Antithrombin-binding oligosaccharides: structural diversities in a unique function? Marco Guerrini & Pierre A. J. Mourier & Giangiacomo Torri & Christian Viskov # Springer Science+Business Media New York 2014 Abstract Heparin-antithrombin interaction is one of the most documented examples of heparin/protein complexes. The spe- cific heparin sequence responsible for the binding corresponds to a pentasaccharide sequence with an internal 3-O-sulfated glucosamine residue. Moreover, the position of the pentasac- charide along the chain as well as the structure of the neighbor units affects the affinity to antithrombin. The development of separation and purification techniques, in conjunction with physico-chemical approaches (mostly NMR), allowed to char- acterize several structural variants of antithrombin-binding oligosaccharides, both in the free state and in complex with antithrombin. The article provides an overview of the studies that lead to the elucidation of the mechanism of interaction as well as acquiring new knowledge in heparin biosynthesis. Keywords Antithrombin . Heparin . Oligosaccharides . NMR . Protein-ligand interaction Introduction Heparin (UFH) and low-molecular-weight heparins (LMWHs) are the most common anticoagulant and antithrom- botic drugs used in medicine [1]. Heparin and its low molec- ular weight versions are composed by linear polysaccharidic chains constituted by alternating disaccharide sequences of either L-iduronic acid (IdoA) or D-glucuronic acid (GlcA), and glucosamine (GlcN). The GlcN residues can be N-sulfated (GlcNS) or N-acetylated (GlcNAc) at position 2, O-sulfated at position 6 (GlcN6S), and more rarely, also at position 3 (GlcNS,3S, designated A*). The uronic acid residues, IdoA and more rarely GlcA, can be O-sulfated at position 2 (IdoA2S/GlcA2S). LMWHs can present further structural het- erogeneity due to the presence of different reducing and nonre- ducing residues at the site of cleavage, characteristic of each depolymerization procedure [2]. Although the dependence of the chain sulfation, degree, and distribution related to the animal and organ source is known, the exact sequence of each heparin polymer chains is still not easy to fully disclose [3, 4]. The activity of heparin is principally based on the binding and activation of antithrombin (AT), the major heparin cofactor in the inhibition of several serine proteases of the coagulation system, particularly factors Xa, IXa, and IIa (thrombin) [5]. On the other hand, LMWHs exert their antithrombotic and antico- agulant activities principally via factor-Xa enzyme, whereas their inhibition of factor-IIa is considerably reduced, since it needs the formation of an AT-thrombin-heparin ternary complex involving oligosaccharide chains of at least 18 residues contain- ing AGA*IA (defined later) toward the nonreducing end [6]. Consequently, the anti-factor-Xa/anti-factor-IIa ratio, defined as 1 for UFH, increases up to 4 for LMWH, with a decreasing risk of bleeding and heparin-induced thrombocytopenia [ 7]. The mechanism of interaction of heparin and LMWHs with AT is one of the best studied examples of heparin/protein complexes [8, 9], representing currently the unique evidence of a highly selective recognition between a glycosaminogly- can and a protein. The specific heparin sequence responsible for the binding corresponds to the pentasaccharide A NAc,6S -G- A NS,3,6S -I 2S -A NS,6S [AGA*IA] that, inducing a conformation- al change in AT, enhances by several hundred fold the rate at which serine proteinases can be inhibited. The interaction of the pentasaccharide is mainly governed by specific electro- static interactions between its sulfate groups and positively charged amino acids of the heparin AT-binding [ATB] region. The molecular basis of the AT-pentasaccharide interaction was M. Guerrini (*) : G. Torri Istituto di Ricerche Chimiche e Biochimiche “G. Ronzoni”, via G. Colombo 81, 20133 Milan, Italy e-mail: guerrini@ronzoni.it P. A. J. Mourier : C. Viskov Sanofi, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France Glycoconj J DOI 10.1007/s10719-014-9543-9