Effect of oversulfated chondroitin-6-sulfate or oversulfated fucoidan in the activation of glutamic plasminogen by tissue plasminogen activator: role of lysine and cyanogen bromide-fibrinogen.

Fucoidan and chondroitin-6-sulfate were oversulfated using chlorosulfonic acid-pyridine complex and were isolated as the sodium salt. Infrared analysis of oversulfated compounds showed introduction of sulfate groups in new positions, and in-vitro studies of the compounds showed a significant increase in the anticoagulant property. Addition of 28.6 microg/ml oversulfated compound gave a two-fold to four-fold increase in the rate of enhancement of activation of glutamic plasminogen by tissue plasminogen activator using 0.05 mol/l Tris buffer (pH 7.35) containing physiological concentrations of NaCl (0.9%). Under these conditions, unfractionated heparin was not active and the native compounds gave less than 30% enhancement. In the present study, the effect of lysine or cyanogen bromide-treated fibrinogen, alone or in combination with the oversulfated compounds, on the activation of glutamic plasminogen by tissue plasminogen activator was investigated. Addition of 16.2 mmol/l L-lysine gave three-fold to four-fold enhancement of activation, which was further enhanced to five-fold to six-fold by addition of 2.86 microg/ml oversulfated chondroitin-6-sulfate or oversulfated fucoidan. Cyanogen bromide-treated fibrinogen (50 microg/ml) gave a 10-fold enhancement of activation by itself, and addition of 2.86 microg/ml oversulfated compounds amplified this to 15-fold. A 25-fold to 35-fold enhancement of activation of glutamic plasminogen was obtained when 2.86 microg/ml oversulfated compounds were combined with 16.2 mmol/l lysine and 50 microg/ml cyanogen bromide-treated fibrinogen. Dilution studies showed that the amplification of the enhancement of lysine by 2.86 microg/ml oversulfated compound was related to interaction of the cofactors with both glutamic plasminogen and tissue plasminogen activator.

Effect of oversulfation on the chemical and biological properties of chondroitin-4-sulfate.

Chondroitin-4-sulfate was oversulfated using chlorosulfonic acid-pyridine complex and was isolated as the sodium salt. A comparison of the infrared analysis of the native (N-2) and oversulfated (S-2) compounds showed that the two spectra were identical except for a new peak in S-2 at 825 cm corresponding to the equatorial C-6 position of galactosamine. There was a 2.7-fold increase of sulfate content in S-2 and a generation of a significant anticoagulant activity as measured by doubling of the prothrombin time of normal citrated human plasma using 7.5 microg, while N-2 was inactive even at 2,000 microg. The result of the in-vitro studies of the activation of glutamic plasminogen by tissue plasminogen activator (t-PA) or by high-molecular-weight urokinase using 0.05 mol/l Tris buffer (pH 7.35) containing a physiological concentration of NaCl (0.9%) showed that 28.6 microg/ml S-2 enhanced the activation by three-fold to four-fold by t-PA or by urokinase, while the same concentrations of N-2 or unfractionated heparin gave less than 30% enhancement of t-PA and no enhancement of urokinase. The mechanism of enhancement by S-2 was investigated by dilution studies. The results showed that S-2 interacted with both urokinase or t-PA and glutamic plasminogen favoring a template model, while N-2 or unfractionated heparin interacted only with t-PA.

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.

The effect of fucoidan, heparin and cyanogen bromide-fibrinogen on the activation of human glutamic-plasminogen by tissue plasminogen activator.

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.

Effect of in-vitro addition of sulfated xylans, glucosans or chondroitins on the prothrombin time of human plasma and on the enhancement of activation of glutamic plasminogen by tissue plasminogen activator.

Three each of sulfated xylans, glucosans or chondroitins were investigated. A comparison of the infrared analysis of the native and sulfated amylopectin showed the presence of peaks corresponding to 800/cm for S-O and 1200-1300/cm for S=O only in the sulfated amylopectin and earlier results using C NMR showed sulfation of hyroxymethyl groups (C-6) of amylopectin. Based upon % sulfate the three classes of compounds following sulfation contained more than one sulfate group per sugar unit. All of them exhibited significant in-vitro anticoagulant property by inhibiting thrombin generation at very low concentrations. In general sulfated xylans or glucosans were better anticoagulants than the chondroitins. The results of in-vitro studies of the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05 mol/l Tris buffer (pH 7.35) containing physiological concentration of NaCl (0.9%), showed that oat spelts xylan sulfate and amylopectin sulfate gave a 20-fold enhancement of the activation in a synergistic manner when used in combination with 32.4 mmol/l of lysine. The mechanism of enhancement was investigated by dilution studies. Sulfated amylopectin interacted with Glu-Plg but not with t-PA and lysine which interacted with both Glu-Plg and t-PA enhanced the activation in a synergistic manner using low concentrations of t-PA.

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.