No abstract

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 1 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-LyspNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 mg/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 K m but a 10-fold increase of K cat suggesting a template mechanism for the attenuation when both cofactors are used.

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.