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)

Thromboelastography (TEG) has been used increasingly as an intraoperative hemostasis monitoring device. Low-molecular-weight heparins are given increasingly to reduce the development of antibodies against the heparin-platelet factor 4 complex, and heparinoids are given to patients who have developed the antibody. We studied the effect of unfractionated heparin, a low-molecular-weight heparin (enoxaparin sodium [Lovenox]), and a heparinoid (danaparoid sodium [Orgaran]) on blood clotting assayed with TEG (TEG clotting) in vitro and the efficacy of protamine sulfate and heparinase for reversing the effect. Heparin, enoxaparin, and danaparoid all caused a dose-dependent inhibition of TEG clotting of normal blood. Concentrations of enoxaparin and danaparoid that totally inhibited TEG clotting only minimally prolonged the activated partial thromboplastin time. While inhibition of TEG clotting by heparin and enoxaparin was reversed by protamine sulfate and heparinase, inhibition by danaparoid was reversed only by heparinase. Abnormal TEG clotting was observed in patients receiving enoxaparin whose plasma level of the drug was more than 0.1 antiXa U/mL. However, the degree of TEG abnormality did not always coincide with plasma levels of the drug.
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PMID:Effects of unfractionated heparin, low-molecular-weight heparin, and heparinoid on thromboelastographic assay of blood coagulation. 1080 Apr 6

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.
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PMID:Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I. 1119 Sep 4

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

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.
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PMID:Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88). 1129 91

Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.
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PMID:Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site. 1525 29

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

To investigate the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and danaparoid (DPD) added to whole blood in vitro on standard and heparinase-modified thromboelastogram (TEG) parameters compared with conventional assays of coagulation. The effects of UFH, LMWH and DPD on standard TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-activated factor X (anti-FXa) activity, at concentrations of these anticoagulants ranging from 0.025 to 1 U/ml. In the second part of the study, the effects of very low concentrations (0.005-0.05 U/ml) of UFH, LMWH and DPD on the difference between standard and heparinase-modified TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-FXa activity. Standard TEG parameters were outside the reference range at lower concentrations of UFH, LMWH and DPD than most conventional coagulation assays were able to detect. Only anti-FXa activity was more sensitive to the presence of these anticoagulants than the standard TEG alone. The lowest concentration of UFH, LMWH and DPD used in this study (0.005 U/ml) caused significant differences between the standard and heparinase-modified alpha-angles of the TEG. In addition, the difference between standard and heparinase-modified TEG parameters distinguished between low concentrations (0.005-0.05 U/ml) of UFH with greater sensitivity than anti-FXa activity, but were less sensitive to LMWH and DPD. The standard TEG is more sensitive to UFH, LMWH and DPD than most conventional coagulation tests, with the exception of anti-FXa activity. Calculation of the difference between standard and heparinase-modified TEG parameters greatly increases the sensitivity of the assay for the effects of these anticoagulants, and is more sensitive to very low quantities of UFH than anti-FXa activity.
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PMID:The effects of unfractionated heparin, low molecular weight heparin and danaparoid on the thromboelastogram (TEG): an in-vitro comparison of standard and heparinase-modified TEGs with conventional coagulation assays. 1647 91

The currently available brand-name low-molecular-weight heparins (LMWHs) in the United States include dalteparin (Pfizer), enoxaparin (Aventis), and tinzaparin (Pharmion). Other products available, in Europe, include certoparin (Novartis), reviparin (Abbott), nadroparin (GlaxoSmithkline), and parnaparin (Alpha-Wasserman). Each of these LMWHs has a characteristic molecular weight profile and biological activity in terms of an anti-FXa and anti-FIIa potency. The mean molecular weight of these drugs ranges from 4.0 kDa to 7.0 kDa and the anti-FXa:anti-FIIa ratio ranges from 1.5 to 3.5. These agents may also be characterized by the presence of specific chemical end groups such as 2-O-sulfo-4-enepyranosuronic acid at the nonreducing terminus (enoxaparin) or 2,5-anhydro-D-mannose at the reducing terminus (dalteparin). Further, the component oligosaccharide chains exhibit product-specific distribution profiles. It is now widely accepted that individual LMWHs are chemically unique agents and cannot be interchanged therapeutically. Each commercial LMWH has been individually developed for specific clinical indications, which are dose and product dependent. Recently, several generic LMWHs have become available in India (Cutenox and Markaparin) and South America (dilutol, clenox, dripanina), and three companies have filed for regulatory approval of a generic version of enoxaparin in the United States. As the primary aim of a generic drug is to reduce cost without compromising patient care, a generic drug is required to be chemically and biologically equivalent to the pioneer drug. Because LMWHs represent complex natural mucopolysaccharide drugs that have undergone chemical and enzymatic modifications, physicochemical and biological information in addition to molecular weight and anti-FXa:anti-FIIa ratio should be used to determine generic equivalency to the branded drug. We have utilized a previously reported approach to systematically compare three generic versions of enoxaparin obtained from India and Brazil with the branded enoxaparin (Lovenox) available in the United States. Testing included molecular and structural profiling, evaluation in clot-based and amidolytic anti-FXa and anti-FIIa assays, and heparinase-I digestion profiles. While the molecular profiles (4.8 +/- 1.8 kD) and anticoagulant potencies as determined by activated partial thromboplastin time (APTT) were comparable for all four agents, the generic products showed variations in the thrombin time (TT) and Heptest assays. Two generic and the branded enoxaparin were readily digested by heparinase-I, losing most of their anticoagulant activity, but one generic product resisted digestion. This may have been due to a unique structural feature in this product. These studies show that, while generic LMWHs may exhibit acceptable molecular weight and anti-FXa profiles, they can exhibit assay-based differences and digestion profiles. Testing in animal models to determine safety, efficacy, and pharmacodynamic parameters may be important to verify equivalence. In order to assure that the generic LMWHs are equivalent to branded LMWHs such that equivalent clinical results are obtained, there is a need to develop clear stepwise guidelines that will establish equivalency in terms of physical, chemical, biochemical, pharmacokinetic, and pharmacodynamic properties for these anticoagulant drugs.
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PMID:Product individuality of commercially available low-molecular-weight heparins and their generic versions: therapeutic implications. 1695 78

Endogenous heparinoids impair coagulation, evidenced by thrombelastography in cirrhotic patients with bacterial infection, but it is not clear which glycosaminoglycans can be detected by native and heparinase-modified thrombelastography. To assess the effects of different glycosaminoglycans on thrombelastography parameters and the reversibility of these effects by heparinase-I-modified thrombelastography. Twenty volunteers were enrolled. Solutions of heparan sulphate, dermatan sulphate, and chondroitin-4-sulphate were prepared at 'equivalent' concentrations, based on the composition and anticoagulant activity of danaparoid. Serial dilutions of each glycosaminoglycan were prepared to achieve 1.0, 0.5, 0.1, and 0.05 U/ml. Native and heparinase-modified thrombelastography, anti-activated factor X activity and heparin cofactor II activity were evaluated at each concentration. A statistically significant heparin-like effect was seen with 1 and 0.5 U/ml heparan sulphate, and 1 and 0.5 U/ml dermatan sulphate, which was completely reversed by heparinase-modified thrombelastography. Anti-activated factor X activity was significantly increased in samples containing heparan and dermatan sulphates. The heparin cofactor II activity decreased with 1.0 and 0.5 U/ml dermatan sulphate and chondroitin-4-sulphate, but not with heparan sulphate. Heparan and dermatan sulphates affect haemostasis when added to whole blood in vitro, detectable by native thrombelastography and completely reversed by heparinase-I-modified thrombelastography. They may therefore be responsible for the heparin-like effect seen by thrombelastography in patients with cirrhosis and bacterial infection.
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PMID:The effects of glycosaminoglycans on coagulation: a thromboelastographic study. 1741 58

Glycosaminoglycans in normal human plasma, mainly represented by chondroitin sulfates and heparan sulfates/heparin (HSGAGs), show a specific distribution in the Cohn-Oncley fractions of human plasma. In the present study we investigated their effects on coagulation. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated for extraction of glycosaminoglycans after extensive proteolysis; the anticoagulant activity in the extracted samples was measured by activated partial thromboplastin time (APTT). The effects of the samples containing HSGAGs on factor II and factor X activities, before and after treatment with heparinase I, were also measured. The molecular weight of HSGAGs was determined by polyacrylamide gel-electrophoresis. Cryoprecipitate and fraction I, fraction II+III, and fraction IV-1 (the fractions containing HSGAGs) prolonged the APTT, whereas fractions IV-4 and V had no effect on the APTT. Fractions containing HSGAGs showed effects on factor II and factor X activities that were sensitive to heparinase I treatment. The molecular weight of HSGAGs recovered in cryoprecipitate and fraction I was 15-18 kDa; that of HSGAGs recovered in fraction IV-1 was 12.0 kDa. In conclusion, these results demonstrate that HSGAGs of different molecular weight, endowed with anticoagulant activity, circulate in normal human plasma in association with specific proteins involved in the regulation of hemostasis; and that endogenous HSGAGs play a role in maintaining the antithrombotic/hemostatic balance in normal human plasma.
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PMID:Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits. 1860 81


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