Structural basis for the anticoagulant activity of heparin. 1. Relationship to the number of charged groups

Abstract

This study was undertaken to provide further information concerning the chemical heterogeneity of heparins and the relationships between the anticoagulant activity (USP assay) and the anionic density of the heparin. A sample of commercial heparin was fractionated into 13 fractions by sequential extraction in a two-phase system of 1-butanol-aqueous NaCl containing excess hexadecylpyridinium chloride. The anionic density distribution was characterized by the fractional distribution of uronate among the fractions. The fractions were characterized by several molar ratios of constituents, molecular weight, charge density, and anticoagulant activity in recalcified sheep plasma. The heparin was broadly distributed among the last 10 fractions; the first three contained impurities which were completely separated from the heparin fractions. The heparin fractions differ systematically in anionic density but are of substantially the same molecular weight. Anticoagulant activity increased markedly with anionic density, ranging from 81 units/mg for the heparin fraction with the lowest anionic density up to a high of 243 units/mg. The relationship between anticoagulant activity and either anionic density or its square is nonlinear. However, in the latter case an initial linear relationship was observed for anticoagulant activities of less than 200 units/mg.

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

“…Hog mucosal heparin (176 U. S. Pharmacopeia, units per milligram; Diosynth BV, Holland) was fractionated by two-phase partition (10,12) to obtain heparin fractions, previously characterized in detail (9)(10)(11)(12), that vary systematically in linear charge density, Z. The highly purified and more homogeneous fractions have similar molecular-weight distributions, with an average molecular weight of 15,000, and contain no detectable dermatan sulfate or other contaminating glycosaminoglycans.…”

“…Five of the nine fractions were further fractionated by AT-agarose affinity chromatography (4) (see Table I). The size distributions of the high affinity (HA) and very low affinity (VLA) subfractions of fraction 3 (Z = 2.71) were compared by passing each through a column (0.8 cm X 60 cm) of 6% agarose (4-0.5 m; Bio-Rad Laboratories, Richmond, CA) and 0.5-ml fractions were collected and analyzed for uronate as described previously (9).…”

“…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).…”

Anticoagulant activity of dermatan polysulfates.

“…et al. 1953;Cifonelli 1974;Hurst et al 1979), the content and distribution of Nsulfated glucosamine (Yosizawa et al 1967;Cifonelli 1974;Nagasawa et al 1977), the proportion of L-iduronic acid or sulfated L-iduronic acid (Cifonelli 1974), the presence of free carboxyl groups (Danishefsky and Siskovic 1971;Danishefsky 1977), molecular size (Rosenberg and Damns 1973;Cifonelli 1974;Laurent et al 1978;Barrowcliffe et al 1979) and so on. Previously we isolated three kinds of dermatan polysulfate, DPS-I (Anno et al 1971), DPS-II (Seno et al 1972) and DPS-III (Seno and Meyer 1963) from hagfish notochord, hagfish skin and shark skin, respectively, and the differences in the degree of sulfation and the position of sulfate groups were found.…”

“…Hurst et al 1979), the content and distribution of Nsulfated glucosamine (Yosizawa et al 1967;Cifonelli 1974;Nagasawa et al 1977), the proportion of L-iduronic acid or sulfated L-iduronic acid (Cifonelli 1974 Barrowcliffe et al 1979) and so on.…”