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 angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM-bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti-bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.
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PMID:Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix. 850 69

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.
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PMID:Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps. 856 35

We examined the ability of unfractionated heparin to modulate the procoagulant activities of stimulated endothelial cells (EC). Confluent human venous umbilical EC were incubated with heparin before or after stimulation, then rinsed extensively to eliminate any heparin in the solution. EC, stimulated for 4 h with endotoxin and interleukin 1 beta, expressed tissue factor and prothrombinase activities. When EC were treated with heparin (6 and 60 micrograms/ml) during the last 10 min of the stimulation period, EC-related procoagulant activities were inhibited in a dose-dependent manner (80-90% inhibition at 60 micrograms/ml). The inhibition was antithrombin-dependent and it disappeared after heparin removal in less than 15 min at 37 degrees C but persisted at 4 degrees C. When EC were treated with heparin (60 micrograms/ml) for 24 h then extensively washed before stimulation, the anticoagulant effect was more modest (50% inhibition). The effect was antithrombin-dependent. Inhibition was maximum after 18-24 h of pretreatment of EC with heparin and was stable for at least 7 h. The cell surface displayed a "heparin-like" activity: treatment by heparin doubled the rate of thrombin-antithrombin complex formation and this effect was heparinase sensitive and chondroitinase ABC insensitive. Thus, heparin modulates the procoagulant properties of stimulated EC according to two distinct mechanisms. The first one is rapid and transient, probably related to the presence of heparin molecules bound at the membrane surface. The second is delayed and persistent, and our results suggest that it is mediated by an increase in the membrane heparan sulfate molecules.
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PMID:Heparin reverses the procoagulant properties of stimulated endothelial cells. 871

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

In a multicentric study the influence of heparinase (Hepzyme) was evaluated on activated partial thromboplastin time, thrombin clotting time and prothrombin time using the recombinant human tissue factor and synthetic phospholipid (phosphatidylcholine and phosphatidyl-serine reagent). Hepzyme itself does not have any influence on normal coagulation values of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays whereas thrombin clotting time was prolonged by 10% (n = 60). In patients treated with unfractionated heparin for recent deep vein thrombosis (n = 47), plasma levels of aPTT, PT and thrombin clotting time (TCT) returned to the normal range in 100%, 97% and 91% after treatment with heparinase, respectively. Plasma samples of patients on coumarin were spiked with 2IU heparin/ml (n = 40) and were treated with heparinase. aPTT returned to baseline levels in 97.5%, PT in 99% and TCT in 69% of the samples. Plasma samples of patients receiving both heparin and coumarin were treated with heparinase (n = 18). aPTT and TCT values were shortened substantially and displayed the prolongation due to the effect of oral anticoagulants. PT values in these patients were also shortened. Freezing of plasma samples after treatment with heparinase resulted in a prolongation of the coagulation times in 15% of PT, 7% of aPTT and not of TCT values. The results show that treatment of plasma samples with heparinase abolishes the effect of unfractionated and low molecular weight heparin in vitro and ex vivo in patients during simultaneous treatment with oral anticoagulants. The use of heparinase may be of significance in patients with concomitant treatment of heparin and oral anticoagulants.
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PMID:Multicentric evaluation of heparinase on aPTT, thrombin clotting time and a new PT reagent based on recombinant human tissue factor. 883 97

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.
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PMID:Heparin cofactor II-dependent antithrombin activity of calcium spirulan. 887 66

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin-serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.
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PMID:Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers. 916 57

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.
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PMID:Specific involvement of glypican in thrombin adhesive properties. 917 91

Heparin, the most widely used antithrombin, suffers several limitations, including high inter-individual variability of anticoagulant response, a nonlinear dose-response curve, inability to inactivate clot-bound thrombin, a requirement for endogenous cofactors and inactivation by platelet factor 4 and heparinase. These shortcomings may explain its suboptimal efficacy and safety in the prevention of arterial vessel occlusion. Heparin's drawbacks may be overcome by direct thrombin inhibitors. The development of these specific antithrombins has been a major therapeutic goal of the past decade. The high expectations generated by the use of these compounds in experimental models of arterial thrombosis appeared to be confirmed by the initial phase I and II clinical studies. However, large phase III trials have been highly discouraging: three trials with hirudin have been interrupted as a result of a high incidence of serious adverse events. Two of these trials were subsequently restarted at lower doses and did not support an incremental efficacy of hirudin over heparin. Two trials in the setting of angioplasty (one with hirudin and one with hirulog) have also failed to demonstrate the superiority of these compounds over heparin. Is this the result of a very narrow therapeutic range of these agents or the consequence of poor design of the phase II studies leading to the selection of inappropriate doses for the comparative efficacy trials? This review focuses on the clinical development of two specific antithrombins: hirudin and hirulog. The experimental pharmacology and human studies of Argatroban are discussed in a review by Fitzgerald and Murphy.
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PMID:Direct thrombin inhibitors in cardiovascular disease. 921 Oct 51

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.
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PMID:Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated. 922 67


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