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)

An indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator. The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.
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PMID:A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. 689 41

Specimens of human fibrinogen mixed with Fab fragments of antibodies that were specific for various portions of the fibrinogen molecule were tungsten shadow-cast and examined by electron microscopy. Typical trinodular fibrinogen molecules were observed when Fab fragments were omitted or when fragments from nonimmune sera were used. In the experimental fibrinogen-Fab preparations, a significant number of molecules were found with an extra nodule. In the case of Fab fragments from antibodies directed to fragment E, the additional nodule was attached to the central sphere of the fibrinogen molecule. Similarly, anti-fragment D preparations yielded molecules that were derivatized on the terminal spheres. Fragments from antibodies raised against a cyanogen bromide fragment of fibrinogen alpha chains (residues 241-476) also led to exclusive derivatization of the terminal domains, although in these cases the additional material was often separated discretely from the terminal sphere by a gap. These experiments confirm longstanding notions that the central domain of a trinodular fibrinogen molecule corresponds to the plasmin-derived fragment E and that the terminal spheres correspond to fragments D. Moreover, the carboxy-terminal two-thirds of alpha chains protrude from the extremities of the molecule, as had been inferred on the basis of indirect biochemical data.
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PMID:Shadow-cast electron microscopy of fibrinogen with antibody fragments bound to specific regions. 694 Dec 44

A panel of monoclonal antibodies to human vitronectin was used to define some epitopes for the multifunctions of this protein. Separate antibodies were identified which strongly inhibited cell spreading activity and which prevented binding to collagen. A third group interfered with the ability of vitronectin to inhibit complement-mediated guinea pig erythrocyte reactive lysis. None of the antibodies from these three groups prevented heparin binding, providing evidence that this reaction occurs at a fourth location; a different monoclonal antibody partially inhibited the binding of heparin. The relative accessibility of each biologically active epitope was assessed by the differential binding of the monoclonals to native and denatured vitronectin. Reactivity of the antibody which inhibited heparin binding greatly increased upon denaturation of vitronectin, implying that this region is normally inaccessible in the native form of the molecule. By contrast, epitopes for cell spreading, collagen binding, and inhibition of terminal complement complex lysis were destroyed by denaturation. On the basis of denaturation data and epitope mapping by competitive exclusion of monoclonal antibodies, a Venn diagram was constructed to represent overlap of monoclonal antibody epitopes in the tertiary structure. Linear epitopes for the antibodies were identified using cyanogen bromide and plasmin-derived peptides from vitronectin. The antibody which strongly inhibited cell binding reacted with a region containing the RGD site, whereas linear epitopes for collagen binding and complement inhibition appeared to reside in a 43-kDa peptide representing the internal region of the amino acid sequence, excluding the heparin-binding site. The latter two epitopes were differentiated from each other by reactivity of the antibody which inhibited collagen binding toward a smaller 20-kDa plasmin-derived peptide.
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PMID:Relative topography of biologically active domains of human vitronectin. Evidence from monoclonal antibody epitope and denaturation studies. 752 35

The M(r) = 94,000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[14C]acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of the disulfide bridges in factor Va heavy chain. 794 16

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na2EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.
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PMID:Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis. 832 Feb 79

The alpha C domain of fibrinogen (A alpha-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the A alpha chain were employed as structural probes to: 1) isolate the human alpha C domain, 2) compare the topography of the eight epitopes within the alpha C domain of intact fibrinogen and in purified alpha C fragments, and 3) explore the degree to which the alpha C domain's role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated alpha C fragments. Five antibodies were raised against small, synthetic peptide immunogens (A alpha-(220-230), A alpha-(425-442), A alpha-(487-498), and A alpha-(603-610)), and three were generated against larger cyanogen bromide (A) alpha chain derivatives with each epitope subsequently localized to discrete A alpha chain sequences (A alpha-(259-276), A alpha-(529-539), and A alpha-(563-578)). Human alpha C preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-A alpha-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of alpha C fragments was either A alpha-220 or A alpha-231 and that, although the extreme COOH-terminal region, A alpha-(603-610), was absent, all molecules were intact at least through A alpha-(563-578). Solution phase competitive assays indicated that the release of the alpha C domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of A alpha-(220-230), A alpha-(259-276), A alpha-(487-498), and A alpha-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. A alpha-(425-442) and A alpha-(563-578). Immunoblotting analysis of alpha C cross-linking in vitro revealed that isolated alpha C fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the A alpha chain proteolysis that occurs during thrombolytic therapy indicated that alpha C fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen alpha C domain and its functional implications and also draw attention to the as yet unexplored role of alpha C fragments in the pathophysiology of thrombosis and hemostasis.
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PMID:Comparative structural and functional features of the human fibrinogen alpha C domain and the isolated alpha C fragment. Characterization using monoclonal antibodies to defined COOH-terminal A alpha chain regions. 857 16

The intrapulmonary thrombi that form after the cessation of circulation are thought to be one of the major causes of graft function failure. We evaluated the effect of recombinant tissue-type plasminogen activator (rt-PA) in a canine cadaver lung transplant model. Donor dogs were killed by the intravenous administration of pancuronium bromide without heparinization, and left for 2 h at room temperature. The donor lungs were then flushed with low potassium dextran glucose (LPDG) solution, being subjected to a total ischemic time of 3 h. Following left lung transplantation, the contralateral pulmonary artery of the recipient dogs was ligated. In group 1 (n = 6), chloride solution was administered from the main pulmonary artery for 90 min, commencing 15 min prior to reperfusion. In group 2 (n = 6), 2.5 microg/kg per min of rt-PA, and in group 3 (n = 6), 5.0 microg/kg per min of rt-PA, were continuously infused in the same manner as in group 1. Lung function, including arterial blood gases and pulmonary hemodynamics, was measured for 3 h. The side effects of rt-PA were evaluated by measuring the prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, alpha2-plasmin inhibitor (alpha2-PI), plasminogen, and fibrin/fibrinogen degradation product (FDP). All of the animals in the three groups survived throughout the observation period. The group 3 animals had significantly better gas exchange than the group 1 animals, and the pulmonary hemodynamics were significantly better in the group 2 and 3 animals than in the group 1 animals. The FDP levels in the group 2 and 3 animals were significantly higher than those in the group 1 animals, while the PT and APTT were significantly prolonged in the group 3 animals. These findings led us to conclude that rt-PA improves early lung function, particularly pulmonary hemodynamics.
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PMID:The effects of recombinant tissue-type plasminogen activator (rt-PA) on canine cadaver lung transplantation. 1048 50

Earthworm fibrinolytic enzyme III-1 (EFE-III-1) was prepared to couple with cross-linking agarose activated by 1,'-Carbonyl- diimidazole (CDI) in this study. Although the activity of the immobilized protease decreased to approximately 64% of the native enzyme, the activity of EFE-III-1 coupled with the resin activated by CDI was higher than that activated by cyanogen bromide (CNBr). The immobilized protease was experimentally demonstrated to hydrolyze IgG, albumin and creatine kinase, besides fibrin(ogen) and plasmin(ogen), suggesting that EFE had a broad substrate specificity.
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PMID:Immobilized earthworm fibrinolytic enzyme III-1 with carbonyldiimidazole activated-agarose. 1214 27

Earlier studies on the stimulatory effect of fucoidan, heparin, and cyanogen bromide (CNBr)-fibrinogen digest on the in-vitro activation of glutamic type plasminogen by tissue plasminogen activator, which were performed using subphysiologic ionic strengths of buffers, gave inconsistent results because of the variation in the ionic strengths of the buffers used. Studies were therefore conducted on the effect of these cofactors using 0.05 mol/l Tris buffer containing a physiologic concentration of sodium chloride. The double reciprocal plots of the activation of glutamic type plasminogen by tissue plasminogen activator in the presence of fucoidan and 6-aminohexanoic acid (6-AH) or heparin and 6-AH showed a four- to six-fold increase in K(cat), while the K(m) remained unchanged. On the other hand, there was greater than six-fold lowering of K(m) from 0.213 to 0.035 micromol/l in the presence of CNBr-fibrinogen, while K(cat) was only slightly increased. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of glutamic type plasminogen or of tissue plasminogen activator after serial dilution. The results suggested that the enhancements by fucoidan and 6-AH or CNBr-fibrinogen were due to their interactions directed towards glutamic type plasminogen, while for heparin and 6-AH, the interaction was directed towards tissue plasminogen activator. Circular dichroism studies in the near ultraviolet range (250-308 nm) showed that 6-AH enhanced the circular dichroism spectra of glutamic type plasminogen around certain chromophores, while fucoidan and heparin had no effect, suggesting that the enhancement by the cofactors may be related to the favorable conformational changes of glutamic type plasminogen by 6-AH.
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PMID:The effect of fucoidan, heparin and cyanogen bromide-fibrinogen on the activation of human glutamic-plasminogen by tissue plasminogen activator. 1269 44

Eczema is widely considered to be an exacerbation of alkaline stress to the skin. Epidermal barrier dysfunction is a feature of eczema pathology, which predisposes affected individuals to distressing morbid symptoms. At least two serine proteases, stratum corneum chymotryptic enzyme (kallikrein 7 [KLK7]) and stratum corneum tryptic enzyme (kallikrien 5 [KLK5]), have increased activity levels in eczematous lesions and both have been implicated in the destruction of corneodesomosomes, which are crucial to epidermal integrity. The present in vitro study investigated whether transcriptional gene silencing after siRNA transfection could influence the activity of these signature enzymes in an in vitro model of eczema induced by alkaline shock. HaCaT epithelial cells were subjected to alkaline stress by the addition of 1,1,3,3-tetramethyl guanidine "superbase" (TMG) to the culture media. The culture media were subsequently tested for chymotryspin, trypsin, plasmin, and urokinase activity using colorimetric peptide assays and for reactive oxygen species using WST1 cell viability reagent. Cells that had been transfected with small interfering ribonucleic acid (siRNA) against KLK5 and KLK7 for 24 h before alkaline shock did not exhibit the increase in serine protease levels observed in untreated controls. Moreover, an endpoint MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) confirmed that detachment of cells from the culture substrate observed in alkaline-stressed cells did not occur in siRNA-treated cells. This in vitro study has established the proof-of-principle that siRNA therapy appears to mitigate the consequences of alkaline shock to the serine protease-associated fragility of epithelial cells that is characteristic of eczema.
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PMID:Transcriptional gene silencing of kallikrein 5 and kallikrein 7 using siRNA prevents epithelial cell detachment induced by alkaline shock in an in vitro model of eczema. 2209 88


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