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Abstract: The novel low-molecular-weight chitosan polysulfate (MW 5120–26,200Da) 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, 13C NMR, and 1H 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. [Copyright &y& Elsevier]

Highlights • 1H-NMR of heparins from different animals show distinct clusters in PCA and k means analysis. • Disaccharides components of heparins from different animals show distinct clusters in PCA and k means. • Tetrasaccharides components of heparins from different animals show distinct clusters in PCA and k means. • Chemometric analysis could help predict the heparin animal source and detect blending. Abstract 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. [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]

Abstract: 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. [Copyright &y& Elsevier]

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]