The anti-factor VIII (fVIII) C2 domain monoclonal antibody ESH8 inhibits fVIII activity only when fVIII is bound to von Willebrand factor (vWf). However, ESH8 binds with similar affinity to fVIII and fVIII.vWf complex, and it does not affect the kinetics of thrombin cleavage at positions 372 and 740 within the fVIII heavy chain and at 1689 within the light chain. The latter is required for fVIII release from vWf. We showed that ESH8 reduced the initial rate of thrombin-activated fVIII (fVIIIa) release from vWf by 4.3-fold compared to that in the absence of antibody. The complex of vWf. fVIII.ESH8 was activated, and the rate constant determined for fVIIIa dissociation from vWf was 4 x 10(-3) s-1. We constructed a mathematical model incorporating the measured rates for fVIIIa release from vWf and for inactivation of heterotrimeric fVIIIa due to the spontaneous loss of the A2 subunit and found that the decreased release rate is sufficient to explain our experimentally observed inhibition of fVIII activity by ESH8. We hypothesize that the slowed rate of fVIIIa release from vWf in the presence of ESH8 allows time for inactivation of unstable fVIIIa prior its participation in the formation of the factor Xase complex. The relevance of these findings is illustrated by our observation that reduction of fVIIIa release from vWf represents an additional mechanism of fVIII inhibition by an anti-C2 domain antibody (epitope 2218-2307) from a hemophilia A patient. This rare antibody binds to a more amino-terminal epitope than other human anti-C2 inhibitors, resulting in its lack of inhibition of fVIII binding to vWf but not to phospholipid. These two fVIII ligands therefore bind to C2 sites which do not overlap completely.