von Willebrand factor (VWF), a protein present in our circulatory system, is necessary to stop bleeding under high shear-stress conditions as found in small blood vessels. The results presented here help unravel how an increase in hydrodynamic shear stress activates VWF's adhesion potential, leading to the counterintuitive phenomena of enhanced adsorption rate under strong shear conditions. Using a microfluidic device, we were able to mimic a wide range of bloodflow conditions and directly visualize the conformational dynamics of this protein under shear flow. In particular, we find that VWF displays a reversible globule-stretch transition at a critical shear rate ␥ crit in the absence of any adsorbing surface. Computer simulations reproduce this sharp transition and identify the large size of VWF's repeating units as one of the keys for this unique hydrodynamic activation. In the presence of an adsorbing collagen substrate, we find a large increase in the protein adsorption at the same critical shear rate, suggesting that the globule unfolding in bulk triggers the surface adsorption in the case of a collagen substrate, which provides a sufficient density of binding sites. Monitoring the adsorption process of multiple VWF fibers, we were able to follow the formation of an immobilized network that constitutes a ''sticky'' grid necessary for blood platelet adhesion under high shear flow. Because areas of high shear stress coincide with a higher chance for vessel wall damage by mechanical forces, we identified the shear-induced increase in the binding probability of VWF as an effective self-regulating repair mechanism of our microvascular system. blood flow ͉ mechanical activated proteins ͉ polymer physics O ur circulatory system is exposed to an amazingly wide span of shear rates, ranging from 1 to 10 5 s Ϫ1 (1). This range calls for adhesion mechanisms during blood clotting that are different for different regimes of shear rates. At small shear rates, which are found in rather wide vessels, objects such as vesicles or cells bind to vessel walls once the contact area and the adhesion strength is large enough (2). At high shear rates, which are found in small arteries, hydrodynamic lift forces inhibit formation of a sufficiently large contact area, which makes adsorption of soft objects from the blood onto vessel walls increasingly difficult. In fact, theoretical analyses predict that adhering compact and soft objects (such as vesicles or platelets) will roll and detach from a surface at a particular shear stress and, more importantly, will remain unbound if the shear rate is increased further (3, 4). Experiments on leukocyte adhesion confirmed these theoretical predictions quite nicely (2). However, the experimental finding for blood platelet adhesion in small arteries contradicts this scenario: von Willibrand factor (VWF)-mediated platelet adhesion, which is necessary to stop bleeding in small vessels, is strongly enhanced under high shear-flow conditions (5). Therefore, this example of shear-induced adsorption m...

Key Points• Tumor-derived VEGF-A mediates endothelial cell activation, VWF release, and platelet aggregation provoking coagulation in tumor patients.• Local ADAMTS13 inhibition promotes VWF fiber formation in tumor microvessels.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 disintegrinlike 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. (Blood. 2015;125(20):3153-3163) IntroductionTo form new metastatic lesions, circulating melanoma cells have to interact with endothelial cells (ECs) and migrate through the vessel wall.1,2 In this context, our own in vitro studies show that melanoma cells activate ECs by an indirect, tissue factor (TF)-mediated thrombin generation.3 Next to this indirect melanoma-induced EC activation, recent findings identified melanoma-derived vascular endothelial growth factor-A (VEGF-A) as main mediator of direct EC activation. 4,5 Both the thrombin-and the VEGF-A-dependent pathways induce EC activation followed by Weibel-Palade body (WPB) exocytosis and the release of inflammatory cytokines and the highly procoagulatory glycoprotein von Willebrand factor (VWF), linking inflammation and coagulation. 6 On the one hand, luminally released VWF fibers are involved in hemostasis and vessel repair as mediators of platelet adhesion to the endothelium. 7,8 On the other hand, we showed that tumor cell-induced ultra-large VWF (ULVWF) fibers have the highest potential for platelet binding and aggregation.9,10 This effect may contribute not only to pathophysiologic vessel occlusion, 11 but also to the establishment of metastasis as platelets facilitate tumor cell extravasation. 12-14Indeed, it is well-known that cancer pati...

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