Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

Abstract

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm 2 for 3 min at 37 • C). We conclude that under the shear conditions used (100 dynes/cm 2 for 3 min at 37 • C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

VWF/ADAMTS13 AND Ischemic Stroke

“…Usually, when newly released into the blood, ULVWF first attaches to the endothelial surface through P-selectin and binds platelets by forming strands with other molecules. ULVWF becomes very heavy after adhering to platelets, when drafted by the sheer force and torsional force from strong blood flow, the binding sites of A1 and A3 domains to ADAMTS13 will be exposed [5]. The A2 domain is not involved in the binding process, which is due to the change of spatial structure after platelet binding to ULVWF and the change of capture speed between ADAMTS13 and ULVWF at high velocity.…”

A biophysical view on von Willebrand factor activation

“…In addition, it should be noted that, both in the AFM and in the flow experiments, A1 domains were immobilized on a surface and not subjected to significant forces prior to binding, in contrast to the situation in vivo, where A1 domains within elongated VWF multimers in flow can be expected to experience forces that are large enough to potentially induce relevant conformational changes (Kim, Hudson, & Springer, 2015;Zhang et al, 2009). In two further AFM-based studies, Wijeratne et al (2013Wijeratne et al ( , 2016 performed force measurements on VWF multimers and dimers after exposure to high shear stress. They reported that peaks in the obtained force-extension traces, which were interpreted as domain unfolding events, occurred at higher forces for sheared than for nonsheared multimers.…”

“…In two further AFM‐based studies, Wijeratne et al (, ) performed force measurements on VWF multimers and dimers after exposure to high shear stress. They reported that peaks in the obtained force‐extension traces, which were interpreted as domain unfolding events, occurred at higher forces for sheared than for non‐sheared multimers.…”

Dependence of Membrane Tether Strength on Substrate Rigidity Probed by Single-Cell Force Spectroscopy

“…Cells were allowed to recover for 4 s between each F−D curve cycle. 36,37 Figure 2 shows typical F−D curves from AFM pulling of single cells. The F−D curve begins with a rapid rise to its maximum and forms a high force region within one micrometer.…”

“…The cantilever was then retracted at a constant speed of 3 μm/s and a force versus distance ( F – D ) curve was recorded. Cells were allowed to recover for 4 s between each F – D curve cycle. , …”