Determination of the kinetic parameters for nonproteolytic and thrombin-mediated dissociation of FVIII and variants from monoclonal antibody ESH8.

The monoclonal antibody ESH8 was covalently immobilized to a biosensor chip at 20 ng/mm². FVIII WT,^SER289^LEU, and pdFVIII (A); FVIII WT and^ALA284^GLU (B); or BDD-FVIII and BDD-^SER289^LEU (C) (2.5 nM) were bound to ESH8 at 1.12 ng/mm². A resonance response of 200 Arc seconds corresponds to 1 ng protein bound per mm² of the biosensor chip surface. The kinetics of plasma-derived FVIII, FVIII WT,^SER289^LEU, ^ALA284^GLU, BDD-FVIII, or BDD-^SER289^LEU 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.