title: New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.
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.
authors.affiliation: Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France. pierre.mourier@sanofi.com.
authors.forename: Pierre A J
authors.initials: PA
authors.lastname: Mourier
authors.affiliation: Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France. olivier.guichard@sanofi.com.
authors.forename: Olivier Y
authors.initials: OY
authors.lastname: Guichard
authors.affiliation: Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France. frederic.herman@sanofi.com.
authors.forename: Fréderic
authors.initials: F
authors.lastname: Herman
authors.affiliation: Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France. philippe.sizun@sanofi.com.
authors.forename: Philippe
authors.initials: P
authors.lastname: Sizun
authors.affiliation: Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France. christian.viskov@sanofi.com.|In Memoriam: The authors would like to respectfully dedicate this article to Pr. B. Casu, a brilliant pioneer in glycosaminoglycan chemistry and analysis, who passed away on 11 November 2016.. christian.viskov@sanofi.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Viskov
mesh_terms: D002853:Chromatography, Liquid; D004789:Enzyme Activation / Q000187:drug effects; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry*; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D066244:Proton Magnetic Resonance Spectroscopy; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D006495:Heparin, Low-Molecular-Weight; D009844:Oligosaccharides; D013917:Thrombin
keywords: LMWH; antithrombin; heparin oligosaccharides; ternary complex; thrombin inhibition