Single-molecule mechanics and regulatory conformational transitions of the force-sensing protein von Willebrand factor

The formation of hemostatic plugs at sites of vascular injury represents a first essential step in the blood coagulation cascade. This process crucially relies on the large, linear multimeric glycoprotein von Willebrand factor (VWF) and its ability to stably bind and recruit platelets to the damaged vessel wall even under conditions of high shear stress. Remarkably, VWF’s hemostatic activity is regulated by force. Forces on VWF multimers in the bloodstream result from the interplay of their immense lengths (up to ≈ 15 µm) with the hydrodynamic flow they encounter. While being inactive under normal blood flow conditions, VWF is activated for its hemostatic function by increased hydrodynamic forces that result from changes in the blood flow profile in the wake of vascular injury, especially due to an elevated elongational flow component. This force-regulation of VWF’s hemostatic activity is not only highly intriguing from a biophysical perspective, but also of eminent physiological importance. On the one hand, it prevents undesired activity of VWF in intact vessels that could lead to thromboembolic complications. On the other hand, it provides a mechanism to facilitate effcient VWF-mediated platelet aggregation exactly where needed. Prerequisite for activation of a VWF multimer is the force-induced, abrupt transition from a rather compact, overall globular conformation to an elongated, string-like conformation. Importantly, VWF’s elongation behavior is governed by several specific intramolecular interactions and force-induced conformational transitions within VWF’s dimeric subunits. By regulating the effective multimer length, these intramolecular interactions also govern VWF’s initial force sensitivity, as hydrodynamic forces strongly scale with dimension. However, despite their central role in the mechano-regulation of VWF’s hemostatic function, these intramolecular interactions and further regulatory force-induced conformational transitions are for the most part not well understood and characterized. In the framework of this thesis, in order to dissect regulatory conformational transitions governing VWF’s hemostatic activity, the mechanical response and the conformational ensemble of VWF dimers –the smallest repeating subunits of multimers– were investigated at the single-molecule level. Using a combination of atomic force microscopy (AFM) imaging and AFM-based single-molecule force measurements, it was shown that even minor pH changes from the physiologic pH of 7.4, especially acidification, result in a markedly decreased mechanical resistance of VWF’s dimeric subunits. This effect could be traced back to destabilization of a specific, strong intermonomer interaction mediated by VWF’s D4 domains. This pH dependence might represent a mechanism to promote activation of VWF in response to local pH changes, which may occur at sites of vascular injury. In addition, further pH-dependent, but mechanically very weak interactions in the C-terminal stem region of VWF dimers could be inferred from the imaging results. To enable direct investigation of interactions in VWF that dissociate at very low, but physiologically highly relevant forces down to < 1 pN, a novel approach for single-molecule protein force spectroscopy based on magnetic tweezers (MT) was developed. This approach, which enables highly parallel and stable measurements at constant forces, was validated using the well-characterized protein domain ddFLN4 as a model system. In this context, also the lifetime of single biotin–streptavidin bonds was investigated and, by measurements with streptavidin variants of different valencies, it was shown that the bond lifetime strongly depends on the pulling geometry. Applying the MT assay to dimeric VWF constructs, several force-induced conformational transitions in VWF could be characterized. For instance, the impact of calcium binding on the kinetics of unfolding and refolding of the VWF A2 domain, a process relevant both for VWF’s activation and down-regulation, was elucidated. Furthermore, mechanically very weak interactions in the C-terminal stem region of VWF dimers, which had previously only been inferred indirectly, were observed directly at a force of ≈ 1 pN. These interactions can be expected to have important physiological implications, as their dissociation likely represents the first specific step of force-induced elongation of VWF. Moreover, a previously unknown transition within VWF’s N-terminal D’D3 assembly was discovered that likely plays a regulatory role in VWF’s biosynthesis. Finally, single-molecule AFM imaging was introduced as a tool to determine the multimer size distribution of VWF, which, due to the positive relation between multimer length and hydrodynamic force, is highly important for VWF’s overall activity. This approach confirmed the previously described exponential size distribution of VWF and, in particular, yielded insights into clinically relevant multimerization defects that could not be gained by established methods of multimer analysis. Taken together, the findings presented in this thesis help to gain a deeper understanding of the complex interplay of interactions and conformational transitions underlying the force-regulation of VWF’s hemostatic function

Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p. Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p. Cys2771Arg and p. Cys2773Arg, we found the majority of monomers (87 +/- 5% and 73 +/- 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 +/- 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p. Cys2771Arg, p. Cys2773Arg, and p. Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini;a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis

Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF’s mechano-activation. Probing the force-dependent lifetime of biotin–streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime

Vascular surveillance by haptotactic blood platelets in inflammation and infection

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets

pH-Dependent Interactions in Dimers Govern the Mechanics and Structure of von Willebrand Factor

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is activated for hemostasis by increased hydrodynamic forces at sites of vascular injury. Here, we present data from atomic force microscopy-based single-molecule force measurements, atomic force microscopy imaging, and small-angle x-ray scattering to show that the structure and mechanics of VWF are governed by multiple pH-dependent interactions with opposite trends within dimeric subunits. In particular, the recently discovered strong intermonomer interaction, which induces a firmly closed conformation of dimers and crucially involves the D4 domain, was observed with highest frequency at pH 7.4, but was essentially absent at pH values below 6.8. However, below pH 6.8, the ratio of compact dimers increased with decreasing pH, in line with a previous transmission electron microscopy study. These findings indicated that the compactness of dimers at pH values below 6.8 is promoted by other interactions that possess low mechanical resistance compared with the strong intermonomer interaction. By investigating deletion constructs, we found that compactness under acidic conditions is primarily mediated by the D4 domain, i.e., remarkably by the same domain that also mediates the strong intermonomer interaction. As our data suggest that VWF has the highest mechanical resistance at physiological pH, local deviations from physiological pH (e.g., at sites of vascular injury) may represent a means to enhance VWF’s hemostatic activity where needed

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 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