Heparin is a highly sulfated linear poly-saccharide that was discovered in

1916 as an anticoagulant and has been used clinically since 1935 (see ref. 1 for review). The anticoagulant activity of heparin results from its binding to the serine protease inhibitor, antithrombin III (ATIII)(2, 3). Heparin binding causes a conformational change in ATIII that results in enhanced inhibition of thrombin and other serine proteases involved in the blood clotting cascade (Fig. 1 A and B). Whereas the clinical reagent heparin is found nearly exclusively in mast cells, it represents a specialized member of the widely distributed class of compounds known as heparan sulfate. Heparan sulfate is found throughout all tissues in virtually every animal species examined, most prominently on cell surfaces and within extracellular matrices. Heparin and heparan sulfate are characterized by repeating units of disaccharides containing a uronic acid (glucuronic or iduronic acid) and glucosamine, which is either N-sulfated or N-acetylated. The sugar residues may be further O-sulfated at the C-6 and C-3 positions of the glucosamine and the C-2 position of the uronic acid. Thus, there are at least 32 potential unique disaccharide units that together make this class of compounds one of the most information dense in biology. The high degree of structural complexity likely underlies the ability of heparan sulfate to play critical roles in a large and diverse number of biological processes (1). However, the excitement generated by the growing list of functions attributed to heparan sulfate is often met by the sobering reality that the methodology for carbohydrate structural analysis has lagged well behind that for proteins and nucleic acids. It has been only in recent years that methods for structural analysis of heparan sulfate have begun to become widely available. Separation techniques such as HPLC and capillary electrophoresis, combined with the use of specific enzymatic and chemical methods to depolymerize heparan sulfate, have provided important information on composition and domain structure (4–6). Approaches using NMR and mass spectrometry have allowed accurate sequencing of small saccharides (7, 8). More recently, methods have been developed that use integrated strategies with chemical and enzymatic steps coupled to accurate separation methods (9–11). One of the most promising of these approaches is the recently described use of a property-encoded nomenclature in conjunction with matrix assisted laser desorption mass spectrometry (PEN-MALDI)(11). In two papers in this issue of PNAS, Sasisekharan and his colleagues put these methods to practice (12, 13). As a test of their method, they used PEN-MALDI to sequence a decasaccharide (AT10), derived from heparin, which was believed to be of known sequence (14). However, the PEN-MALDI analysis suggested a different sequence (Fig. 2)(13). To reconcile this apparent conflict, the authors used several analytical techniques, including integral glycan sequencing and one-dimensional proton NMR to converge on the structure of AT10. Armed with definitive knowledge of the structure of AT10, Shriver et al.(12) were then able to probe the anticoagulant mechanisms of a heparin fragment containing only a partial ATIII binding site, as well as to evaluate potential ways to generate low molecular weight heparin for clinical applications. Together, these papers provide an excellent paradigm for what will undoubtedly become a large number of studies aimed at defining the structure–function relationships for specific heparin-and heparan sulfate-derived oligosaccharides. The reaction of heparin with antithrombin and proteases has …