Introduction
The heparan sulfate (HS) family of polysaccharides found throughout metazoan lifeforms comprises the most anionic polysaccharides in nature ranging from 20 to 200 monosaccharide units in length, and HS is ubiquitously expressed on cell surfaces and in the extracellular matrix of mammals. HS is essential for life in all mammalian species and modulates numerous biological activities involving growth and development, inflammation and immune system regulation, angiogenesis and metabolism, as well as disease pathologies of cancer, infection, and neurodegenerative disorders. The degree and patterns of their sulfation represent huge diversity for informational cues to direct and tightly regulate biological functions. They achieve this through selective interactions with protein partners via divergent sulfated binding motifs that bind to cognate protein-binding sites. HS is produced by a complex biosynthetic machinery that initially creates a repeating disaccharide unit of uronic acid (UA) and N-acetylglucosamine (GlcNAc), where the UA is either iduronic acid (IdoA) or glucuronic acid (GlcA). The glucosamines can be modified with an N-sulfate (NS) or remain as an N-acetyl (NAc) moiety [via action of N-deacetylase/N-sulfotransferases 1 to 4 (NDST1 to NDST4)]. UAs can subsequently be modified with an O-sulfate at the carbon-2 position by 2-O-sulfotransferase (HS2ST1). Further O-sulfates can be added to glucosamine residues at the carbon-6 position [via 6-O-sulfotransferases 1 to 3 (HS6ST1 to HS6ST3)] and more rarely at the carbon-3 position [via 3-O-sulfotransferases 1 to 6 (HS3ST1 to HS3ST6)]. Divergent patterns of sulfation created by the orchestration of these enzyme families are the key hallmarks of functionally specific protein-binding sites in HS. Deciphering the details of these sulfation patterns, including the apparently less common 3-O-sulfate modification, remains a major hurdle.
Heparin, a member of the HS family, is a widely used anticoagulant and is the world’s most sold biopharmaceutical by weight, yet it remains a poorly characterized heterogeneous animal-sourced product. Most unfractionated heparin (UFH) is purified from porcine intestinal mucosa, with low–molecular weight heparins (LMWHs) being fractionated from UFH. In addition, the supply and quality of heparins are causes for concern due to infection outbreaks in animal stocks, such as the ongoing swine flu in China, and the contamination of crude heparin with oversulfated glycosaminoglycans (GAGs) in 2007 that resulted in many deaths.
The mechanism of heparin’s anticoagulant activity involves predominantly heparin binding and activation of antithrombin III (ATIII), which is then able to complex and inactivate thrombin, factor Xa (FXa), and other proteases. Heparin binds to ATIII through a specific pentasaccharide sequence, GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S, whereas the interaction of ATIII and thrombin requires heparin chains of at least 18 monosaccharide units in length. In contrast, FXa activity via ATIII activation requires only the pentasaccharide sequence, and a synthetic heparin mimetic (fondaparinux) has been created on the basis of this structure. Removal of the 3-O-sulfate group on the 3-O-sulfated glucosamine (GlcNS3S6S) within the pentasaccharide sequence was shown to result in limited ATIII activity, demonstrating the essential requirement for 3-O-sulfation for potent anticoagulant activity.
Major complications of heparin and LMWH in clinical use include both bleeding and thrombosis. The structural heterogeneity of heparins provides the avidity to complex with large numbers of proteins including plasma proteins, which can lead to adverse consequences of unpredictable anticoagulation and also life-threatening heparin-induced thrombocytopenia (HIT). HIT can be nonimmune or immune-mediated, both resulting in decreased platelet counts. Platelets produce a protein called platelet factor 4 (PF4; also called CXCL4), which is capable of forming large heparin-PF4 complexes; in immune HIT, antibodies to these complexes are induced and platelets are activated, resulting in the formation of blood clots and low platelet levels. Heparin has the highest incidence of HIT at around 5% of patients, whereas LMWH has an incidence of around 1%. Heparin/LMWH binding to PF4 has previously been demonstrated to require N-sulfation of the glucosamine (GlcNS) and 2-O-sulfation of the UA (UA2S). This raises the possibility of better targeting of heparin therapeutics with reduced side effects by tailored generation of optimized structures.
Sources：SCIENCE IMMUNOLOGY, 24 Jun 2022, Vol 7, Issue 72,
DOI: 10.1126/sciimmunol.abo5407
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