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.

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.