Mechanical Regulation of Von Willebrand Factor A1 Domain

Abstract

Regulation of the bond between platelet Glycoprotein Ib (GPIb) and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and/or tensile mechanical force, but the structural basis of this mechanical regulation remained unknown. To address this question, we expressed a number of A1 constructs that differed in one or more residues or domains and anchored them to the surface at known concentrations and orientations. We then measured rolling velocities of platelets or GPIb-coated microspheres to determine the importance of different structural regions and the manner in which force is applied. We show that an intrinsically disordered peptide region N-terminal to the A1 domain has strong inhibitory activity. In addition, the A1-GPIb bond is much stronger when the A1 domain is anchored via the C-terminus rather than the N-terminus. This suggests that shear stress activates A1-GPIb binding in two ways. First, it applies tensile force across multimeric VWF, activating the A1 domain by dislodging this inhibitory region. Second, it applies tensile force across the A1-GPIb bond, inducing a conformational change in the C-terminal region of VWF that allosterically activates the A1 domain, perhaps in a way analogous to that seen in integrins.