Activation Of Glutamic Plasminogen Research Articles

Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase

Earlier studies of addition of naturally sulfated polysaccharides including unfractionated heparin showed a significant enhancement of the in-vitro activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). However, supplementing of physiological concentration of NaCl (0.9%) to the buffer reversed the enhancement. To overcome this reversal attempts were made to oversulfate the compounds and re-evaluate their biological properties. Chondroitin-6-sulfate (N-2) was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared and H-NMR studies of the oversulfated compound showed introduction of new sulfate groups with the formation of 60% of chondroitin-4-6-disulfate. In-vitro studies were conducted on comparing the effect of oversulfated chondroitin-6-sulfate (S-2) with native compound (N-2) and unfractionated heparin in enhancing the activation of Glu-Plg by t-PA or u-PA using 0.05 mol/l Tris buffer (pH 7.3) containing 0.9% of NaCl. The enhancement of activation of Glu-Plg by t-PA or u-PA was measured by formation of plasmin using H-D-Glu-Phe-Lys-pNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 microg/ml of S-2, 4-6-fold by addition of 32.4 mmol/l of lysine or 5.4 mmol/l of 6-aminohexanoic acid (6-AH) and 14-16-fold enhancement by addition of both S-2 and lysine or S-2 and 6-AH showing a synergistic effect, whereas unfractionated heparin alone gave no enhancement and in conjunction with lysine or 6-AH gave no additional enhancement. Similar studies using u-PA in place of t-PA gave identical results. During the activation of Glu-Plg to plasmin, lysine plasminogen (Lys-Plg) is reported to be an intermediate. Therefore we investigated the role of S-2, lysine and 6-AH in the activation of Lys-Plg to plasmin. The results showed that S-2 enhanced this activation, whereas lysine or 6-AH which were active in enhancing the activation of Glu-Plg were not active using Lys-Plg indicating that the site of enhancement by lysine or 6-AH was during the initial phase. Double reciprocal plot of Glu-Plg activation by t-PA with or without S-2 and lysine showed no change in Km but a 10-fold increase of Kcat suggesting a template mechanism for the attenuation when both cofactors are used.

Effect of native fucoidan, sulfated fucoidan, heparin and 6-aminohexanoic acid on the activation of glutamic-plasminogen by urokinase: role of NaCl

Addition of a physiological concentration of NaCl (0.9%) to 0.05 mol/l Tris buffer (pH 7.4) reversed the enhancement of the activation of glutamic-type plasminogen (Glu-Plg) by low molecular weight urokinase by fucoidan and heparin, while addition of aminohexanoic acid (6-AH) enhanced the activation. Native fucoidan (N-2), sulfated fucoidan (S-2) and heparin alone and in the presence of 6-AH were investigated to determine the effect of NaCl addition to 0.05 mol/l Tris (pH 7.4) on the activation of Glu-plg by high molecular weight urokinase (HMW u-Pa). Heparin alone and in conjunction with 6-AH enhanced 4.5 to 5.5-fold the initial rate of activation of Glu-plg, while N-2 alone or in conjunction with 6-AH gave 3 to 5.5-fold enhancement and S-2 gave no enhancement of activation using 0.05 mol/l Tris buffer (pH 7.4). Addition of 0.9% NaCl to the buffer reversed the enhancement by the cofactors but, in the presence of 6-AH, N-2 gave two-fold and S-2 gave three-fold enhancement of activation while heparin gave 25% enhancement. The mechanism of enhancement by S-2 in the presence of 6-AH was investigated by dilution and enzyme kinetic studies. The results show that the enhancement was due to interaction with HMW u-PA and not with Glu-plg.

Effect of oversulfation on the chemical and biological properties of fucoidan

Fucoidan was sulfated using chlorosulfonic acid–pyridine complex and isolated as the sodium salt. Infrared analysis of the native and sulfated fucoidans gave identical results in respect to the O–H stretching, hemiacetal stretching and S O stretching. Absorption around 840 cm −1 was also present in both the fucoidans representing the sulfate at the axial C-4 position except for a shoulder at 820 cm −1, which was present only in the sulfated compound indicating the presence of sulfate groups at the equatorial C-2 position. The sulfated compound showed four times higher anticoagulant activity in doubling prothrombin time of normal citrated human plasma in comparison with native fucoidan. Earlier studies using native fucoidan showed that the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or by urokinase (u-PA) was enhanced when the in vitro studies were conducted using 0.05 M Tris buffer (pH 7.4) and that the addition of physiological concentration of NaCl (0.9%) to the buffer reversed the activation. The results of the current studies showed that sulfated fucoidan gave higher stimulations of Glu-Plg activation by u-PA and by t-PA in comparison to native fucoidan when the in vitro studies are conducted using Tris buffer containing 0.9% NaCl.

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

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.

The effect of 6-aminohexanoic acid and fucoidan on the activation of glutamic plasminogen by streptokinase.

Studies were conducted on the effect of 6-aminohexanoic acid (6-AH) or fucoidan on the activation of glutamic plasminogen (glu-plg) by streptokinase using 0.05 mol/l Tris buffer containing a physiological concentration of NaCl. In contrast to the earlier reports where no NaCl was added to the buffer solution, addition of 6-AH enhanced the initial rate while the inhibition by fucoidan was not affected. Double reciprocal plots of the activation of glu-plg by streptokinase in the presence of 6-AH showed an increase in Vmax, but no change in Km. However, the addition of fucoidan showed a decrease in Vmax, but no change in Km. To determine whether the stimulatory effect of 6-AH was specifically directed towards glu-plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of 6-AH 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 glu-plg, but not of streptokinase, influenced the ratios, suggesting an interaction of 6-AH with glu-plg. Similar experiments were conducted to determine the mechanism of inhibition of streptokinase by fucoidan. The results indicated that fucoidan was interacting with streptokinase, but not with glu-plg. Circular dichroism studies of glu-plg in the near-ultraviolet spectra (250-308 nm) showed that addition of 6-AH enhanced the spectra in the region around certain chromophores, which reflected conformational changes. On the contrary, the far-ultraviolet spectra were almost identical.

Effect of fucoidan during activation of human plasminogen

Fucoidan [sulfated poly (L-fucopyranose)] was compared with 6-aminohexanoic acid (6-AH) or CNBr-cleaved fibrinogen (CNBr-Fbg) alone or in combination in enhancing the activation of glutamic plasminogen (Glu-Plg) or lysine plasminogen (Lys-Plg) by two-chain tissue plasminogen activator (t-PA) or LMwt-urokinase or by streptokinase. Fucoidan enhanced the t-PA activation of Glu-Plg or Lys-Plg at Plg concentrations greater than 75nM, while stimulation by CNBr-Fbg of t-PA activation followed saturation kinetics of Michaelis-Menton. During t-PA activation of Glu-Plg, a high degree of synergism was observed between 6-AH and fucoidan while the enhancement by CNBr-Fbg was not influenced by fucoidan and was reversed by 6-AH. Fucoidan alone at higher concentrations was effective in enhancing the activation of Glu-Plg by urokinase while the combination of fucoidan and 6-AH showed additive effect in enhancing the activation of Lys-Plg. The activation of Glu-Plg by streptokinase was reversed by fucoidan in a manner similar to that reported for 6-AH. The results are interpreted to suggest that CNBr-Fbg and 6-AH compete with each other for the same lysine binding sites (LBS) on the Plg molecule while fucoidan acted synergistically with 6-AH in enhancing the t-PA activation of Glu-Plg by a different mechanism. The double reciprocal plot for the interaction of Glu-Plg and urokinase also showed a significantly higher affinity between the two in presence of fucoidan.