*2.1. Heparin and Heparan Sulfate*

Both Hep and HS chains are synthesized as a modification of a PG protein core, sharing a biosynthetic scheme but exhibiting some disparities [35,36]. Thus, initially, the sequential addition of four sugar residues by different glycosyltransferases will give rise to the linker tetrasaccharide (for Hep/HSXyl-Gal-Gal-GlcA) connected to the core protein's serine residue as a linker region [37]. Notably, the linkage region also serves as a primer for the initiation of the CS chains biosynthesis. In the case of HS, the members of the EXTL family of glycosyltransferases trigger chain creation by transferring an *N*-acetylglucosamine (GlcNAc), whereas in the case of CS chains, a β-*N*-acetylgalactosamine (β-GalNAc) residue is attached to the linkage primer by a CSGalNAc-transferase [37]. Polymerization of HS then takes place by the alternating addition of GlcAβ1,4 and GlcNAcα1,4 residues through the action of designated glycosyltransferases [38].Modifications, such as N-deacetylation and N-sulfation of glucosamine, and O-sulfations are subsequently performed. The GlcA residues can, on some occasions, be epimerized to iduronic acid (IdoA)[35,36].

The two GAGs differ, as the main HS disaccharide unit comprises a GlcA and Nacetylated GlcN(GlcNAc). In contrast, the main Hep disaccharide consists of sulfated, at the carbon 2 IdoA(IdoA2S), and N-sulfated GlcN also sulfated at C6 (GlcNS6S). Due to the high Hep sulfation level, this GAG is characterized as a biomacromolecule with the highest negative charge density [39]. The functionalization with sulfate is uniformly distributed along the Hep chain, whereas HS chains exhibit alternatively exchanging regions of high sulfation with lower or non-sulfated sequences [40]. Indeed, Sulf-1 and Sulf-2, sulfatase enzymes, are active at the extracellular compartment and trim the 6-O-sulfates partially from HS, but do not affect Hep, which is not located at the cells' membranes [41]. As a result, the Hep chain mainly comprises trisulfated disaccharides (80%) consisting of sulfated IdoA and sulfated GlcN.

The HS chains predominantly consist of disaccharide repeats comprised of GlcA and GlcNAc, with a much lower sulfation level [42]. Notably, the "fully sulfated" HS sequences, denominated as S domains, commonly exhibit the highest binding propensity to Hep/HS-binding proteins [43]. Indeed, the binding between proteins and HS/Hep is most commonly executed by charge–charge interactions between the proteins' basic amino acids and the anionic sulfate and/or carboxylate [18,44]. The interaction between respective binding proteins and HS is likewise affected by the GAG heterogeneity and cationic association [19]. Moreover, posttranslational modifications, such as N-glycosylation, of the HS/Hep binding proteins can regulate ligand and HS/Hep binding as shown for the fibroblast growth factor receptor 1 [45]. Notably, its disaccharide unit's extensive modifications render HS the most complex animal polysaccharide [19].

HS chains are synthesized by almost all mammalian cells in the forms of HSPG and are localized to the cell membrane (e.g., syndecans) and pericellular space/basement membranes (e.g., perlecan) or extracellular matrices. Despite the HS chain's extensive functionalization, its fine structure is notably conserved in a given cell type [46,47]. HS's composition varies both spatially and temporally during development and in a celltypedependent manner. The involved regulating mechanisms remain poorly elucidated.

Significant changes occur in HS composition during carcinogenesis, and vitally, both tumor growth and tumor-dependent angiogenesis depend on HS growth factor interactions [48].

Hep is synthesized only in connective tissue-type mast cells or basophils [49]. The Hep chain is synthesized during the core protein modification of the PG, seglycin. Seglycine exhibits a small protein core but undergoes extensive glycosylation, resulting in a molecular weight up to 750 kDa [50]. The bound Hep chains' molecular weight varies between 60 KDa and 75 kDa. These Hep chains are cleaved into 5–25 kDa fragments when mast cells and basophils are degranulated [51,52]. Mast cells release Hep by exocytosis upon binding specific antigens to the IgE antibodies attached to their cell-surface receptors [53]. However, Mast cell serglycin can also be decorated by other GAG chains, such as CS and DS [54].

Hep, however, can be uptaken by various cells, including endothelial cells, as the primary site for removing unfractionated Hep from the circulation is the liver sinus endothelial cells [55].

In mammalians, HS/Hep are enzymatically degraded by heparanase, a strict endo-βglucuronidase [56].
