Linhardt R J, Loganathan D, al-Hakim A, Wang H M, Walenga J M, Hoppensteadt D, Fareed J
Division of Medicinal and Natural Product Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
J Med Chem. 1990 Jun;33(6):1639-45. doi: 10.1021/jm00168a017.
Low molecular weight heparins from a variety of commercial sources were examined. These had been prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical beta-elimination, enzymatic beta-elimination, and chromatographic fractionation. The molecular weight and polydispersity of these low molecular weight heparins showed greater differences than were observed for typical commercial heparin preparations. Considerable differences were also observed in the antithrombin III mediated anti factor Xa activity, the heparin cofactor II mediated antifactor IIa activity, and the USP activity of these low molecular weight heparins. An oligosaccharide-mapping technique (comparable to the peptide mapping of proteins) was applied to these low molecular weight heparins in an effort to understand the structural features responsible for their activity differences. Heparin lyase from Flavobacterium heparinum was first used to depolymerize the low molecular weight heparin into its constituent oligosaccharides. The oligosaccharides present in the resultant mixture were identified and quantitated by using standard oligosaccharides of defined structure on gradient polyacrylamide gel electrophoresis and strong anion exchange high pressure liquid chromatography. Six of the oligosaccharide products have been identified and represent nearly 90 wt % of heparin's mass. Even though all the low molecular weight heparins showed these six oligosaccharide components, their content in each varied greatly, accounting for 20 to over 90% of their mass. The antithrombin III mediated anti factor Xa activities of the low molecular weight heparins correlated only poorly to the concentration of a hexasaccharide containing a portion of heparin's antithrombin III binding site. The heparin cofactor II mediated antifactor IIa activity, however, could not be correlated to these six oligosaccharides of known structure nor to the molecular weight or charge density of these low molecular weight heparins. The low molecular weight heparins prepared by different methods each showed a new distinctive oligosaccharide in their maps. Their isolation and structural characterization, which included two-dimensional NMR and fast atom bombardment mass spectrometry, indicated that these unusual oligosaccharides result from end-sugar modification during chemical depolymerization. Both gel electrophoresis and high-pressure liquid chromatography mapping techniques showed a greater structural diversity between low molecular weight heparins than had previously been observed between similarly analyzed commercial heparins.
对来自多种商业来源的低分子量肝素进行了检测。这些低分子量肝素是通过多种方法制备的，包括过氧化裂解、亚硝酸裂解、化学β-消除、酶促β-消除和色谱分级分离。与典型的商业肝素制剂相比，这些低分子量肝素的分子量和多分散性表现出更大的差异。在抗凝血酶III介导的抗Xa因子活性、肝素辅因子II介导的抗IIa因子活性以及这些低分子量肝素的美国药典活性方面也观察到了相当大的差异。一种寡糖图谱技术（类似于蛋白质的肽图谱）被应用于这些低分子量肝素，以试图了解导致其活性差异的结构特征。首先使用来自肝素黄杆菌的肝素裂解酶将低分子量肝素解聚成其组成寡糖。通过在梯度聚丙烯酰胺凝胶电泳和强阴离子交换高压液相色谱上使用具有确定结构的标准寡糖，对所得混合物中存在的寡糖进行鉴定和定量。已鉴定出六种寡糖产物，它们占肝素质量的近90%。尽管所有低分子量肝素都显示出这六种寡糖成分，但它们在每种低分子量肝素中的含量差异很大，占其质量的20%至90%以上。低分子量肝素的抗凝血酶III介导的抗Xa因子活性与含有肝素部分抗凝血酶III结合位点的六糖浓度之间的相关性很差。然而，肝素辅因子II介导的抗IIa因子活性与这些已知结构的六种寡糖以及这些低分子量肝素的分子量或电荷密度均无相关性。通过不同方法制备的低分子量肝素在其图谱中各自显示出一种新的独特寡糖。它们的分离和结构表征，包括二维核磁共振和快原子轰击质谱，表明这些不寻常的寡糖是化学解聚过程中末端糖修饰的结果。凝胶电泳和高压液相色谱图谱技术均显示，低分子量肝素之间的结构多样性比之前在类似分析的商业肝素之间观察到的更大。
