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Abstract

Brain metastasis is associated with a high risk for venous thromboembolism (VTE), which determines the prognosis of the patient. We postulated that von Willebrand factor (VWF), a procoagulant glycoprotein stored in endothelial cells (ECs) and platelets, contributes to VTE and promotes metastasis in the brain. Previously, it was shown that EC activation and the subsequent formation of luminal VWF fibers mediate the recruitment of platelets promoting tumor-associated vessel occlusion and pulmonary metastasis. However, little is known about the function of VWF within the specialized vascular bed of the blood-brain barrier. The aim of this project is to determine the contribution of VWF in the pathophysiology of brain metastasis. Using in vitro approaches significant lower levels of VWF were detected in primary human microvascular brain ECs compared to macrovascular human umbilical vein ECs. This was reflected by a restricted release of VWF and low numbers of luminal VWF fibers upon tumor cell-induced brain EC activation. In line, brain microvessels of wild type mice showed low amounts of stored VWF and few VWF-platelet aggregates. However, immunofluorescence analyses of brain tissue from patients with brain metastasis revealed a strong formation of luminal VWF-platelet aggregates mediating vessel occlusions. These findings were confirmed in the ret transgenic mouse model, which develops spontaneous melanoma with metastasis in the brain: metastatic ret mouse brains showed a strong formation of intravascular VWF-platelet aggregates. Importantly, this phenomenon was already observed in brains of ret transgenic mice without visible cerebral metastasis, suggesting that luminal VWF fibers are involved in initial steps of the brain metastatic cascade. High resolution microscopy techniques revealed the contribution of activated platelets in the formation of luminal VWF networks in the brain. Consistent with this, aggregometry assays and in vitro microfluidic model of brain microcirculation showed that the lack of VWF in platelets (VWF-/-) resulted in a reduced platelet aggregation. Additionally, impedance measurements and transmigration assays demonstrated that blocking platelet activation with the low molecular weight heparin Tinzaparin reduces the impact of platelet-mediated endothelium disruption and the transmigration of tumor cells. Furthermore, systemic anticoagulation using Tinzaparin reduced platelet accumulation and VWF networks in ret mouse brains. The impact of anticoagulation on brain metastasis formation was examined after intracardiac injection of melanoma cells in mice treated with Tinzaparin. Quantification of brain metastasis showed that Tinzaparin attenuates the metastatic load compared to non-treated control mice. In summary, our findings provide new insights into the mechanisms by which platelet-derived VWF promotes cerebral thrombosis and identifies platelet activation as a promising therapeutic target for the prevention of brain metastases.