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3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life.

ABSTRACT: INTRODUCTION:Heparins are common blood anticoagulants that are critical for many surgical and biomedical procedures used in modern medicine. In contrast to natural heparin derived from porcine gut mucosa, synthetic heparins are homogenous by mass, polymer length, and chemistry. MATERIALS & METHODS:Stable cell lines expressing the human and mouse Stabilin receptors were used to evaluate endocytosis of natural and synthetic heparin. We chemoenzymatically produced synthetic heparin consisting of 12 sugars (dodecamers) containing 14 sulfate groups resulting in a non-3-O sulfated structure (n12mer). Half of the n12mer was modified with a 3-O sulfate on a single GlcNS sugar producing the 3-O sulfated heparin (12mer). Wildtype (WT), Stabilin-1 knock-out (KO), and Stabilin-2 KO C57BL/6 mice were developed and used for metabolic studies and provided as a source for primary liver sinusoidal endothelial cells. RESULTS & CONCLUSIONS:Human and mouse Stabilin-2 receptors had very similar endocytosis rates of both the 12mer and n12mer, suggesting that they are functionally similar in primary cells. Subcutaneous injections of the n12mer and 12mer revealed that the 12mer had a much longer half-life in circulation and a higher accumulation in liver. The n12mer never accumulated in circulation and was readily excreted by the kidneys before liver accumulation could occur. Liver sinusoidal endothelial cells from the Stabilin-2 KO mice had lower uptake rates for both dodecamers, whereas, the Stabilin-1 KO mice had lower endocytosis rates for the 12mer than the n12mer. 3-O sulfation of heparin is correlated to both a longer circulatory half-life and hepatotropism which is largely performed by the Stabilin receptors.

SUBMITTER: Miller CM

PROVIDER: S-EPMC6242777 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life.

Miller Colton M CM Xu Yongmei Y Kudrna Katrina M KM Hass Blake E BE Kellar Brianna M BM Egger Andrew W AW Liu Jian J Harris Edward N EN

Thrombosis research 20180517

<h4>Introduction</h4>Heparins are common blood anticoagulants that are critical for many surgical and biomedical procedures used in modern medicine. In contrast to natural heparin derived from porcine gut mucosa, synthetic heparins are homogenous by mass, polymer length, and chemistry.<h4>Materials & methods</h4>Stable cell lines expressing the human and mouse Stabilin receptors were used to evaluate endocytosis of natural and synthetic heparin. We chemoenzymatically produced synthetic heparin c ...[more]

PMID: 29793137

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Project description:The structure of heparin and heparan sulfate (Hep/HS) oligosaccharides, as determined by the length and the pattern of sulfation, acetylation, and uronic acid epimerization, dictates their biological function through modulating interactions with protein targets. But fine structural determination is a very challenging task due to the lability of the sulfate modifications and difficulties in separating isomeric HS chains. Previously, we reported a strategy for chemical derivatization involving permethylation, desulfation, and trideuteroperacetylation, combined with standard reverse phase LC-MS/MS that enables the structural sequencing for heparin/HS oligosaccharides of sizes up to dodecasaccharide by positionally replacing all sulfates with more stable trideuteroacetyl groups, allowing for robust MS/MS sequencing. However, isomeric oligosaccharides that contain both N-sulfation and N-acetylation become isotopomers after labeling, differing only in the sites of deuteration. This prevents chromatographic separation of these different mixed domain sequences post-derivatization, and makes sequencing by MS/MS difficult due to co-fragmentation of the isotopomers leading to chimeric product ion spectra. In order to improve chromatographic separation of mixed domain oligosaccharides, we have introduced a propionylation step in place of trideuteroacetylation for labeling of sites of sulfation. HS standard disaccharides have been used to evaluate the efficiency of this improved chemical derivatization. The results show that we can quantitatively replace sulfation with propionyl groups with the same high efficiency as the previously reported trideuteroacetylation. After derivatization, we demonstrate the ability to chromatographically separate two mixed domain tetrasaccharide isomers differing solely by the order of N-sulfation and N-acetylation, allowing for full sequencing of each by MS/MS. These results represent a marked improvement in the ability of our previously reported derivatization strategy to analyze complex mixtures of Hep/HS oligosaccharides without a decrease in sensitivity.

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3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life.

Publications

3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life.

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