Fractionation of heparin using antithrombin III reversibly bound to concanavalin A-Sepharose

Denton, J. J.; Lewis, Walter; Nieduszynski, Ian A.; Phelps, C. F.

doi:10.1016/0003-2697(81)90598-4

Cited by 24 publications

(11 citation statements)

References 10 publications

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“…Washed platelets were prepared as previously described [14]. Fractionation of heparan sulphate and heparin over ATIII immobilized on concanavalin A (conA) Sepharose were made as described previously [15].…”

Section: Methodsmentioning

confidence: 99%

“…Heparan sulphate was chromatographed over ATIII immobilized upon ConA-Sepharose [15] and fractions pooled separately and concentrated to represent no affinity (NA), low-affinity (LA) and high-affinity (HA) fractions, respectively. The yield of each was measured and found to be, as a percentage Of the total, 97.4°7o for NA, 2.0% for LA and 0.6% for HA.…”

Section: Fractionation Of Heparan Sulphate Upon Immobilized a Tiiimentioning

confidence: 99%

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Heparan sulphate with no affinity for antithrombin III and the control of haemostasis

Scully¹,

Ellis²,

Kakkar³

1988

FEBS Letters

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Heparan sulphate with no affinity for antithrombin III (ATIII) was observed to cause acceleration of the factor Xa:ATIII interaction by 1100-fold (k2, 7 × 107 M-l.min -1) and the prothrombinase:ATllI interaction by 2900-fold (k2, 2.5 x l0 T M-1 .min-1). Although high-affinity heparan sulphate catalyzed higher acceleration and at lower concentration, in natural mixtures of the two forms the activity of the no affinity form predominated. Heparan sulphate had no significant effect on the thrombin:ATIII interaction but inhibited its potentiation by heparin (Kd 0.3/~M). From the estimated concentration of heparan sulphate on the endothelial cell surface it is proposed that the non-thrombogenic property of blood vessels is due to the acceleration of the factor Xa or prothrombinase:ATIIl interaction by the greater mass of surface-bound heparan sulphate rather than by the much smaller proportion of heparin-like molecules (with high affinity for antithrombin III) which may be present.

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Section: Methodsmentioning

confidence: 99%

Section: Fractionation Of Heparan Sulphate Upon Immobilized a Tiiimentioning

confidence: 99%

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis

Scully¹,

Ellis²,

Kakkar³

1988

FEBS Letters

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“…The fractions with low (fraction 2) and high (fraction 3) charge density were used in the study. Affinity chromatography on immobilized antithrombin III was performed as described elsewhere [6]. Briefly, about 1 g human antithrombin III (Kabi-Vitrum.…”

Section: Preparation O F Heparin Fractionsmentioning

confidence: 99%

Basic-Fibroblast-Growth-Factor-Mediated de novo Angiogenesis Is More Effectively Suppressed by Low-Molecular-Weight than by High-Molecular-Weight Heparin

Norrby

Østergaard

1996

Int J Microcirc

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We recently reported that the subcutaneous (s.c.) administration of a low-molecular-weight heparin (LMWH) fraction significantly inhibited de novo angiogenesis in the mesentery induced by the intraperitoneal (i.p.) injection of saline to adult rats compared with unfractionated heparin and high-molecular-weight heparin (HMWH) fractions. The present study assesses the effect on basic fibroblast growth factor (bFGF)-mediated de novo angiogenesis in the mesentery of the systemic administration of a LMWH fraction (2.6 kD) and a series of four HMWH fractions (about 20 kD) with varying degrees of polydispersity, charge density and anticoagulant activity. bFGF, a prototypic heparin-binding angiogenic growth factor, was injected i.p. at 220 pM on days 0-4. The heparins were given s.c. on days 0-13 or 0-14 at doses which were approximately within the range used clinically. Angiogenesis was assessed by microscopic morphometry and image analysis in groups of animals killed on days 14 and 15. Compared with the saline control, the LMWH and three fo the HMWHs significantly inhibited angiogenesis in terms of microvascular length (MVL), a measure of microvascular density. Interestingly, the vascularized area (VA), a measure of microvascular spatial extension, and the total microvascular length (VA × MVL) were significantly lower in the LMWH-treated animals than in the animals treated with one of the HMWHs. The total microvascular length was, moreover, significantly reduced in the LMWH-treated animals compared with the combined data of all the HMWH-treated animals. No significant effects were related to the degree of charge density and anticoagulant activity of the heparins. In view of the putative significant angiogenic role of bFGF in human angiogenesis diseases, the present findings may have implications for the choice of anticoagulant treatment modality for patients suffering from cancer and other angiogenesis diseases.

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“…These stock solutions were stored at -70°C and dilutions were made for uronic acid assay, aPTT, antithrombin-mediated anti-FXa and anti-FIla assays, DS assay, and heparinase-catalysed depolymerization. Affinity fractionation of heparin Heparin was fractionated based on its affinity to ATIII (Denton et al, 1981). A concanavalin A-Sepharose column (1 cm x 10 cm) at 4°C was equilibrated with 0.02 M-Tris/HCl containing 50 mM-NaCl, 1 mM-MgCl2, 1 mM-manganese chloride and 1 mM-CaCl2 at pH 7.4.…”

Section: Preparation Of Commercial Heparinsmentioning

confidence: 99%

Mapping and quantification of the major oligosaccharide components of heparin

Linhardt

Rice

Kim

et al. 1988

Biochemical Journal

147

126

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A new method of determining the oligosaccharide composition of commercial glycosaminoglycan heparin is described in which heparin was first depolymerized using heparin lyase (EC 4.2.2.7), and then analysed by a single h.p.l.c. step. All 20 of the porcine and bovine heparins examined were found to contain a small number of major oligosaccharide components, which on average comprised 86% of their mass. The five most abundant oligosaccharides have defined chemical structures. Although the relative abundance of oligosaccharides varied, the heparins examined were surprisingly similar. Porcine, bovine, low-Mr, and high and low antithrombin III (ATIII)-affinity heparins, however, each had distinctly different proportions of these major oligosaccharide components. The concentrations of one of these five oligosaccharides, containing a portion of the ATIII binding site, correlated with the anticoagulant activity of the ATIII-affinity-fractionated porcine-mucosal heparins from which it was derived. An additional oligosaccharide of undetermined structure was found in significant quantities in both bovine heparin and high ATIII-affinity porcine-mucosal heparin. The correlation between oligosaccharide concentration and anticoagulant activity suggests that the oligosaccharide is derived from a structural variant of the ATIII-binding site. Finally, for the heparins examined chondroitin/dermatan sulphate formed 0.6-7.4% of their mass.

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Fractionation of heparin using antithrombin III reversibly bound to concanavalin A-Sepharose

Cited by 24 publications

References 10 publications

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis

Basic-Fibroblast-Growth-Factor-Mediated de novo Angiogenesis Is More Effectively Suppressed by Low-Molecular-Weight than by High-Molecular-Weight Heparin

Mapping and quantification of the major oligosaccharide components of heparin

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