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)

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

Fibronectin is a major glycoprotein component of normal fibroblasts in culture. External fibronectin is predominantly present in a pericellular fibrillar matrix that mediates distant cell-cell and cell-substratum contacts. A small proportion of external fibronectin is closely associated with the plasma membrane. In the matrix, fibronectin is partially disulfide bonded into complexes. Plasma transglutaminase, activated by thrombin, also cross-links external fibronectin into high-molecular-weight covalent complexes. In cultures of normal fibroblasts, pericellular matrix fibronectin displays extensive codistribution with (pro)collagens types I and III. Transformed adherent cells show decreased formation of the fibronectin-collagen matrix. The deficient synthesis of fibronectin and other matrix components and abnormal interactions with the matrix may account for several phenotypic characteristics of transformed cells. The pericellular matrix structure has been prepared by use of deoxycholate and hypotonic medium to solubilize the cells. The matrix contains glycosaminoglycans, procollagens, and fibronectin. The fibronectin codistributes with the procollagens. The matrix may be considered to be an in vitro equivalent of the connective tissue matrix and basal laminae found in vivo. Human sarcoma cells spread rapidly on the prepared matrix and assume an elongated morphology characteristic of normal fibroblasts. The prepared matrix may provide a general tool to study the effects of matrix on cellular behavior and differentiation.
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PMID:Fibronectin and the pericellular matrix of normal and transformed adherent cells. 29 68

Platelets are cells which develop adhesive properties following stimulation. Since fibronectin (fn) mediates adhesive properties of several cells, we sought evidence for platelet associated fn. Lysates of suspensions of washed human platelets containing less than or equal to 50 ng soluble fn/10(9) cells contained 2.85 micrograms fn antigen per 10(9) cells. The platelet fn antigen competition curve showed a similar slope to the curve for purified plasma fn suggesting antigenic identity. Immunofluorescent staining for fn was minimal in intact cells suggesting that the majority of fn antigen is intracellular. In permeable platelets, fluorescent staining for fn was seen in a punctate distribution suggesting a granule localization. Stimulation of platelet secretion by thrombin released platelet fn antigen. Suramin, a drug which inhibits platelet secretion, inhibited fn release. The apparent secretion of platelet fn, taken with the immunofluorescent data, support the localization of a portion of platelet fn antigen in a storage granule.
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PMID:The detection, immunofluorescent localization, and thrombin induced release of human platelet-associated fibronectin antigen. 39 28

Platelets lysed with Triton X-100 contain 3.44 +/- 1.27 (SD) microgram of fibronectin (cold-insoluble globulin) per 10(9) platelets. Fibronectin was partially released from washed whole platelets by collagen or thrombin, and its release by collagen was inhibited by aspirin. Analysis of subcellular fractions obtained by density-gradient centrifugation of disrupted platelets indicated that fibronectin was contained in the alpha granules. Fibrinogen depleted of fibronectin (less than 2 microgram/mg) supported ADP-induced aggregation as effectively as fibrinogen contaminated with this protein, thus reinforcing the generally held view that fibrinogen itself is the necessary protein cofactor in this reaction.
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PMID:Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation. 44 75

The surface proteins of cultured human skin fibroblasts were iodinated and then exposed to one or more of the following blood coagulation proteins: thrombin, fibrinogen, and factor XIII (plasma protransglutaminase). Radiolabeled polypeptides were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. After exposure to physiological concentrations of activated factor XIII (XIIIa), the band of radioactivity corresponding to the major labeled surface protein (fibronectin, molecular weight = 2.2 x 10(5) daltons) was cross-linked to a very high molecular weight complex. The cross-linking reaction was inhibited by fibrin (which is known to bind the catalytic subunit of XIIIa). Cross-linking of labeled cell surface fibronectin to fibrin could not be demonstrated. The fibrillar pattern of surface fibronectin appeared unaffected by cross-linking when studied by immunofluorescence. Cross-linking of cell surface fibronectin by XIIIa requires highly specific enzyme-substrate and protein-protein interactions, and may be an important physiological reaction.
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PMID:Cross-linking of a major fibroblast surface-associated glycoprotein (fibronectin) catalyzed by blood coagulation factor XIII. 97 39

The source of angiogenic activity of fibrin degradation products has been sought in a series of experiments, applying degradation products from different types of fibrin and fibrinogen to the chick chorioallantoic membrane. The presence of platelets or fibronectin during clotting was not essential for activity, and neither was crosslinking. Fibrinogen degradation products were non-stimulatory, as was serum. Molecular sieve column chromatography indicated a range of active fragments. Admixture of active fibrin degradation products with antifibrin fragment E, but not D, antiserum neutralized activity. Preparations containing only fibrin fragment E retained activity. A commercial preparation of fibrinogen fragment E was inactive until treated with thrombin. These experiments point to fibrin fragment E being the source of angiogenic activity, with thrombin cleavage being the essential step in generating activity.
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PMID:Angiogenic activity of fibrin degradation products is located in fibrin fragment E. 128 Jun 77

Microvascular endothelial cells express a variety of cell-surface integrins in vivo and in vitro with varying affinities for matrix proteins. The vitronectin receptor (VnR), a complex of the alpha v and beta 3 integrin chains, is capable of binding to a variety of matrix proteins that are deposited in injured tissues, including vitronectin, fibrinogen, and thrombin. Staining of frozen sections of human skin with antibodies recognizing the VnR and examination by immunofluorescence microscopy demonstrates staining in a vascular pattern suggesting in vivo expression of the vitronectin receptor on endothelial cells. Examination of pure cultures of human dermal microvascular endothelial cells (HDMEC) by flow-cytometric analysis and enzyme-linked immunosorbent assay confirmed that HDMEC also express cell surface VnR complex in vitro. Stimulation of human dermal microvascular endothelial cells in vitro with agents that stimulate protein kinase C resulted in dose- and time-dependent increases in expression of alpha v and beta 3 integrin chains. Additionally, stimulation with basic fibroblast growth factor induced similar increases, but stimulation with transforming growth factor-beta or interleukin-1 alpha failed to increase VnR expression. Increases in cell-surface VnR expression also correlated with an increased ability of microvascular endothelial cells to bind to vitronectin, but not fibronectin-coated surfaces. Although increases in cell-surface expression of beta 3 paralleled increases in expression of cell-surface alpha v, regulation of mRNA expression was distinct for each chain. These data suggests that microvascular endothelial cells express the VnR complex in vivo, that the cell-surface expression of this integrin on dermal microvascular endothelial cells can be regulated, and that this regulation may be important in cell adherence, cell migration, and wound healing.
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PMID:Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells. 128 60

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

The association between blood coagulation and cancer growth and metastatic dissemination is not yet completely understood. In this study we demonstrate that thrombin is capable of enhancing tumor cell adhesive properties and thereby increases tumor cell metastatic potential. Following exposure to alpha-thrombin, Walker 256 carcinosarcoma cells and B16 amelanotic melanoma cells became more adherent to both endothelial cell monolayers and the subendothelial matrix component, fibronectin. Preincubation of W256 and B16a cells with doses of alpha-thrombin from 0.01 to 10.0 U/ml produced a bell shape dose-response curve with the maximal effect (a 2-5-fold increase in adhesion) observed at 0.1 U/ml (corresponding to 0.8 nM). Complexes of alpha-thrombin with its inhibitors, hirudin and antithrombin III-heparin, diminished its effect on tumor cell adhesion. The effect of thrombin on tumor cell adhesion may be mediated by the alpha IIb beta 3 integrin as thrombin increased cell surface expression of the alpha IIb beta 3 complex. The significance of the in vitro observations was further substantiated by results of in vivo studies. Pretreatment of B16a cells with alpha-thrombin resulted in a 2-fold increase in the number of metastatic lung colonies in an experimental metastasis model. The data indicate a new role for thrombin in the metastatic spread of cancer.
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PMID:Thrombin enhances tumor cell adhesive and metastatic properties via increased alpha IIb beta 3 expression on the cell surface. 128 30

The effect of circulating lupus anticoagulant on platelet interaction with collagen and other proteins was tested, with the aim of understanding the role of membrane phospholipids in platelet function. Plasma samples from 26 systemic lupus erythematosus (SLE) patients, containing circulating lupus anticoagulant (LAC), were examined for their effect on adhesion and aggregation of normal human platelets. We find that SLE plasma, but not normal plasma, inhibits platelet adhesion to collagen in a concentration-dependent manner. At a plasma concentration of 1% the inhibition was 73 +/- 9% (mean +/- SD). In sharp contrast, there was no effect on platelet adhesion to fibronectin. Purified IgG from the same plasma samples also had an inhibitory effect. At 15 micrograms/ml (comparable in IgG concentration to 0.1% plasma) it inhibited adhesion to collagen by 33 +/- 11%. Inhibition could be abolished by preincubation of the LAC-containing plasma with cardiolipin (CL), phosphatidylinositol (PI), and, to a lesser extent, phosphatidylserine (PS) but not with phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Inhibition could also be abolished by preincubation of the LAC-containing plasma with a 10-fold excess of washed normal platelets. The effect of 1% LAC plasma on platelet aggregation was as striking, showing 79 +/- 26% inhibition of collagen-induced aggregation, and it could also be abolished by preincubation of the LAC plasma with cardiolipin. In contrast, the effect of LAC plasma on thrombin-induced aggregation was rather modest. Our results indicate that antiphospholipid antibodies interfere with platelet adhesion and stimulation by collagen in vitro and point to an important role of external plasma membrane phospholipids, particularly PI, in collagen-induced platelet activation.
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PMID:Lupus anticoagulant antibodies inhibit collagen-induced adhesion and aggregation of human platelets in vitro. 128 33


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