The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor

A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). Time acidic region of the LCh, residues 1649-1689, arise controls fVIII vWf binding by an unknown mechanism, Although anti-acidic region monoclonal antibodies prevent formation of the fVIII-vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated, By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14- and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively, Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K-d 72 nM) was 19-fold lower than that of LCh, This was not due to an altered conformation of the acidic region within the 14-kDa fragment,since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K-d 564-660 nM) compared with LCh (K-d 3.8 nM). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2, The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170-2327) indicated that removal of the acidic region leads to a conformational change within C2, This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site, Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.