Kinetics and affinity of VWFpp binding to immobilized multimeric VWF. (A) Three hundred twenty response unit (RU) VWF was immobilized on the SPR sensor via anti-VWF mAb. Different concentrations of VWFpp (indicated in nanomolar units in panels A-C) were perfused over the substrate in MES (pH 6.2) binding buffer with 10mM CaCl2. (B) Studies identical to those in panel A except that 920 RU of VWF was immobilized and the running buffer was HEPES (pH 7.4) with 2.5mM CaCl2. (C) 320 RU of VWF was immobilized and running buffer was HEPES with 2.5mM EDTA(pH 7.4). Solid lines in panels A through C represent experimental data and dashed lines are fits to the 1:1 interaction model. Kinetic constants from fits are summarized in Table 1. Equilibrium KD analysis for panels A and B are shown in panels D and E, respectively.
