EFFECT OF SULFATED XYLANS ON COAGULATION OF HUMAN PLASMA

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Mechanism of Action of Sulfated Xylans on the Coagulation of Human Plasma: Therapy with heparin is commonly used for the prevention or the treatment of post-operative venous thrombosis. It is now recognized that commercial heparin is not homogenous and there is a need for a substitute. Xylans will be isolated from delignified holocelluloses obtained from corn cobs, larchwood, esparto grass and oatspelts. These will be sulfated by heating with chlorosulfonic acid-pyridine complex and further purified by DEAE or ECTEOLA cellulose chromatography. Agarose gel electrophoresis and ultracentrifugation will be carried out to determine their homogeneity. The sulfated xylans will be separated into high and low affinity fractions using antithrombin III (AT-III)-CH sepharose and will be investigated in regard to specific rotations, molecular weights, nature of sugar units and the location of the sulfate groups. To determine the mechanism of their action, the effect of xylan sulfates will be studied in the following systems: 1) Amidolytic cleavage of specific chromogenic peptide substrates by coagulation factor(s) alone or in presence of AT-III 2) Interaction with thrombin (IIa) or an acceleration of an interaction between AT-III and IIa as measured by fluorescence polarization of the IIa- dansylarginine N-3-ethyl-1,5-pentanediyl) amide (DAPA) complex. 3) Interaction with AT-III as measured by enhancement of fluorescence due to an alteration in the intrinsic spectral properties of AT-III 4) Inhibition of activated partial thromoplastin time, prothrombin time and thrombin time of pooled normal human plasma. To determine structure-function relationships various oligosaccharide fragments will be prepared from the xylans either by chemical techniques or by enzymatic digestion using purified beta-xylosidase from A. Niger, characterized and sulfated. The avidities of these components for AT-III will be measured by equilibrium dialysis or by using AT-III-CH sepharose column. The complexation of the sulfated xylans with specific coagulation factor or AT-III will be studied by using acrylamide gel electrophoresis or ultracentrifugation. The binding site(s) between the sulfated xylans and the coagulation factor(s) or AT- III will be studied by using specific protein modification reagents. The results of the above studies will provide a better understanding of the structural components of sulfated xylans essential for optimum AT-III inhibition of coagulation factors and may lead to the development of a more selective anticoagulant.