Abstract
Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.
Highlights
IntroductionLow Molecular Weight Heparins (LMWH) are lifesaving anticoagulant drugs which have been used for several decades in the prevention and treatment of venous and arterial thromboembolism [1]
Low Molecular Weight Heparins (LMWH) are lifesaving anticoagulant drugs which have been used for several decades in the prevention and treatment of venous and arterial thromboembolism [1].LMWH are industrially derived from the starting material heparin, a complex mixture of mammalian polysaccharides extracted from porcine mucosa
We have demonstrated that for octadecasaccharide regioisomers, built up from the same AGA*IA sequences and Is flanking disaccharides, thrombin inhibition potency may dramatically differ
Summary
Low Molecular Weight Heparins (LMWH) are lifesaving anticoagulant drugs which have been used for several decades in the prevention and treatment of venous and arterial thromboembolism [1]. LMWH are industrially derived from the starting material heparin, a complex mixture of mammalian polysaccharides extracted from porcine mucosa. The centenary of the discovery of heparin has been widely celebrated around the world and many symposia, such as the 24th Glycosaminoglycans. Heparin is a mixture of heteropolymeric chains having a mean molecular weight around 15,000 Dalton. It consists of alternating units of 2-deoxy-2-sulfamido-α-D-glucopyranose
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