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)

The syndecan family of transmembrane heparan sulfate proteoglycans is abundant on the surface of all adherent mammalian cells. Syndecans bind and modify the action of various growth factors/cytokines, proteases/antiproteases, cell adhesion molecules, and extracellular matrix components. Syndecan expression is highly regulated during wound repair, a process orchestrated by many of these effectors. Each syndecan ectodomain is shed constitutively by cultured cells, but the mechanism and significance of this shedding are not understood. Therefore, we examined (i) whether physiological agents active during wound repair influence syndecan shedding, and (ii) whether wound fluids contain shed syndecan ectodomains. Using SVEC4-10 endothelial cells we find that certain proteases and growth factors accelerate shedding of the syndecan-1 and -4 ectodomains. Protease-accelerated shedding is completely inhibited by serum-containing media. Thrombin activity is duplicated by the 14-amino acid thrombin receptor agonist peptide that directly activates the thrombin receptor and is not inhibited by serum. Epidermal growth factor family members accelerate shedding but FGF-2, platelet-derived growth factor-AB, transforming growth factor-beta, tumor necrosis factor-alpha, and vascular endothelial cell growth factor 165 do not. Shed ectodomains are soluble, stable in the conditioned medium, have the same size core proteins regardless whether shed at a basal rate, or accelerated by thrombin or epidermal growth factor-family members and are found in acute human dermal wound fluids. Thus, shedding is accelerated by activation of at least two distinct receptor classes, G protein-coupled (thrombin) and protein tyrosine kinase (epidermal growth factor). Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces. Regulated shedding of syndecans suggests physiological roles for the soluble proteoglycan ectodomains.
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PMID:Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation. 916 35

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

Vascular smooth muscle (VSM) cells express transmembrane proteoglycans of the syndecan gene family. We reported previously that the expression of syndecans by VSM cells is regulated by mitogens such as serum, platelet-derived growth factor, and basic fibroblast growth factor and that syndecan expression is induced after balloon injury in vivo. We now show that thrombin is a potent inducer of syndecan-1 expression in VSM cells. Transient transfection experiments with a rat syndecan-1 promoter construct demonstrated that thrombin stimulates transcription of the syndecan-1 gene. Syndecan expression in response to thrombin was not inhibited by downregulation of protein kinase C. Thrombin-induced syndecan-1 expression was dependent on tyrosine kinase activity. Calcium was necessary for syndecan-1 expression, but increasing the intracellular calcium levels was not sufficient to induce syndecan-1 expression. Analysis of antithrombin III (AT III) binding activity revealed that thrombin caused an increase in the synthesis of syndecan-1 molecules that exhibited high-affinity AT III binding. These results suggest that VSM cells could play an important role in controlling local thrombus formation subsequent to vascular injury, via a feedback mechanism that involves thrombin-induced stimulation of an inhibitor of thrombin activity.
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PMID:Thrombin stimulates syndecan-1 promotor activity and expression of a form of syndecan-1 that binds antithrombin III in vascular smooth muscle cells. 940 33

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.
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PMID:Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding. 994 92

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.
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PMID:Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase. 1068 61

The serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane-associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8-induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III-induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.
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PMID:Cell-surface heparan sulfate proteoglycan-mediated regulation of human neutrophil migration by the serpin antithrombin III. 1115 40

Antithrombin (AT) is a plasma-derived, single-chain glycoprotein with a molecular weight of 58 kDa. It is a serine protease inhibitor (serpin), sharing about 30% homology in amino acid sequence with other serpins. AT is a complex molecule with multiple biologically important properties. It is a potent anticoagulant that has been demonstrated to provide benefit in animal models and small cohorts of patients with coagulation disorders. AT also has remarkable anti-inflammatory properties, several of which result from its actions in the coagulation cascade. Activated coagulation proteases like activated factor X and thrombin contribute to inflammation; for instance, by the release of pro-inflammatory mediators. Inhibition of these proteases by AT prevents their specific interaction with cells and subsequent reactions. Anti-inflammatory properties of AT independent of coagulation involve direct interactions with cells leading to the release of, for instance, prostacyclin. Binding of AT to a recently identified cellular receptor, syndecan-4, leads to the interference with the intracellular signal induced by mediators like lipopolysaccharides and, thereby, to a down-modulation of the inflammatory response. AT has been shown to be effective in prospective and well-controlled small-scale studies of patients with inflammatory conditions, including sepsis. Although AT did not decrease overall patient mortality in a double-blind, placebo-controlled, phase III trial of patients with sepsis, it is important to note that AT improved the survival of individuals in this study not receiving heparin as a prophylactic regimen, which can be explained by the impaired interaction of AT with its cellular receptor in the presence of heparin, resulting in the reduction of the anti-inflammatory properties. Accordingly, the supplementation of AT without concomitant heparin may be beneficial in disorders with inflammatory characteristics, which has to be demonstrated in further clinical studies. Finally, recent results suggest that latent AT can induce apoptosis of endothelial cells by disrupting cell-matrix interactions. Further investigations will have to demonstrate whether latent and/or cleaved AT are physiological means to control angiogenesis. A potential prophylactic or therapeutic use as an anti-angiogenic and antitumor agent merits further exploration, including whether the growth of vessels in tumor tissues or close to tumors can be controlled by latent AT without affecting the formation of blood vessels during wound healing processes.
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PMID:Antithrombin: a new look at the actions of a serine protease inhibitor. 1244 4

Circulating endotoxin is elevated in sepsis and plays a role in endothelial dysfunction whereas antithrombin is decreased by virtue of its consumption during complex formation with clotting factors and by proteolytic degradation by granulocyte elastase. Dysfunction of endothelium results in enhanced leukocyte rolling and diapedesis into tissues leading to edema formation and injury. Antithrombin exerts beneficial effects on endothelial function in sepsis. A direct anti-inflammatory action of anti-thrombin in inflammatory cells is exerted via heparan sulfate proteoglycans. In this study, we investigated whether antithrombin affects endotoxin-induced adhesion of neutrophils to human endothelial cells in vitro and whether glycosaminoglycans are involved in its signaling. Adhesion of human neutrophils to monolayers of umbilical vein endothelial cells was tested under static conditions. Endothelial cells were pretreated with endotoxin, interleukin-1, heparinase-I, chondroitinase-ABC or anti-syndecan-4-antibody. Endotoxin and interleukin-1 increased neutrophil adherence to human umbilical vein endothelial cells which was inhibited by antithrombin. Concomitant incubation with pentasaccharide abolished this effect of antithrombin. Treatment of endothelial cells with heparinase or chondroitinase led to higher adhesion and prevented effects of antithrombin. With antibodies to syndecan-4, enhanced adhesion of neutrophils was observed. As studied by Western blotting, endotoxin-induced signaling was diminished by antithrombin and the effect was reversible by chondroitinase or heparinase. From our results, we can conclude that endotoxin-induced adhesion of leukocytes to endothelium can be reversed by ligation of syndecan-4 with antithrombin's heparin-binding site and interferences with stress response signaling events in endothelium.
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PMID:Syndecan-4-dependent signaling in the inhibition of endotoxin-induced endothelial adherence of neutrophils by antithrombin. 1465 50

The accumulation of extracellular matrix components such as proteoglycans is a hallmark of an atherosclerotic lesion. A large heparan sulfate proteoglycan, perlecan, dramatically increases in the advanced lesion, and vascular smooth muscle cells are the cell type responsible for the accumulation. In this study, we investigated the effects of thrombin on the proteoglycan synthesis in cultured human coronary smooth muscle cells to determine the interrelationship between the accumulation of proteoglycans and the procoagulant state of blood in atherosclerosis. The cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of thrombin, and the labeled proteoglycans were characterized by Sepharose CL-4B molecular sieve chromatography and DEAE-Sephacel ion-exchange chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was determined by SDS-polyacrylamide gel electrophoresis before and after digestion with chondroitinase ABC or papain. The results indicate that thrombin increases the cell layer-associated heparan sulfate proteoglycan with a core protein size of approximately 400 kDa without any change in the length of the glycosaminoglycan chains when the cell density is high. The heparan sulfate proteoglycan was identified as perlecan by Western blot analysis. In addition, quantitative reverse transcription-polymerase chain reaction showed that thrombin elevated the steady-state level of perlecan mRNA but not that of versican, decorin, and syndecan-1 mRNAs, although that of biglycan mRNA was moderately elevated. Furthermore, the percentage of disaccharide units that compose perlecan heparan sulfate chains remained unaffected by thrombin. Therefore, it is suggested that thrombin induces the perlecan core protein synthesis without influencing the formation of the heparan sulfate chains in human coronary smooth muscle cells at a high cell density. The regulation of proteoglycan synthesis by thrombin may be involved in the accumulation of perlecan in advanced lesions of atherosclerosis.
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PMID:Induction of synthesis of a large heparan sulfate proteoglycan, perlecan, by thrombin in cultured human coronary smooth muscle cells. 1571 25

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.
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PMID:Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells. 1573 Nov


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