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)

It has previously been shown, that large differences exist between the effects of 6-aminohexanoic acid or alpha1-antitrypsin on fibrinolysis caused by a porcine tissue plasminogen activator or by human urokinase, while insignificant differences exist between the effects of a number of natural protease inhibitors on fibrinolysis caused by the two types of plasminogen activator. The present study shows that changes in substrate composition (pH, ionic strength fibrinogen concentration, plasminogen concentration) may influence to different degrees the fibrinolytic activities of human urokinase and the porcine tissue plasminogen activator. It is suggested, that this finding is partly related to marked differences in affinity for fibrin of the two activators.
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PMID:Differences in the reactivities of human urokinase and the porcine tissue plasminogen activator. 1 58

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.
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PMID:Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells. 132 62

The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The cationic locus on the recombinant kringle 2 domain of tissue-type plasminogen activator that stabilizes its interaction with omega-amino acids. 133 68

Plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) is regulated by the vascular components fibrin(ogen) fragments, plasminogen and lipoprotein (a). Kinetic analysis demonstrates that CNBr-derived fibrinogen fragments completely protect plasmin from alpha 2AP. Plasminogen and 6-aminohexanoic acid decrease the rate of inhibition by 5- and 10-fold respectively. These studies show that CNBr-derived fibrinogen fragments and 6-aminohexanoic acid bind plasmin kringle(s) with binding constants of 2 micrograms/ml and 120 microM respectively, and that plasminogen binds to alpha 2AP with an affinity of 0.5 nM. The unmodulated inhibition is not effected by the presence of lipoprotein (a), but in the presence of protective CNBr-derived fibrinogen fragments the rate of inhibition is increased by the presence of the lipoprotein. The kinetics demonstrate that lipoprotein (a) binds to CNBr-derived fibrinogen fragments with an affinity of 4 nM, displacing plasmin from the protective surface. In addition, tissue-type plasminogen activator and trypsin inhibition by alpha 2AP is not slowed by the presence of CNBr-derived fibrinogen fragments or plasminogen (Pg), respectively. These kinetics suggest that the initial reversible interaction between plasmin and alpha 2AP is mediated by binding of the inhibitor to the kringle 1 domain of plasmin, with a reversible inhibition constant (Ki) of 5.0 x 10(-10) M. Under conditions where this kringle-inhibitor interaction is blocked, the reversible inhibition still occurs between the plasmin and alpha 2AP, but the initial Ki is increased to 5.0 x 10(-9) M. These data suggest that, in the circulation, plasmin inhibition by alpha 2AP may be down-regulated by fibrin, fibrin(ogen) fragments and Pg, but up-regulated by lipoprotein (a) in the presence of fibrin or fibrin(ogen) fragments. The lipoprotein (a)-mediated promotion of plasmin inhibition may provide an additional mechanism by which the lipoprotein impairs fibrinolysis and promotes atherosclerosis.
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PMID:Lipoprotein (a) promotes plasmin inhibition by alpha 2-antiplasmin. 138 85

We have previously isolated a monoclonal antibody, designated as 1-3-1, specific for tissue-type plasminogen activator (t-PA). We have shown that t-PA dissociates from 1-3-1 in the presence of the lysine analogue 6-aminohexanoic acid (6-AHA). Here we describe a method for the one-step immunoaffinity purification of t-PA from conditioned melanoma cell medium, using 1-3-1 immobilised on Sepharose under mild elution conditions, favourable for t-PA. The yield of t-PA (antigen or total protein) from a 1-3-1-Sepharose column, when eluted using a buffer supplemented with 0.2 M 6-AHA at neutral pH, was as effective as other buffers that involve a strong pH-change, i.e., pH 2-3. However, the enzymatic activity of the t-PA purified with 6-AHA was 25 to 30% higher, as compared with t-PA eluted using a pH change. This resulted in a markedly higher specific activity of t-PA purified with 0.2 M 6-AHA, as compared with t-PA purified using a strong pH-change. The purity of t-PA, purified using the present method, was very high, as determined by gel electrophoresis. An additional advantage of the present procedure is that the mild elution conditions prolong the column life.
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PMID:One-step purification of tissue-type plasminogen activator using affinity chromatography with a special monoclonal antibody under mild conditions. 152 79

Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.
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PMID:Binding of human plasminogen to basement-membrane (type IV) collagen. 159 90

The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-aminohexanoic acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure. All three sets of structures are well defined, with averaged atomic root-mean-square deviations between individual structures and mean set structures of 0.77, 0.99 and 0.70 A for backbone atoms, and 1.36, 1.55 and 1.41 A for all atoms, respectively. Kringle 2 is an oblate ellipsoid with overall dimensions of approximately 34 A x 30 A x 17 A. It exhibits a compact globular conformation characterized by a number of turns and loop elements as well as by one right-handed alpha-helix and five (1 extended and 4 rudimentary) antiparallel beta-sheets. The extended beta-sheet exhibits a right-handed twist. Close van der Waals' contacts between the Cys22-Cys63 and Cys51-Cys75 disulfide bridges and the central hydrophobic core composed of the Trp25, Leu46, His48a and Trp62 side-chains are among the distinguishing features of the kringle 2 fold. The binding site for 6-AHA appears as a rather exposed cleft with a negatively charged locus defined by the Asp55 and Asp57 side-chains, and with an aromatic pocket structured by the Tyr36, Trp62, His64 and Trp72 side-chains. The Trp62 and His64 rings line the back surface of the pocket, while the Tyr36 and Trp72 rings confine it from two sides. The Trp62 and Trp72 indole rings conform a V-shaped groove. The methyl groups of Val35 also contribute lipophilic character to the ligand-interacting surface. It is suggested that the positively charged side-chains of Lys34 and, potentially, Arg69 may favor interactions with the carboxylate group of the ligand. The Trp25 and Tyr74 aromatic rings, although conserved elements of the binding site structure, seem not to undergo direct contacts with the ligand.
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PMID:Solution structure of the tissue-type plasminogen activator kringle 2 domain complexed to 6-aminohexanoic acid an antifibrinolytic drug. 176 44

The binding of plasminogen to preformed human plasma clots immersed in citrated human plasma was measured and correlated with the sensitivity of these clots to lysis with recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA) or two chain urokinase-type plasminogen activator (tcu-PA, urokinase). When 0.15 ml plasma clots were compressed mechanically to about 1% of their original weight, and immersed in 0.15 ml plasma, 131I-labeled native plasminogen (Glu-plasminogen) adsorbed progressively from the plasma milieu onto the clot; binding was 3 +/- 1% (n = 10) after 1 h, 7 +/- 1% after 12 h and 12 +/- 1% after 48 h. This was associated with an increased sensitivity of the clot to lysis; 50% clot lysis in 4 h was obtained with 65 +/- 5 ng/ml (n = 3) rt-PA before and 30 +/- 5 ng/ml (n = 3) after 48 h preincubation in plasma (p less than 0.01), with corresponding values of 660 +/- 55 ng/ml (n = 3) and 280 +/- 25 ng/ml (n = 3) for rscu-PA, (p less than 0.01), and 800 +/- 85 ng/ml (n = 3) and 270 +/- 35 ng/ml (n = 3) for urokinase (p less than 0.01). Additional binding of plasminogen and increased sensitivity to lysis were reduced or abolished when the clot was preincubated in plasminogen-depleted or in t-PA-depleted plasma, or when 20 mM 6-aminohexanoic acid or 2,000 KIU/ml aprotinin were added.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Correlation between progressive adsorption of plasminogen to blood clots and their sensitivity to lysis. 209 91

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.
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PMID:Kinetic studies on the effect of heparin and fibrin on plasminogen activators. 244 77


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