Structural Determinants of Enoxaparin Oligosaccharides for the Down-Regulation of Tissue Factor Induced Factor VIIa Generation In Human Plasma.
Authors: Gerotziafas Grigoris T , Chakroun Tahar , Galea Vassiliki , Samama Meyer Michel , Elalamy Ismail 
Published: October 2019

Abstract
Abstract 1106

Introduction.
Low molecular weight heparins are multitargeted drugs that exert their antithrombotic effect primarily via the binding of pentasaccharide domain on antithrombin. The structural and functional fingerprint of enoxaparin regarding the affinity for AT and the inhibition of FXa and thrombin has been extensively studied. The depolymerization process used for manufacturing enoxaparin results in the formation of a 1,6 anhydro ring (bicyclic) structure at the reducing end of all oligosaccharides bearing 6-0-sulfo-groups on the glucosamine moiety. The influence of this particular structure on the antithrombotic activity of enoxaparin is not known. In previous studies our group demonstrated that enoxaparin and the synthetic pentasaccharide inhibit factor VIIa generation after triggering tissue factor pathway in human plasma. In patients suffering from acute coronary syndromes treatment with enoxaparin induces inhibition of FVIIa in plasma.

Aim.
We investigated the effect of structural characteristics of enoxaparin oligosaccharide on the generation of FVIIa.

Methods.
Oligosaccharides (hexa- octa- dodeca- saccharides) with the 1,6 anhydro ring structure (40%-50%) or without it (<7%) and one octasaccharide with high affinity for antithrombin (AT) were provided by Sanofi-Aventis France. Normal platelet poor plasma (PPP) from 5 healthy donors was spiked with increasing concentrations of the studied oligosaccharides (0.625 to 10 μg/ml) or enoxaparin or saline (control). Factor VIIa generation was studied after TF pathway activation in PPP according to previously described assay sensitive to detect the inhibitory effect of low concentrations of enoxaparin and pentasaccharide (Gerotziafas et al Blood Coagul Fibrinolysis. 2003;14:633-8). Briefly in polystyrene tubes containing one volume of normal human PPP was added one volume of diluted (1:250) non calcified thromboplastin (Recombiplastin). After a 3 min incubation, coagulation was triggered by adding one volume of CaCl2 solution (0.025 M). FVIIa levels were determined with the one-stage clotting assay using recombinant thromboplastin (TF1–218) truncated to interact only with FVIIa (Staclot FVIIa-rTF; Diagnostica Stago, Asnières, France), with clotting times determined by chronometric method.

Results.
At 2.5 μg/ml enoxaparin all studied oligosaccharide significantly inhibited FVIIa generation. The octasaccharide with the higher affinity for AT inhibited FVIIa generation significantly more than enoxaparin or the other oligosaccharides. Oligosaccharides possessing the 1,6 anhydro ring structure induced more pronounced inhibition of FVIIa generation compared to those without this structure. Octa- and dodeca- saccharides induced higher inhibition of FVIIa generation compared to the hexasaccharide. This difference was not influenced by the presence of the 1,6 anhydro ring structure.

Conclusion.
The FVIIa generation in PPP is a sensitive experimental system for the evaluation of structural/functional relationships in oligosaccharides contained in LMWH preparations. The present study is the first to report that the length of the oligosaccharide chain the affinity to AT and the presence of the 1,6 anhydro ring structure are structural characteristics enoxaparin which in addition to the pentasaccharide domain dowregulate TF triggered factor VIIa generation.

Disclosures:
No relevant conflicts of interest to declare.