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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We and other researchers have previously found that colony-stimulating factors (CSFs), which generally include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), promote invasion by lung cancer cells. In the present study, we studied the effects of these CSFs on gelatinase production, urokinase plasminogen activator (uPA) production and their activity in human lung cancer cells. Gelatin zymographs of conditioned media derived from human lung adenocarcinoma cell lines revealed two major bands of gelatinase activity at 68 and 92 kDa, which were characterized as matrix metalloproteinase (MMP)-2 and MMP-9 respectively. Treatment with CSFs increased the 68- and 92-kDa activity and converted some of a 92-kDa proenzyme to an 82-kDa enzyme that was consistent with an active form of the MMP-9. Plasminogen activator zymographs of the conditioned media from the cancer cells showed that CSF treatment resulted in an increase in a 48-55 kDa plasminogen-dependent gelatinolytic activity that was characterized as human uPA. The conditioned medium from the cancer cells treated with CSFs stimulated the conversion of plasminogen to plasmin, providing a direct demonstration of the ability of enhanced uPA to increase plasmin-dependent proteolysis. The enhanced invasive behaviour of the cancer cells stimulated by CSFs was well correlated with the increase in MMPs and uPA activities. These data suggest that the enhanced production of extracellular matrix-degrading proteinases by the cancer cells in response to CSF treatment may represent a biochemical mechanism which promotes the invasive behaviour of the cancer cells.
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PMID:Granulocyte, granulocyte-macrophage, and macrophage colony-stimulating factors can stimulate the invasive capacity of human lung cancer cells. 1040 91

The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen alpha-Folate Receptor (alpha-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.
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PMID:Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro. 1041 Nov 5

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.
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PMID:Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing. 1041 51

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP: MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin: ACP: MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP: MMP-9 and fibrin: ACP: MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5-2.5 microM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 microM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 microM) by higher phosphate (500 microM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 microM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP: MMP-9 complexes (50-100 microM). Neither POP nor PCP had any effect on preformed fibrin: ACP: MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin: ACP: MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.
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PMID:Amorphous calcium phosphate-mediated binding of matrix metalloproteinase-9 to fibrin is inhibited by pyrophosphate and bisphosphonate. 1044 97

The plasmin activation system plays a key role in extracellular matrix degradation in many malignant tumors. Because no data are available on the involvement of the plasmin activation system in matrix degradation by thyroid carcinoma, the present study was performed using follicular thyroid carcinoma cell lines obtained from a primary tumor (FTC-133) and metastases (FTC-236 and FTC-238) of one patient. Matrix degradation by these cell lines was studied assessing the release of radioactivity from S35-methionine labeled extracellular matrix coated onto plastic. The involvement of constituents of the plasmin activation system as well as matrix metalloproteinases (MMPs), another class of proteolytic enzymes, which can be activated by plasmin, were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and zymography. In the matrix degradation experiment, S35 release by FTC-133 was significantly higher than FTC-236 and FTC-238. S35 degradation could be inhibited by the plasmin inhibitor aprotinin and by anti-human urokinase-type plasminogen activator (uPA) antibody, indicating the involvement of the plasmin activation system. Matrix degradation could also be inhibited by the MMP inhibitor marimastat, thus demonstrating the involvement of MMPs in matrix degradation by these cell lines. Zymographic assays revealed activity of uPA in all cell lines. However, in contrast with FTC-236 and FTC-238, no plasminogen activator inhibitor (PAI) or PAI1 mRNA were found in FTC-133. Therefore, the differences in PAI activity as observed between the cell lines may originate from differences in PAI1 gene transcription. Differences in PAI1 expression did not affect the attachment of these cell lines to vitronectin. We conclude that the plasmin activation system is involved in extracellular matrix degradation by these metastatic follicular thyroid carcinoma cell lines. Differences in extracellular matrix degradation between the cell lines correspond with differences in PAI1 gene expression, indicating the significance of PAI1 in extracellular matrix degradation by metastatic follicular thyroid carcinoma.
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PMID:Degradation of extracellular matrix by metastatic follicular thyroid carcinoma cell lines: role of the plasmin activation system. 1052 70

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation ( approximately 186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg ( approximately 94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 102-Asn 103. Degradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 123-Leu 124, beta Asn 137-Val 138, and beta Glu 141-Tyr 142, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.
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PMID:Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments. 1052 39

Clinical situations with release of proteinases from blood cells or tissues into the circulation may result in a marked decrease of blood proteinase inhibitor content which in turn may result in a capillary leak syndrome, shock and even in exitus letalis. Replenishment of blood proteinase inhibitors is of benefit in such situations. In this study the inhibitory potential of fresh plasma, fresh frozen plasma and the liquid plasma protein preparation Biseko has been tested with the following enzymes: human leukocyte elastase, human plasmin, human matrix metalloproteinase-9, bovine trypsin, bovine chymotrypsin and rat liver lysosomal cathepsins. The concentration of the blood protein preparations resulting in 50% inhibition of constant amounts of each of the enzymes has been determined by plotting residual activity vs. log of concentration of blood protein preparation in enzyme assays. From these IC50 values inactivation ratios for 1 mg and 1 ml of the blood protein preparations was calculated. These inactivation ratios show an equal suitability of fresh plasma, fresh frozen plasma or full plasma proteins for replenishment of plasma proteinase inhibitory potential in vitro. As additional finding, commercial preparations of human serum albumin exert a surprisingly high inhibitory potential to lysosomal cathepsins.
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PMID:Comparative determination of the antiproteolytic potential of therapeutically used blood protein preparations. 1056 70

A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-.) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser(441)-Val(442) peptide bond in human plasmin(ogen), with additional cleavage of the Lys(78)-Val(79) and Pro(447)-Val(448) peptide bonds, and a secondary cleavage at Lys(615)-Val(616). Thereby, plasminogen is converted into an angiostatin-like fragment containing kringles 1-4 (K1-4) and miniplasminogen (kringle 5 and the serine proteinase domain). The purified K1-4 fragment showed a comparable cytotoxicity toward endothelial cells as the elastase-derived K1-3 fragment (12.7% versus 10.6% at a concentration of 10 microg/mL). Plasminogen, bound to monocytoid THP-1 cells, was also cleaved by the 24k-endopeptidase, resulting in generation of an angiostatin-like fragment and in a decreased capacity to generate cell-associated plasmin following activation by urokinase. The 24k-endopeptidase was not efficiently neutralized by specific inhibitors against the serine, cysteine, aspartic, or matrix metalloproteinase classes of enzymes. In human plasma or serum, however, it induced only very limited plasminogen degradation, apparently due to neutralization of its activity by alpha(2)-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.
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PMID:Specific proteolysis of human plasminogen by a 24 kDa endopeptidase from a novel Chryseobacterium Sp. 1063 Oct 10

Ligneous conjunctivitis (LC) is a rare disease of unknown etiology characterized by the growth of "woody" plaques on ocular and extraocular mucosa. These lesions are comprised of fibrin and both direct and indirect evidence implicates hypofibrinolysis as the primary defect in LC. To further elucidate the pathophysiology of LC we investigated the biochemical aspects of ligneous lesions with respect to the fibrinolytic system. Ligneous lesions were obtained from the right eye of a 15 year-old female patient with longstanding LC since age 2.5 year. Ligneous conjunctivitis in this patient has exhibited a chronic recurrent coarse and has involved multiple muscosal sites. Samples analyzed included an abundant mucoid thread from the conjunctival fornix and the ligneous plaque attached to the inferior tarsus. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to characterize protein profiles and by a variety of zymographic methods to visualize fibrinolytic enzymes. We found that mucoid and ligneous samples were distinct entities. Specifically, ligneous samples contained polypeptides with electrophoretic profiles characteristic of intact fibrin, and were replete in fibrin-bound tissue plasminogen activator (t-PA). Despite the presence of ample t-PA, ligneous samples were essentially devoid of fibrinolytic activity. In contrast, neither proteins nor t-PA could be detected in mucoid samples when fractionated by 7.5-15% SDS-PAGE or analyzed by fibrin zymography, respectively. Despite the absence of t-PA, mucoid samples were replete in fibrinolytic activity. This activity was plasminogen independent, heterogenous and inhibited by PMSF. Degradation profiles suggested that this activity represented in part alpha-chymotrypsin, consistent with this patient's treatment regime, as well as plasmin, elastase and an unidentified neutrophil-derived activity. Interestingly, ligneous samples contained both latent and activated forms of matrix metalloproteinase-9 (MMP-9), whereas mucoid samples contained predominantly activated forms of MMP-9. LC is characterized by defective fibrinolysis, despite the presence of ample t-PA and intact fibrin, and by an abundant mucoid thread which binds both endogenous and exogenous enzymes including serine protease(s) and collagenase(s). The implications of these results with respect to a role for exuberant mucus production or abnormal mucins in the development of a relative mucosal-site specific plasmin(ogen) deficiency is discussed.
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PMID:Ligneous conjunctivitis: biochemical evidence for hypofibrinolysis. 1070 63

We examined the role of the serine proteinase plasmin in regulating fibroblast-mediated tissue remodeling during wound healing. As an in vitro model system, collagen lattices were seeded with human dermal fibroblasts, and various concentrations of plasmin were added to the medium of the contracting lattices. Within 16 h, fibroblast-populated collagen lattices treated with plasmin rapidly contracted from approximately 20 mm to less than 2 mm in diameter. Measurements of collagen lattices with radiolabeled collagen indicated that, when these lattices included either fibroblasts or conditioned medium derived from fibroblast-populated collagen lattices, exogenous plasmin induced collagen degradation and rapid lattice contraction. Western blot analyses of conditioned medium demonstrated that fibroblasts in collagen lattices secreted the latent matrix metalloproteinase, MMP-1, which was subsequently cleaved by plasmin. Additionally, rapidlattice contraction and collagen degradation were blocked when collagen lattices were treated simultaneously with plasmin and aprotinin or a tissue inhibitor of metalloproteinases, TIMP-1. These results provide strong evidence that plasmin regulates rapid contraction of collagen lattices by activating fibroblast-secreted MMP-1 that triggers collagen degradation. The findings from this study suggest that fibroblast-populated collagen lattices can be used as an in vitro model system to investigate the mechanisms by which plasmin and cell-secreted plasminogen activators control MMP-1 mediated extracellular lattice degradation and remodeling during wound healing.
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PMID:Plasmin triggers rapid contraction and degradation of fibroblast-populated collagen lattices. 1073 68


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