## 2 Results
### An Increased Number of GAG-Binding Motifs Results in Higher Affinity for HS
The replacement of Ala by Gln residues in the GAG-binding motifs, generating the KKQKASAS-3 mer, resulted in increased affinity of the peptide for LMWH, dsLMWH and HS. In contrast, a decreased affinity of the KKQKASAS-3 mer for HA was observed. Furthermore, the KKQKASAS- and KKQK-3 mers were compared to evaluate the contribution of Ala and Gln residues in the linker sequences to GAG-binding affinity. The KKQK-3 mer (Figure 3C) showed higher affinity for LMWH, dsLMWH and HS, whereas the KKQKASAS-3 mer showed higher affinity for HA. In conclusion, amino acids in both GAG-binding motifs and linker sequences appear to affect GAG-binding affinity. Moreover, hydrophilic Gln residues in GAG-binding motifs and linker sequences seem to increase the binding affinity for LMWH, dsLMWH and HS. However, the presence of hydrophobic Ala residues in GAG-binding motifs and linker sequences improves the affinity for HA. Figure 3: Gln residues in both GAG-binding motifs and linker sequences increase trimer peptide binding affinity for (ds)LMWH and HS, but not HA. Isothermal fluorescence titration (IFT) measurements were performed to study the interactions between TAMRA-labeled trimer peptides (KKAK-, KKOKASAS- and KKQK-3 mer) and GAGs ((ds)LMWH, HS and HA). (**A–C**) Binding isotherms for the KAK-3 mer (**A**), KKQKASAS-3 mer (**B**) and KKQK-3 mer (**C**) are displayed. The change in fluorescence intensity (\(\Delta\)F) divided by the intrinsic fluorescence of the peptide (F\({}_{0}\)) is shown on the y-axis. \(\Delta\)F is calculated by subtracting F\({}_{0}\) from the fluorescence emission measured at a certain GAG concentration (F). The concentration of the added GAG is shown on the x-axis. Data points represent mean \(\pm\) SEM (\(n=3\)). (**D**) K\({}_{\text{d}}\) values (\(\pm\)SD) that are derived from the binding isotherms in (**A–C**) are visualized in the bar plot. TAMRA-labeling is indicated with [*] and the GAG-binding motifs are underlined in the amino acid sequences of the peptides. Dimer Peptides with Long versus Short GAG-Binding Motifs Are Characterized by Differential Affinity for LMWH and dsLMWH  
The contribution of short versus long GAG-binding motifs to GAG-binding affinity was investigated by comparing the KKQK-2 mer and the KKKQQK-2 mer, containing two short and two long GAG-binding motifs, respectively. Figure 4A,B represent the binding isotherms of these peptides, and their K\({}_{\text{d}}\) values are displayed in Figure 4C. The KKKQK-2 mer was characterized by a higher affinity for LMWH and dsLMWH in comparison with the KKQK-2 mer, indicating a possible role of long GAG-binding motifs for binding to these GAGs. For HS and HA, only minor differences between the two peptides were observed. A summarizing overview of all the K\({}_{\text{d}}\) values, derived from the IFT measurements, is depicted in Figure 5. This comparison shows that the chemokine-derived CXCL9(74-103) has the highest affinity for GAGs and that, in contrast to almost all other peptides, CXCL9(74-103) has a high preference for binding to HS compared with other GAGs. Figure 4: Dimer peptides with either short or long GAG-binding motifs show differential affinity for GAGs. Isothermal fluorescence titration (IFT) measurements were performed to study the interactions between TAMRA-labeled dimer peptides (KKQK-2 mer and KKKQK-2 mer) and GAGs ((ds)LMWH, HS and HA). (**A**,**B**) Binding isotherms for the KKQK-2 mer (**A**) and the KKKQK-2 mer (**B**) are displayed. The change in fluorescence intensity (\(\Delta\)F) divided by the intrinsic fluorescence of the peptide (F\({}_{0}\)) is shown on the y-axis. \(\Delta\)F is calculated by subtracting F\({}_{0}\) from the fluorescence emission measured at a certain GAG concentration (F). The concentration of the added GAG is shown on the x-axis. Data points represent mean \(\pm\) SEM (\(n\) = 3). (**C**) K\({}_{\text{d}}\) values (\(\pm\)SD) that are derived from the binding isotherms in (**A**,**B**) are visualized in the bar plot.