Previous work  suggested that HIP peptide-1 could enrich for anticoagulantly active Hp on the basis of a bio-specific interaction with the ATIII binding pentasaccharide. The goal of the current study was to substantiate this hypothesis. However, an overwhelming amount of data presented here demonstrates that the underlying hypothesis is invalid. Although we can enrich for anticoagulantly active Hp by HIP peptide-1 affinity chromatography using tritiated heparin or Hp byproducts as a starting material, this enrichment is not due to bio-specific interactions. Furthermore, repeated chromatography experiments with unlabelled conventional Hp as the starting material failed to significantly enrich for ATIII binding Hp. Lastly, Zhang et al.  have recently reported that HIP peptide-1 does not protect anticoagulantly active HS from complete heparitinase digestion.
It is clear from the current data that our samples of unlabelled and tritiated Hp are not biochemically similar. A significant proportion (38%) of tritiated Hp does not bind an anion exchange column while 100% of unlabelled Hp does. We hypothesize that this sample of tritiated Hp contains 38% free label and thus has no apparent negative charge. Since the fractionation of this particular preparation of tritiated Hp over HIP peptide-1 is similar to the previously published observations, we can only conclude that the initial preparation also contained significant amounts of "free label". The presence of "free label" would explain the repeated enrichment of ATIII-dependent anti FXa ability seen in tritiated Hp. Rather than enriching for the ATIII binding pentasaccharide by HIP peptide-1 chromatography of tritiated Hp, this chromatography enriches for Hp, leaving behind "free label". This would result in an apparent enrichment of activity per DPM in bound tritiated Hp materials and a depletion of activity per DPM in unbound tritiated Hp materials.
When the starting material used for fractionation on a HIP peptide-1 column is Hp byproducts, a charge dependent increase in ATIII-dependent anti FXa activity for bound materials is seen. Hp byproducts are a very heterogeneous mixture of GAGs. Due to the nature of the Hp/HS biosynthetic pathways, the ATIII binding pentasaccharide is formed preferentially in highly negatively charged regions . Our hypothesis is that HIP peptide-1 chromatography does not enrich for anticoagulant activity from Hp byproducts on the basis of a bio-specific interaction with the ATIII binding pentasaccharide but rather due to non-specific charge interactions. This hypothesis was substantiated by comparing the charge characteristics of Hp byproducts depleted of HIP peptide-1 or ATIII binding activity. Also, the binding ability of HIP peptide-1 for Hp byproducts depleted of ATIII binding activity was determined. Firstly, elution profiles from anion exchange chromatography show that ATIII binding species have an increased charge profile when compared to starting material, but the ATIII-depleted materials are only slightly shifted to a less negatively charged profile compared to starting material. In contrast, HIP peptide-1 depleted Hp byproducts are essentially depleted of its most negatively charged species. This shows that highly negatively charged species within Hp byproducts are associated with, but not sufficient for ATIII interaction while HIP peptide-1 has a simple charge requirement. Secondly, HIP peptide-1 binds some ATIII depleted Hp byproducts indicating that the ATIII binding pentasaccharide is not essential for HIP peptide-1 binding. The inherently high negative charge of ATIII binding species [11, 12] and the charge heterogeneity of Hp byproducts makes the HIP peptide-1 separation of Hp byproducts into pools with low and high anticoagulant activity possible. In contrast, the relatively homogeneously charged Hp preparations tested are refractory to a similar separation.
Binding studies suggest that HIP peptide-1 has a selectivity for GAGs of the Hp/HS subclass . This is demonstrated by the inability of CS-A, DS, CS-C, KS, and HA to act as effective competitors for HIP peptide-1 binding to tritiated Hp. Additionally, HIP peptide-1 is found to bind subsets of cell surface JAR cell HS and DS, suggesting differentiation within a class of GAG. Binding potential is increased in pools of JAR cell HS or DS that have longer length and higher sulfation content. Likewise, Hp byproducts bound in the presence of 0.20 M have increased size (D.E.H. and M.H. unpublished observations) and charge when compared to Hp byproducts bound in 0.15 M NaCl. The new interpretation of this data is that HIP peptide-1 binds GAGs via a threshold charge and not due to inherent bio-specificity. This is further supported by the determined hierarchy of binding potential; Hp > CS-E > DS > CS-C = BK-HS, which mimics the charge density order of the GAGs; suggesting that the sulfation content is the most important factor in the interaction with HIP peptide-1 and not the subclass of GAG.
Theoretically, it should be possible to create linear peptides that can specifically bind to 'sequences' within the linear GAG chain. Our current knowledge on Hp/HS binding motifs has come from an examination of Hp/HS binding proteins that identified the XBBXBX and XBBBXXBX motifs where X is an uncharged or hydrophobic amino acid and B is a basic amino acid . It is important to note that HIP peptide-1 (CRPKAKAKAKAKDQTK) does not exactly correspond to either of these motifs yet binds Hp/HS. However, studies have shown that concatamers of peptides that conform to these consensus motifs have binding affinity proportional to the number of subunits  and can reverse the antithrombotic activity of Hp in vivo . A natural example of a concatamer of Hp/HS binding motifs is human HIP/RPL29. This protein is highly basic with 29.5% of Lys/Arg content distributed evenly throughout the protein. Studies with deletion mutants of human HIP/RPL29 show that Hp/HS binding ability increases with the length of deletion mutant, irrespective of domain . Concatamers of Hp/HS binding sequences may be a common mechanism of protein/GAG interactions. Future work will be aimed at identifying novel proteins and peptide sequences that specifically interact with ATIII binding Hp/HS.