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

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

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

Plasmin is a labile enzyme destroyed by a process termed autodigestion. Studied by a kinetic assay on the substrate Tos-Gly-Pro-Lys-pNA this process is shown to follow a bimolecular mode of reaction, which is retarded by plasmin degradation products. Plasmin is protected by fibrinogen, by epsilon-aminocaproic acid (6-aminohexanoic acid), by increasing ionic strength, and by glycerol. CNBr fragments of fibrinogen did not protect. Lack of substrate protection of plasmin may give rise to errors in a two-stage plasminogen activator assay, while the presence of substrate in a one-stage method prevents degradation of the generated plasmin.
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PMID:The autodigestion of human plasmin follows a bimolecular mode of reaction subject to product inhibition. 293 86

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.
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PMID:A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis. 294 88

The catalytic efficiency (kcat/Km) of high-molecular-mass urokinase for the activation of Glu-plasminogen is increased about 10-fold in the presence of CNBr-digested fibrinogen. This stimulation is similar to that observed with 6-aminohexanoic acid, and yields kinetic parameters comparable to those for the activation of Lys-plasminogen by urokinase. The increase of the activation rate of Glu-plasminogen by urokinase in the presence of CNBr-Fg can thus be explained by a conformational change in the plasminogen molecule similar to that observed upon conversion of Glu-plasminogen to Lys-plasminogen and upon binding of 6-aminohexanoic acid to Glu-plasminogen. Stabilization of the Michaelis complex between urokinase and plasminogen by formation of a cyclic ternary complex with CNBr-Fg, which has been invoked to explain the dramatic stimulatory effect of CNBr-Fg on the activation of plasminogen by tissue-type plasminogen activator, does not appear to play a significant role in the increased activation rate.
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PMID:Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase. 648 41

The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4 degrees C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd > 350 nmol/L). At 37 degrees C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid-inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan-inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor-associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan-inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t-PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA.
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PMID:Degradation of tissue-type plasminogen activator by human monocyte-derived macrophages is mediated by the mannose receptor and by the low-density lipoprotein receptor-related protein. 757 46

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.
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PMID:Complement component C7 is a plasminogen-binding protein. 781 88

It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligand-binding studies were performed to characterize the interactions between t-PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t-PA to immobilized FCB-2 (Kd = 0.70 +/- 0.10 nM) and FCB-5 (Kd = 0.47 +/- 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 1 +/- 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit t-PA binding to solid-phase fibrin (Ki = 3.3 microM and 6.4 microM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (Ki = 123 +/- 47 microM and 364 microM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t-PA binding, thus confirming the existence of both a lysine-dependent and a lysine-independent mechanism of binding of t-PA to both fibrin and FCB-2. In contrast, the binding of t-PA to FCB-5 was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences present in FCB-5 and FCB-2; contribution to binding by a lysine-dependent interaction was detected only in FCB-2 and is probably of minor relevance as suggested by the limited effect of 6-aminohexanoic acid.
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PMID:Study of tissue-type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen. 811 48

A series of conservative and radical mutations have been made at an aromatic residue, Y76, of the isolated kringle 2 domain of tissue-type plasminogen activator ([K2tPA]) in order to assess the importance of this residue in the ligand binding properties and structural stability of this protein domain. We have successfully expressed in Escherichia coli r-[K2tPA] variants with the following amino acid mutations at Y76: Y76-->A, Y76-->E, Y76-->F, Y76-->K, Y76-->L, Y76-->Q, and Y76-->W. The binding constants of 6-aminohexanoic acid (EACA) and 7-aminoheptanoic acid (7-AHpA) to each of these mutants were investigated by titration of the alterations in intrinsic fluorescence of the mutant kringles with these amino acid ligands. Compared to the wild-type kringle (r-[K2tPA]), which possessed dissociation constants (Kd) of 43 and 6 microM, respectively, for EACA and 7-AHpA, only the Y76-->E mutant displayed a substantially increased Kd value for these amino acids, viz., 117 microM for 7-AHpA. More moderate increases in this parameter were observed for the Y76-->A and Y76-->K variants (2-3-fold increases in the Kd), with no significant differences noted in the cases of Y76-->L, Y76-->Q, and Y76-->W. A most interesting observation was made with the Y76-->F mutant, which showed a 4-6-fold reduction in the Kd for these amino acid ligands. The conformations of all of the mutants were less stable than that of wtr-[K2tPA], as revealed by thermal denaturation studies, suggesting that a Y at sequence position 76 is of importance to the conformational stability of this kringle domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of tyrosine-76 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability and its omega-amino acid ligand binding site. 814 48

The 1H-NMR spectrum of the kringle 1 domain of human plasminogen complexed with 6-aminohexanoic acid, an antifibrinolytic drug, has been assigned. Elements of secondary structure have been identified on the basis of sequential, medium and long-range dipolar interactions, back-bone amide spin-spin couplings (3JHN-H alpha) and 1H-2H exchange rates. The kringle contains scarcely any repetitive secondary structure: eight reverse turns and two short beta-sheets. These comprise 40% and 12% of the domain, respectively. No alpha-helix was found. An aromatic cluster formed by His31, Phe36, Trp62, Phe64, Tyr72 and Tyr74 is indicated by several inter-residue Overhauser connectivities. Contacts between the methyl groups of Leu46 and the side chains of Phe36, Trp62 and Trp25 are observed. A second hydrophobic cluster formed by Tyr9, Ile77 and Leu78 is also indicated. A comparison of secondary structure elements among plasminogen kringles 1 and 4 and tissue-type plasminogen activator kringle 2 suggests that there is variability in the position and number of reverse turns on going from one kringle to another; however, the beta-sheets are conserved among the homologs.
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PMID:1H-NMR assignments and secondary structure of human plasminogen kringle 1. 818 75


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