The D' domain of von Willebrand factor requires the presence of the D3 domain for optimal factor VIII binding.

The D'-D3 fragment of von Willebrand factor (VWF) can be divided into TIL'-E'-VWD3-C8_3-TIL3-E3 subdomains of which TIL'-E'-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D', D3 and monomeric C-terminal subdomain truncation variants of D'-D3. Competitive binding assays and surface plasmon resonance analysis revealed that D' requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D'-D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D'-D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D'-D3 is indispensable for effective interaction between D' and FVIII explaining why specific mutations in D3 can impair FVIII binding.