The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one-stage and a two-stage assay. SPR-analysis revealed that the equilibrium dissociation constants (K(D)) of ESH8 and ESH4 are low and in a similar range (ESH8: K(D(ESH8)) = 0.542 nM; ESH4: K(D(ESH4)) = 0.761 nM). A 5.7 times higher K(D) than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest K(D) of 28.8 nM. In accordance with the lowest K(D), ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one-stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 microg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 microg/ml of ESH4. Despite its attenuated K(D), ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 microg/ml in the one-stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity.
- blood coagulation