Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Experimental and clinical data suggest that tissue factor (TF), the major initiator of blood coagulation cascade, as well as proteases and components of the fibrinolytic system are involved in tumor growth at least in some solid tumors via effects on angiogenesis. Whereas the pro- and anti-angiogenic effects of the plasminogen/plasmin system and plasminogen kringle domains, respectively, are well characterized, the pathways responsible for the pro-angiogenic properties of TF remain poorly understood. To learn more about the biological significance of the recently described binding of plasminogen to the extracellular domain of TF, we examined the effects of soluble TF (sTF) on angiostatin-inhibited proliferation of endothelial cells. In solid phase binding assays, we found that sTF binds specifically to plasminogen, to the plasminogen kringle domains K1-3, K1-5, K4, as well as to mini-plasminogen. Inhibition of binding of plasminogen and its kringle domains to sTF by the lysine analog 6-aminohexanoic acid (AHA) suggests that lysine-binding sites are involved in plasminogen interaction with TF. Moreover, in the presence of sTF, the inhibitory effect of K1-5 on bFGF-mediated HUVEC proliferation was dose-dependently and saturably abolished. This suggests that TF can interfere with the antagonistic effect of K1-5 on endothelial cell proliferation. In contrast, sTF by itself had no effect on the endothelial cell proliferation. Whereas the interference of TF with K1-5-mediated effect was prevented by AHA, this lysine analog did not abolish the proliferation inhibition of K1-5. In conclusion, the binding of sTF to the plasminogen fragment K1-5 seems to antagonize the anti-angiogenic effects of this plasminogen fragment.
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PMID:Soluble tissue actor interferes with angiostatin-mediated inhibition of endothelial cell proliferation by lysine-specific interaction with plasminogen kringle domains. 1252 59

Earlier studies on the stimulatory effect of fucoidan, heparin, and cyanogen bromide (CNBr)-fibrinogen digest on the in-vitro activation of glutamic type plasminogen by tissue plasminogen activator, which were performed using subphysiologic ionic strengths of buffers, gave inconsistent results because of the variation in the ionic strengths of the buffers used. Studies were therefore conducted on the effect of these cofactors using 0.05 mol/l Tris buffer containing a physiologic concentration of sodium chloride. The double reciprocal plots of the activation of glutamic type plasminogen by tissue plasminogen activator in the presence of fucoidan and 6-aminohexanoic acid (6-AH) or heparin and 6-AH showed a four- to six-fold increase in K(cat), while the K(m) remained unchanged. On the other hand, there was greater than six-fold lowering of K(m) from 0.213 to 0.035 micromol/l in the presence of CNBr-fibrinogen, while K(cat) was only slightly increased. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of glutamic type plasminogen or of tissue plasminogen activator after serial dilution. The results suggested that the enhancements by fucoidan and 6-AH or CNBr-fibrinogen were due to their interactions directed towards glutamic type plasminogen, while for heparin and 6-AH, the interaction was directed towards tissue plasminogen activator. Circular dichroism studies in the near ultraviolet range (250-308 nm) showed that 6-AH enhanced the circular dichroism spectra of glutamic type plasminogen around certain chromophores, while fucoidan and heparin had no effect, suggesting that the enhancement by the cofactors may be related to the favorable conformational changes of glutamic type plasminogen by 6-AH.
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PMID:The effect of fucoidan, heparin and cyanogen bromide-fibrinogen on the activation of human glutamic-plasminogen by tissue plasminogen activator. 1269 44

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.
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PMID:Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin. 1270 62

Studies were conducted on the effect of heparin or 6-aminohexanoic acid (6-AH) on the activation of glutamic plasminogen (Glu-Plg) by streptokinase in the presence of different concentrations of buffer, NaCl and divalent cations. Heparin and 6-AH inhibited streptokinase-mediated activation of Glu-Plg using 10 mM Tris-HCl buffer pH 7.4. This inhibition was partially reversed by the addition of 0.2-1.0 mM of Mg ions. Increasing the ionic strength of Tris-HCl buffer from 10 to 50 mM or addition of 50-150 mM of NaCl to 50 mM Tris-HCl pH 7.4 inhibited the activation of Glu-Plg by streptokinase while decreasing the % inhibition by heparin over the control samples. Double reciprocal plot of the activation of Glu-Plg by streptokinase using 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl showed that the addition of heparin lowered Vmax by 50% without affecting Km. To determine whether the inhibitory effect of heparin was specifically directed towards Glu-Plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of heparin over the controls were plotted against the inverse of the volume fraction of Glu-Plg or streptokinase after serial dilutions. The results indicated that the dilutions of streptokinase but not of Glu-Plg influenced the ratios, suggesting an interaction of heparin with streptokinase. Addition of 6-AH reversed the inhibitory effect of NaCl on the activation of Glu-Plg by streptokinase and the results of the near UV CD spectra of Glu-Plg showed that addition of 6-AH enhanced the spectra in this region with an increase in the ellipticity which was not affected by addition of NaCl.
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PMID:Ionic modulation of the effects of heparin and 6-aminohexanoic acid on plasminogen activation by streptokinase: the role of ionic strength, divalent cations and chloride. 1287 76

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.
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PMID:[Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex]. 1291 37

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.
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PMID:[Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin]. 1292 19

Studies were conducted on the mechanism of the stimulatory effect of 6-aminohexanoic acid (6-AH) during the in vitro activation of human glutamic plasminogen (Glu-Plg) by streptokinase or by tissue plasminogen activator (t-PA) and the possible role of the addition of physiological concentrations of NaCl to the buffer solution. Enhancement by 6-AH was investigated by measuring the rate of plasmin generation using chromogenic substrate H-D-glu-phe-lys-pNA (S-2403). Control studies using plasmin showed that the addition of 6-AH at concentrations below 20 mM did not significantly affect the initial rate of the amidolytic activity of plasmin with or without the addition of NaCl to 0.05 M Tris buffer (pH 7.4). On the other hand, addition of NaCl to the buffer slowed down the initial rate of activation of Glu-Plg by streptokinase or by t-PA while increasing the percent enhancement by 6-AH when compared with the controls. The ratios of the initial rates of plasmin generation in the presence or in the absence of 6-AH were plotted against the inverse of the volume fraction of Glu-Plg, streptokinase or t-PA after serial dilutions. The results showed that when the activation reactions were performed in 50 mM of Tris buffer (pH 7.4), the enhancements by 6-AH were related to its interaction with streptokinase or t-PA, while using the same Tris buffer containing 0.6 % NaCl, the enhancements by 6-AH were related to its interaction with both Glu-Plg and streptokinase or t-PA. However, upon increasing the NaCl to 0.9%, the results showed that the enhancements by 6-AH of the activation of Glu-Plg by streptokinase or t-PA were related to its interaction with Glu-Plg. The results suggested that changes in the concentrations of NaCl play a regulatory role during the activation process.
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PMID:Mechanism of the stimulatory effect of 6-aminohexanoic acid on plasminogen activation by streptokinase or tissue plasminogen activator: the role of chloride. 1474 74

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.
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PMID:Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity. 1476 98

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg </=2-fold, but surprisingly, rendered both interactions near totally LBS-dependent. The LBS-independent affinity of SK for [Lys]Pm, 3000-fold higher compared with [Lys]Pg, was reduced dramatically by alpha-domain truncation. Thermodynamic analysis demonstrates that the SK alpha-domain contributes substantially to affinity for all Pg/Pm species solely through LBS-independent interactions, and that the higher affinity of SK for [Lys]Pm compared with [Lys]Pg involves all three SK domains. The residual affinity of the SK betagamma-fragment for all Pg/Pm species was increased by an enhanced contribution to complex stability from LBS-dependent interactions or free energy coupling between LBS-dependent and -independent interactions. Redistribution of the free energy contributions accompanying alpha-domain truncation demonstrates the interdependence of SK domains in stabilizing the SK-Pg/Pm complexes. The flexible segments connecting the SK alpha, beta, and gamma domains allow their rearrangement into a distinctly different bound conformation accompanying loss of the constraint imposed by interactions of the alpha-domain.
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PMID:Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin. 1562 24

Native Fucoidan and unfractionated heparin enhanced by 6-fold the in vitro activation of human glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05M Tris buffer pH 7.4, while sulfated fucoidan inhibited the activation under these conditions. Double reciprocal plots of these interactions showed that sulfated fucoidan inhibited the activation in a noncompetitive manner while the enhancements by heparin or native fucoidan were due to an increase of Vmax without affecting Km. To determine whether the stimulatory effect of the individual cofactor was due to its interaction with Glu-Plg or with t-PA, experiments were performed at a fixed level of the cofactor and either varying in a serial fashion the concentration of Glu-Plg or of t-PA. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of Glu-Plg or of t-PA. The results showed that heparin interacted with Glu-Plg while native fucoidan and sulfated fucoidan interacted with t-PA. Studies were also conducted on the effect of the two fucoidans and heparin on the activation of Glu-Plg by t-PA using 0.05M Tris buffer pH 7.4 containing 0.1 M NaCl. Under these conditions, sulfated fucoidan was most effective in enhancing the activation followed by native fucoidan and heparin respectively. The results of this study showed that in presence of the buffer containing 0.1 M NaCl, heparin was interacting with t-PA while the two fucoidans were interacting with both t-PA and Glu-Plg. A comparison of the double reciprocal plots of the rate of enhancement by the cofactors using 0.05M Tris buffer pH 7.4 containing 0.1M NaCl or in presence of buffer alone showed that the cofactors were more effective using 0.05M Tris buffer pH 7.4 alone and that addition of NaCl to the buffer slowed down the reactions by decreasing Vmax without affecting Km. Circular Dichroism (CD) studies of Glu-Plg in the far ultraviolet (UV) range showed that addition of NaCl destabilized the beta sheet structure which was reversed by addition of 6-aminohexanoic acid (6-AH) or one of the cofactors, while the near UV CD spectra of Glu-Plg in presence of 0.1 M NaCl was enhanced by the cofactors by increasing its ellipticity as reported earlier for 6-AH.
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PMID:Mechanism of the stimulatory effect of native fucoidan, highly sulfated fucoidan and heparin on plasminogen activation by tissue plasminogen activator: the role of chloride. 1572 89


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