Fucosylated Chondroitin Sulfates from the Sea Cucumbers Paracaudina chilensis and Holothuria hilla: Structures and Anticoagulant Activity

Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [→3)-β-d-GalNAc (N-acethyl galactosamine)-(1→4)-β-d-GlcA (glucuronic acid)-(1→]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1→2)-Fuc3S4S-(1→ at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.

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Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor

Thrombosis and Haemostasis ◽

10.1160/th06-06-0308 ◽

2007 ◽

Vol 97 (01) ◽

pp. 139-145

Author(s):

Mercedes López ◽

Goetz Nowak ◽

Thomas Bitter

Keyword(s):

Serine Proteases ◽

Anticoagulant Activity ◽

Tight Binding ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Coagulation Assays ◽

Competitive Kinetics ◽

Inhibition Of Thrombin

SummaryThe design of small chimeric thrombin inhibitors based on the structure of dipetalogastin II has been previously described. These proteins are effective inhibitors of thrombin showing slow binding or slow, tight-binding kinetics. We report here about dipetacompinR10H, a new dipetalogastin II-derived chimeric thrombin inhibitor, which exhibits classical competitive kinetics. The dissociation constant Ki of dipetacompinR10H was determined to be 17.1 ± 0.8 pM. In various coagulation assays it showed a comparable anticoagulant activity like r-hirudin and r-dipetalogastin II. DipetacompinR10H’s inhibition of thrombin was specific, since no inhibition of other serine proteases like factor Xa, plasmin, trypsin or chymotrypsin has been observed.

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Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646437 ◽

1991 ◽

Vol 66 (04) ◽

pp. 453-458 ◽

Cited By ~ 15

Author(s):

John T Brandt

Keyword(s):

Specific Activity ◽

Anticoagulant Activity ◽

Factor Xa ◽

Clinical Importance ◽

Potential Method ◽

Quantitative Expression ◽

Increased Risk ◽

Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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Enzymatic Synthesis and Characterization of Different Families of Chitooligosaccharides and Their Bioactive Properties

Applied Sciences ◽

10.3390/app11073212 ◽

2021 ◽

Vol 11 (7) ◽

pp. 3212

Author(s):

Noa Miguez ◽

Peter Kidibule ◽

Paloma Santos-Moriano ◽

Antonio O. Ballesteros ◽

Maria Fernandez-Lobato ◽

...

Keyword(s):

High Performance ◽

Chemical Characterization ◽

Amperometric Detection ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Enzymatic Production ◽

Bioactive Properties ◽

Substrate Properties ◽

Chitin Hydrolysis

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry, is critical for structure–function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharides.

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Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽

10.1182/blood.v63.4.927.bloodjournal634927 ◽

1984 ◽

Vol 63 (4) ◽

pp. 927-934

Author(s):

MJ Rabiet ◽

M Jandrot-Perrus ◽

JP Boissel ◽

J Elion ◽

F Josso

Keyword(s):

High Performance ◽

Fluorescence Enhancement ◽

Antithrombin Iii ◽

Factor Xa ◽

Competitive Inhibitor ◽

Amidolytic Activity ◽

Direct Inhibition ◽

High Affinity ◽

Factor Va

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

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Biological Characterization of a Pituitary Monkey Lutropin Preparation after Chromatofocusing or after High Performance Anion Exchange Chromatography

Journal of Liquid Chromatography ◽

10.1080/01483918808067171 ◽

1988 ◽

Vol 11 (6) ◽

pp. 1261-1271

Author(s):

Per Hallin ◽

Shafiq A. Khan

Keyword(s):

Anion Exchange ◽

High Performance ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Biological Characterization

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Novel anticoagulants based on direct inhibition of thrombin and factor Xa

Behavioural Pharmacology ◽

10.1097/00008877-199802000-00003 ◽

1998 ◽

Vol 9 (2) ◽

pp. 75???82

Author(s):

Stephen J. Gardell ◽

Philip E.J. Sanderson

Keyword(s):

Factor Xa ◽

Direct Inhibition ◽

Novel Anticoagulants ◽

Inhibition Of Thrombin

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Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽

10.1182/blood.v80.4.942.942 ◽

1992 ◽

Vol 80 (4) ◽

pp. 942-952 ◽

Cited By ~ 59

Author(s):

L Zhang ◽

A Jhingan ◽

FJ Castellino

Keyword(s):

Protein C ◽

Anticoagulant Activity ◽

Coagulation Factor ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Factor Ix ◽

Human Protein ◽

Complete Disappearance ◽

Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

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A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

10.1055/s-0039-1687438 ◽

1979 ◽

Author(s):

A.S. Bhargava ◽

J. Heinick ◽

Chr. Schöbel ◽

P. Günzel

Keyword(s):

Blood Clotting ◽

Anticoagulant Activity ◽

Factor Xa ◽

Thrombin Time ◽

Beagle Dogs ◽

Clotting Time ◽

Relative Potencies ◽

Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

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Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

Natural Product Communications ◽

10.1177/1934578x1801301232 ◽

2018 ◽

Vol 13 (12) ◽

pp. 1934578X1801301

Author(s):

Huiqin Wang ◽

Guanzhen Gao ◽

Lijing Ke ◽

Jianwu Zhou ◽

Pingfan Rao

Keyword(s):

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Scutellaria Baicalensis ◽

Dependent Manner ◽

Scutellaria Baicalensis Georgi ◽

Isolation And Characterization ◽

Pas Staining ◽

Ph Range ◽

Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

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