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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In an attempt to obtain a biological marker for the enigmatic and fatal neurologic disorder, amyotrophic lateral sclerosis (ALS), several laboratories have explored alterations in various extracellular matrix components in both skeletal muscle and skin. We have studied the distribution of fibronectin, laminin, heparan sulfate proteoglycan (HSPG) and collagen types I, III and IV, along with the platelet alpha-granule glycoprotein, thrombospondin (TSP), by immunofluorescence in frozen sections of muscle from control denervating conditions and ALS patients. In ALS and control muscle, types I and III collagen were localized to the endomysium and the perimysium. Type IV collagen and laminin precisely delineated each muscle fiber (endomysium or basement membrane) but did not stain the perimysium. We found no marked quantitative or qualitative differences in the distribution of collagen types I, III and IV, laminin, fibronectin or HSPG in ALS patients compared to controls. However, when polyclonal antisera for TSP was used we found a marked increase in the deposition of this multi-domain glycoprotein in ALS patients' muscle compared to control muscle. Quantitative analysis of soluble extracts from control and ALS patients' muscle by ELISA also indicated that TSP was increased in ALS. TSP is released from platelet alpha-granules in response to thrombin stimulation. TSP elevation implies coagulation activity via the extravascular thrombolytic system in ALS and may correlate with regeneration. Other studies have indicated decreased circulating protease inhibitors and increased serine proteases in this disorder.
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PMID:Thrombospondin, a platelet alpha-granule and matrix glycoprotein, is increased in muscle basement membrane of patients with amyotrophic lateral sclerosis. 128 72

We have studied two natural anticoagulant pathways in normal and in transplanted human hearts. The first is the thrombomodulin pathway. Our immunocytochemical results show thrombomodulin localized to endothelium in heart biopsy specimens before transplantation. This reactivity persists in the absence of cellular rejection, but the infiltration of immune cells is associated with a lack of endothelial thrombomodulin. The second pathway is composed of antithrombin III (ATIII) bound to heparan sulfate proteoglycan (HSPG) molecules on endothelial cells. These ATIII-HSPG complexes bind and inactivate thrombin at the endothelial surface. Our immunocytochemical results show ATIII localized to endothelium in heart biopsy specimens before transplantation. This reactivity is present in the absence of vascular rejection as defined by either angiography or microscopy. The absence of thrombomodulin and ATIII is always associated with fibrin deposition within the microcirculation. Thrombomodulin and ATIII pathways appear to be independent, for cellular rejection often is associated with thrombomodulin-negative ATIII-positive endothelium, and vascular rejection often is associated with thrombomodulin-positive ATIII-negative endothelium. Cytokines from activated macrophages down-regulate endothelial thrombomodulin without generally affecting the ATIII-HSPG pathway. Immunosuppressive therapy depletes cytokine-producing cells that affect thrombomodulin, but there presently is no therapy to protect endothelium in vascular rejection. It is possible that heparin could interact with endothelium and bind ATIII to maintain a state of thromboresistance.
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PMID:Natural anticoagulant pathways in normal and transplanted human hearts. 131 72

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

Proteoglycans were extracted from bovine lung gas exchange tissue, pleura, and bronchioles with 4.0 M guanidinium chloride at 5 degrees C in the presence of protease inhibitors. Preliminary purification of the proteoglycans was achieved by an initial CsCl isopycnic centrifugation (rho 0 = 1.33) and through precipitation with cetylpyridinium chloride in 0.5 M KCl. Further purification and fractionation of proteoglycans was achieved by a second CsCl isopycnic centrifugation (rho 0 = 1.45) in 4.0 M guanidinium chloride. Based on the ultracentrifuge profiles and electrophoretic behavior, the major fractions were pooled. They were purified further by gel filtration on Sepharose CL-2B and characterized by extensive analyses. A heparan sulfate proteoglycan was the major proteoglycan identified in the gas exchange tissue and in the pleura. The major proteoglycan component from the bronchioles was a chondroitin sulfate proteoglycan. Approximate molecular weight of 2 x 10(6) for the heparan sulfate proteoglycan from the pleura and chondroitin sulfate proteoglycan from the bronchioles and 1 x 10(6) for the heparan sulfate proteoglycan from the gas exchange tissue were estimated from gel filtration analyses. After incubation with hyaluronic acid, the chondroitin sulfate proteoglycans from the bronchioles showed an increase in specific viscosity and a higher molecular weight compound eluting near the void volume in Sepharose CL-2B column chromatography. The proteoglycans exhibited varied anticoagulant activities in Stypven, partial thromboplastin and thrombin clotting times and inhibited thrombin-induced platelet aggregation.
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PMID:Isolation and characterization of proteoglycans from bovine lung. 740 Jan 34

Adhesive interactions between cells and the subendothelial extracellular matrix take place at several stages during tumor progression and metastasis. We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which can be exposed in the presence of low concentrations of plasmin and cell-associated heparan sulfate proteoglycan. Thus, thrombin may act as a matrix-adhesive molecule via activation of the alpha v beta 3 integrin. We have now identified a 31 amino acid fragment as the minimal thrombin-generated cleavage product, which contains an active RGD site, following gel filtration analysis on FPLC Superdex 75 column. The role of membrane-associated heparan sulfate in thrombin conversion to an adhesive protein was demonstrated by using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Incubation of both thrombin and a low concentration of plasmin on the surface of wild type CHO cells resulted in a typical digestion cleavage profile upon gel filtration. No cleavage products were observed when thrombin and a suboptimal plasmin concentration were incubated on monolayers of CHO cell mutants lacking heparan sulfate. Next, we examined the possible role of the thrombin RGD site during the progression of tumor development and metastasis. Toward this, we tested murine melanoma cells expressing low (B16-F1 cells) and high (B16-BL6 cells) lung colonization potentials in cell adhesion assays in vitro. Differential adherence capability of the cells was observed: while high attachment levels of B16-BL6 cells were obtained, the low metastatic B16-F1 cells did not adhere to thrombin RGD. Antibodies raised against the RGD site in thrombin specifically recognized thrombin digested with plasmin, but were unable to interact with native thrombin or prothrombin and inhibited potently B16-BL6 melanoma cell adhesion. Furthermore, the antibodies failed to recognize RGD in other adhesive plasma proteins such as vitronectin, fibrinogen, or fibronectin. Provided that the RGD-containing fragments of thrombin are widely distributed throughout the vascular system, they may have a significant role during tumor progression and dislodgement of metastatic cells. The development of RGD mimetics and/or specific antibodies might thus be applied to inhibit a critical step in metastatic spread.
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PMID:The involvement of thrombin RGD in metastasis: characterization of a cryptic adhesive site. 774

Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.
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PMID:Regulation of protease nexin-1 target protease specificity by collagen type IV. 800 28

Basic fibroblast growth factor (bFGF) has been shown to stimulate cell proliferation after vascular injury. The mitogenic activity of bFGF requires interactions with both a high affinity receptor and a cell-surface heparan sulfate proteoglycan. We tested the ability of platelet factor 4 (PF 4) and other platelet heparin-binding proteins to modulate bFGF-stimulated [3H]thymidine incorporation into fibroblasts. The supernatant of thrombin-stimulated platelets contained an inhibitor of bFGF-induced mitogenesis; this activity coeluted with PF 4 upon gel filtration, heparin-agarose, and ion-exchange chromatography. Purified thrombospondin and beta-thromboglobulin did not inhibit the mitogenic activity of bFGF. PF 4 inhibited the activity of 5 pM bFGF with 50% inhibitory concentration of 75 nM. Purified PF 4 also inhibited the basal incorporation of [3H]thymidine into 3T3 fibroblasts and the increased [3H]thymidine incorporation occurring after wounding of a cell monolayer. PF 4 did not affect the mitogenic activity of serum. Inhibition of bFGF activity by PF 4 could be overcome by exogenous heparin or chondroitin-4-sulfate, suggesting that inhibition of mitogenesis is caused by binding of PF 4 to cell-surface glycosaminoglycans. These results indicate that an important role of PF 4 released at sites of vascular injury and platelet activation is to control cellular proliferation caused by the release of bFGF from ruptured cells.
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PMID:Platelet factor 4 modulates the mitogenic activity of basic fibroblast growth factor. 804 Feb 68

Rat-liver-derived BRL-3A cells, which express both type-II phospholipase A2 (PLA2) and cytosolic PLA2 (cPLA2), generated prostaglandin E2 (PGE2) in the presence of fetal calf serum. When the cells were treated with tumor necrosis factor (TNF), PGE2 generation was greatly stimulated. The production of PGE2 observed in both cases was suppressed by a type-II PLA2-specific inhibitor, thielocin A1. Appreciable amounts of type-II PLA2 were released into the medium from the TNF-stimulated cells when heparin was added extracellularly. The release of type-II PLA2 from TNF-stimulated cells was also found in the presence of heparan sulfate or dextran sulfate, whereas other glycosaminoglycans showed no effects under the same conditions. These findings suggest that type-II PLA2 expressed in BRL-3A cells mostly associates with the cell surface by binding to cellular heparan sulfate proteoglycan. Removal of cell-surface-associated type-II PLA2, by either extracellular addition of heparin or by prior treatment of the BRL-3A cells with heparitinases, resulted in marked reduction of PGE2 synthesis in the cells. Exposure of BRL-3A cells to thrombin also induced the apparent secretion of type-II PLA2, and thrombin-stimulated PGE2 generation was suppressed by heparin effectively. Type-II PLA2 secreted and attached to heparan sulfate on the cell surface may therefore play an essential role in PGE2 synthesis by BRL-3A cells.
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PMID:Participation in cellular prostaglandin synthesis of type-II phospholipase A2 secreted and anchored on cell-surface heparan sulfate proteoglycan. 828 31

Vascular heparan sulfate proteoglycan (vHSPG) is an important functional component of the microvasculature. Previous studies have demonstrated autoimmunity to vHSPG in systemic lupus erythematosus (SLE). In the current studies, we further investigated the immunospecificity of anti-vHSPG antibodies in SLE sera by enzyme-linked immunoassay (ELISA). In direct binding assays, SLE sera contained IgG antibodies reactive with native vHSPG and with heparan sulfate (HS) glycosaminoglycan in significantly higher titers than controls. Employing purified SLE IgG in liquid-phase competitive immunoinhibition ELISAs, SLE IgG anti-HS antibodies cross-reacted with heparin and DNA, but not with other glycosaminoglycans or anionic phospholipid antigens. Immunochemical studies demonstrated that the immunodominant site on HS recognized by SLE IgG contained 2-O-sulfated uronic acid. Removal of N-sulfated and 6-O-sulfated residues primarily on N-acetyl-glucosamine had no effect on antigenicity, further demonstrating that nonspecific charge interactions which are the result of sulfation do not solely account for the antigenicity of HS. SLE IgG from patients with active SLE was further affinity purified on DNA-cellulose and HS-Sepharose columns for immunospecificity studies. After affinity purification of both anti-DNA and anti-HS antibodies, significant enhancement of direct binding reactivity with HS was noted. In addition, anti-DNA and anti-HS IgG antibody reacted with the cell surface of endothelial cells by a cellular ELISA (CELISA). Immunoinhibition studies of CELISA reactivity confirmed that affinity-purified SLE IgG anti-DNA anti-HS antibody were reactive with endothelial cell surface HS antigens. Furthermore, SLE IgG anti-DNA antibody reactivity with endothelial cells was not reduced by DNase treatment of the cells, but was significantly reduced by heparitinase digestion. Since HS plays an important role in the maintenance of normal anticoagulation on the endothelial cell surface by binding antithrombin III, we investigated the inhibition of heparin-accelerated thrombin-antithrombin III complex formation by SLE IgG. Purified IgG from patients with active SLE, but not from normal controls, inhibited heparin-accelerated formation of TAT complexes. These studies demonstrate the presence of IgG autoantibodies to HS in patients with SLE. Anti-HS antibodies recognize an antigenic site also present in heparin, but not other glycosaminoglycans, bind to the endothelial cell surface, and inhibit the formation of TAT complexes. SLE IgG anti-HS antibodies recognize a sulfated uronic acid epitope containing 2-O-sulfate which is important in certain functions of HS, including antithrombin III binding. Thus, anti-HS antibodies may promote a procoagulant state at the endothelial cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Autoantibodies to vascular heparan sulfate proteoglycan in systemic lupus erythematosus react with endothelial cells and inhibit the formation of thrombin-antithrombin III complexes. 829 26

A lysate of unstimulated human umbilical vein endothelial cells (HUVEC) exhibited phospholipase A2 (PLA2) activity, which hydrolyzed phospholipids bearing arachidonate more preferentially than those bearing linoleate at the sn-2 position. An anti-rabbit cytosolic PLA2 monoclonal antibody absorbed the activity, whereas an anti-human type II PLA2 monoclonal antibody did not. HUVEC treated with thrombin generated prostaglandin I2 (PGI2), and the PLA2 activity of the thrombin-stimulated cells was absorbed almost completely by the anti-cytosolic PLA2 antibody. HUVEC treated with tumor necrosis factor (TNF) also generated PGI2. PGI2 generation by TNF-treated cells was suppressed partially by extracellular addition of the anti-type II PLA2 antibody. PLA2 activity in a lysate of TNF-stimulated cells was increased about 2-3-fold, and about half of the increased activity was suppressed by the anti-type II PLA2 antibody. Addition of heparin together with TNF resulted in release of type II PLA2 in the medium. Thus, both cytosolic and type II PLA2s may be involved in agonist-stimulated PGI2 synthesis in HUVEC. Furthermore, exogenously added type II PLA2 was bound to the cell surface and synergistically enhanced PGI2 generation in TNF-stimulated HUVEC. This binding was blocked by either heparin or a monoclonal antibody recognizing the heparin-binding domain of type II PLA2. Taken together, type II PLA2 generated endogenously as well as added exogenously may be captured on the HUVEC surface via heparan sulfate proteoglycan and may contribute to cellular arachidonate metabolism.
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PMID:Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2. 841 61


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