DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.

von Willebrand factor is dimerized by protein disulfide isomerase.

Multimeric von Willebrand factor (VWF) is essential for primary hemostasis. The biosynthesis of VWF high-molecular-weight multimers requires spatial separation of each step because of varying pH value requirements. VWF is dimerized in the endoplasmic reticulum by formation of disulfide bonds between the C-terminal cysteine knot (CK) domains of 2 monomers. Here, we investigated the basic question of which protein catalyzes the dimerization. We examined the putative interaction of VWF and the protein disulfide isomerase PDIA1, which has previously been used to visualize endoplasmic reticulum localization of VWF. Excitingly, we were able to visualize the PDI-VWF dimer complex by high-resolution stochastic optical reconstruction microscopy and atomic force microscopy. We proved and quantified direct binding of PDIA1 to VWF, using microscale thermophoresis and fluorescence correlation spectroscopy (dissociation constants KD = 236 ± 66 nM and KD = 282 ± 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar KD (258 ± 104 nM) measured for PDI interaction with the isolated CK domain and the atomic force microscopy images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. On the basis of protein-protein docking and molecular dynamics simulations, combined with fluorescence microscopy studies of VWF CK-domain mutants, we suggest the following mechanism of VWF dimerization: PDI initiates VWF dimerization by forming the first 2 disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond, Cys2811-2811', is formed, presumably to protect the first 2 bonds from reduction, thereby rendering dimerization irreversible. This study deepens our understanding of the mechanism of VWF dimerization and the pathophysiological consequences of its inhibition.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Gain-of-Function Variant p.Pro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics.

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target.

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation.

ADAMTS13 regulates the hemostatic activity of von Willebrand factor (VWF). Determined by static assays, proteolytic activity <10IU/dL in patient plasma, in absence of ADAMTS13 autoantibodies, indicates Upshaw-Schulman syndrome (USS); the congenital form of Thrombotic Thrombocytopenic Purpura (TTP). We have recently functionally characterized sixteen USS-associated ADAMTS13 missense variants under static conditions. Here, we used two assays under shear flow conditions to analyze the activity of those seven mutants with sufficiently high residual secretion plus two newly identified variants. One assay determines cleavage of VWF strings bound to the surface of endothelial cells. The other, light transmission aggregometry-based assay, mimics degradation of VWF-platelet complexes, which are likely to be present in the circulation during TTP bouts. We found that 100 ng/ml of all variants were able to cleave about 80-90% of VWF strings even though 5 out of 9 exhibited activity ≤1% in the state-of-the-art static assay at the same concentration. These data indicate underestimation of ADAMTS13 activity by the used static assay. In simulated circulation, two variants, with missense mutations in the vicinity of the catalytic domain, exhibited only minor residual activity while all other variants were able to effectively break down VWF-platelet complexes. In both assays, significant proteolytic activity could be observed down to 100 ng/ml ADAMTS13. It is thus intriguing to postulate that most variants would have ample activity if secretion of 10% of normal plasma levels could be achieved.

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.

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations.

The platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and ß3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF-GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor.

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, "Mutual A domain interactions in the force sensing protein von Willebrand Factor" (Posch et al., 2016) [1].