The protective effect of von Willebrand factor (VWF) toward activated protein C (APC)-catalyzed inactivation of factor VIII (FVIII) has been attributed mainly to inhibition of FVIII binding to phospholipid. In the present study, we demonstrated that VWF-mediated FVIII protection from APC also results from direct inhibition of FVIII binding to APC. Inhibition of FVIII binding to anhydro-APC by VWF would be consistent with partial or complete overlap of the FVIII binding sites for APC and VWF. We examined, therefore, the inhibitory effects of 6 synthetic peptides spanning residues 1996 to 2028 around the previously localized APC binding region (FVIII residues 2009-2018). Peptide 2009 to 2018 inhibited FVIII binding to anhydro-APC by 83% (50% inhibition, 55 microM). Similarly, peptide 2013 to 2022 inhibited FVIII binding to VWF by 84% (50% inhibition, 25 microM). It was also found that peptides 2009 to 2018 and 2013 to 2022 optimally bound to anhydro-APC and VWF, respectively. A rabbit antipeptide IgG, raised against peptide 2009 to 2022, blocked the binding of both anhydro-APC and VWF to FVIII. This immunoglobulin G inhibited proteolytic cleavage of FVIII by APC. Our results indicate that the essential regions for the binding of APC and VWF to FVIII overlap and that the protective effect of VWF on APC-catalyzed FVIII inactivation includes competitive inhibition of APC binding to FVIII by VWF.