Determination of the kinetic parameters for nonproteolytic and thrombin-mediated dissociation of FVIII WT and^ARG531^HIS from MoAb ESH8. MoAb ESH8 was covalently immobilized to a biosensor chip at 20 ng/mm². FVIII WT or ^ARG531^HIS (2.5 nmol/L) were bound to ESH8 at 1.12 ng/mm². A resonance response of 200 Arc seconds corresponds to 1 ng of protein bound per square millimeter of the biosensor chip surface. The kinetics of FVIII WT or^ARG531^HIS nonproteolytic dissociation from ESH8 was recorded after replacement of the ligand by dissociation buffer (at arrow). At the second arrow, thrombin (1 U/mL) was added and thrombin-mediated dissociation of the A2 subunit from immobilized dimers was followed. The k_off values for nonproteolytic and thrombin-mediated dissociation were derived from dissociation kinetic curves as described under Materials and Methods.