Xylan Sulfate Research Articles

Mechanism of potentiation of antithrombin III and heparin cofactor II inhibition by sulfated xylans

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or BC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.

Mechanism of potentiation of antithrombin III [at-III]inhibition by sulfated xylans

Anticoagulant properties of three sulfated compounds prepared from xylans isolated from corn cobs, larchwood and oatspelts were compared with heparin and sodium pentosan polysulfate (SP-54) by studying their effects on activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using pooled normal human plasma. These compounds were more effective than SP-54 in delaying coagulation by all the three procedures while oatspelts xylan sulfate was as effective as heparin in inhibiting APTT and PT and more effective than heparin in inhibiting TT on a molar basis. The sulfated xylans were more effective than heparin or SP-54 in potentiating the AT-III inhibition of amidolysis of H-D-Phe-Pip-Arg-pNa (S-2238) by thrombin (IIa) or amidolysis of Bz-Ile-Glu-Gly-Arg-pNa (S-2222) by Xa. Study of the high affinity binding of the xylan sulfates to AT-III-Sepharose column showed that the amount of the xylan sulfate recovered in the eluates from this peak was greatly increased with an increase in molecular weight (MW). A buffered mixture of IIa, AT-III and dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) was used to study the inactivation of IIa by AT-III. Larchwood xylan sulfate (2–10 μg) was found to accelerate this inactivation which was neutralized by human platelet factor 4(PF 4). The results also suggested an interaction between larchwood xylan sulfate and IIa which may potentiate an interaction between AT-III and IIa.

Isolation and anticoagulant properties of a new sulfated xylan: Comparison with heparin and a sodium pentosan polysulfate (SP-54)

Larchwood xylan was purified by diaminoethylaminoethyl (DEAE) cellulose chromatography, sulfated by heating with chlorosulfonic acid -pyridine complex and the sulfated polysaccharide was isolated by epichlorohydrin triethanolamine (ECTEOLA) cellulose chromatography as the sodium salt. It's molecular weight was ∼25000 and the specific rotation was [α] D20–70°. Electrophoretic analysis of the sulfated xylan along with heparin and SP-54 using lithium acetate-agarose technique showed that the charge density of the xylan sulfate was similar to heparin and it moved as a single component in contrast to commercial heparin which resolved into two while SP-54 moved at the same rate as the marker dye. Anticoagulant properties of the sulfated xylan were compared with heparin and SP-54 by studying its effect on the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) using pooled normal human plasma. The sulfated xylan was more effective than SP-54 in delaying coagulation by all the three measurements while it was less active than heparin in inhibiting APTT and PT.

Studies on the anticoagulative activities of sulfated polysaccharides and inhibitory effect on experimental blood-borne tumor metastasis

Blood coagulation plays a very important role for the attachment of tumor cells to the capillary endothelium at an early stage of hematogenous metastasis. Attempts were made to find more reliable antimetastatic agents by studying the effect of five sulfated polysaccharides having different molecular weight and sulfur content on intravenously induced blood-borne pulmonaly metastasis in rats. Xylan sulfate and dextran sulfate strongly inhibited the development of metastatic nodules on the pulmonary surface. Chondroitin polysulfate was less inhibitory, whereas chondroitin sulfate and glucose polysulfate were not inhibitory. Their antimeta-static effect depended on the dose and time of administration, being greatest when they were injected intraperitoneally 1hr. before intravenous inoculation of AH-109A cells.The mechanism of inhibition of blood-borne metastasis by these sulfated polysaccharides was studied in relation to the blood coagulation system. We examined the coagulative activity of three sulfated polysaccharides, xylan sulfate, chondroitin polysulfate and chondroitin sulfate which have strong, weak and no inhibitory effect on metastasis, respectively.The coagulative activity of blood was measured by determining whole blood clotting time, partial thromboplastin time, prothrombin time and thrombin time. Xylan sulfate had the strongest anticoagulative activity, and showed the dose dependence of it. Anticoagulative activity of chondroitin polysulfate was less than that of xylan sulfate. Chondroitin sulfate had no anticoagulative activity. These results indicated that the antimetastatic activity of sulfated polysaccharides were correlated with their anticoagulative activity.Subsequently, we investigated the effect of three compounds on blood coagulation factors. The surface contact factors, factor XI and XII in the plasma of rats injected with xylan sulfate were markedly inactivated. Moderate inactivations of factor II, V, VII, VIII, IX and X were noted in these plasma. Marked inactivations of factor XI and XII, and moderate inactivation of factor VIII were noted in the plasma of rats injected with chondroitin polysulfate. Chondroitin sulfate did not cause any inactivation of blood coagulation factors. The results indicated that the inactivation of surface contact factor was also closely related with inhibitory effects on blood-borne metastasis.Intravascular coagulation concerning mainly with the attachment of transported tumor cells to the endothelium of capillaries might be thought to be induced by 1) obstruction of blood flow by tumor cell emboli, 2) release of the tissue thromboplastin from injured endothelium, 3) activation of contact factors by injured endothelium due to tumor cells, 4) activation of the extrinsic coagulation factors by tumor cells. Xylan sulfate interfered with intrinsic thromboplastin formation due to in-activation of contact factors in intrinsic blood coagulation mechanism, and showed the antithrombin activity.

Effect of sulfated polysaccharides on blood-borne pulmonary metastasis in rats.

The inhibitory effect of sulfated polysaccharides on blood-borne metastasis was examined. As a model of blood-borne metastasis, the ascitic form of hepatoma AH-109A tumor was injected intravenously into Donryu strain rats. Examination of the pulmonary metastatic nodules developed 2 weeks later showed inhibitory effect of the five sulfated polysaccharides tested. Xylan sulfate was the most inhibitory, and exerted its inhibitory effect when the tumor cells were in the pulmonary capillary beds. However, fromthe rapid disappearance of radioactivity from the lungs after injection of 125IUDR-labeled AH-109A cells, tumor cells seemed to be retained in the lungs for only a very short time. Measurement of the anticoagulative and fibrinolytic activities of three sulfated polysaccharides showed that the inhibitory effect of these compounds on blood-borne metastasis was proportional to their anticoagulative and fibrinolytic activities, xylan sulfate showing the highest activities. These results suggest that sulfated polyaccharides may inhibit blood-borne pulmonary metastasis by inhibiting the lodging of tumor cells in the pulmonary capillary beds.