Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Structural requirements for binding to the bone calcitonin (CT) receptor and for CT bioactivity both in vitro and in vivo were assessed for a series of N-terminally truncated, N alpha-acetylated, fragments of salmon calcitonin (sCT). Sequential deletion of amino acid residues from the amino-terminus of [Ala7]sCT-(2-32) peptide amide first led to partial agonists and, upon deletion of residues 1 to 7, to a high affinity antagonist, N alpha-acetyl-sCT-(8-32)-NH2. The presence of two separate domains within the sCT sequence is proposed: (I) a binding domain comprising residues 9-32 and (II) an activation domain requiring residues 3 to 6. N alpha-acetyl-sCT-(8-32)-NH2, in several bioassays including plasminogen activator release from LLC-PK1 cells (pA2 = 7.31), cAMP production in UMR-106-06 cells (pA2 = 7.81) and in the fetal rat long bone resorption assay showed potent antagonistic properties.
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PMID:N-terminal truncation of salmon calcitonin leads to calcitonin antagonists. Structure activity relationship of N-terminally truncated salmon calcitonin fragments in vitro and in vivo. 132 97

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.
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PMID:Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin. 147 36

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.
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PMID:Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide. 153 63

We present evidence that over-expression of human plasminogen, the precursor to the serine protease plasmin, can be cytotoxic to mammalian cells. When an expression vector containing plasminogen cDNA is transfected into baby hamster kidney cells, the number of drug-resistant colonies as well as the levels of plasminogen secreted by those colonies is lower than observed in similar transfections of other protease precursor genes. The recombinant plasminogen accumulates intracellularly as degraded NH2-terminal fragments. In contrast, a mutant of plasminogen that produces inactive plasmin (active site Ser740 changed to Ala) is synthesized by these cells as a full-length plasminogen molecule, and the colony numbers and expression levels are normal. Thus, the generation of plasmin activity is responsible for the cytotoxic phenomena and the degradation associated with plasminogen expression. In addition, experiments using a plasminogen mutant that cannot be activated to plasmin (activation cleavage site Arg560 to Gly) or using coexpression of antisense urokinase RNA indicate that an endogenous plasminogen activator is responsible for converting newly synthesized plasminogen to plasmin. Finally, coexpression of plasminogen with alpha 2-plasmin inhibitor, a serpin which is the physiologic inhibitor of plasmin, prevents the toxic effects of intracellular plasmin activity and allows the synthesis and secretion of native human plasminogen.
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PMID:Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity. 183 Dec 1

Laminin is a large multidomain glycoprotein with diverse biological activities which include stimulation of neurite outgrowth, enhancement of tumor metastasis, and promotion of cell growth, adhesion, and differentiation. A 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg- NH2) was identified which promotes metastasis and stimulates collagenase IV activity in the culture medium of B16 melanoma cells (Kanemoto et al., 1990). We report that this peptide, here designated LamA2091-2108, is also a potent stimulator of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, resulting in a 22-fold increase in the kcat/Km of the activation reaction. The activity of purified type I and type IV collagenase was inhibited by LamA2091-2108 with IC50 values of 3 and 43 microM, respectively. These data support an alternative mechanism for the appearance of collagenase activity in the culture media of melanoma cells, namely, that the peptide stimulates plasminogen activation, subsequently generating collagenase activity.
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PMID:Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain. 184 24

The heparin-binding p30 protein amphoterin is proposed to mediate adhesive interactions of the advancing plasma membrane in migrating and differentiating cells. Since the NH2-terminal part of amphoterin is exceptionally rich in lysine residues, we have studied its interactions with plasminogen and tissue plasminogen activator (t-PA). On immunostaining of N18 neuroblastoma cells, amphoterin and t-PA showed a close co-localization in the filopodia of the leading membrane and in the substrate-attached material. In purified systems, both t-PA and plasminogen bound to immobilized amphoterin, and their binding was inhibited by the lysine analogue epsilon-aminocaproic acid. Plasminogen bound to immobilized amphoterin was activated by t-PA, and this resulted in effective degradation of the immobilized amphoterin. Correspondingly, amphoterin-bound t-PA activated plasminogen. In solution amphoterin accelerated t-PA-catalyzed plasminogen activation maximally 46-fold. The results indicate that t-PA and plasminogen form through their lysine-binding sites a complex with amphoterin, which results in acceleration of plasminogen activation and effective degradation of amphoterin. We suggest that local acceleration of t-PA-catalyzed plasminogen activation by amphoterin at the leading membrane enhances the penetration of growing cytoplasmic processes through extracellular materials during cell migration, differentiation and regeneration. The amphoterin-mediated adhesion at the leading membrane may be transient in nature, because the protein also enhances its own breakdown by accelerating t-PA-catalyzed plasminogen activation.
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PMID:Interactions of plasminogen and tissue plasminogen activator (t-PA) with amphoterin. Enhancement of t-PA-catalyzed plasminogen activation by amphoterin. 190 31

Fibrinogen-NDSK complex is a model of protofibril having some features of the fibrin polymer structure. This complex has been studied for its ability to stimulate the plasminogen activation by t-PA. The fibrinogen-NDSK complex have increased the rate of plasminogen activation by t-PA as compared to fibrinogen or NDSK taken separately. This acceleration had slow and fast phases. Lys-plasminogen was activated more effectively as compared to glu-plasminogen. The kinetic parameters of glu- and lys-plasminogen activation at fast phase were: Km--0.18 and 0.015 mu/M, Kkat--0.27 and 0.06 s-1, respectively. Fibrinogen X2--fragments, deprived of alpha C-domains and NH2-end peptides of bB-chains, formed complexes with NDSK, which however did not stimulate the plasminogen activation by t-PA. These findings have shown that the fibrinogen-NDSK complex is an effective stimulator of the plasminogen activation by t-PA. The activating ability of the complex may be due to structures formed in the course of fibrinogen and NDSK polymerization as a result of alpha C-domain interaction.
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PMID:[Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex]. 192 82

Phospholipases A2 (PLA-2) are conserved enzymes that can vary widely in their activity toward certain biological targets. Activity of PLA-2 toward Escherichia coli treated with the bactericidal/permeability-increasing protein (BPI) of granulocytes has been detected only in "Group II" PLA-2 (lacking Cys11-Cys77) and correlates with overall basicity and the presence of a cluster of basic amino acids within a variable surface region near the NH2 terminus (including residues 6, 7, 10, 11, and 15). We now show that of five pancreatic PLA-2 ("Group I" enzymes) tested from different species of mammals, the human enzyme that is most basic both globally (pI 8.7) and locally (Arg-6, Lys-7, and Lys-10) is active toward BPI-treated E. coli (approximately 1-2% activity of the most active Group II PLA-2) whereas the other four PLA-2 are essentially inactive (less than 0.1%). The cDNA of the pig pancreatic PLA-2 (pI 6.4; Arg-6, Ser-7, Lys-10) has been modified by site-specific mutagenesis and the wild-type and mutant PLA-2 have been expressed in and purified from either E. coli or Saccharomyces cerevisiae to determine more precisely the structural determinants of PLA-2 activity toward BPI-treated E. coli. The single substitution of lysine (or arginine) for Ser-7 transformed the pig pancreatic PLA-2 into an active enzyme toward BPI-treated E. coli possessing 25-50% the activity of the human PLA-2. Additional modifications to increase global basicity (increase in net charge up to +4) caused a further (up to 2-fold) increase in activity. All mutant PLA-2 still containing Ser-7 possessed little or no activity toward BPI-treated E. coli. Changes in activity toward BPI-treated E. coli were accompanied by parallel changes in enzyme binding to this target. In contrast, substitution of lysine (or arginine) for Ser-7 caused little or no alteration of enzyme activity toward either autoclaved E. coli or egg yolk lipoproteins indicating no major effects on the catalytic properties of the PLA-2. This study demonstrates directly the role of NH2-terminal basic residues in the action of PLA-2 on BPI-treated E. coli and suggests that these properties mainly facilitate PLA-2 binding to this biological target.
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PMID:Conversion of pig pancreas phospholipase A2 by protein engineering into enzyme active against Escherichia coli treated with the bactericidal/permeability-increasing protein. 199 11

The effect of fibrin-targeting of urokinase-type plasminogen activator (u-PA) on its fibrinolytic potency was studied using recombinant fusion proteins of u-PA with the NH2-terminal region of tissue-type plasminogen activator (t-PA/u-PA) and chemical complexes of u-PA with F(ab')2 fragments of a fibrin specific monoclonal antibody (u-PA/MA-15C5-F(ab')2). Two chain derivatives of a low Mr variant of u-PA comprising amino acids Leu144-Leu411 (tcu-PA-32k), obtained by cleavage of recombinant single-chain u-PA (rscu-PA-32k) with thrombin (rtcu-PA-32k/T) or plasmin (rtcu-PA-32k/P) were investigated. The plasmin-derived two chain u-PA moieties, rtcu-PA-32k/P, rt-PA/tcu-PA-32k/P and rtcu-PA-32k/MA-15C5-F(ab')2/P had high specific activities in amidolytic and fibrin plate assays (130,000 and 150,000 IU/mg u-PA, 43,000 and 71,000 IU/mg u-PA and 32,000 and 56,000 IU/mg u-PA respectively). The thrombin-derived two chain u-PA moieties had a very low amidolytic activity, corresponding to less than or equal to 1 percent of that of their plasmin-derived counterparts. On fibrin plates, however, rtcu-PA-32k/T had a negligible activity, whereas rt-PA/tcu-PA-32k/T and rtcu-PA-32k/MA-15C5-F(ab')2/T had specific activities of 12,000 and 25,000 IU/mg u-PA respectively. The catalytic efficiency for plasminogen activation of rtcu-PA-32k/MA-15C5-F(ab')2/T is 4,000-fold lower than that of rtcu-PA-32k/MA-15C5-F(ab')2/P, but its concentration required for 50 percent lysis in 2 hours of a 125I-fibrin labeled plasma clot in human plasma (C50) is only 25-fold higher. The catalytic efficiency of rt-PA/tcu-PA-32k/T is 1,600-fold lower and the C50 100-fold higher than that of rt-PA/tcu-PA-32k/P. The catalytic efficiency and the fibrinolytic potential of rtcu-PA-32k/T are negligible as compared to that of rtcu-PA-32k/P. These observations may be explained by conversion of the thrombin derived two chain u-PA moieties to their plasmin-derived analogues at the fibrin surface. This conversion appears to be most efficient for the antibody conjugate which has a high fibrin-affinity, less efficient for the t-PA/u-PA chimera which has only moderate fibrin-affinity, and negligible for the unconjugated u-PA moiety which has no fibrin-affinity. These findings illustrate the importance of plasmin-mediated positive feedback mechanisms in u-PA mediated clot lysis.
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PMID:Effect of fibrin-targeting on clot lysis with urokinase-type plasminogen activator. 210 93

Recombinant variants of tissue plasminogen activator (t-PA) containing either substitutions or deletions of amino acids within the fibronectin finger-like domain (residues 6-50) were found to exhibit widely varying in vivo clearance profiles in rats and fibrinolytic activity in 125I-fibrin clot lysis assays. Clearance was not significantly affected by changes in the densely charged region of amino acid residues 7-10. Deletions or substitutions of amino acids in the region 14-32 decreased both fibrinolytic activity and the clearance of the enzyme. Modifications within the predicted omega loop of residues 37-41 affected clearance only to a small degree, whereas amino acid alterations in the region of residues 42-49 resulted in as much as a 6-fold decrease in the rate of clearance with only relatively minor decreases in the fibrinolytic activity of the variants. The cumulative results distinguish discrete sections of the NH2-terminal region of the enzyme as determinants of in vivo clearance and fibrinolytic activity of t-PA. In addition, the fibrinolytic activity of a variant containing the substitutions Gln42----Asn, His44----Glu, and Asn117----Gln, when compared with wild-type t-PA in an in vivo rabbit venous clot lysis model, was found to have similar lytic efficacy at approximately one-fourth the dose. We conclude that decreases in the in vivo clearance of t-PA can result in more potent thrombolytic agents in vivo, even though the in vitro fibrinolytic activity of the enzyme may be somewhat impaired.
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PMID:Site-directed mutagenesis in human tissue-plasminogen activator. Distinguishing sites in the amino-terminal region required for full fibrinolytic activity and rapid clearance from the circulation. 210 43


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