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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site. 249 49

We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Biochem. 104, 579-586], we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions. Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli. 249 71

This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affinity chromatography and gel filtration in the presence of aprotinin. The final product (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70,000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at Mr 70,000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37,000 and 33,000 under reduced conditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic activity.
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PMID:Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle. 249 95

We have used differential scanning calorimetry to measure the effect of replacements of valine 65 on thermal stability of the isolated kringle-2 domain of tissue plasminogen activator (t-PA). The role of this site in stability was examined because a human t-PA variant having this valine (residue 245 in t-PA numbering) replaced with a methionine has been described [Johnston, M.D., & Berger, H. (1987) U.K. Patent Application GB 2176702A]. Mutants of kringle-2 having valine 65 replaced with Met, Leu, Ile, Thr, Ala, or Ser were constructed by using site-directed mutagenesis in conjunction with a restricted site selection strategy. Isolated kringle-2 domains were expressed in Escherichia coli and purified as previously described for the wild-type domain [Cleary, S., Mulkerrin, M.G., & Kelley, R.F. (1989) Biochemistry 28, 1884-1891]. None of these substitutions results in a significant perturbation of the native conformation of kringle-2 as judged by far-UV circular dichroism and equilibrium dialysis measurements of L-lysine affinity. A two-state analysis of the heat capacity profile observed for heating a solution of wild-type (w-t) kringle-2 containing 100 mM citrate, pH 4.5, provides values of 64.3 +/- 0.8 degrees C for Tg (melting temperature), 81 +/- 5 kcal/mol for delta H g, and 1.2 +/- 0.9 kcal/(mol-deg) for delta C p. Thermal denaturation of w-t kringle-2 is reversible in the pH range 3-6 as indicated by the observation of similar heat capacity profiles for consecutive heating cycles and also recovery of spectroscopic and lysine binding properties upon cooling the heat-denatured protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain. 250 77

Bovine serum albumins (BSA), modified with a variety of carboxyl group agents, stimulated the tissue-type plasminogen activator (t-PA)-catalyzed activation of human plasminogen. Modification with taurine (tau) and putrescine (put) provided the best stimulants. The tauBSA and putBSA were effective at a concentration of 5 micrograms/ml and enhanced the Lys-plasminogen activation by two-chain t-PA in a dose-dependent manner to a maximum of 44- to 46-fold at 200 micrograms/ml. The Km values for the activation of Glu-plasminogen by t-PA in the presence of tauBSA and putBSA (100 micrograms/ml) were 1.7 and 1.8 microM, while the kcat values were 0.059 and 0.062 s-1, respectively. T-PA was bound to both tauBSA and putBSA, which were immobilized on agarose beads, with KD values of 163 and 138 nM, respectively. The two modified BSAs were good substrates for plasmin and were hydrolyzed by the enzyme to small peptides. All of these modified BSA-related actions were inhibited by lysine analogs (e.g. tranexamic acid) which were adjusted to the concentrations required for the inhibition of the plasminogen (Kringle 1 domain) binding to fibrin. On the other hand, acetylation or succinylation of the amino groups of BSA was not effective, while alkylation of the thiol groups of this protein resulted in a moderate stimulation of the plasmin generation. The present results show that t-PA and plasminogen form complexes with certain charge-modified BSAs via their lysine-binding sites. The different stimulation potency of modified BSAs may provide a model for in vivo counterparts of fibrin.
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PMID:A comparison of bovine serum albumins, modified with a variety of carboxyl group agents, as stimulators of tissue-type plasminogen activator-catalyzed activation of plasminogen. 250 19

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.
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PMID:Production in eukaryotic cells and characterization of four hybrids of tissue-type and urokinase-type plasminogen activators. 250 71

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.
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PMID:Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator. 250 19

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator. 250 59

The fibrinolytic (fibrin dissolving) properties of several anionic, cationic, nonionic and zwitterionic detergents were assessed in an in vitro fibrin agarose assay. Of the 4 anionic detergents tested, only sodium dodecyl sulfate (SDS) was found to be fibrinolytic. SDS was fibrinolytic either in the absence or presence of factor XIII. Four other cationic detergents were found to possess similar fibrinolytic properties. These cationic detergents were cetyltrimethylammonium bromide (CTAB), mix alkyltrimethyl ammonium bromide (MTAB), hexadecyltrimethylammonium bromide (HTAB) and cetylpyridium chloride (CPC). The nonionic (digitonin, triton X-100/tween 20) and zeitterionic (CHAPS, zeittergent 3-08) detergents were not fibrinolytic. Detergents mediated fibrinolysis, unlike that of tissue type plasminogen activator and urokinase, was independent of the presence of plasminogen. Non-detergents such as polyethylene glycol and highly charged compounds such as poly-1-lysine and poly-1-glutamic acid were not fibrinolytic. Fibrinolytic activity was observed for SDS and the cationic detergents at concentrations ranging from 0.1-10 percent. The effects of these fibrinolytic detergents (SDS, CTAB, MTAB, HTAB and CPC) on clot formation and on pre-formed clots were then assessed, using freshly drawn human venous blood. Incorporation of these detergents into blood inhibited the formation of clots in a concentration dependent manner. The detergents were also able to dissolve pre-formed clots in a similar fashion. SDS was found to be most potent in these properties.
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PMID:Fibrin solubilizing properties of certain anionic and cationic detergents. 251 Mar 56

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.
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PMID:Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments. 251 Aug 23


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