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)

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.
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PMID:Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma. 275 13

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.
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PMID:Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan. 295 57

The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted.
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PMID:Anticoagulant activity in cell-free peritoneal fluid of an experimental pancreatic ascites tumor. 300 55

Thrombomodulin isolated from rabbit lung was separated by ion-exchange chromatography on DEAE-cellulose into a retarded (acidic) and a nonretarded (nonacidic) fraction. Both fractions contained the cofactor required for the activation of protein C. In addition, the acidic fraction (but not the nonacidic fraction) prevented the clotting of fibrinogen by thrombin ("direct" anticoagulant activity) and accelerated the inhibition of thrombin by antithrombin (effect corresponding to 2-10 international units of heparin per mg of protein). Both of these activities were readily neutralized by the synthetic polycation Polybrene, which did not appreciably affect protein C activation. They were also eliminated by digestion of thrombomodulin with bacterial heparinase, which, in addition, converted the acidic form of the protein C activation cofactor to a nonacidic form. Similar conversion observed during storage of thrombomodulin was attributed to endogenous proteinase activity. Density-gradient centrifugation of the acidic form of thrombomodulin in CsCl/4M guanidinium chloride failed to separate either of the direct or antithrombin-dependent anticoagulant activities from the protein C activation cofactor, which showed a buoyant density of 1.31-1.34 g/ml. The nonacidic cofactor had a lower density, 1.26-1.28 g/ml. Unreduced thrombomodulin yielded two major fractions of protein C activation cofactor on NaDodSO4/PAGE, with apparent Mr of approximately 68,000 and 57,000, respectively. The larger component contained essentially all of the direct and antithrombin-dependent anticoagulant activities. We propose that these activities as well as the negative charge and the higher buoyant density of the acidic, Mr 68,000 form of thrombomodulin are due to a heparin-like polysaccharide and, further, that this component can be separated from the major portion of the molecule, which contains the protein C activation site, through the action of a proteinase.
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PMID:Functional domains of rabbit thrombomodulin. 301 29

Studies were conducted to define the location of components and sequences in heparin with respect to their distance from the peptide linkage in the native proteoglycan. A purified heparin-oligopeptide was linked via its amino terminus to a matrix containing an azo bond and an activated carboxyl group. The polysaccharide chain was maximally degraded, either with heparinase or nitrous acid, and the soluble products were removed. The heparin-oligopeptide fragments that remained on the matrix were released by reductive cleavage of the azo linkage and characterized. The fragments, as well as heparin released without prior degradation, contained serine and glycine as the principal amino acids; the ratio of galactose to xylose was 2:1. The ratio of glucosamine to serine of 33:1 in the undegraded heparin was reduced to 6:1 and 1:1 in the heparinase-treated and nitrous acid-treated products, respectively. The undegraded sample and the fragments contained phosphate in equivalent amounts, demonstrating its presence in the heparin-protein linkage region. The heparin-oligopeptide preparation was also fractionated by gel filtration and high and low molecular weight fractions thus obtained were each linked to the insoluble matrix. The products that were subsequently released were subfractionated on a molecular weight-calibrated column of Sephadex G-200, and eluates were assayed for activity in promoting the neutralization of thrombin and factor Xa by antithrombin. The results revealed a sharp decrease in specific activity in heparin-oligopeptide fractions below Mr = 15,000 indicating that the anticoagulant-conferring segment is located at about 20 disaccharide units away from the peptide linkage region.
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PMID:Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan. 333 97

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.
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PMID:Activation of heparin cofactor II by heparin oligosaccharides. 337 65

To study the structural requirements in heparin for interaction with heparin cofactor II (HC II) we have analyzed the properties of oligosaccharide fractions obtained after digestion of heparin by heparinase and gel filtration. No activation of HC II was detected in the presence of di-, tetra-, hexa-, octa-, deca-, or do-decasaccharides. The hexasaccharide pool was fractionated by ion-exchange chromatography, and the structure of the major species, obtained in a homogeneous state, was investigated by NMR. All the resonances were unambiguously assigned using correlation by homonuclear and heteronuclear scalar coupling. The six monosaccharide residues of this hexasaccharide were thus easily identified. The sequence was established through two-dimensional nuclear Overhauser effect experiments. The results indicate that this product is a hexasaccharide recently described by Linhardt et al. (Linhardt, R. J., Rice, K. G., Merchant, Z. M., Kim, Y. S., and Lohse, D. L. (1986) J. Biol. Chem. 261, 14448-14454). However, we could not confirm the anticoagulant activity observed by these authors. Moreover, none of the individual components obtained after fractionation of the hexasaccharide pool was able either to activate HC II against thrombin or to inhibit HC II activation by heparin. Thus, our data led us to conclude that no unique sequence is involved in heparin for binding to HC II and inactivation of thrombin. The interaction merely results from the highly anionic character of heparin.
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PMID:Is there a unique sequence in heparin for interaction with heparin cofactor II? Structural and biological studies of heparin-derived oligosaccharides. 337 40

To assess the contribution of mast cells to the maintenance of blood fluidity, the hindlimb vasculature of mast cell-deficient mice (W/Wv) and littermates containing normal levels of mast cells (+/+), were perfused with purified human thrombin and antithrombin. Enzyme-inhibitor complex generation within the vasculature was enhanced to a comparable extent for W/Wv and +/+ mice over the uncatalyzed rate, that level of complex produced within a similar time interval in the absence of heparin. Perfusion of purified Flavobacterium heparinase prior to infusion of the hemostatic components, or perfusion of antithrombin modified at the heparin-binding domain, reduced W/Wv and +/+ hindlimb thrombin-antithrombin complex formation to the uncatalyzed rate. To further define the cellular source of the vascular-associated heparin-like molecules, endothelial cells isolated from epididymal fat pads of W/Wv and +/+ mice were grown in vitro. The acceleration of thrombin-antithrombin interactions in the presence of endothelial cell-derived glycosaminoglycans was similar for W/Wv and +/+ mice, was abolished with purified bacterial heparinase, and was expressed to only a minor extent when utilizing modified antithrombin. The biologically active mucopolysaccharides appear to be present on the cell surface.
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PMID:Anticoagulantly active heparin-like molecules from mast cell-deficient mice. 370 60

To control blood levels of heparin during extracorporeal therapy, the use of a blood filter containing heparinase, a heparin-specific enzyme that cleaves heparin to small fragments with less anticoagulant activity, has been proposed. These fragments have anti-factor Xa activity but no anti-thrombin activity. The potential toxicity of heparin fragments as compared to heparin was examined in rats by identifying presumptive sites of drug-related toxicity by whole-body autoradiography and by histological examination of major organs. Radioautograms of rats sacrificed 5 hr after dosing with [35S]heparin fragments indicated no potential targets different from what was observed in rats dosed with [35S]heparin. In addition, the faster urinary clearance of heparin fragments resulted in a lower concentration of these fragments than of heparin in all common target organs. No hemorrhages or other lesions were found in rats injected intravenously with heparin fragments (100 mg/kg) and sacrificed after 5 hr. In addition, no mortality or delayed toxic effects were observed in a similar group of animals sacrificed 2 weeks after dosing. In contrast, 80% of rats injected with heparin (100 mg/kg) showed hemorrhages of the lungs at the time of necropsy.
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PMID:Comparative studies of heparin and heparin fragments: distribution and toxicity in the rat. 373 75

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.
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PMID:Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells. 379 24


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