NOX-A12 competes with the GAG heparin for binding to CXCL12. (A) MS-5 cells were incubated with high concentrations of heparin (200 µg/mL) for the indicated time leading to the release of CXCL12. Results indicate the concentration of CXCL12 in supernatants quantified by ELISA. The dashed line indicates the basal CXCL12 secretion levels. (B) MS-5 cells were incubated with various concentrations of heparin for 3 hours. Results indicate the concentration of CXCL12 in supernatants quantified by ELISA. Heparin induced a concentration-dependent release of CXCL12 in MS-5 cells with an EC₅₀ of about 180 µg/mL (12.4 µM based on a molecular weight of 14.5 kilodalton). (C) CXCL12 showed a dose-dependent binding to immobilized biotinylated heparin with dissociation constant (K_D) of 407 nM. (D) Simultaneous injection of 125 nM of CXCL12 in the presence of various heparin concentrations in solution led to a dose-dependent competition of CXCL12 binding to the immobilized heparin with an IC₅₀ of 21 µg/mL (1.45 µM based on a molecular weight of 14.5 kilodalton). (E) Simultaneous injection of 125 nM of CXCL12 together with NOX-A12 led to a competition of CXCL12 binding to immobilized heparin with an IC₅₀ of 67.93 nM. (F) NOX-A12 mobilized heparin-bound CXCL12 in a Biacore flow system. Injection of 2.5 µM of human CXCL12 led to binding to immobilized heparin. Injection of 100 nM of NOX-A12 for 30 seconds during the dissociation phase of the binding event led to a release of heparin-bound CXCL12 from the surface, whereas revNOX-A12 showed no significant influence on the dissociation of CXCL12 from immobilized heparin compared with buffer.