EFFECT OF SULFATED XYLANS ON COAGULATION OF HUMAN PLASMA

硫酸化木聚糖抗凝血作用机制的研究 血浆：肝素治疗通常用于预防 或手术后静脉血栓的治疗。现在是时候了 认识到商业肝素不是均一的，而且有 需要一位替代者。木聚糖将从脱木素中分离出来 从玉米棒、落叶松、西班牙草中获得的综纤维素 还有燕麦卷。这些将通过加热来进行硫酸盐化 氯磺酸-吡啶络合物及其DEAE进一步纯化 或ECTEOLA纤维素层析法。琼脂糖凝胶电泳法 并将进行超速离心以确定它们的 同质性。硫酸化的木聚糖将被分离成高和 抗凝血酶III(AT-III)-CH琼脂糖凝胶的低亲和力组分 并将就特定的旋转、分子 重量、糖单位的性质和硫酸盐的位置 组。为了确定它们的作用机制，它们的作用 木聚糖硫酸酯将在以下体系中进行研究：1) 氨解裂解特定的发色肽底物 凝血因子(S)单独或与AT-Ⅲ相互作用 与凝血酶(IIa)或加速相互作用 AT-III和IIa由IIa-的荧光偏振测量 丹磺精N-3-乙基-1，5-戊二基酰胺(DAPA)络合物。 3)与AT-III的相互作用，通过增强 由于本征光谱的变化而产生的荧光 AT-III的性质4)活化部分的抑制作用 凝血酶原时间、凝血酶原时间和凝血酶时间 正常的人体血浆。确定结构-功能关系 各种低聚糖片段将从木聚糖中制备 或通过化学技术或通过酶消化使用 从黑曲霉中提纯的β-木糖苷酶，鉴定和硫酸盐化。 这些组件对AT-III的亲和力将通过以下方式测量 平衡透析法或AT-III-CH琼脂糖柱。这个 硫酸盐化木聚糖与特定凝聚的络合作用 因子或AT-III将用丙烯酰胺凝胶进行研究 电泳法或超速离心法。结合部位(S) 硫酸木聚糖与凝血因子(S)或AT- III将通过使用特定的蛋白质修饰进行研究 试剂。上述研究的结果将提供更好的 对硫酸化木聚糖结构组成的认识 对最佳AT-III抑制凝血因子和 可能导致一种更具选择性的抗凝血剂的开发。

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.