VON WILLEBRAND FACTOR
Using techniques in single-molecule manipulation, we have illuminated a fundamental feedback mechanism that the body uses to regulate the clotting of blood. Small tensile forces, such as those experienced in the circulation, can unfold the von Willebrand factor A2 domain, enabling its cleavage by the ADAMTS13 enzyme. This, in turn varies the body’s hemostatic potential.
MECHANOENZYMATIC CLEAVAGE OF THE ULTRALARGE VASCULAR PROTEIN
VON WILLEBRAND FACTOR
We are interested in understanding the how hemostasis and blood clotting are regulated by mechanical forces in the blood stream. Specifically, we have been studying the effect of forces and flows on the vascular protein von Willebrand factor (VWF). Regarding down-regulation of hemostasis by force, the A2 domain of VWF is the key to this largely mechanical molecular feedback loop. Using optical tweezers, we have characterized the unfolding and refolding kinetics of the A2 domain, and have shown that force acts as an “cofactor”, enabling the cleavage of A2 by the ADAMTS13 enzyme. Furthermore, the hydrodynamic tensile forces encountered by VWF in the circulation are sufficient to enable cleavage, which in turn down regulates the body’s hemostatic potential. Here is the abstract of work that has recently been published in Science Magazine, in collaboration with the Springer group.
Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.
FLOW-INDUCED ELONGATION OF VON WILLEBRAND FACTOR PRECEDES TENSION-DEPENDENT ACTIVATION
Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor–ligand binding.
Molecular model of flow-induced VWF activation: a Schematic of extended VWF and platelet surface drawn to scale, including average spacing between GPIbα/GPIbβ 1:2 complexes and length of mucin-like regions shown as brush-like curves. “+” and “−” indicate net electrostatic charge at plasma pH; sialic acid makes mucins negatively charged. b Model of VWF activation under flow. VWF is tethered on a vessel wall. Three values of shear stress (σ) are shown that do not extend, extend but do not activate, or extend VWF and are above the threshold shear stress required for activation (σ50, a function of NVWF). Tension is proportional to the number of VWF monomers downstream of the tether point. Activation of A1 is schematized as disruption by mechanical tension of hydrogen bonds involving residues external to the A1 disulfide, converting A1 from a low-affinity square shape to a high-affinity round shape. c, d Views of the GPIbα–A1 complex crystal structure with similar orientations in the upper portion of each panel. c Ribbon cartoons of GPIbα in gray and A1 in rainbow from N (blue) to C-terminus (red). Spheres mark N and C-termini. The lower panel shows the force-bearing region at the N and C-termini near the long-range disulfide (gold stick). Hydrogen bonds involving residues external to the disulfide are shown as black dashes. d Electrostatic surface potentials colored according to the key. In the lower, open-book view, GPIbα and A1 are rotated 90° towards the viewer around the dashed axis to show their highly electrostatic interfaces. e Shear and elongational flows. (Left) Shear flow can be represented as the combination of elongational flow and rotational flow. Arrows show flow streamlines and dots indicate no-flow regions. (Right) Effects of vasoconstriction and bleeding on flow. VWF concatemers (red) are more compact in shear flow, and as the elongational component of flow increases, they are more extended. When tethered to the vessel wall, as shown in the constriction site, VWF is more easily extended when tethered than when in free solution since tension exerted on it cannot be minimized by movement with the flow—rather, the tension in the molecule must resist the total drag force induced by the flow on the downstream portions of VWF
RELEVANT GROUP PUBLICATIONS
Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging
von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our labs had shown using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically we found that the required shear stress was higher than reported for free-in-flow VWF—an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF’s molecular mechanism, we measured free VWF extension in shear flow using PULSIS—Pulsed Laser Stroboscopic Imaging of Single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame...
Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association
Von Willebrand factor (VWF) is an ultra-long concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to zero at the downstream, free end. Using single-molecule fluorescence microscopy, we directly visualize the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules and show that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF.... Read more ›
Electrostatic Steering Enables Flow-Activated Von Willebrand Factor to Bind Platelet Glycoprotein, Revealed by Single-Molecule Stretching and Imaging
Von Willebrand factor (VWF), a large multimeric blood protein, senses changes in shear stress during bleeding and responds by binding platelets to plug ruptures in the vessel wall. Molecular mechanisms underlying this dynamic process are difficult to uncover using standard approaches due to the challenge of applying mechanical forces while monitoring structure and activity. By combining single-molecule fluorescence imaging with high-pressure, rapidly switching microfluidics, we reveal the key role of electrostatic steering in accelerating the binding between flow-activated VWF and GPIbα, and in rapidly immobilizing platelets under flow. We measure the elongation and tension-dependent activation of individual VWF multimers under a range of ionic strengths and pH levels, and find that the association rate is enhanced by 4 orders of magnitude by... Read more ›
Flow-induced elongation of von Willebrand factor precedes tension-dependent activation
Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions... Read more ›
Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand Factor
Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13... Read more ›
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