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.

Public Health Relevance

Low molecular weight heparin (LMWH) is the most widely form of heparin utilized as a blood thinner and anticoagulant. We are investigating the interaction of synthetic (custom-made) LMWH with liver endothelium Stabilin receptors, the site of heparin and LMWH elimination from the blood. It is hoped that synthetic heparin will eventually replace today's heparin and LMWHs, an animal-derived product.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Intercellular Interactions Study Section (ICI)
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Chang, Henry
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University of Nebraska Lincoln
Schools of Earth Sciences/Natur
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Miller, Colton M; Xu, Yongmei; Kudrna, Katrina M et al. (2018) 3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life. Thromb Res 167:80-87
Miller, Colton M; Wan, W Brad; Seth, Punit P et al. (2018) Endosomal Escape of Antisense Oligonucleotides Internalized by Stabilin Receptors Is Regulated by Rab5C and EEA1 During Endosomal Maturation. Nucleic Acid Ther 28:86-96
Bano, Fouzia; Tammi, Markku I; Kang, David W et al. (2018) Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins. Biophys J 114:2910-2922
Miller, Colton M; Tanowitz, Michael; Donner, Aaron J et al. (2018) Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver. Nucleic Acid Ther 28:119-127
Pirie, Elaine; Ray, Shayoni; Pan, Calvin et al. (2018) Mouse genome-wide association studies and systems genetics uncover the genetic architecture associated with hepatic pharmacokinetic and pharmacodynamic properties of a constrained ethyl antisense oligonucleotide targeting Malat1. PLoS Genet 14:e1007732
Gaus, Hans; Miller, Colton M; Seth, Punit P et al. (2018) Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor. Biochemistry 57:2061-2064
Cabral, Fatima; Miller, Colton M; Kudrna, Katrina M et al. (2018) Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion. J Vis Exp :
Baggenstoss, Bruce A; Harris, Edward N; Washburn, Jennifer L et al. (2017) Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif. Glycobiology 27:154-164
Xu, Yongmei; Chandarajoti, Kasemsiri; Zhang, Xing et al. (2017) Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins. Sci Transl Med 9:
Natarajan, Vaishaali; Harris, Edward N; Kidambi, Srivatsan (2017) SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis. Biomed Res Int 2017:4097205

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