Structure-function relationships of heparin species

Abstract

We have fractionated porcine heparin species of low molecular weight, with an average specific anticoagulant activity of 96 units/mg by affinity chromotography. Highly active and relatively inactive preparations of similar size were obtained with specific anticoagulant activities of 360 and 4 units/mg, respectively. The highly active heparin fraction possesses 1.1 additional residues of glucuronic acid and 1.5 fewer residues of N-sulfated glucosamine per molecule compared to the relatively inactive species. This decrease in N-sulfated glucosamine appears to be secondary to a corresponding increase in N-acetylated glucosamine. This form also contains a tetrasaccharide sequence with a N-sulfated glucosamine at its reducing end as well as equivalent amounts of glucuronic acid and iduronic acid. Furthermore, the internal glucosamine residue of this sequence appears to be N-acetylated. Sufficient amounts of this tetrasaccharide sequence are present within the highly active preparation such that each molecule may be endowed with this structure. The relatively inactive product contains a significantly decreased quantity of this tetrasaccharide sequence such that only £-20% of these molecules may possess this structure. The mean distance between nonsulfated uronic acid residues of the highly active species is smaller than that separating similar residues of the relatively inactive product. In addition, a larger number of the nonsulfated uronic acid residues of the highly active material appears either to be present in a restricted region of the molecule separated only by glucosamine residues or to be located at penultimate positions within the polysaccharide chain.Heparin functions as an anticoagulant by binding to antithrombin and accelerating the rate at which this protein inactivates the serine proteases of the hemostatic mechanism (1). During the last 60 years, a number of investigators have attempted to define the chemical and physical features of heparin that are responsible for its anticoagulant action. However, the precise relationship between the structure of the mucopolysaccharide and its biologic properties has remained elusive. A communication from our laboratory provided evidence that only a small fraction (25-35%) of a given heparin preparation binds tightly to antithrombin and is responsible for 85-95% of the anticoagulant activity (2). This finding engendered the hope that, once the active fraction was carefully examined, a unique structure-function relationship for heparin would emerge. The existence of active and relatively inactive heparin species has been confirmed by Lindahl and his coworkers (3). Unfortunately, these investigators were unable to find any chemical parameters that distinguished between their two heparin fractions (3). MATERIALS AND METHODSHuman antithrombin and human thrombin were both isolated by methods previously reported from our laboratory (4). Two porcine heparin products were utilized as starting material forThe costs of publication of this article were defrayed in pa...

The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

“…5. Residues 1-4 are those that give rise, on exhaustive deamination, to the 'Rosenberg tetrasaccharide' (Rosenberg et al, 1978;Rosenberg & Lam, 1979), implying that the unique A* residue is at position 4 of the original octasaccharide. The 13C signals from the 'regular' disaccharide sequence 1I-As should therefore account for residues 5 and 6, and those for the sequence Is-am for residues 7 and 8.…”

“…G, I and Aa residues forming an irregular region were found to be significantly more represented in high-affinity than in low-affinity pig mucosal heparin (Rosenberg et al, 1978). Heparin fragments with high affinity for antithrombin III have been isolated by various processes.…”

Effect of Heparin and Heparin Fractions on Platelet Aggregation

“…The '4C-labeled platelets in PRP or GFP wvere then exposed to 5 ,um/ml thrombin or "stock" heparin for 15 (44). The resultant products attained maximal anticoagulant potency when molar ratios of inhibitor to mucopolysaccharide were set at <0.10.…”

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery

“…When the lipoplexes interact with heparin and heparin sulphate, the negative charge of the polyelectrolytes determines NA release from the lipoplex through the same type of cooperative process that is responsible for lipoplex formation [136][137][138].…”