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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is well established that cultured endothelial cells are induced to generate tissue factor activity when incubated with either endotoxin or thrombin. In this study a perfusion system was used on 3-4 cm long human saphenous veins. The veins were perfused with thrombin (2.5 U/ml), endotoxin (30 ng/ml) or just medium for 3 h at 37 degrees C. After the perfusion, the veins were treated with collagenase, and EC were collected and subjected to tissue factor activity measurements. Some perfused veins were examined for tissue factor activity on the vessel wall by allowing factor VII and factor X to interact with the lumen of the intact vessels, followed by quantitation of generated factor Xa in a chromogenic assay. No formation of tissue factor activity could be found after perfusion in either collagenase-dissolved endothelial cells or in the coupled chromogenic assay for tissue factor activity performed in the lumen of the vessel. Our data strongly suggest that endothelial cells in intact endothelium may behave quite differently from isolated endothelial cells stimulated in cell cultures.
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PMID:Lack of ability to synthesize tissue factor by endothelial cells in intact human saphenous veins. 213 38

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.
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PMID:The activation of factor X by hepatocyte plasma membranes. 261 30

Purified acid-soluble and insoluble human collagen accelerated the clotting of plateletpoor plasma in silicone-treated tubes. The clot-promoting effect did not appear to be due to thromboplastic activity since the collagen preparations did not activate factor X in the presence of factor VII and calcium. Instead, collagen appeared to accelerate clotting by activating Hageman factor (factor XII) on the basis of the following findings: collagen increased the clot-promoting activity of partially purified Hageman factor but exerted no further effect in the presence of kaolin, a known activator of Hageman factor; clot-promoting eluates were obtained from collagen exposed to normal, hemophilic, or PTC-deficient plasma but not from collagen exposed to Hageman or PTA-deficient plasma. The collagen molecule itself appeared to be required for the clot-promoting activity since digestion with collagenase or thermal denaturation at pH 2.5 (about 35 degrees C) resulted in very marked reduction in clot-promoting activity. Since thermal denaturation is associated with transformation of collagen structure from triple helical to random coil form, it is suggested that the native form of collagen is essential for the ability to activate Hageman factor. Blockage of the free amino groups by treatment with nitrous acid or dinitrofluorobenzene only slightly reduced the clot-promoting activity of collagen. In contrast, since addition of cationic proteins to collagen markedly reduced pro-coagulant activity it is suggested that negatively charged sites on the collagen molecule are critical for Hageman factor activation. This suggestion is supported by the finding that pepsin treatment of collagen, which removes the predominantly negatively charged telopeptides, results in significant decrease in coagulant activity. Esterification of collagen, which neutralizes 80-90% of the free carboxyl groups, reduced coagulant activity by over 90% and it is suggested that the free carboxyl groups of glutamic and aspartic acids provide the negatively charged sites critical for Hageman factor activation.
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PMID:Activation of Hageman factor by collagen. 430 76

Endothelial cells grown on filters developed junctional complexes that reduced diffusional transport and increased electrical resistance over the cell layer. Induction of tissue factor by recombinant interleukin-1 beta led to a highly polarized tissue factor expression on the apical cell surface only. After prolonged growth to allow deposition of matrix, removal of the endothelial cells by collagenase or by 0.1 mol/L NH4OH left behind some cellular material as well as tissue factor, which was only detectable in the upper compartment. A human bladder carcinoma cell line, which does not form tight junctions and expresses tissue factor constitutively, showed essentially no polarity. Endothelial cell secretory compounds like von Willebrand factor, tissue plasminogen activator, and plasminogen activator inhibitor-1 were constitutively released to both sides. The added secretion due to recombinant interleukin-1 beta stimulation of the endothelial cells observed for von Willebrand factor and tissue plasminogen activator was, however, localized to the apical surface. The availability of tissue factor on the luminal surface of endothelial cells, ie, allowing contact with factor VII in the flowing blood, has potentially very significant pathophysiological consequences.
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PMID:Polar expression of tissue factor in human umbilical vein endothelial cells. 794 8

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH(2)-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys(20)-Leu(21)(close to Kunitz I domain and producing a 35-kDa band), Arg(83)-Ile(84) (between Kunitz I and II domains), and Ser(174)-Thr(175) (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys(20)-Leu(21) with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.
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PMID:Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation. 1085 19

One of the limitations to hepatocyte transplantation is the restricted availability of donor liver tissue. The aim of this study was to evaluate livers from non-heart-beating donors (NHBDs) as a source of hepatocytes for cell transplantation. A total of 20 livers/segments obtained from NHBD were perfused under good manufacturing practices using a standard collagenase digestion method. The donor liver median warm ischemia time was 15 minutes (range, 11-40 minutes), and cold ischemia time was 13 hours (range, 6-30 hours) prior to cell isolation. The cell viability of the hepatocytes obtained was 52% (1-81%), with a yield of 2.2 x 10(6)(0.2-29.7 x 10(6)) cells per gram of tissue. There was a significant negative correlation between hepatocyte viability and length of both warm ischemia (r = -0.544, P = 0.013) and cold ischemia (r = -0.510, P = 0.022). Preliminary experiments were performed on the viability testing of NHBD livers based on digestion of needle biopsies with collagenase and assessment of the hepatocytes produced. Two of the NHBD cell preparations, which had been cryopreserved, were used as part of a series of cell infusions for hepatocyte transplantation. A 3.5-yr-old girl with Crigler-Najjar syndrome type I received 9.7 x 10(8) NHBD hepatocytes (viability on thawing, 65%), and a 4-month-old boy with inherited clotting factor VII deficiency received 5.0 x 10(8) hepatocytes (viability, 57%). In conclusion, hepatocytes suitable for cell transplantation can be obtained from NHBD livers. Higher viability values may be obtained if both warm and cold ischemia times of donor liver can be reduced prior to processing.
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PMID:Isolation of hepatocytes from livers from non-heart-beating donors for cell transplantation. 1652 14

Neurological deterioration during the first day after intracerebral hemorrhage (ICH) is associated with early hematoma growth in 18 to 38% of patients. While clinical studies continue to evaluate efficacy of activated recombinant factor VII (rFVlla) for reducing frequency of early hematoma growth, there have been no studies investigating the effect of rFVIIa on early hematoma growth. We used a collagenase-induced ICH model in the rat to evaluate the effects of rFVIIa on early hematoma growth. Two hours after injection of 0.14 U of type IV bacterial collagenase in 10 microL of saline into the basal ganglia, a small amount of blood collected in the striatum. The ICH gradually increased in size, extending posteriorly to the thalamus by 24 hours after injection. Intravenous administration of rFVIIa immediately after collagenase injection decreased average hematoma volume at 24 hours compared with vehicle-treated group (168.1 +/- 13.4 mm3 vs. 118.3 +/- 23.0 mm3, p < 0.01). There was also a decrease in total hemoglobin content in rats treated with rFVlla compared with vehicle-treated rats (optical density at 550 nm: 0.87 +/- 0.08 vs. 0.71 +/- 0.09, p < 0.05). There was no difference in cortical brain water content overlying the hematoma between the rFVlla- and vehicle-treated groups (81.4 +/- 0.7% vs. 81.7 +/- 0.4%). Our study indicates that treatment with rFVIla may be useful in reducing the frequency of early hematoma growth in ICH patients.
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PMID:Early hemostatic therapy using recombinant factor VIIa in a collagenase-induced intracerebral hemorrhage model in rats. 1667 57

Hepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patient's liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at King's College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler-Najjar syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.
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PMID:Hepatocyte transplantation for liver-based metabolic disorders. 1676 14

Objective To develop and establish a simple and repeatable method of primary culture of mouse brain microvascular endothelial cells (BMECs) of relatively high purity. Methods Isolated and cultured from the brain tissue of ICR mice aged 2-3 weeks old, the mouse BMECs were gained through physical fragmentation methods such as cutting up and passing through cell sieve, BSA density gradient centrifugation and chemical enzyme digestion. The digestion time of 1 g/L type II collagenase was strictly controlled within 15-20 minutes. The cultured cells were identified by cell morphological observation and immunocytochemical staining to detect factor VII-associated antigen. Results In 24 hours after seeding, the spindle-shaped or polygon-shaped cells began to migrate from the micro vessel segment and gradually gathered and grew in clusters; after 7 days, the fused cells showed a typical single layer and arranged in the shape of paving stone. VII-associated antigen showed positive in the cytoplasm which was brown red. The purity of microvascular endothelial cells was above 95%. Conclusion We have successfully established a simple and repeatable primary culture method for mouse BMECs of relatively high purity.
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PMID:[Primary culture and identification of mouse brain microvascular endothelial cells]. 3251 70

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