Shear stress triggers conformational stretching of von Willebrand factor (VWF), responsible for its self-association and binding to the platelet receptor GpIbα. This phenomenon supports primary haemostasis under flow. Type 2B-VWF natural mutants are considered having increased affinity for platelet GpIbα.
To assess the mechanism responsible for the enhanced interaction of the p.R1306W- VWF mutant with the platelet receptor.
The interaction of GpIbα with WT and p.R1306W-VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. The analysis of static VWF conformation in solution was carried out by dynamic light scattering spectroscopy. The shear-stress induced self-association of VWF multimers was investigated by atomic force microscopy (AFM) over a 0-60 dyn/cm2 range.
WT-VWF does not interact with GpIbα under static conditions, whereas the mutant at ≈2 μg/ml already binds to the receptor. By contrast, the WT- and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GpIbα (Kd ≈ 20 nM). The hydrodynamic diameter of resting R1306W -VWF multimers was significantly higher than the WT (210 ± 60 nm vs. 87 ± 22 nm). At shear forces<14 dyn/cm2, the p.R1306W multimers rapidly change conformation entering a regime of self-aggregation, which, at variance, is induced for WT-VWF by shear forces>30 dyn/cm2. Mechanical stretching AFM experiments showed that p.R1306W multimers need less energy/length unit (≈ 10 pN) to be stretched compared to WT protein.
The increased avidity of p.R1306W-VWF for GpIbα arises mostly from a higher sensitivity to shear stress that facilitates exposure of GpIbα binding sites.
- Atomic Force Microscopy
- Shear stress
- Surface Plasmon Resonance
- Von Willebrand Disease