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)

The purpose of this study was to determine the prevalence and effect of heparin contamination in samples drawn from implanted venous access devices (VADs, ports) on factor VIII activity and Bethesda inhibitor assay (BU) and the efficacy of heparinase to neutralize heparin. Plasma samples containing 85, 45, and 2 U/dL factor VIII were spiked in vitro with heparin from 0 to 3 U/mL. Factor VIII activity was assayed with a one-stage clotting assay on paired samples before and after heparinase, 25 mg/mL plasma. Paired patient samples drawn from VADs were assayed for heparin concentration, factor VIII, and BU before and after heparinase. At all three concentrations of factor VIII in vitro, the addition of heparin at 0.12 to 0.25 U/mL decreased assayed factor VIII activity. Heparinase neutralized up to 2 U/mL heparin and resulted in accurate factor VIII determination. Of 105 VAD samples, 47 (45%) had heparin contamination >0.05 U/mL. Of 47 heparin-contaminated samples, 42 showed decreased factor VIII activity in before/after comparisons. False-positive BU results were detected in 6 of 47 heparin-contaminated samples. Heparin contamination occurs frequently in samples drawn from VADs and could increase costs through excessive factor concentrate use. We recommend that all VAD samples be pretreated with heparinase before the assay of factor VIII activity or Bethesda inhibitor titers.
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PMID:Heparin neutralization is essential for accurate measurement of factor VIII activity and inhibitor assays in blood samples drawn from implanted venous access devices. 1088 30

Because the subcutaneous absorption of heparin depends on its molecular weight, high performance size exclusion chromatography methods, among others, have been developed for its determination. Because heparin consists of polymer chains of varying lengths, a large number of calibrators, 19 in our work, are required for these molecular weight analyses. Because the preparation of that many calibrants is both time-consuming and expensive, other methods requiring fewer calibrants are being sought. In pursuit of these aims, a heparinase-degraded heparin has been accepted as the first international reference preparation low molecular weight heparin for molecular weight calibration. We have previously proposed another calibrant, the Heparin Mass Calibrator. In this article, we introduce another calibrant, called the Heparin Molecular Mass Calibrant. It offers the advantage of a much wider availability because it is a commercial product. In addition to having a superior refractive index chromatography run profile, when used in combination with two Narrow Range Calibrators with molecular weights of 34.4 and 18.4 kDa, this new calibrant covers both the higher and the lower molecular weight ranges. Besides, ultraviolet detection is not needed for molecular weight calculations. Therefore, it is proposed that its feasibility as a universal reference calibrator for both heparins and low molecular weight heparins be considered.
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PMID:Isoamyl nitrite depolymerized heparin as a universal calibrator for heparins and low molecular weight heparins. 1089 78

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.
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PMID:Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin. 1098 27

The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determined ex vivo is a valid measure of the FVII activity in vivo. We measured plasma FVII:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conducted in vitro experiments to investigate the effect of unfractionated heparin and TFPI, which is released from the endothelium by heparin, on FVII:C, FVIIa, and FVII:Ag. Heparin infusion decreased triglycerides and increased FFA and TFPI. This was accompanied by significant reductions in FVIIa, FVII:C and FVII:Ag. In vitro, anti-TFPI antibodies increased FVIIa and FVII:C, and heparin reduced FVIIa. The heparinase Hepzyme was unable to abolish the effect of heparin. There were no in vitro effects on FVII:Ag. We conclude that, due to interference by TFPI and heparin in post-heparin plasma, it is impossible to measure the in vivo FVII activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effects ex vivo. The observed effect of heparin on FVII:Ag should be investigated further.
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PMID:In vitro effects of heparin and tissue factor pathway inhibitor on factor VII assays. possible implications for measurements in vivo after heparin therapy. 1113 52

Monocyte macrophages (Mphi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to Mphi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of, various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca(2+)/Mg(2+) enhanced DV binding. This argues against involvement of beta(2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains.
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PMID:Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains. 1116 46

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

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma. In vivo, HHV-8 DNA and transcripts have been detected in B cells, endothelial cells, macrophages, and epithelial cells. HHV-8 infects a variety of cell lines of human and animal origin, leading to latent or abortive infection. This study shows that the broad cellular tropism of HHV-8 may be in part due to its interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). This conclusion is based on the following findings: (i) HHV-8 infection of human foreskin fibroblast (HFF) cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan closely related to HS. Chondroitin sulfates A and C did not inhibit HHV-8 infection. (ii) Enzymatic removal of HFF cell surface HS with heparinase I and III reduced HHV-8 infection. (iii) Soluble heparin inhibited the binding of radiolabeled HHV-8 to human B cell lines, embryonic kidney epithelial (293) cells, and HFF cells, suggesting interference at the virus attachment stage. (iv) Cell surface adsorbed HHV-8 was displaced by soluble heparin. (v) Radiolabeled HHV-8 also bound to wild-type HS expressing Chinese hamster ovary (CHO-K1) cells. In contrast, binding of virus to mutant CHO cells deficient in HS was significantly reduced. These data show that the gamma2 herpesvirus HHV-8, similar to some members of alpha, beta, and gamma2 herpesviruses, adsorbs to cells by binding to cell surface HS-like moieties. Heparin did not completely prevent the binding and infectivity of HHV-8, suggesting that HHV-8 interactions with HS could be the first set of ligand-receptor interaction leading to the binding with one or more host cell receptors essential for the subsequent viral entry process.
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PMID:Human herpesvirus 8 interaction with target cells involves heparan sulfate. 1128 7

Human cytomegalovirus (HCMV) is known to down-regulate the expression of human leukocyte antigen (HLA) class I, the process of which involves a subset of virus genes. Infection of human foreskin fibroblast (HFF) cells with UV-inactivated HCMV (UV-HCMV), however, resulted in an increase in HLA class I presentation on the cell surface in the absence of HCMV gene expression. Heparin, which inhibits the interaction of virus particles with cell surface heparan sulfate proteoglycans (HSPGs), blocked the effect of UV-HCMV on HLA class I expression. Pretreatment of cells with heparinase I decreased in a dose-dependent manner the effect of UV-HCMV on HLA class I expression enhancement. Sodium chlorate, which is known to inhibit the sulfation of HSPGs, gave a similar result. Pretreatment of UV-HCMV with trypsin or monoclonal antibody reactive with the envelope glycoprotein gB reduced the increase in HLA class I expression on the HFF cell surface by UV-HCMV. RT-PCR analysis demonstrated that the increase in HLA class I presentation on the HFF cell surface was due to an increase in HLA class I transcription. Thus, binding of HCMV particles to cell surface HSPGs appears to be required for the stimulation of HLA class I expression. It is also possible that virus entry, in addition to binding to HSPGs, may be involved in the stimulation of HLA class I expression, since the UV-HCMV entered the cells and all treatments to block virus binding to HSPGs would necessarily prevent virus entry.
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PMID:Human cytomegalovirus binding to heparan sulfate proteoglycans on the cell surface and/or entry stimulates the expression of human leukocyte antigen class I. 1156 34

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
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PMID:Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans. 1175 75

We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.
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PMID:Interaction of an uuter membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans. 1185 41


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