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

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.
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PMID:Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor). 388 54

A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of greater than 20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases.
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PMID:The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains. 608 98

A urokinase-type plasminogen activator secreted by subcultured normal human umbilical vein endothelial cells was purified and compared to urinary urokinase (Mr = 54,000). The enzyme was isolated from serum-free conditioned medium in the presence of 0.1% (v/v) Triton X-100 by p-aminobenzamidine-agarose affinity chromatography, followed by Sephacryl S-200 gel filtration, followed by immunoadsorption chromatography on affinity purified specific anti-urokinase IgG-Sepharose CL-4B. This plasminogen activator form was obtained from the culture medium with a yield of about 47% and specific activity of about 93,000 IU/mg of protein, and represented approximately 18% of the total multiple molecular plasminogen activator activity forms present in endothelial cell conditioned medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single band of plasminogen activator activity with an estimated molecular weight of about 54,000 that was completely inhibited by diisopropyl fluorophosphate (DFP) as well as a single band of radioactivity with similar molecular weight for both the isolated L-[4,5-3H]leucine and [3H]DFP-labeled enzyme. The radiolabeled protein focused as a single major band with a pI value of pH 8.5. The endothelial cell activator and urokinase appeared to be identical in terms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, location of the [3H]DFP-labeled active site in the Mr = 33,000 heavy chain and [3H]DFP-labeled active site tryptic peptide, and two-dimensional 125I-labeled tryptic peptide maps. In quenching experiments of the fibrinolytic activities using affinity purified specific anti-urokinase IgG the endothelial cell-derived activator and urokinase appeared to be immunochemically identical, but unrelated to tissue plasminogen activator. These results indicate that the Mr = 54,000 urokinase-type plasminogen activator from cultured normal human endothelial cells is similar to, or identical with, Mr = 54,000 urinary urokinase.
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PMID:Isolation and characterization of a urokinase-type plasminogen activator (Mr = 54,000) from cultured human endothelial cells indistinguishable from urinary urokinase. 653 33

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.
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PMID:Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein. 749 70

Sequencing of two internal peptides from the putative human endothelial cell tissue plasminogen activator (t-PA) receptor identified an analog of the calcium- and phospholipid-binding protein, annexin II (Ann-II). The polymerase chain reaction-derived, full-length cDNA revealed complete sequence identity with the heavy chain of Ann-II, and ligand-precipitated receptor protein immunoreacted specifically with a monoclonal antibody to Ann-II. Transfected 293 cells bound plasminogen (Kd = 114 nM; Bmax = 347,000) as well as t-PA (Kd = 48 nM; Bmax = 380,000). Antisense oligonucleotides directed against endothelial cell Ann-II mRNA inhibited binding of both t-PA and plasminogen by 49% and 38%, respectively. The K307T mutant of Ann-II expressed on 293 cells failed to bind plasminogen, while the K328I mutant bound this ligand in a manner equivalent to the wild-type. Binding of plasminogen to both the wild-type and the K328I mutant was blocked by pretreatment of 293 cells with carboxypeptidase B. These data suggest a novel mechanism whereby a plasmin-like serine protease may cleave Ann-II at Lys307-Arg308, exposing a new carboxyl-terminal lysine residue (Lys307) for binding and efficient activation of plasminogen.
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PMID:An endothelial cell receptor for plasminogen/tissue plasminogen activator. I. Identity with annexin II. 806 40

The human urokinase (uPA) kringle (K) domain has been characterized via high resolution NMR spectroscopy. The 1H spectrum is analogous to that of the K2 domain of tissue-type plasminogen activator (tPA) and other homologous domains from the plasminogen (Pgn) heavy chain. This indicates a similar folding for the uPA/K. Comparisons of the high-field methyl and aromatic regions of the uPA/K and tPA/K2 spectra against those from the Pgn/K1 and K4 homologues afford the immediate assignment of signals stemming from conserved residues, such as the characteristic high-field shifted Leu46 delta, delta'-methyl doublets, and the aromatic side chains at the hydrophobic core, in particular those from Trp25, His48a, Tyr50, and Trp62. Resonances unresolved due to spectral overlaps in the 1H-1H correlated two-dimensional spectra were identified via a natural abundance 1H-13C single/multiple quantum correlated experiment. Spin systems unique to the uPA/K, such as His7, His37, His40, and His78, were assigned from Overhauser experiments and sequence information. Acid/base titrations of His imidazole signals in 2H2O yielded pKa* (pKa determined from acid/base titration in 2H2O, uncorrected for deuterium isotope effects) values of 6.2 for His7, 6.3 for His37, 6.4 for His40, 4.1 for His48a, and 6.2 for His78, which suggests a significant structural protection for His48a, consistent with a buried location within the hydrophobic core. Binding of low molecular weight heparin to the uPA/K in 2H2O affects mainly the His37, His40, His48a, and Tyr50 resonances, in a concerted and saturable fashion (association constant approximately 58 mM-1). The absence of perturbation of the His7 and His78 side chains indicates that segment 37-50 is selectively sensitive to heparin binding. Thus, the kringle outer B-loop is likely to be proximal to the basic residues responsible for the interaction with the polyanion ligand.
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PMID:1H NMR characterization of the urokinase kringle module. Structural, but not functional, relatedness to homologous domains. 831 53

We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).
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PMID:High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells. 844 52

Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.
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PMID:Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin. 862 99

Factor Va is the essential cofactor in prothrombinase-dependent activation of prothrombin. Resistance of Factor VaLeiden to inactivation by activated protein C (APC) contributes to thrombotic tendencies in subjects with the variant due, in part, to the inability to terminate thrombin production which increases both fibrin accretion and the frequency of thrombus formation. A reduced ability to inhibit thrombin generation, however, may lead to the stabilization of a clot through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). This hypothesis was tested by determining the profibrinolytic effect of APC on lysis time using clots formed with plasma from either homozygous normal (n = 4) or homozygous factor VLeiden (n = 4) subjects. Clots were formed in the presence of tissue-type plasminogen activator, thrombin, phosphatidylcholine/phosphatidylserine vesicles, Ca2+, and various concentrations of APC. Approximately 10-fold more APC was required to reduce lysis time from 140 to 50 min in clots containing factor VLeiden compared to normal factor V. This effect was specific to the form of factor V present in plasma since identical results were obtained in an appropriately reconstituted purified system, which included both TAFI and either form of factor V purified from pooled plasma. In the absence of TAFI, APC did not affect clot lysis in experiments with either normal factor V or factor VLeiden. During the various lysis assays performed with purified components, clots were solubilized and the proteolytic alterations in factor V/Va were assessed by Western blotting using a specific factor Va heavy chain monoclonal antibody. The heavy chain of factor VaLeiden persisted for as long as 60 min, in the presence of 6.3 n APC indicating sustained activity of factor VaLeiden during the lysis assay. In contrast, no factor Va heavy chain was present after the first 5.0 min in clots formed in the presence of normal factor V and 6.3 n APC. These combined data indicate that factor VaLeiden specifically attenuates the profibrinolytic effect of APC. Thus, an impaired TAFI-dependent profibrinolytic response to APC in APC-resistant individuals appears to be an additional factor contributing to the prothrombotic tendencies in subjects with factor VLeiden.
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PMID:An antifibrinolytic mechanism describing the prothrombotic effect associated with factor VLeiden. 879 78

We have previously demonstrated a low-affinity (0.8 microM, non-covalent complex formation between high-molecular-mass kininogen (HK) and plasminogen (Plg) which prevented Plg interaction with glioma and endothelial cells. We have now extended our previous observations by exploring the potential complex formation between Plg and low-molecular-mass kininogen (LK) and between LK and HK with Plg cleaved with human neutrophil elastase (HNE). Plg cleavage by HNE (PlgHNE) yielded kringles 1-3, kringle 4 and mini-plasminogen. PlgHNE was subjected to SDS/PAGE under non-reducing conditions, followed by western blotting, and incubated with either 125I-HK or 125I-LK. Autoradiograms revealed that 125I-HK bound to miniplasminogen and to kringles 1-3 but not to kringle 4 and the presence of 10 mM 6-aminohexanoic acid (Ahx) disrupted only the interaction with kringles 1-3. In contrast, 125I-LK bound to miniplasminogen but not to kringles 1-3 or 4 and Ahx had no effect at all. The complex formation of either HK (0.67 microM) or LK (3 microM) with Plg (1.5 microM) did not affect its conversion to plasmin by tissue plasminogen activator (t-PA) (10 U/ml) in the presence of a tissue plasminogen stimulator (0.14 microM). However, the rate of conversion of plasminogen to plasmin by t-PA was affected when platelets were added to the reaction mixture. Since HK (0.83 microM) has been shown to inhibit plasmin-induced platelet aggregation, we investigated whether this inhibitory property is found within the heavy chain shared by HK and LK. We found that LK inhibited plasmin-induced platelet aggregation, but a 4-fold molar excess was required when compared to HK. Compared to plasmin, 3-5-fold molar excess of miniplasmin is required to induce platelet aggregation, indicating the important role of kringles 1-3 for plasmin interactions with these cells. These results indicate that HK and LK-mediated inhibition of plasmin-induced platelet aggregation is likely due to complex formation with kringle 5 without interfering with plasmin's active site. We found an additional interaction between HK and kringles 1-3 enhancing the inhibitory effect, presumably by interfering with plasmin's interaction with platelets. This HK and LK-associated modulation of plasmin-induced platelet aggregation may serve as a template to develop synthetic peptides as novel therapeutic agents to prevent some of the plasmin-associated thrombocytopenia seen during thrombolytic therapy.
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PMID:High-molecular-mass and low-molecular-mass kininogens block plasmin-induced platelet aggregation by forming a complex with kringle 5 of plasminogen/plasmin. 942 7


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