearance rate) and its interactions with the coagulant factors. It was recently discovered that the Stabilin receptors in the sinusoidal endothelium of liver are the major clearance receptors for unfractionated (UFH) and low-molecular weight (LMWH) heparins along with other ligands that maintain clean blood and healthy circulation. We hypothesize that the clearance rates of heparin are mediated by the Stabilin receptors and directly attributable to specific sulfations along the heparin polymer and length of the polymer. All of the enzymes involved with heparin modification have been cloned resulting in the production of custom-made heparin or de novo low molecular weight heparin (dnLMWH). 35S-labeled UFH, LMWH and homogenous dnLMWH will be tested for binding and clearance in biochemical, cell biological and animal physiological experiments in the Harris lab. We have confirmed that the minimal polymer length of 10 sugars is required and 3-O sulfation is optimized for cellular endocytosis in recombinant cell lines. In this proposal, we will first measure affinity constants (KD) using purified ectodomains from both human Stabilin-1 and Stabilin-2 and probe the heparin binding site(s) by 1) deletion mutagenesis and 2) using an affinity label and analyzed by mass spectrometry. Secondly, using both primary cells from fresh rat livers and recombinant cells, we will determine how the dnLMWHs are degraded and the vesicle trafficking of both Stabilin receptors and cargo to complement the blood clearance data. This information will give us an understanding of the degradative pathway for heparins and how these heparins are presented to blood and kidney from liver endothelium. Lastly, we will perform rodent experiments to determine whole body clearance rates (in mice), distribution within the liver, and the bioactivity of each dnLMWHs through monitoring Factor Xa activity. We will test homogenous dnLMWHs in Stab1/-2 KO mice to a) confirm that bulk clearance is mediated by the Stab receptors, b) monitor clearance rates in the absence of individual and both Stab receptors, and c) physiological binding profiles for each receptor. Our goals are to determine which heparin modification(s) are essential for fast and slow systemic clearance. This research may provide the means to produce custom heparin for specific patient applications, without the safety concerns of today's porcine-derived heparin batches.

描述（由申请人提供）：猪源性肝素是世界上最常用的抗凝血药物之一，用于侵入性手术、透析、血栓形成/凝血治疗以及涉及血液处理的许多其他程序。肝素的作用取决于血液中的半衰期（清除率）及其与凝血因子的相互作用。最近发现，稳定素受体。肝窦内皮细胞中的肝素是普通 (UFH) 和低分子量 (LMWH) 肝素以及其他维持清洁血液和健康循环的配体的主要清除受体。我们发现肝素的清除率是由稳定剂介导的。受体并直接归因于沿着肝素聚合物和聚合物长度的特定硫酸化，所有与肝素修饰有关的酶都已被克隆，从而产生定制肝素或从头低分子量肝素 (dnLMWH) 35S 标记的 UFH、LMWH 和均质 dnLMWH 将在 Harris 实验室进行生化、细胞生物学和动物生理实验的结合和清除测试。需要 10 个糖的最小聚合物长度，并且针对重组细胞系中的细胞内吞作用优化了 3-O 硫酸化。在本提案中，我们将首先测量亲和力。使用来自人 Stabilin-1 和 Stabilin-2 的纯化胞外域确定常数 (KD)，并通过 1) 缺失诱变和 2) 使用亲和标记并通过质谱分析来探测肝素结合位点。从新鲜大鼠肝脏和重组细胞中，我们将确定 dnLMWH 如何降解以及稳定蛋白受体和货物的囊泡运输，以补充血液清除数据。这些信息将使我们了解肝素的降解途径以及这些肝素如何从肝内皮呈递到血液和肾脏。最后，我们将进行啮齿动物实验以确定全身清除率（在小鼠中）、肝脏内的分布和。通过监测因子 Xa 活性，我们将在 Stab1/-2 KO 小鼠中测试同质 dnLMWH，以 a) 确认大量清除是由 Stab 受体介导的。在缺乏单个和两种 Stab 受体的情况下监测清除率，以及 c) 每种受体的生理结合特征。我们的目标是确定哪些肝素对于修饰快速和缓慢的全身清除至关重要。为特定患者应用生产定制肝素，无需担心当今猪源肝素批次的安全问题。