The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

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