Objective—Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion. Approach and Results—Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA–VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA–VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA–VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA–VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains. Conclusions—Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation

50S taphylococcus aureus is a major pathogen responsible for various infections in humans and is the most frequent cause of infective endocarditis in industrialized countries. 1 Prerequisite for the pathogenesis of infective endocarditis is infection of the endocardium.2 Therefore, one of the first and essential steps is bacterial adhesion to the endothelium. In contrast to other pathogens causing infective endocarditis, S. aureus appears to be able to bind not only to damaged but also to intact endothelium. [3][4][5] Fibrinogen, fibronectin, and platelets, in combination with S aureus clumping factor and fibronectin-binding proteins, are involved in the pathogenesis of infective endocarditis. [6][7][8] In addition, wall teichoic acids of S aureus have been implicated in adhesion to the endothelial cell (EC) layer. 9 However, the molecular mechanisms of the very first adhesion steps of S aureus to the undamaged endothelium are still largely unknown.Background-During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Methods and Results-Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). Conclusions-Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureusinduced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis. Clinical Perspective on p 59Recruitment of host cells to the endothelium is a crucial step during inflammation and coagulation. It has been shown previously that under shear von Willebrand factor is a potent bindi...

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The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανβ3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber–dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non‐ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non‐ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin‐mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co‐culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma.

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Background: IgA-vasculitis (IgAV) encompasses a systemic form involving kidneys, gut, skin or joints, and a skin-limited form. One characteristic feature of systemic IgAV is deposition of galactosedeficient IgA1 (GD-IgA1) in kidneys (as in IgA-nephropathy). The relevance of GD-IgA1 for cutaneous vasculitis is unknown. Objective: We investigated if GD-IgA1 is deposited perivascularly in systemic and also skin-limited IgAV, and if its serum levels differ between both forms. Methods: In a case control study, deposition of GD-IgA1 was analysed immunohistochemically by KM55-antibody in skin biopsies from 12 patients with skin-limited and 4 with systemic IgAV. GD-IgA1levels were compared by ELISA in sera from 15 patients each with skin-limited and systemic IgAV and from 11 healthy subjects. Results: All biopsies from systemic, but also from skin-limited IgAV revealed perivascular GD-IgA1deposition. The average GD-IgA1-level in serum was significantly higher in systemic than in skinlimited IgAV, despite overlap between both groups. Limitations: Although high GD-IgA1-levels may be predictive of systemic IgAV, patient numbers were too low to determine cutoff values for systemic versus skin-limited IgAV. Conclusion: While perivascular GD-IgA1-deposition is a prerequisite for systemic and skin-limited IgAV, high GD-IgA1-levels in some patients with systemic IgAV suggest a dose-dependent effect of GD-IgA1 in IgAV.

In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)–mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.