Binding to antithrombin of heparin fractions with different molecular weights

Abstract

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34 700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

Structure-activity relationships of heparin. Independence of heparin charge density and antithrombin-binding domains in thrombin inhibition by antithrombin and heparin cofactor II.

“…For example, molecular-weight fractionation leads to concomitant fractionation by AT affinity and vice versa (5). It is interesting that AT-affinity fractionation of charge-density fractionated heparins apparently produces much less concomitant molecular-weight fractionation than was observed for heparins that were heterogeneous in charge density (6).…”

“…A short sequence of defined structure (2) is necessary for AT-heparin binding and, hence, activity with AT (3,4). Because additional properties such as molecular weight (5,6), thrombin affinity (6)(7)(8), and charge density (9)(10)(11)(12), can also markedly affect the activity of a heparin, the molecule must have multiple functional domains (6,7).…”

Analysis of affinity and structural selectivity in the binding of proteins to glycosaminoglycans: development of a sensitive electrophoretic approach.

“…A portion of the 125I-labeled fluoresceinamine-heparin (125I-F-heparin) was fractionated by gel filtration [on Sephadex G-100 in 50 mM sodium 3-(Nmorpholino)-2-hydroxypropanesulfonate) (Mopso), pH 7.0/ 125 mM sodium acetate] and the last 10.8% of the radioactive material to elute (0.57 < Ka, < 0.76) was pooled as a low molecular weight (LMW) fraction. This fraction was estimated to contain heparin chains of Mr -6000 (3,(15)(16)(17)(18).…”

Orientation of Heparin-binding Sites in Native Vitronectin

“…These quantitative analyses argue against the contribution of secondary sites to binding after unfolding of vitronectin. For further consideration of the ways in which protein:ligand binding stoichiometries can be influenced by multiple binding sites on either heparin or the protein, the reader is referred to published work on antithrombin or thrombin binding to heparin (45,52,53).…”