Erratum to "Rigor and reproducibility instruction in academic medical libraries," 2022;110(3):281-93.

[This corrects the article DOI: 10.5195/jmla.2022.1443.].

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Introduction: Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Methods: Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Results: Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Conclusion: Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Heparan sulfates and heparan sulfate binding proteins in sepsis.

Heparan sulfates (HSs) are the main components in the glycocalyx which covers endothelial cells and modulates vascular homeostasis through interactions with multiple Heparan sulfate binding proteins (HSBPs). During sepsis, heparanase increases and induces HS shedding. The process causes glycocalyx degradation, exacerbating inflammation and coagulation in sepsis. The circulating heparan sulfate fragments may serve as a host defense system by neutralizing dysregulated Heparan sulfate binding proteins or pro-inflammatory molecules in certain circumstances. Understanding heparan sulfates and heparan sulfate binding proteins in health and sepsis is critical to decipher the dysregulated host response in sepsis and advance drug development. In this review, we will overview the current understanding of HS in glycocalyx under septic condition and the dysfunctional heparan sulfate binding proteins as potential drug targets, particularly, high mobility group box 1 (HMGB1) and histones. Moreover, several drug candidates based on heparan sulfates or related to heparan sulfates, such as heparanase inhibitors or heparin-binding protein (HBP), will be discussed regarding their recent advances. By applying chemical or chemoenzymatic approaches, the structure-function relationship between heparan sulfates and heparan sulfate binding proteins is recently revealed with structurally defined heparan sulfates. Such homogenous heparan sulfates may further facilitate the investigation of the role of heparan sulfates in sepsis and the development of carbohydrate-based therapy.

Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis.

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Investigation of the pharmacokinetic properties of synthetic heparan sulfate oligosaccharides.

Heparan sulfate (HS) is a sulfated polysaccharide with a wide range of biological activities. There is an increasing interest in the development of structurally homogeneous HS oligosaccharides as therapeutics. However, the factors influencing the pharmacokinetic properties of HS-based therapeutics remain unknown. Here, we report the pharmacokinetic properties of a panel of dodecasaccharides (12-mers) with varying sulfation patterns in healthy mice and uncover the pharmacokinetic properties of an octadecasaccharide (18-mer) in acutely injured mice. In the 12-mer panel, 1 12-mer, known as dekaparin, is anticoagulant, and 3 12-mers are nonanticoagulant. The concentrations of 12-mers in plasma and urine were determined by the disaccharide analysis using liquid chromatography coupled with tandem mass spectrometry. We observed a striking difference between anticoagulant and nonanticoagulant oligosaccharides in the 12-mer panel, showing that anticoagulant dekaparin had a 4.6-fold to 8.6-fold slower clearance and 4.4-fold to 8-fold higher plasma exposure compared to nonanticoagulant 12-mers. We also observed that the clearance of HS oligosaccharides is impacted by disease. Using an antiinflammatory 18-mer, we discovered that the clearance of 18-mer is reduced 2.8-fold in a liver failure mouse model compared to healthy mice. Our results suggest that oligosaccharides are rapidly cleared renally if they have low interaction with circulating proteins. We observed that the clearance rate of oligosaccharides is inversely associated with the degree of binding to target proteins, which can vary in response to pathophysiological conditions. Our findings uncover a contributing factor for the plasma and renal clearance of oligosaccharides which will aid the development of HS-based therapeutics.

Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Analysis of 3-O-Sulfated Heparan Sulfate Using Isotopically Labeled Oligosaccharide Calibrants.

The 3-O-sulfated glucosamine in heparan sulfate (HS) is a low-abundance structural component, but it is a key saccharide unit for the biological activities of HS. A method to determine the level of 3-O-sulfated HS is lacking. Here, we describe a LC-MS/MS based method to analyze the structural motifs. We determined the levels of 3-O-sulfated structural motifs from pharmaceutical heparin manufactured from bovine, porcine, and ovine. We discovered that saccharide chains carrying 3-O-sulfation from enoxaparin, an FDA-approved low-molecular weight heparin, displayed a slower clearance rate than non-3-O-sulfated sugar chains in a mouse model. Lastly, we detected the 3-O-sulfated HS from human brain. Furthermore, we found that a specific 3-O-sulfated structural motif, tetra-1, is elevated in the brain HS from Alzheimer's disease patients (n = 5, p = 0.0020). Our method offers a practical solution to measure 3-O-sulfated HS from biological sources with the sensitivity and quantitative capability.

Errata for Issue 110 (3).

[This corrects the article DOI: 10.5195/jmla.2022.1443.][This corrects the article DOI: 10.5195/jmla.2022.1447.][This corrects the article DOI: 10.5195/jmla.2022.1579.].

Improving the Sensitivity for Quantifying Heparan Sulfate from Biological Samples.

Heparan sulfates (HSs) are widely expressed glycans in the animal kingdom. HS plays a role in regulating cell differentiation/proliferation, embryonic development, blood coagulation, inflammatory response, and viral infection. The amount of HS and its structural information are critically important for investigating the functions of HS in vivo. A sensitive and reliable quantitative technique for the analysis of HS from biological samples is under development. Here, we report a new labeling reagent for HS disaccharides analysis, 6-amino-N-(2-diethylamino)ethyl quinoline-2-carboamide (AMQC). The AMQC-conjugated disaccharides are analyzed by LC-MS/MS in positive mode, significantly improving the sensitivity. The use of AMQC coupled with authentic 13C-labeled HS disaccharide internal standards empowered us to determine the amount and the disaccharide composition of the HS on a single histological slide. We used this method to profile the levels of HS in the plasma/serum and tissues/organs to assist the disease prognosis in two animal models, including the acetaminophen (APAP)-induced acute liver injury mouse model and the burn injury mouse model. The method may uncover the roles of HS contributing to the diseases as well as provide a potential new set of biomarkers for disease diagnosis and prognosis.

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Investigation of the biological functions of heparan sulfate using a chemoenzymatic synthetic approach.

Heparan sulfate (HS) is a highly sulfated polysaccharide playing essential physiological and pathophysiological roles in the animal kingdom. Heparin, a highly sulfated form of HS, is a widely used anticoagulant drug. Isolated from biological sources, both heparin and HS are polysaccharide mixtures with different sugar chain lengths and sulfation patterns. Structural heterogeneity of HS complicates the investigation of HS-related biological activities. The availability of structurally defined HS oligosaccharides is critical in understanding the contribution of saccharide structures to the functions. The chemoenzymatic synthetic approach is emerging as a cost-effective method to synthesize HS oligosaccharides. Structurally defined oligosaccharides are now widely available for biologists. This review summarizes our efforts in using this new synthetic method to develop new anticoagulant therapeutics and discover the role of HS to protect liver damage under pathological conditions. The synthetic method also allows us to prepare reference saccharide standards to improve structural analysis of HS.

Potential Use of Anti-Inflammatory Synthetic Heparan Sulfate to Attenuate Liver Damage.

Heparan sulfate is a highly sulfated polysaccharide abundant on the surface of hepatocytes and surrounding extracellular matrix. Emerging evidence demonstrates that heparan sulfate plays an important role in neutralizing the activities of proinflammatory damage associate molecular patterns (DAMPs) that are released from hepatocytes under pathological conditions. Unlike proteins and nucleic acids, isolation of homogenous heparan sulfate polysaccharides from biological sources is not possible, adding difficulty to study the functional role of heparan sulfate. Recent advancement in the development of a chemoenzymatic approach allows production of a large number of structurally defined oligosaccharides. These oligosaccharides are used to probe the physiological functions of heparan sulfate in liver damage under different pathological conditions. The findings provide a potential new therapeutic agent to treat liver diseases that are associated with excessive inflammation.

Synthetic anticoagulant heparan sulfate attenuates liver ischemia reperfusion injury.

Heparan sulfate (HS) is a sulfated glycosaminoglycan abundant on the cell surface and in the extracellular matrix and has several biological activities including anticoagulation and anti-inflammation. Liver ischemia reperfusion injury is associated with coagulation and inflammatory responses. Here, we synthesized HS oligosaccharides with defined sulfation patterns and show that synthetic anticoagulant HS oligosaccharides limit liver ischemia reperfusion injury in a mouse model. Using a small targeted HS library, we demonstrate that an oligosaccharide that possesses both anticoagulant activity and binding affinity to HMGB1, the inflammatory target, decreases injury greater than oligosaccharides that only bind to HMGB1 or only have anticoagulant activity. HS oligosaccharides may represent a potential new therapeutic option for decreasing liver damage resulting from ischemia reperfusion injury.

Quantitative analysis of heparan sulfate using isotopically labeled calibrants.

Heparan sulfate is a sulfated polysaccharide that displays essential physiological functions. Here, we report a LC-MS/MS-based method for quantitatively determining the individual disaccharide composition and total amount of heparan sulfate. Using eight 13C-labeled disaccharide calibrants and one 13C-labeled polysaccharide calibrant, we complete the analysis in one-pot process. The method is both sensitive and has the throughput to analyze heparan sulfate from mouse tissues and plasma.

Using heparin molecules to manage COVID-2019.

The coronavirus disease 2019 (COVID-19) pandemic is becoming one of the largest global public health crises in modern history. The race for an effective drug to prevent or treat the infection is the highest priority among health care providers, government officials, and the pharmaceutical industry. Recent evidence reports that the use of low-molecular-weight heparin reduces mortality in patients with severe coronavirus with coagulopathy. Although the full scope of the benefits from heparin for COVID-19 patients is unfolding, encouraging clinical data suggest that heparin-like molecules may represent a useful approach to treat or prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The intent of this article is to offer our opinions on the mechanism(s) by which heparin may attenuate the course of SARS-CoV-2 infection. Furthermore, we propose a novel strategy to treat or prevent SARS-CoV-2 infection using "designer" heparin molecules that are fabricated using a synthetic biology approach.

Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure.

Acetaminophen/paracetamol (APAP) overdose is the leading cause of drug-induced acute liver failure (ALF) in the United States and Europe. The progression of the disease is attributed to sterile inflammation induced by the release of high mobility group box 1 (HMGB1) and the interaction with receptor for advanced glycation end products (RAGE). A specific, effective, and safe approach to neutralize the proinflammatory activity of HMGB1 is highly desirable. Here, we found that a heparan sulfate (HS) octadecasaccharide (18-mer-HP or hepatoprotective 18-mer) displays potent hepatoprotection by targeting the HMGB1/RAGE axis. Endogenous HS proteoglycan, syndecan-1, is shed in response to APAP overdose in mice and humans. Furthermore, purified syndecan-1, but not syndecan-1 core protein, binds to HMGB1, suggesting that HMGB1 binds to HS polysaccharide side chains of syndecan-1. Last, we compared the protection effect between 18-mer-HP and N-acetyl cysteine, which is the standard of care to treat APAP overdose. We demonstrated that 18-mer-HP administered 3 hours after a lethal dose of APAP is fully protective; however, the treatment of N-acetyl cysteine loses protection. Therefore, 18-mer-HP may offer a potential therapeutic advantage over N-acetyl cysteine for late-presenting patients. Synthetic HS provides a potential approach for the treatment of APAP-induced ALF.

Using structurally defined oligosaccharides to understand the interactions between proteins and heparan sulfate.

Heparan sulfate (HS) is widely present on the animal cell surface and in the extracellular matrix. HS achieves its biological functions by interacting with proteins to change proteins' conformation, oligomerization state and cellular location. The challenging question to study HS is how to dissect the relationship between the structures of HS and the biological activities. In the past several years, crucial techniques have been developed to overcome this challenge. A novel chemoenzymatic method to synthesize structurally defined HS oligosaccharides has offered a key access to this class of sulfated carbohydrate molecules. Recent rapid progress of HS microarray technology allows screening of the interaction of a target protein with a large number of HS oligosaccharides. The improved availability of HS oligosaccharides and HS microarray analysis will undoubtedly accelerate the investigation of the contribution of the specific sulfated carbohydrate structures of HS in a wide range of biological contexts.

Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards.

Enoxaparin is a low-molecular weight heparin used to treat thrombotic disorders. Following the fatal contamination of the heparin supply chain in 2007-2008, the U.S. Pharmacopeia (USP) and U.S. Food and Drug Administration (FDA) have worked extensively to modernize the unfractionated heparin and enoxaparin monographs. As a result, the determination of molecular weight (MW) has been added to the monograph as a measure to strengthen the quality testing and to increase the protection of the global supply of this life-saving drug. The current USP calibrant materials used for enoxaparin MW determination are composed of a mixture of oligosaccharides; however, they are difficult to reproduce as the calibrants have ill-defined structures due to the heterogeneity of the heparin parent material. To address this issue, we describe a promising approach consisting of a predictive computational model built from a library of chemoenzymatically synthesized heparin oligosaccharides for enoxaparin MW determination. Here, we demonstrate that this test can be performed with greater efficiency by coupling synthetic oligosaccharides with the power of computational modeling. Our approach is expected to improve the MW measurement for enoxaparin.