The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo.

Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant. SUMMARY: Background Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF