Fluid Shear Induces Conformation Change in Human Blood Protein von Willebrand Factor in Solution

Abstract

Many of the physiological functions of von Willebrand Factor (VWF), including its binding interaction with blood platelets, are regulated by the magnitude of applied fluid/hydrodynamic stress. We applied two complementary strategies to study the effect of fluid forces on the solution structure of VWF. First, small-angle neutron scattering was used to measure protein conformation changes in response to laminar shear rates (G) up to 3000/s. Here, purified VWF was sheared in a quartz Couette cell and protein conformation was measured in real time over length scales from 2-140 nm. Second, changes in VWF structure up to 9600/s were quantified by measuring the binding of a fluorescent probe 1,1'-bis(anilino)-4-,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) to hydrophobic pockets exposed in the sheared protein. Small angle neutron scattering studies, coupled with quantitative modeling, showed that VWF undergoes structural changes at G < 3000/s. These changes were most prominent at length scales <10 nm (scattering vector (q) range >0.6/nm). A mathematical model attributes these changes to the rearrangement of domain level features within the globular section of the protein. Studies with bis-ANS demonstrated marked increase in bis-ANS binding at G > 2300/s. Together, the data suggest that local rearrangements at the domain level may precede changes at larger-length scales that accompany exposure of protein hydrophobic pockets. Changes in VWF conformation reported here likely regulate protein function in response to fluid shear.

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

“…Furthermore, threefold prolonged measurement times were needed to achieve a reliable signal-to-noise ratio. The urea-containing buffer could only partly unfold the A2 domains and not all of them, thereby not all the cleavage sites may have been fully exposed (37). FCS measurements of the diffusion coefficients of rVWF-wt dimer and rVWF-G1629E dimer in nondenaturing and denaturing buffer confirmed the observation of partial unfolding (Fig.…”

The effects of shear flow on protein structure and function

“…Furthermore, real-time measurements of the conformational dynamics of vWF in response to laminar shear flow in a Couette flow-cell, using a combination of small angle neutron scattering (SANS) spectroscopy and quantitative modeling, showed an irreversible conformational change in vWF, under hydrodynamic shear stress, at shear rates 3000 s 21 . 27 The observed conformational change was found to involve the exposure of hydrophobic domains at shear rates [2300 s 21 , as indicated by an increase in the binding affinity of the hydrophobic dye 1,10-bis(anilino)-4-,40-bis(naphtalene)-8,80-disulfonate (bis-ANS). 27 The shear-effect was more pronounced with increasing shear rate.…”

“…27 The observed conformational change was found to involve the exposure of hydrophobic domains at shear rates [2300 s 21 , as indicated by an increase in the binding affinity of the hydrophobic dye 1,10-bis(anilino)-4-,40-bis(naphtalene)-8,80-disulfonate (bis-ANS). 27 The shear-effect was more pronounced with increasing shear rate.…”

“…109 Interestingly, it has been shown that molecular confinement enhances the deformation of entangled polymers under squeeze flow, 110 a condition which models pulsatile blood flow. Similarly, there are a considerably number of reports 27,34,35,42,59,60,[111][112][113] citing haemodynamic shear stress as the primary facilitator of the proteolytic cleavage of large vWF multimers freshly released from endothelial cells as well as vWF-mediated platelet adhesion during haemostasis. The accumulation of large vWF multimers in the plasma may lead to disease states including thrombotic thrombocytopenic purpura, microvascular occlusion and atherosclerosis, due to the development of microvascular thrombi resulting from the complexation of the hyperactive vWF multimers and platelets at regions of low physiological shear.…”

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

“…VWF multimers can then mediate the adhesion of platelets. It has been shown that VWF multimers change from a globular to an elongated conformation under shear stress [10][11][12] and remain * chkiang@rice.edu in their activated, more adhesive state for hours [13]. Prolonged activation may coincide with the lateral association of VWF multimers into a fibrillar form of VWF [3,8,14].…”