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.