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Background: An increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct role of key coagulatory proteins and platelets in cancer initiation, angiogenesis and progression. The aim of this study was to evaluate the prognostic value of von Willebrand factor (vWF) and its regulatory enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), D-dimers and platelets in a cohort of patients with metastatic melanoma receiving ICI., Methods: In a prospective cohort of 83 patients with metastatic melanoma, we measured the systemic levels of vWF-antigen (vWF:Ag), ADAMTS13 activity, D-dimers and platelets, before the beginning of the treatment (baseline), and 6, 12 and 24 weeks after. In parallel, we collected standard biological parameters used in clinical routine to monitor melanoma response (lactate deshydrogenase (LDH), S100). The impact of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) on overall survival (OS) in patients receiving ICI was assessed. Univariable and multivariable Cox proportional models were then used to investigate any potential association of these parameters to clinical progression (progression-free survival (PFS) and OS). Baseline values and variations over therapy course were compared between primary responders and resistant patients., Results: Patients with melanoma present with dysregulated levels of vWF:Ag, ADAMTS13 activity, D-dimers, LDH, S100 and CRP at the beginning of treatment. With a median clinical follow-up of 26 months, vWF:Ag interrogated as a continuous variable was significantly associated with PFS in univariate and multivariate analysis (HR=1.04; p=0.007). Lower values of vWF:Ag at baseline were observed in the primary responders group (median: 29.4 µg/mL vs 32.9 µg/mL; p=0.048) when compared with primary resistant patients. As for OS, we found an association with D-dimers and ADAMTS13 activity in univariate analysis and vWF:Ag in univariate and multivariate analysis including v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and Eastern Cooperative Oncology Group (ECOG) performance status. Follow-up over the course of treatment depicts different evolution profiles for vWF:Ag between the primary response and resistance groups., Conclusions: In this prospective cohort, coagulatory parameters such as ADAMTS13 activity and D-dimers are associated with OS but baseline vWF:Ag levels appeared as the only parameter associated with response and OS to ICI. This highlights a potential role of vWF as a biomarker to monitor ICI response of patients with malignant melanoma., Competing Interests: Competing interests: J-CS, LK, ATB, CM, AA, SVS, MS, WH, CS, BD, ILH, RR, TH, JK, IH, GG, KP and SWS declare no competing interest. CG is a member of the advisory board and has received honoraria and travel expenses from Almirall, Amgen, Beiersdorf, BioNTech, Bristol-Myers Squibb, Immunocore, Janssen, MSD Sharp & Dohme, Novartis, Pierre-Fabre, Roche, Sanofi Genzyme, SUN Pharma, Sysmex/Inostics, outside the submitted work. CG holds shares of Dermagnostix., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis., Results: Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively 'masks' the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics., Conclusions: These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell-derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer-derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell-derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A-mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. IMPLICATIONS: We identified the VEGF-A-mediated release of VWF from endothelial cells to be associated with bladder cancer progression., (©2020 American Association for Cancer Research.)

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands' blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism., (© 2019 by The American Society of Hematology.)

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., (© 2018 by The American Society of Hematology.)

Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.

The protein von Willebrand factor (VWF) is essential in primary hemostasis, as it mediates platelet adhesion to vessel walls. VWF retains its compact (globule-like) shape in equilibrium due to internal molecular associations, but is able to stretch when a high enough shear stress is applied. Even though the shear-flow sensitivity of VWF conformation is well accepted, the behavior of VWF under realistic blood flow conditions remains poorly understood. We perform mesoscopic numerical simulations together with microfluidic experiments in order to characterize VWF behavior in blood flow for a wide range of flow-rate and hematocrit conditions. In particular, our results demonstrate that the compact shape of VWF is important for its migration (or margination) toward vessel walls and that VWF stretches primarily in a near-wall region in blood flow making its adhesion possible. Our results show that VWF is a highly optimized protein in terms of its size and internal associations which are necessary to achieve its vital function. A better understanding of the relevant mechanisms for VWF behavior in microcirculation provides a further step toward the elucidation of the role of mutations in various VWF-related diseases.

Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation., (© 2015 by The American Society of Hematology.)

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., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Co-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 trisphosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimer-platelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

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., (© 2014 American Heart Association, Inc.)

The specific interactions of von Willebrand factor (VWF) with the vessel wall, platelets or other interfaces strongly depend on (a shear-induced) VWF activation. Shear flow has been shown to induce a conformational transition of VWF, but is modulated by its thermodynamic state (state-function relationship). The state in turn is determined by physical (e.g. vessel geometry), physico-chemical (e.g. pH) and molecular-biological (e.g. mutants, binding) factors. Combining established results with recent insights, we reconstruct VWF biology and its state-function relationship from endothelial cell release to final degradation in the human vasculature. After VWF secretion, endothelial-anchored and shear activated VWF multimers can rapidly interact with surrounding colloids, typically with platelets. Simultaneously, this VWF activation enables ADAMTS13 to cleave VWF multimers thereby limiting VWF binding capacity. The subsequent cell-surface dissociation leads to a VWF recoiling to a globular conformation, shielding from further degradation by ADAMTS13. High local concentrations of these soluble VWF multimers, transported to the downstream vasculature, are capable for an immediate reactivation and re-polymerisation initiating colloid-binding or VWF-colloid aggregation at the site of inflamed endothelium, vessel injuries or pathological high-shear areas. Focusing on these functional steps in the lifecycle of VWF, its qualitative and quantitative deficiencies in the different VWD types will facilitate more precise diagnostics and reliable risk stratification for prophylactic therapies. The underlying biophysical principles are of general character, which broadens prospective studies on the physiological and pathophysiological impact of VWF and VWF-associated diseases and beares hope for a more universal understanding of an entire class of phenomena.

Von Willebrand factor (VWF), a key player in hemostasis, is increasingly recognized as a proinflammatory protein. Here, we found a massive accumulation of VWF in skin biopsies of patients suffering from immune complex (IC)-mediated vasculitis (ICV). To clarify the impact of VWF on cutaneous inflammation, we induced experimental ICV either in mice treated with VWF-blocking antibodies or in VWF(-/-) mice. Interference with VWF led to a significant inhibition of the cutaneous inflammatory response. We confirmed the major findings in irritative contact dermatitis, a second model of cutaneous inflammation. In vivo imaging of cutaneous inflammation in the dorsal skinfold chamber revealed unaffected leukocyte rolling on anti-VWF treatment. However, we identified that reduced leukocyte recruitment is accompanied by reduced vascular permeability. Although VWF-mediated neutrophil recruitment to the peritoneum was described to require the VWF receptor on platelets (glycoprotein Ibα (GPIbα)), the VWF/GPIbα axis was dispensable for cutaneous inflammation. As assessed in tail bleeding assays, we could exclude interference of VWF blockade with hemostasis. Of particular importance, anti-VWF treatment was effective both in prophylactic and therapeutic administration. Thus, VWF represents a promising target for the treatment of cutaneous inflammation, e.g., leukocytoclastic vasculitis.

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 aureus-induced 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.

Nanoparticle-induced endothelial cell (EC) dysfunction, due to the induction of inflammation and/or the activation of the coagulation system, is associated with pulmonary and ischemic cardiovascular diseases. Although it is contigent on several mechanisms, involving formation of reactive oxygen species and inflammatory cytokines such as interleukin (IL)-6 and 8, the involvement of the coagulation system is not well understood. The results of toxicity assays using the tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) release showed that silica NP-induced cytotoxicity depends on the size and the dose of applied NP. Moreover, propidium iodide (PI) stainings and caspase 3/7 assays identified increased necrosis in ECs. Exposing human umbilical vein endothelial cells (HUVECs) to SiO(2) NP with diameters of 304 nm and 310 nm led to significant increase of Weibel-Palade body (WPB) exocytosis, associated with the release of von Willebrand factor (VWF) and the formation of ultralarge fibers (ULVWF). High resolution microscopy techniques revealed that internalization and perinuclear localization of perylene-labeled NP with a size of 310 nm affect not only viability, but also cell migration and proliferation. In conclusion, our data indicate that NP-induced activation and dysfunction of ECs is reflected by release of VWF and necrotic cell death., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollate-induced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier.

Activation of the coagulation system in malignancy enables tumor spreading and is thus associated with poor prognosis for the patient. In this study, we analyzed the in vitro mechanisms by which two human metastatic melanoma cell lines, MV3 and WM9, transform the vascular endothelium into a prothrombotic activated state. We show that both melanoma cell lines activate prothrombin due to tissue factor (TF) expression by showing that thrombin generation was blocked with a TF-neutralizing antibody and TF-siRNA. In addition, using the cysteine protease inhibitor E-64, we excluded the formerly described cancer procoagulant (CP) as a major factor contributing to thrombin generation. Furthermore, we describe a direct thrombin-independent response of endothelial cells (ECs) to MV3-derived supernatant as measured by rapid release of VWF. We also show that two clinically approved LMWHs, tinzaparin and enoxaparin, are effective inhibitors of thrombin generation and thrombin activity in plasma. Furthermore, our data indicate a protective effect of heparins on EC activation as shown by reduced VWF release in response to MV3 supernatant. These promising effects of heparins on the melanoma-induced thrombotic conditions justify further clinical investigations in the field of oncology.

Neoplastic diseases are often associated with thromboembolic events, however the precise mechanism underlying this observation is a matter of ongoing investigation. It is known that matrixmetalloproteinase-1 (MMP-1) canonically activates the thrombin receptor (PAR-1) and we recently reported that highly metastatic tumor cells of melanoma and colon cancer are secreting matrixmetalloproteinase-1. This tumor-derived MMP1 was shown to be a major activator of endothelial PAR-1, thus leading to endothelial cell activation. As tumor-induced thrombosis is a characteristic of metastazing tumors, we investigated whether tumor-derived supernatant (TUSN) from melanoma and colon cancer may induce adhesion of circulating platelets, an initial step in thrombus formation. A time-course study revealed that TU-SN induces a rapid secretion of von Willebrand factor (VWF) within minutes. Using a novel microfluidic device we analyzed platelet-endothelial interactions in a closed circuit. Immunofluorescence imaging showed that TU-SN rapidly induces platelet-string formation via secreted VWF. We demonstrated that tumor-derived supernatant is a potent agonist inducing platelet adhesion under flow conditions.

The large glycoprotein von Willebrand factor (VWF) is involved in the initial haemostatic reaction mediating the interaction between platelets and the injured vessel wall. It has been demonstrated that unusually large VWF (ULVWF) multimers after being released from endothelium are capable of developing elongated membrane-anchored strings that are hyperactive to bind platelets. In the present study we investigated whether soluble plasma-derived VWF is competent to develop similar thrombotically active multimers. We demonstrated that soluble VWF multimers isolated from human plasma self-assemble to a network of fibers immobilized on a collagen matrix and are functionally active to bind platelets. Formation of these VWF fibers depends on shear flow, concentration of soluble VWF, and a suitable binding surface. Self-assembly of soluble VWF does not require the presence of cellular membrane ligands. The network of fibers is subjected to rapid degradation by proteolytic activity of plasma ADAMTS-13. Atomic force microscopy images elucidate the nanostructure of VWF fibers and illustrate self-association and -aggregation of several filamentous multimers. Together, these results suggest that circulating VWF can contribute to a formation of hyperactive VWF fibers on exposed subendothelial collagen during vascular injury.

Ingenol mebutate (IM) is highly effective in the treatment of human papillomavirus (HPV)-induced anogenital warts (AGW) leading to fast ablation within hours. However, the exact mode of action is still largely unknown. We performed dermoscopy, in vivo confocal microscopy (CLM), histology, immunohistochemistry, and immunofluorescence to gain insights in mechanisms of IM treatment in AGW. In addition, we used in vitro assays (ELISA, HPV-transfection models) to further investigate in vivo findings. IM treatment leads to a strong recruitment of neutrophils with thrombosis of small skin vessels within 8 h, in a sense of immunothrombosis. In vivo and in vitro analyses showed that IM supports a prothrombotic environment by endothelial cell activation and von Willebrand factor (VWF) secretion, in addition to induction of neutrophil extracellular traps (NETosis). IM superinduces CXCL8/IL-8 expression in HPV-E6/E7 transfected HaCaT cells when compared to non-infected keratinocytes. Rapid ablation of warts after IM treatment can be well explained by the observed immunothrombosis. This new mechanism has so far only been observed in HPV-induced lesions and is completely different from the mechanisms we see in the treatment of transformed keratinocytes in actinic keratosis. Our initial findings indicate an HPV-specific effect, which could be also of interest for the treatment of other HPV-induced lesions. Larger studies are now needed to further investigate the potential of IM in different HPV tumors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Primary hemostasis and blood clotting is known to be influenced by the red blood cell volume fraction (hematocrit) in blood. Depressed or elevated levels of red blood cells can lead to vascular perfusion problems ranging from bleeding to thrombus formation. The early stage of hemostasis and thus blood clotting in all vessel sections from the arterial to the venous system involves the adhesion of platelets to von Willebrand factor. Here we present experimental and theoretical results showing that the adhesion probability of platelets to von Willebrand factor is strongly and nonlinearly dependent on hematocrit and flow rate. Interestingly, the actual binding forces are not markedly different, which suggest that the origin of such behavior is in the distribution of platelets. Using hydrodynamic simulations of a simple model, we explicitly show that the higher the hematocrit and the flow rate, the larger the amount of platelets residing close to the wall. Our simulation results, which are in excellent agreement with the experimental observations, explain why such phenomena occur. We believe that the nonhomogeneous red blood cell distribution as well as the shear dependent hydrodynamic interaction is key for the accumulation of platelets on the vessel wall. The work we present here is an important step forward from our earlier work on single molecules and extends into the collective cellular behavior of whole blood. It sheds new light on the correlation between hematocrit and the initial steps in hemostasis and thrombosis, and outlines advances for the treatment of vascular diseases associated with high levels of red blood cells. These results are not only highly relevant for the field of hemostasis and the physics of blood clotting but are also of powerful impact in applied science most obviously in drug delivery and colloidal science. [ABSTRACT FROM AUTHOR]

[Display omitted] • Clinical data suggest that thrombosis limits efficacy of ICIs (e.g. anti-PD-1). • Coagulation suppresses ICIs via platelets, leukocytes and the complement system. • Molecular mechanisms of CAT envision future therapeutic targets to support ICIs. • ICIs combined with anticoagulant LMWHs may improve clinical outcome. Advances in understanding the molecular mechanisms of tumor progression have achieved impressive progress in the treatment of cancer and so-called immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Indeed, antibody-based drugs blocking immune escape of tumor cells by modulation of T cell responses are increasingly utilized for a wide range of tumor entities. Nonetheless, response rates remain limited, and the development of secondary resistance is a common problem. In addition, by increasing the immune response a variety of severe side effects are provoked. Next to autoimmune responses, activation of the complement system and skin toxicity, an increased incidence for thrombotic complications has been observed associated with an increased mortality rate. Based on this, it can be postulated that the interplay of coagulation with inflammation in the tumor microenvironment is relevant for each step in the tumor life cycle. This review focuses on the coagulation as central player fostering mechanisms associated with tumor progression. Thus, a better understanding of the molecular pathways involved in the complex interaction of circulating tumor cells, the plasmatic coagulation and immune cells may help to improve therapeutic concepts reducing mortality and morbidity associated with cancer. [ABSTRACT FROM AUTHOR]

Advances in understanding the molecular mechanisms of tumor progression have achieved impressive progress in the treatment of cancer and so-called immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Indeed, antibody-based drugs blocking immune escape of tumor cells by modulation of T cell responses are increasingly utilized for a wide range of tumor entities. Nonetheless, response rates remain limited, and the development of secondary resistance is a common problem. In addition, by increasing the immune response a variety of severe side effects are provoked. Next to autoimmune responses, activation of the complement system and skin toxicity, an increased incidence for thrombotic complications has been observed associated with an increased mortality rate. Based on this, it can be postulated that the interplay of coagulation with inflammation in the tumor microenvironment is relevant for each step in the tumor life cycle. This review focuses on the coagulation as central player fostering mechanisms associated with tumor progression. Thus, a better understanding of the molecular pathways involved in the complex interaction of circulating tumor cells, the plasmatic coagulation and immune cells may help to improve therapeutic concepts reducing mortality and morbidity associated with cancer. [ABSTRACT FROM AUTHOR]

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pHin) and intracellular free calcium concentration ([Ca2+]in), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pHex of 6.4 to a physiologic pHex of 7.4. Extracellular realkalinization was accompanied by pHin recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na+/H+ exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca2+]in was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pHin recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pHin recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion. J. Cell. Physiol. 211: 399–409, 2007. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]