Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.
 ...
PMID:Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland. 389 62

Pure cultures of bovine endothelial cells (EC) produce and secrete large amounts of plasminogen activators (PA). Cocultivation of EC with vascular smooth muscle cells (SMC) resulted in a significant decrease of PA activities secreted by the EC, whereas the cellular PA activities remained unaffected. Secreted PA activities were absent in the growth medium as long as the SMC to EC ratio was 2:1 or higher. The PA inhibitory activity of the SMC was rapid and cell-to-cell contact was not necessary. The PA inhibitory activity was present in homogenates of SMC as well as in the medium conditioned by them but not in the extracellular matrix elaborated by these cells. Serum free medium conditioned by SMC neutralized both tissue type (t-PA) and urokinase like (u-PA) plasminogen activators. Gel electrophoretic analysis of SMC conditioned medium followed by reverse fibrin autography demonstrated PA inhibitory activities in the molecular weight (Mr) range of 50,000 to 52,000 similar to those present in media conditioned by bovine endothelial cells or fibroblasts. Regular fibrin zymography of SMC conditioned medium incubated with u-PA or t-PA revealed the presence of a component with a calculated approximate Mr of 45,000 to 50,000 which formed SDS resistant complexes with both types of PA. These data demonstrate that vascular SMC produce and secrete (a) inhibitor(s) of PAs which may influence the fibrinolytic potential of EC.
 ...
PMID:Vascular smooth muscle cells inhibit the plasminogen activators secreted by endothelial cells. 392 3

Porcine tissue-type plasminogen activator (t-PA) increases the binding of 125I-glu-plasminogen to clots made from human plasma or purified fibrinogen in a time and t-PA concentration dependent fashion. The accumulation of plasminogen was faster and greater on noncrosslinked plasma clots than on clots which had been crosslinked by Factor XIIIa. Furthermore, the uptake of plasminogen to crosslinked fibrin clots occurred at a slower rate in the presence of alpha 2-plasmin inhibitor (alpha 2 PI) than in its absence. The kinetics of the uptake of 125I-plasminogen were analyzed using SDS-polyacrylamide gel electrophoresis and radioautography of solubilized plasma clots formed in the presence of t-PA. During the initial phase there was a decrease of clot-bound glu-plasminogen; simultaneously, there was a slight increase in clot-bound glu-plasmin and in plasmin complexed to alpha 2 PI that was crosslinked to alpha-chain polymers of fibrin. This was followed by a marked increase in clot-bound plasminogen having glutamic acid as NH2-terminal (glu-plasminogen) and gluplasmin. t-PA-induced enhancement of glu-plasminogen uptake appears to be mediated by plasmin but does not require the conversion of glu-plasminogen to plasminogen having lysine or methionine as NH2-terminal. The described mechanism assures an adequate supply of clot-bound plasmin, which is the enzyme ultimately involved in the degradation of fibrin.
 ...
PMID:Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots. 621 Mar 7

Plasminogen activator-inhibitor complexes were analyzed by SDS-polyacrylamide gel electrophoresis and enzymography. The complexes appeared as fibrinolytically active bands in the fibrin-indicator gel. A high-molecular-weight t-PA form comigrating with a t-PA-inhibitor complex (Mr 95 000-135 000) from cultured human endothelial cells was purified from plasma by immunoadsorption on anti-t-PA-Sepharose followed by gel filtration on Sephadex G-150. The high-molecular-weight t-PA form was fibrinolytically inactive when assayed by the fibrin-plate method. It was converted to a form with the same electrophoretic mobility as t-PA (Mr 72 000) when treated with 1.5 M NH4OH/39 mM SDS. These observations suggested that the plasma high-molecular-weight t-PA form was an enzyme-inhibitor complex. The complex did not show immunological cross-reactivity with a number of known plasma serine proteinase inhibitors. Both t-PA and u-PA rapidly formed complexes with an inhibitor which was present in plasma in pmolar concentrations. p-Aminobenzamidine blocked the reaction, indicating that the active center of the activator was indeed implicated in complex formation. The complex between the plasma inhibitor and t-PA and the high-molecular-weight t-PA had the same electrophoretic mobilities. The rapid plasminogen activator inhibitor in plasma showed remarkable similarity to a plasminogen activator inhibitor from cultured human endothelial cells. In addition to the high-molecular-weight t-PA form described above, three other t-PA forms were isolated from plasma. Our results indicated that they represented free t-PA and t-PA in complex with respectively C1-esterase inhibitor and alpha 2-antiplasmin.
 ...
PMID:Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor. 643 84

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95 000-135 000). This t-PA form was converted to Mr-72 000 t-PA by 1.5 M NH4OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and Mr-72 000 t-PA comigrated with high-molecular-weight t-PA. From the increase in Mr of t-PA or u-PA upon complex formation, the Mr of the endothelial cell component was estimated to be 50 000-70 000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH4OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involved in the complex formation. It was further noted that serum-free conditioned medium or endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.
 ...
PMID:Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex. 643 88

In human umbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70 degrees C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.
 ...
PMID:Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium. 654 Oct 58

Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (+/- 16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (Mr 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanilide. The fibrinolytic activity as measured by clot lysis in only slightly increased. The physiological significance of the cleavage is discussed.
 ...
PMID:Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin. 689 Dec 67

Human tissue-type plasminogen activator was produced in centigram quantities from 50-60 1 batches of conditioned medium of a human melanoma cell culture. The yields of the procedure remained essentially unchanged during 50 subsequent preparations. The final products were of high purity as assessed by SDS gel electrophoresis. These materials, after filtration on 0.22 microM Milliporefilters were sterile and free of viruses and pyrogens. Their stability was very good in the fluid form at temperatures up to 56 degrees C. Material obtained by the present procedure has been used to investigate the biological and thrombolytic properties of tissue-type plasminogen activator.
 ...
PMID:Purification of human tissue-type plasminogen activator in centigram quantities from human melanoma cell culture fluid and its conditioning for use in vivo. 689 42

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.
 ...
PMID:Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein. 749 70

We have analyzed the proteolytic activity of a recombinant form of apolipoprotein(a) [r-apo-(a)]. A mutant 17-kringle from of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (tPA) as determined by SDS-PAGE and fluorography and by Western blot analysis. However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated into r-Lp(a) particles, were uniformly inactive against a variety of chromogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteolytic activity, we generated truncated forms of the Ser-->Arg proteolytic activity, we generated truncated forms of the Ser-->Arg mutant containing one or 10 kringle IV repeats. These truncated versions of r-apo(a) were susceptible to cleavage by tPA but were inactive against the plasmin substrate S-2251. Treatment of the Ser-->Arg mutant of the 17-kringle r-apo(a) with tPA and diisopropylflurophosphate (DFP) did not result in modification of the mutant protease domain by DFP. Finally, we incubated r-apo(a) or r-Lp(a) particles formed in vitro with purified human LDL; no degradation of LDL was observed after 16 h at 37 degrees C. The results of this study suggest that one or more of the substitutions present in the protease domain of apo(a), in addition to the Arg-->Ser substitution, render apo(a) proteolytically inactive.
 ...
PMID:Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a). 749 9


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>