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Thrombosis is the main cause of many cardiovascular diseases. The conventional pharmaceuticals used for preventing thrombosis can induce significant adverse side effects, especially bleeding risk. Hence, the development of more effective and safe antithrombotic agents is strongly demanded to improve clinical treatment. In order to rapidly screen and identify novel antithrombotic substances from natural marine products, a multi-target screening strategy is reported to quantify the antithrombotic activity with high-performance liquid chromatography – mass spectrometry (HPLC-MS). Five key thrombosis-related targets, including thrombin, coagulation factors Xa, XIa and XIIa and platelet phosphodiesterase 3, were selected to establish the multi-target antithrombotic activity screening assay. The Michaelis-Menten constants (Km) were 8.16 µM, 58.85 µM, 120.03 µM, 35.24 µM and 79.67 µM, respectively. Five known inhibitors, namely argatroban, rivaroxaban, benzamidine, 3-carboxamide-coumarinand, and milrinone, were used for validation. The reproducibility of the screening strategy was verified by calculating the overall average Z′ value, which was 0.88. Subsequently, this method was assessed by screening a small marine library of 78 compounds. Five coumarin compounds were selected, and their antithrombotic activities were further validated in standard assessments of enzyme inhibition and on the coagulometer. According to the validation, the multi-target assay based on dried blood spots and HPLC-MS was suitable for screening antithrombotic substances with considerable reliability. [ABSTRACT FROM AUTHOR]

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1. [ABSTRACT FROM AUTHOR]

An accurate, rapid, and cost-effective methodology for enzyme assay is highly demanded to screen the effect of compounds on target at the molecular level. Thrombin (EC 3.4.21.5) and factor Xa (FXa, EC 3.4.21.6) have been identified as the critical targets for the development of potential drugs with anticoagulant activity. In this study, a rapid, sensitive and accurate assay based on UHPLC-MS/MS method has been developed for inhibitor screening against thrombin and factor Xa simultaneously. For thrombin and factor Xa, the Michaelis-Menten constants ( K m ) were calculated to be 6.14 and 57.27 μM, respectively. The inhibition constants ( K i ) for two known inhibitors, argatroban and rivaroxaban, were determined to be 16.23 and 0.41 nM, respectively. The assay was further validated through the determination of a high Z′ factor value of 0.89. Finally, the developed assay was applied to screen a chemical library against two enzymes. Three hit compounds belonging to a class of sulfated polysaccharides were identified and their targets of inhibition action were further evaluated. The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents. [ABSTRACT FROM AUTHOR]

Abstract: Fifteen steroids were isolated from the soft coral Dendronephthya sp., of which five are determined as new compounds, namely (22E)-3-O-β-formylcholest-5,22-diene (1), (22E)-3-O-β-formyl-24-methyl-cholest-5,22-diene (2), 2-ethoxycarbonyl-2-β-hydroxy-A-nor-cholest-5-ene-4-one (3), (22E)-2-ethoxycarbonyl-2-β-hydroxy-A-nor-cholest-5,22-diene-4-one (4), and (22E)-2-ethoxycarbonyl-2-β-hydroxy-24-mthyl-A-nor-cholest-5,22-diene-4- one (5). 1 and 2 belonged to 3-O-formylated cholesterol analogues, and 3 to 5 are unique ring A-contracted steroids. Their structures were elucidated by extensive 2D NMR in association with IR, MS analysis. [Copyright &y& Elsevier]

Bacterial exopolysaccharides (EPSs) are widely applied in food, cosmetic, and medical industries. The EPS produced by Phyllobacterium sp. 921F was a novel polysaccharide, which exhibits attractive characteristics of high yield, favorable rheological properties, and excellent moisture retention ability. Considering the complexity of polysaccharide structures, specific enzymatic hydrolysis was employed here to resolve the structure of the EPS. End-products including tetra-, hexa- and octa-saccharides were isolated. According to their mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectra, the backbone of the EPS was found to be mainly comprising a → 4)-β- d -Glc p -(1 → 3)-α- d -Gal p (4,6-S-Pyr)-(1 → disaccharide repeating units. Based on atomic force microscopy results, EPS exhibited characteristics that were consistent with a stiff, elongated molecule with no branches. The length and height of the single molecular chain were approximately 600 and 0.7 nm, respectively. Our clarification of structure and molecular morphology of EPS from Phyllobacterium sp. 921F provide a foundation for the industrial application of this potential moisture-retaining material. • Structure of the exopolysaccharide from Phyllobacterium sp. 921F was investigated. • Three oligosaccharides were isolated and resolved by MS and NMR. • Repeating unit was composed by glucose, galactose and pyruvate in a ratio of 1:1:1. • The morphology of single molecular chain and aggregates was observed by AFM. [ABSTRACT FROM AUTHOR]

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with 0,2A- and 2,5A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Zint-ions (Zint − 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [2,5Ared]/[0,4Ared] (red: denotes reducing terminal). [Copyright &y& Elsevier]