Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF’s hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above  ～ 50 pN and provided  ～ 80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF’s effective length contributing to force sensing, they can be expected to tune VWF’s sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF’s function in hemostasis and thrombosis.Force-sensing molecules are critically involved in a variety of biological processes, such as regulation of muscle gene expression or assembly of the cytoskeleton (1–4). In the vasculature, activation of the plasma glycoprotein von Willebrand factor (VWF) for hemostasis crucially depends on its distinct ability to sense hydrodynamic forces (5–7). These forces result from the interplay between hydrodynamic flow and VWF’s extraordinary length (8–10), which can exceed 15 μm in the plasma (6). VWF’s length arises from its linear multimeric nature. Linear multimers (concatamers) are composed of a variable number of dimers, which are linked N-terminally via disulfide bonds. Dimers, the smallest repeating subunits of VWF with a molecular mass of  ～ 500 kDa, consist of two monomers that are linked via C-terminal disulfide bonds (11, 12).Under static conditions, VWF was reported to adopt a collapsed conformation (6). When subjected to sufficiently high forces, as for instance at sites of vascular injury, vasoconstriction, or stenosis, VWF undergoes an abrupt transition from the collapsed to a stretched conformation (Fig. 1A) (6). This transition was shown to correlate with an increased adhesiveness to collagen and platelets (6, 13), enabling stretched VWF to recruit platelets to an injured vessel wall and to promote the formation of a platelet plug. VWF’s physiological importance is underlined by mutations that can cause von Willebrand disease (14), the most common hereditary bleeding disorder.Open in a separate windowFig. 1.Single-molecule force measurements on VWF dimers. (A) Illustration of VWF’s ability to sense hydrodynamic forces in blood vessels. When subjected to sufficiently high forces, for instance at sites of vascular injury, vasoconstriction, or stenosis, VWF undergoes an abrupt transition from a collapsed to a stretched conformation and promotes hemostasis. (B) Schematic representation of pulling recombinant VWF dimers. A ybbR-tag at the N terminus of one of the monomers allowed for covalent anchoring, and a Twin-Strep-tag at the N terminus of the other monomer enabled specific pulling via a Strep-Tactin functionalized AFM cantilever. (C) Denoised force–extension traces of dimers showing A2 unfolding peaks (blue arrows) at low (type I traces) or at high extension values (type II traces). Type I traces repeatedly exhibited a peak (dimer opening, green arrow) at higher force. The final peak (brown arrow) corresponds to the unbinding of the Twin-Strep-tag from Strep-Tactin. (D) Bimodal distribution of the position of the first A2 unfolding event.Down-regulation of VWF’s hemostatic potential is achieved by the cleavage of long concatamers into shorter ones by the enzyme ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) (15). Notably, the specific cleavage site is buried in the A2 domain and exposed by A2 unfolding (8, 16). The interplay of force-induced A2 unfolding and enzymatic cleavage has been investigated in detail at single-molecule level (8). In this context, unfolding of an isolated A2 domain was shown to occur at forces of ∼ 7–14 pN at loading rates ranging from 0.35 to 350 pN⋅s⁻¹. In the presence of neighboring A1 and A3 domains, A2 unfolding was observed at slightly higher forces of ∼ 20 pN and comparable loading rates (17).VWF’s activation for hemostasis correlates with its elongation above a critical force threshold (6, 7). However, the current understanding of the underlying molecular mechanisms is limited. Clearly, A2 unfolding is likely to contribute significantly to the elongation of VWF, as the induced length increment of roughly 45 nm (at 20 pN) is almost as large as the end-to-end length of a static monomer (8, 17–19). Stabilization of all other domains in VWF through disulfide bonds was predicted (20), but lacks experimental evidence.Besides domain unfolding, separation of potential intramonomer and intermonomer interactions may play a crucial role for VWF’s elongation. For example, interactions between monomers may tune VWF’s force-sensing ability by promoting compactness, as suggested by various computational studies (6, 21, 22). Experimentally, self-association of VWF molecules was reported, but has not been assigned to individual domains (23, 24).In this study, we report on force-induced conformational changes of VWF and present a strong intermonomer interaction that is expected to tune VWF’s force-sensing ability in the bloodstream. Evidence for this interaction comes from force–extension traces of dimers, which were probed in atomic force microscopy (AFM)-based single-molecule force measurements. Complementarily, we characterized the static conformation of VWF by AFM imaging and small-angle X-ray scattering (SAXS). From the combination of force and imaging data, we gain a quantitative understanding of the mechanisms underlying the force-sensing ability of VWF.