Liver-Mediated Clearance of Low Molecular Weight Heparins

肝脏介导的低分子量肝素清除率

• 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
• 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

 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药物之一，并且需要用于侵入性手术、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。肝素的有效性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。最近发现稳定蛋白受体 在肝窦内皮中，肝素是普通肝素（UFH）和低分子量肝素（LMWH）沿着维持清洁血液和健康循环的其它配体的主要清除受体。我们假设肝素的清除率由稳定蛋白受体介导，并直接归因于沿肝素聚合物和聚合物长度的特定硫酸化沿着。所有参与肝素修饰的酶都已被克隆，从而产生定制肝素或从头低分子量肝素（dnLMWH）。将在Harris实验室的生物化学、细胞生物学和动物生理学实验中检测35 S标记的UFH、LMWH和均质dnLMWH的结合和清除。我们已经证实，需要10个糖的最小聚合物长度，并且3-O硫酸化对于重组细胞系中的细胞内吞作用是优化的。在该提议中，我们将首先使用来自人稳定蛋白-1和稳定蛋白-2的纯化胞外域测量亲和常数（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。其次，使用来自新鲜大鼠肝脏的原代细胞和重组细胞，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，以通过监测因子Xa活性来确定全身清除率（小鼠中）、肝脏内的分布以及每种dnLMWH的生物活性。我们将在Stab 1/-2 KO小鼠中检测同质dnLMWH，以a）确认总体清除率由Sta B受体介导，B）监测不存在单个和两种Sta B受体时的清除率，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修饰对快速和缓慢的全身清除至关重要。这项研究可以提供生产用于特定患者应用的定制肝素的方法，而没有当今猪源性肝素批次的安全性问题。