Biophysical Characterization of Mechanosensors within the Plasma Protein von Willebrand Factor and its Receptor Platelet Glycoprotein Ib-IX

Abstract

The large, multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. Platelet binding is achieved by interaction between the A1 domain of the Iba chain of platelet surface receptor Glycoprotein (GP) Ib-IX. Both GPIB-IX and VWF are capable of sensing and reacting to tensile forces. Using the single-molecule force measurement approach, we have recently identified a quasi-stable mechano-sensitive domain (MSD) of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. The MSD unfolds at 5-20 pN when subjected to mechanical stretch by the engaged A1. Unfolding of the MSD triggers receptor signaling and stimulates platelets. Similarly, earlier studies by the Springer group have identified the mechanosensor within VWF, the A2 domain (i.e., the domain adjacent to A1). Tensile force at the level of 10-20 pN unfolds the A2 structure, leading to A2's subsequent cleavage by plasma enzyme ADAMTS-13 (Zhang, X. et al. Science, 324:1330-4). Since both MSD and A2 unfold at a similar force level, simultaneous activation of both mechanosensors may occur given that the interacting VWF and GPIb-IX are stretched to provide sufficient force. Herein, we show that optical tweezer-controlled pulling of the full-length of VWF on GPIb-IX indeed induced unfolding of both MSD and A2. Additional studies using recombinant wildtype and mutant VWF suggest that the unfolding of MSD and A2 is dependent on the mechanical strengths of the VWF-GPIba and VWF-collagen interactions, as well as on the activation state of the VWF. Together, the study has revealed the biomechanical properties of VWF and GPIb-IX, with significant implications for the pathogenesis of Von Willebrand Disease and GPIb-IX- related blood diseases.