Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dermatan sulphate does not catalyse the inactivation of factor Xa. However, the low molecular weight (LMW) dermatan sulphate Desmin 370 has been shown to generate circulating anti-Xa activity following administration to humans. Using a single batch of Desmin 370, we measured 3 U/mg of anti-Xa activity by amidolytic assay in vitro. The material responsible for this activity had a lower molecular weight range (6000 and 1800 Da) than Desmin 370 and was more highly sulphated than the bulk of the drug. Heparinase digestion of Desmin 370 eliminated 90% of the in vitro anti-Xa activity without significantly interfering with its ability to potentiate inactivation of thrombin by HCII, suggesting that the anti-Xa activity is not due to dermatan sulphate and is probably heparin. When 125I-labelled Desmin 370 together with 40 mg/kg carrier drug was administered intravenously to a rabbit, anti-Xa activity was readily detectable in the plasma for up to 10 h and had a longer half-life than the sulphated radiolabel. Most of this anticoagulant activity was recovered from the plasma by Polybrene affinity chromatography and was probably a sulphated glycosaminoglycan. Administration of the heparinase-digested drug to a rabbit resulted in 70% less anti-Xa activity than the undigested drug. We conclude that Desmin 370 contains detectable quantities of biologically active low molecular weight heparin, which is responsible for persistent anti-Xa activity following intravenous administration.
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PMID:Low molecular weight heparin is responsible for the anti-Xa activity of Desmin 370. 881 78

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.
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PMID:Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites. 894 54

We investigated the effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa-tissue factor activity by antithrombin III (ATIII) on a human bladder carcinoma (J82) cell line and an ovarian carcinoma (OC-2008) cell line, two tumor cell lines which constitutively synthesize and express high levels of cell surface tissue factor. We observed that ATIII inactivated factor VIIa-tissue factor more readily on OC-2008 cells than on J82 cells in the absence of added heparin. Likewise, factor Xa was more effectively inactivated on OC-2008 cells than on J82 cells. The ability of ATIII to inactivate factor VIIa-tissue factor activity on the OC-2008 cell was reduced following treatment of the cells with heparinase. This indicated that heparin-like GAGs were expressed on the OC-2008 cell surface, and that these GAGs were important for the inhibition of factor VIIa-tissue factor activity by ATIII. In addition, we demonstrated that the ability of ATIII to inactivate factor VIIa-tissue factor activity was markedly reduced following treatment of cells with calcium ionophore (A23187). However, the effect of cell surface GAGs on the inhibition of factor Xa by ATIII remained even after treatment of OC-2008 cells with A23187. In contrast to the manner of inhibition by ATIII/heparin, TFPI effectively inactivated factor VIIa-tissue factor activity on the cell surfaces even after induced physical damage or disruption of the cell by treatment with A23187. Our collective findings suggest that GAGs on cell surfaces play an important role in regulating factor VIIa-tissue factor activity by ATIII under normal conditions, or in the early phases of physical damage or destruction of the cell. However, TFPI may play a more important role than ATIII in regulating the activity of factor VIIa-tissue factor in a vascular trauma site following extensive cell injury.
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PMID:The effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa--tissue factor activity by antithrombin III. 971 70

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.
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PMID:Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis. 1043 45

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).
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PMID:A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana. 1070 86

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 IU/mg) and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase I. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540, graded concentrations (1.25-100 microg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was employed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37 degrees C) at 1 mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide. SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.
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PMID:Synthetic heparin pentasaccharide depolymerization by heparinase I: molecular and biological implications. 1119 Sep 7

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.
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PMID:Structural characterization of human liver heparan sulfate. 1565 73

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.
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PMID:Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion. 2005 99

The objective was to identify the usefulness of heparin level by anti-factor Xa (anti-Xa) assay vs activated partial thromboplastin time (PTT) or activated clotting time (ACT) in neonates undergoing extracorporeal membrane oxygenation (ECMO). A retrospective record review of 21 patients in the neonatal intensive care unit (mean ECMO initiation age, 2 days; range, 0-4 days; male/female ratio, 1:1) undergoing ECMO from 2006 to 2008 was performed. Linear regression correlations between anti-Xa, PTT, and ACT were determined by extrapolating PTT and ACT therapeutic ranges that corresponded with the ECMO heparin target range of 0.3 to 0.6 U/mL. Pearson correlation coefficients between heparin levels and PTT (-0.903 to 0.984), PTT less than 40 seconds after correction using PTT-heparinase (-0.903 to 1.000), and ACT (-0.951 to 0.891) in this patient population were widely variable. Inconsistency of PTT and ACT therapeutic ranges corresponding to heparin levels of 0.3 to 0.6 U/mL prompts a multifactorial approach to ECMO management because no single laboratory test can be used to determine appropriate anticoagulation management.
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PMID:Evaluation of heparin assay for coagulation management in newborns undergoing ECMO. 2108 59

There is need for a rapid assay to determine the efficacy of low-molecular-weight-heparin (LMWH) in whole blood. Heparinase was used to eliminate, and thereby quantify, the anticoagulant activity of the low-molecular-weight-heparin, enoxaparin. The percent change in the clotting time of whole blood in the presence of heparinase yielded the anticoagulant contribution of enoxaparin. A minimally activated assay (MAA) of whole blood clotting time was evaluated for the detection and relative quantification of enoxaparin. The assay cartridge consisted of a plain glass tube and detection magnet, with no additional sources of activation. Comparisons were also made with a point-of-care, activated partial thromboplastin time (aPTT) assay. Plasma or whole blood was spiked with enoxaparin at concentrations ranging from 0.1 to 1.0 anti-factor Xa IU/ml. A commercial preparation of heparinase I was used to digest enoxaparin, and clotting times were determined with and without heparinase incubation. Heparinase digestion caused an average shortening of clotting time of 21.1% (47.3 s) in blood spiked with 0.4 anti-Xa IU/ml enoxaparin, an amount expected in the therapeutic range; also, 0.1 anti-Xa IU/ml of enoxaparin could be reliably detected. The assay performed comparably when transferred to a point-of-care setting with heparinase being added directly to citrated blood collection tubes, followed by either MAA or aPTT assay. Strong correlations were obtained with both assays between the percent change in clotting time (after heparinase) and the added concentration of enoxaparin, or in comparison with the chromogenically measured concentration of enoxaparin. The assays for an individual blood sample can be completed within 10 min.
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PMID:Toward development of a point-of-care assay of enoxaparin anticoagulant activity in whole blood. 2121 19


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