Force-sensitive autoinhibition of the von Willebrand factor is mediated by interdomain interactions

Abstract

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis.

Figure 1

Blockage of the GPIbα binding site in the VWF revealed by MD simulations of the VWF A1 and A2 domains. (A) Scheme illustrating the human VWF-A1A2 fragment (residues 1269 to 1670). The A1 and A2 domains are connected by a 30 residue linker (yellow). GPIbα anchors platelets to VWF by binding to the A1 domain. VWF size is controlled by cleavage of the unfolded A2 domain by ADAMTS13. O-linked (cyan) and N-linked sugars (N-sugars, orange) are found within the fragment. (B) One of the multiple starting conformation used in the MD simulations (protein as cartoon and surface and sugars as sticks). The domain-domain center of mass (A1-A2) separation is indicated with the black arrow. (C) A1-A2 separation along the concatenated MD simulation time. Gray lines separate individual MD runs. The right plot shows the normalized histogram of the A1-A2 separation. Conformations at the bottom show examples with the two domains in contact (cartoon) contrasted to the region occupied by GPIbα when it binds to A1 (red surface), taken at the instants marked with the red symbols. (D) GPIbα binding site accessible surface (GPIbα-BS-AS) as a function of the A1-A2 separation (main panel) and its normalized histogram (right plot), recovered from MD simulations. Reduced GPIbα-BS-AS values indicate blockage of the GPIbα binding site. The GPIbα-BS-AS derived from the VWF A1-GPIbα complex x-ray structure (16) is depicted by the cyan line. The red symbols correspond to the conformations shown in (C). To see this figure in color, go online.

Figure 2

Orientational preferences of the VWF-A1A2 complex in the blocked state. (A) Principal component analysis (PCA) of the structures of the not-covalently linked VWF-A1A2 complex, with the GPIbα binding site blocked, predicted by molecular docking, yielded two main collective vectors (eig1 and eig2). MD trajectories (the last 50 ns) starting from these structures were projected onto the two-dimensional (2D) space created by these two vectors (projections in arbitrary units). Each dot, representing a simulation snapshot, reflects an adopted interdomain orientation. Each run is colored according to its interdomain potential interaction energy, V, and average solvent accessible hydrophobic surface reduction, SAHSR (see B). Representative orientations of runs with both high V and SAHSR (enclosed by the red circle in B) are displayed (A1 domain, white; A2 domain, color; β3 strands, cartoon; A2 β6 strand, ribbon, and A2 C terminus, sphere). The red arrows illustrate the change in orientation of A2 on horizontal changes in the 2D-PCA space. (B) SAHSR as a function of V (time-average $\pm$ standard deviation from the last 50 ns of each run). Colors indicate the projection along a linear fit (black line), with both V and SAHSR ranging from small (light green) to large (blue) values. To see this figure in color, go online.

Figure 3

Force response of the VWF-A1A2 complex from force-probe MD simulations. (A) The N-terminus (Nt) of the A1 domain and the C terminus (Ct) of the A2 domain were pulled away from each other by harmonic springs. The domains were initially in contact but not connected by a linker (domains in cartoon and N-linked sugars in stick representation). (B) Snapshot illustrating a typical dissociation event of the VWF-A1A2 complex induced by the applied force (same representation as in A). Slight unfolding of the C-terminal part of the A2 domain was observed. The disulfide bond Cys1272-Cys1458 (C-C) prevented the A1 domain from unfolding. (C) Cumulative dissociation events (from 17 runs) as a function of the distance $D_{e-e}$ between the pulled N- and C termini at the moment of dissociation. Here, $\Delta D=D_{e-e}-D_{e-e}\left(0\right)$, subtracting the initial distance $D_{e-e}\left(0\right)$, is shown. The Y1605-M1606 (YM) ADAMTS13 cleavage site was exposed after separation of the A2 C-terminal β5 strand from the core of the protein (event indicated by the dotted line). The black circle corresponds to the dissociation event illustrated in (B). To see this figure in color, go online.

Figure 4

Force response of the VWF-A1A2 complex from AFM. (A) Typical approach-retraction force-distance profiles associated to no-binding and binding events. The elongation L, of the A1 and A2 domains, together with the mPN linkers and the 3-aminopropyltriethoxy silane coating molecules, summed to the cantilever deflection (CD) was determined by the difference between approach and retraction curves. (B) (1) Number of binding events between VWF A1 and A2 domains. A1 was connected to the tip of the AFM cantilever (triangle) using malemide-PEG-NHS (mPN) linkers. It was approached to and retracted from the surface carrying mPN-linked A2 domains. Force-distance cycles presented in (A) correspond to this situation. (2–4) Number of binding events measured in control AFM experiments, in which the A1 domain was blocked by soluble A2 domains (2), or either the A2 domains on the surface (3) or the A1 domain connected to the cantilever (4) were replaced by VWF A3 domain. (C) Cumulative distribution of L (black line) and its correction by subtracting the size of A1 and the mPN linkers (gray area). Dotted line indicates the expectation value (EV) of L. To see this figure in color, go online.

Figure 5

Changes in shear-induced fiber and aggregate formation on deletion of the VWF A2 domain. Live-cell fluorescence images of platelet-decorated VWF fibers and platelet-VWF aggregates observed in microfluidic experiments at the indicated shear rates (different columns). Microfluidic channels were perfused with plasmatic wild-type VWF (wt-VWF, top row), VWF with the A2 domain deleted ($\Delta$A2-VWF, middle row), or VWF with the A1 domain deleted ($\Delta$A1-VWF, bottom row). A static image is presented as background, displaying platelets, fibers, and aggregates in black. Moving fibers and aggregates are highlighted in color. Their positions were tracked during 10 s after taking the static image. Flow direction is indicated with the arrow and the line corresponds to 100 μm. To see this figure in color, go online.