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Non-anticoagulant biological functions of heparin-based drugs have drawn increasing attention. However, the exploration into the non-anticoagulant activities of various low molecular weight heparins was associated with bleeding risks in clinical practice and often led to controversial conclusions due to the structural differences. In this study, we aimed to establish a process to produce a library of heparin derivatives with structural diversity and reduced/abolished anticoagulant activity through the combination of chemical modifications and enzymatic cleavage of heparins. The depolymerization characteristics of various selectively modified heparin derivatives by three heparinases were comprehensively analyzed. The order of periodate treatment and heparinase-I depolymerization was proved to significantly change the structural characteristics of the oligosaccharide products. Finally, among several heparin derivatives that screened in the bleomycin-induced cell apoptosis model, the low molecular weight partially 6-O-/N-desulfated heparins showed the strongest anti-apoptotic activities. This study provided a useful approach for future development of novel heparin-derivative medications., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.

Heparin is a polysaccharide anticoagulant drug isolated from animal tissues. There have been concerns on the safety and security of the heparin supply chain since 2007-8 when a contamination crisis led to its disruption. The current study applies a suite of modern analytical techniques to porcine, bovine and ovine intestinal mucosal heparins. These techniques include structural analysis by nuclear magnetic resonance spectrometry, disaccharide compositional analysis, bottom-up analysis of tetrasaccharides corresponding to heparin's antithrombin III binding site. Chemometric analysis was then applied to understand how these structural differences to predict the animal/tissue source of heparin and to help detect blending of heparins from various sources., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. However, heparin-induced thrombocytopenia and thrombosis (HITT) is a serious side effect of heparin therapy, resulting in relatively high risk of amputation and even death. HITT is caused by forming of complexes between heparin and platelet factor 4 (PF4). Enoxaparin, one of the most commonly used low molecular weight heparin (LMWH), were developed in 1980's. The lower molecular weight of enoxaparin reduces the risk of HITT by binding to less PF4. To detect the binding capacity between enoxaparin and PF4 could be an effect way to control this risk before it goes to patients. In this work, a size exclusion chromatography (SEC) method was developed to analyze the patterns of complexes formed between PF4 and enoxaparin. The chromatographic condition was optimized to separate PF4, enoxaparin, ultra-large complexes and small complexes. The linearity and stability of this method were confirmed. The impacts of PF4/enoxaparin mixture ratios and incubation time on the forming complexes were investigated. Four enoxaparin samples were analyzed with this method to verify its practicability. It is a robust, accurate and practicable method, and provides an easy way to monitor the capacity of enoxaparin forming complexes with PF4, suggesting the HITT related quality of enoxaparin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fucoidans are complex sulfated polysaccharides extracted from brown algae. Depending on the concentration, they have been shown to stimulate and inhibit blood coagulation in vitro. Promotion of coagulation is mediated by blocking tissue factor pathway inhibitor (TFPI). We screened fucoidan extracts from four brown algae species in vitro with respect to their potential to improve coagulation in bleeding disorders. The fucoidans' pro- and anticoagulant activities were assessed by global hemostatic and standard clotting assays. Results showed that fucoidans improved coagulation parameters. Some fucoidans also activated the contact pathway of coagulation, an undesired property reported for sulfated glycosaminoglycans. Chemical evaluation of fucoidans' complex and variable structure included molecular weight (Mw), polydispersity (polyD), structural heterogeneity, and organic and inorganic impurities. Herewith, we describe a screening strategy that facilitates the identification of crude fucoidan extracts with desired biological and structural properties for improvement of compromised coagulation like in hemophilia., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field (1)H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by (1)H-NMR to assess the utility of (1)H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field (1)H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5-10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, (13)C NMR, and (1)H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.

Heparin is unique as one of the oldest drugs currently still in widespread clinical use as an anticoagulant, a natural product, one of the first biopolymeric drugs, and one of the few carbohydrate drugs. Recently, certain batches of heparin have been associated with anaphylactoid-type reactions, some leading to hypotension and death. These reactions were traced to contamination with a semi-synthetic oversulfated chondroitin sulfate (OSCS). This Highlight reviews the heparin contamination crisis, its resolution, and the lessons learned. Pharmaceutical scientists now must consider dozens of natural and synthetic heparinoids as potential heparin contaminants. Effective assays, which can detect both known and unknown contaminants, are required to monitor the quality of heparin. Safer and better-regulated processes are needed for heparin production.

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.

Heparin, a widely used anticoagulant, is being rapidly displaced by low-molecular-weight heparins. Recently, certain lots of heparin have been associated with anaphylactoid-type reactions resulting from contamination with oversulfated chondroitin sulfate. This impurity has also contaminated low-molecular-weight heparins obtained by chemical and enzymatic depolymerization of heparin. The sensitivity of oversulfated chondroitin sulfate to five different depolymerization processes similar to ones used in preparing low-molecular-weight heparins is reported.

RATIONALE: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products. [ABSTRACT FROM AUTHOR]

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two fractions (A and C) of GAGs from porcine jejunum and duodenum were purified. • A is a heparan sulfate, and C is composed of β- d -GlcA-(1 → 4) and β- d -GalNAc-(1 → 3). • They possessed controllable anti-coagulant activity in vitro. • Additional sulfate modification enhanced the effect. To make full use of the porcine sources and develop better choice of novel GAGs as anti-coagulants, two fractions of GAGs from the porcine jejunum (A) and duodenum (C) have been separated & purified. The products were further sulfated to give B and D in order to test the influence of sulfate pattern on the bioactivity. The results showed that the relative molecular weight range of A was 3000–50,000 (Mw, g/mol), whereas C had an average molecular weight of 75,885 (Mw, g/mol). A was identified as a novel heparan sulfate through enzymatic hydrolysis analysis. C was a chondroitin like polysaccharide mainly composed of β - d -GlcA-(1 → 4) and β - d -GalNAc-(1 → 3). A possessed controllable anti-coagulant activity (7 IU/mg) in vitro. The activity of D almost achieved the same magnitude of A. This study demonstrated the anticoagulant potential of the polysaccharides, providing solid foundation for development of anti-coagulants from porcine intestine. [ABSTRACT FROM AUTHOR]

Low-molecular-weight polyguluronate sulfate (LPGS) was prepared as a heparinoid by chemical sulfation of polyguluronic acid (PG), which was isolated from the hydrolysate of alginate by pH-fractionated precipitation. The structure of LPGS was elucidated based on FT-IR and 2D NMR spectroscopy. Sulfation was demonstrated to occur at the C-2 and C-3 positions of the guluronic acid residues and the degree of substitution (DS) was 1.53 per monosaccharide residue. The weight-averaged molecular weight of LPGS was 11.4kDa determined by high performance gel permeation chromatography (HPGPC). The in vitro anticoagulant activity and in vivo anti-inflammatory activity of LPGS were evaluated. Results showed that LPGS significantly prolonged the whole blood clotting time and activated partial thrombinplastin time (APTT), and exhibited considerable anti-inflammatory activity in cotton pellet-induced granuloma in rats. [Copyright &y& Elsevier]