Binding was demonstrated by (a) ELISA, (b) glycan microarray, and (c) surface plasmon resonance. Dissociation constant for Tie1, as determined by ELISA: KD,app = 6.16 nM (5.40 to 7.01 nM); value represents mean (95% CI); graphed data represent n = 2 independent replicates. This value is similar to dissociation constants reported for other GAG-protein interactions.33,37 For glycan microarrays, data represent mean ± s.e.m., n = 10 individual spots per glycan concentration. Dissociation constant for Tie1, as determined by SPR: KD,app = 3.14 nM (3.12 to 3.15 nM); value represents mean (95% CI). SPR data were fit using a 1:1 Langmuir model shown in red. (d) Immunofluorescence images of HS GAGs (green) and Tie1-Fc ectodomain (red) binding to HUVECs treated with or without heparinase (HSase), scale bar = 50 μm. Nuclear staining by DAPI is shown in blue. Images are representative of 3 biologically independent samples.