## 4 Materials and Methods
### Isothermal Fluorescence Titration (IFT)  
In order to study the affinity of the interaction between chemically synthesized TAMRA-labeled peptides and GAGs, isothermal fluorescence titration (IFT) experiments were performed [42; 43; 44; 45; 46]. Upon the interaction of a peptide with a GAG, a structural re-arrangement of the peptide is induced, influencing the fluorescence emission. Consequently, a dose-dependent decrease in fluorescence (quenching) could be detected. Hence, the change in fluorescence intensity was used to quantify the binding of the peptide to the GAG. The K\({}_{\mathrm{d}}\) values were calculated from the resulting binding isotherms. A spectrofluorometer (Fluoromax-4; Horiba Scientific; Kyoto, Japan), attached to a temperature controller (LFI-3751; Wavelength Electronics; Bozeman, MT, USA) to ensure a stable temperature of 20 \({}^{\circ}\)C, was used for the measurements. Fluorescence emission spectra were recorded at 560 to 750 nm after excitation at 550 nm. The scan speed was set to 500 nm/min, and the slit widths were set at 3 nm and 5 nm for excitation and emission, respectively. Concentrated stock solutions (100 uM and 500 uM) of GAGs were prepared, and the GAGs used in these experiments included: LMWH (5 kDa; Lovenox; Sanofi; Bridgewater, NJ, USA), HS (22 kDa; Celsus Laboratories; Cincinnati, OH, USA), HA (11.5 kDa; Sigma-Aldrich; St. Louis, MO, USA) and partially desalted LMWH (5 kDa; dsLMWH; 2-O, 3-O desulfated enoxaparin; desulfated in the laboratory in Graz). First, a fluorescence emission spectrum of the TAMRA-labeled peptide at 700 nM in 500 uL PBS without any added GAG was recorded after equilibration at room temperature in the dark for 30 min (intrinsic fluorescence of the peptide). Next, 0.5 uL of a 100 uM GAG stock solution was added, and the next spectrum was recorded after 1 min of equilibration. This step was repeated until a total GAG concentration of 800 nM was reached. Subsequently, the highest concentration (500 uM) of GAG stock solution was added in 0.5 uL steps until a total GAG concentration of 3300 nM was obtained. These titration series were performed three times in total. For each individual measurement, the area under the curve (AUC) was calculated from the fluorescence emission spectrum. To eliminate background signals, fluorescence emission spectra of the titration series without peptide (in PBS only) were also collected, and the corresponding AUC values were subtracted from those of the spectra with peptide [45]. The change in fluorescence intensity (AF) divided by the intrinsic fluorescence of the peptide (F\({}_{0}\) corresponding to the fluorescence emission in the absence of GAG) was plotted against the concentration of added GAG. \(\Delta\)F is acquired by subtracting F\({}_{0}\) from the fluorescence emission measured at a certain GAG concentration (F). Mean values of \(\Delta\)F/F\({}_{0}\) were calculated from three independent measurements. Origin 8.0 software (OriginLab; Northampton, MA, USA) was used to analyze the resulting binding isotherms. Data fitting was performed by applying a nonlinear regression using the following equation:  
\[F=F_{i}+F_{max}\left[\frac{K_{d}\ +\ [protein]\ +\ [ligand]\ -\sqrt{(K_{d}\ +\ [protein]\ +\ [ligand])^{2}\ -\ 4[protein][ligand]}}{2[protein]}\right]\]  
where F\({}_{i}\) and F\({}_{max}\) are the values of the initial fluorescence, which was fixed to 0 for the calculation of the fitting and the maximum fluorescence, respectively; and [protein] and [ligand] represent the total protein (TAMRA-labeled peptide) and ligand (GAG) concentrations [45]. The dissociation constants (K\({}_{d}\) values) were calculated and used for data comparison and are presented as values +/ - standard deviation (SD), where SD refers to the precision of the fitting procedure. This fitting equation assumes an equimolar ratio of binding sites on the peptide and its ligand.