Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.
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PMID:High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis. 141 2

Glucocorticoids ameliorate erosion in animal osteoarthritis (OA) models and suppress synthesis of matrix metalloproteinases (MMP). However, in in vitro studies, their inhibitory effects on matrix degradation of cartilage have not been well documented by monitoring aggrecan. Collagen was monitored in this study to examine the effects of dexamethasone in cartilage explant culture. Dexamethasone clearly blocked collagen degradation induced by the combination of interleukin-1 (IL-1) and plasminogen at the concentration of 10(-9) M, which is much lower than the concentrations reportedly required to inhibit matrix synthesis. In addition, MMP-1 and MMP-3 were suppressed by dexamethasone treatment in a similar range of concentrations. The conversion of plasminogen to plasmin, however, was not blocked by treatment with dexamethasone. These results suggest that the inhibitory effect of dexamethasone on collagen degradation may be due to suppression of MMP production rather than suppression of fibrinolytic cascade. Thus, the ability of glucocorticoids to inhibit matrix degradation in vitro, which could be clearly shown by monitoring collagen degradation, may endorse their efficacy in animal OA models and suggest potential therapeutic effectiveness.
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PMID:Dexamethasone inhibits collagen degradation induced by the combination of interleukin-1 and plasminogen in cartilage explant culture. 1044 72

Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.
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PMID:Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9). 1171 48

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-alpha-inhibitor (IalphaI). Experiments using the purified components of IalphaI indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6.IalphaI complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.
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PMID:Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments. 1591 24

Fibrin is a hydrogel carrier widely used in cartilage tissue engineering. It is rapidly degraded by plasmin, which is produced by the cells. epsilon-Aminocaproic acid (EACA) can be used to inhibit this enzyme and thus save the fibrin carrier. In this study we investigated the effect of EACA on the transforming growth factor beta-1-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). To assess this, we used the standard pellet culture system, and EACA treatment was compared to an untreated chondrogenic control. To investigate differentiation, real-time RT-PCR was used on chondrocytic marker genes: aggrecan, collagen types II and X, and the SRY-related HMG-box gene 9 (SOX9). Also, specific glycosaminoglycan production was measured. Safranin-O/fast green staining was used to localize proteoglycans and collagens within the pellet. All results concur that EACA did not affect the chondrogenic differentiation process at 5 muM concentration, which is adequate to inhibit fibrin degradation. Therefore, it is a useful plasmin inhibitor for cartilage tissue engineering with hMSCs.
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PMID:Epsilon-aminocaproic acid is a useful fibrin degradation inhibitor for cartilage tissue engineering. 1908 6