Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.6.12 (chondroitinase)
 2,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The endothelial cell surface receptor thrombomodulin (TM) displays various anticoagulant functions: it acts as a cofactor for the activation of protein C (PC) by thrombin, prevents the activation of fibrinogen, platelets and Factor V by thrombin. TM was also shown to accelerate the inhibition of thrombin by its physiological inhibitor antithrombin III (ATIII). The studies performed on rabbit lung TM were undertaken in order to provide better understanding, along with the identification and the characterization of functional domains, to the mechanism of action of TM. On the basis of the physical and chemical properties of TM, which were compatible with those of a proteoglycan, the presence of a sulfated polysaccharide chain covalently bound to TM, constituting an acidic domain independent of the protein C activation cofactor site, was suggested. Further enzymatic and chemical characterization showed that rabbit TM was in fact a chondroitin sulfate proteoglycan. Monoclonal antibodies raised against rabbit TM and proteins known for their ability to neutralize the activity of heparin, as well as TM submitted to chondroitinase digestion were used in order to identify the role of the different structural domains of TM. Binding of thrombin to TM at a primary site on the protein part is a prerequisite for all the biological activities of TM. However, while this binding is sufficient for TM to promote the activation of PC by thrombin, the inhibition by TM of thrombin-induced fibrinogen clotting and factor V activation requires the interaction of thrombin at a secondary site with the polysaccharide chain of TM. This interaction with the polysaccharide chain (which carries a highly sulfated trisaccharide at the non-reducing terminus) leads to the inhibition of the procoagulant functions of TM-bound thrombin towards fibrinogen and factor V as well as an increased reactivity of the enzyme with ATIII. These results were rationalized in the functional model proposed for the rabbit TM-proteoglycan. An original aspect of the TM-proteoglycan resides in the fact that the chondroitin sulfate side chain brings new anticoagulant activities, in addition to the PC activation cofactor activity, to the molecule. TM is a new type of proteoglycan with important regulatory function in hemostasis, which anticoagulant properties depend on both the protein core and the polysaccharide chain.
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PMID:[Thrombomodulin: a new proteoglycan. Structure-function relation]. 165 16

The isolation and partial characterization of a novel anticoagulant from the plasma of a patient with metastatic prostate cancer is described. The patient had a prolonged activated partial thromboplastic time, prothrombin time and thrombin time which did not correct by mixing with normal plasma. The reptilase time was normal and the prolonged thrombin time was corrected with protamine sulfate suggesting a heparin-like anticoagulant. A glycosaminoglycan anticoagulant (GAC) was isolated from the patient's plasma. The inhibitory activity of the GAC was destroyed by treatment with chondroitinase ABC. The GAC migrated on agarose gel electrophoresis between keratin sulfate and heparan sulfate. Purified GAC possessed only 2% (W/W) of the antithrombin III cofactor activity of porcine heparin. In assays using purified fibrinogen, the GAC was shown to directly inhibit fibrinogen proteolysis by thrombin. It is concluded that this glycosaminoglycan anticoagulant directly inhibits thrombin clotting of fibrinogen and is a new mechanism for abnormal hemostatic assays in cancer.
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PMID:A glycosaminoglycan inhibitor of thrombin: a new mechanism for abnormal hemostatic assays in cancer. 189 11

To investigate mechanisms regulating intra-alveolar coagulation, we studied monolayers of the A549 human lung epithelial cell line. The surface of A549 cells delayed the onset of prothrombin-to-thrombin conversion and prevented total prothrombin consumption in normal plasma compared to plastic cell-free wells. Similar results were achieved with bovine pulmonary endothelial (CPAE) and rat intestinal epithelial (IEC-6) cell lines, whereas Madin-Darby canine kidney renal epithelial cell line accelerated thrombin formation. The A549 surface catalyzed antithrombin III-thrombin complex formation with no significant increase in thrombin inactivation from heparin cofactor II. The A549 cell surface effects were largely, but not completely, reversed to values obtained for plastic when protein C-deficient plasma was used. Pretreatment of the cell surface with chondroitinase ABC plus heparitinase prior to thrombin generation experiments had no effect on the total prothrombin consumed but decreased the initial delay. Heparan sulfate as well as dermatan sulfate and other chondroitin sulfates were detected on the A549 surface using alcian blue staining. Conditioned media from A549, CPAE, and IEC-6 cells delayed the clot time of recalcified plasma. Use of chondroitinase ABC and heparitinase were both required to obliterate the A549 conditioned media activity. After growing A549 cells in 35SO(2-)4-containing medium, the resultant conditioned medium was found to contain 2,000 kD and 300- to 1,000-kD proteoglycans that yielded chains of less than or equal to 100 kD on reductive elimination with base.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A549 lung epithelial cells synthesize anticoagulant molecules on the cell surface and matrix and in conditioned media. 201

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.
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PMID:Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. 216 32

We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.
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PMID:Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. 279 47

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.
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PMID:Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen. 294 15

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.
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PMID:Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin. 838 42

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