Electrostatic Steering Enables Flow-Activated Von Willebrand Factor to Bind Platelet Glycoprotein, Revealed by Single-Molecule Stretching and Imaging

Abstract

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 electrostatic steering. Under supraphysiologic salt concentrations, strong electrostatic screening dramatically decreases platelet binding to VWF in flow, revealing the critical role of electrostatic attraction in VWF-platelet binding during bleeding.

Keywords: hemostasis; mechanosensing; microfluidics; single-molecule fluorescence; single-molecule force spectroscopy.

Figure 1

VWF multimer can be elongated and activated by flow. (Adapted from Fu et al10]) (A) (i) Schematic of platelet binding to VWF that is tethered to the blood vessel wall, (ii) a VWF multimer, and (iii) the domain lineup of a dimer segment. (B) Views of the GPIbα–A1 complex crystal structure [11] with electrostatic surface potentials colored according to the key. In the open-book view on the right-hand-side, GPIbα and A1 are rotated 90° towards the viewer around the dashed axis to show their highly electrostatic interfaces. (C) Schematic of the TIRF microscope with pressure-actuated flow.

Figure 2

VWF extension does not significantly depend on [NaCl] from 10 to 500 mM. (A) Representative images of a single VWF molecule extending under shear stress following the indicated buffer exchange. For each buffer condition, a typical flow profile consists of cycles of start- and stop-flow in forward (+) and backward (−) directions at multiple different shear stresses, which are indicated below each image (10 ms exposure every second). (B, C) Normalized extension of VWF multimers vs. wall shear stress is presented, with the length of each molecule normalized by its fully-stretched length, as measured during the first instance of 1280 dyn cm⁻² flow (L₀, color key). Extension is presented under forward (solid lines) and backward (dash lines) flow following 150 -> 10 -> 150 mM NaCl (b, N=46 multimers) or 150 -> 500 -> 150 mM NaCl (c, N=44 multimers) buffer exchanges. Black solid and dashed lines in (B) and (C) are the average and the standard deviation of the extension of individual molecules, respectively. They are also overlaid as blue, green and magenta lines in (E) and (F), to aid in comparing the results from the indicated buffer exchange sequences. (D) Percentage of VWF compaction in 10 mM to 1 M NaCl buffer.

Figure 3

VWF-GPIbα association kinetics are salt dependent. (A) Representative time-lapse dual color fluorescence images of VWF extension and binding to GPIbα at 80 mM NaCl after 1280 dyn cm⁻² wall shear flow was turned on and off at 0 and 4 second, respectively. Association rates (B) and dissociation rates (C) between VWF and GPIbα under different [NaCl]. Lines in (b) are two-state model fit. (D) The 50% activation force vs [NaCl] (E) Association rate of the high affinity state vs the square root of ionic strength matches the electrostatic steering model. See equation (3) for details about the x axis. Error bars indicate 95% confidence intervals.

Figure 4

Fixed platelets bind to and roll on VWF-coated surface under flow. (A) Footprints of platelets rolling on VWF-coated surface under 20 dyn cm⁻² wall shear in 150 mM and 500 mM NaCl in the first 8 seconds of flow. Brighter patterns indicate overlapped footprints. The accumulated number of platelets bound to the surface (B) and the average speed of platelet rolling on the surface (C) are plotted as a function of wall shear stress. The inset shows a zoom-in of the rolling speed between 0.5 and 10 dyn cm⁻² for easier viewing. For shear stresses lower than 0.2 dyn cm⁻² and higher than 50 dyn cm⁻² in 500 mM NaCl buffer, fewer than 3 platelets bound to the surface and the rolling speed was not plotted. Error bars indicate standard errors of the mean.

Figure 5

VWF extension slightly depends on pH from 6.2 to 9.8. VWF multimer extension normalized to length during the first 1280 dyn cm⁻² flow (L₀, color key) vs. wall shear stress under forward (solid lines) and backward (dash lines) flow following pH 7.4 -> 6.2 -> 7.4 (a, N=35 multimers) or pH 7.4 -> 9.8 -> 7.4 (b, N=38 multimers) buffer exchanges. Black solid lines and the black dashed lines in (A) and (B) are the average and the standard deviation of the extension of individual molecules. They are also overlaid as blue, green and magenta lines in (C) and (D), to aid in comparing the results from indicated buffer exchanges. (E) Percentage of VWF compaction in pH 5.4 to 9.8 buffer.

Figure 6

VWF-GPIbα association kinetics are pH dependent. Association rates (A) and dissociation rates (B) between VWF and GPIbα at different levels of pH. The lines in (a) are two-state model fits. The 50% activation force (C) and the association rate of the high affinity state (D) vs pH. Error bars indicate 95% confidence intervals.