Platelet-derived von Willebrand Factor Fosters Intracranial Hypercoagulation in Metastatic Brains of Mice and Humans

Scientific Research Question: The occurrence of brain metastases is associated with a high risk for venous thromboembolism (VTE), which in turn defines outcome and prognosis of cancer patients. We postulated that von Willebrand factor (VWF), a multimeric protein stored in endothelial cells (ECs) and platelets contributes to VTE and brain metastasis formation. Own findings demonstrate that VWF is highly adhesive for circulating platelets and is thus responsible for tumor-associated vessel occlusion and tumor progression. However, only few studies have tried to dissect the function of VWF within the specialized vascular bed of the blood brain barrier. In addition, the contribution of platelet-derived VWF in disorders of the brain is still elusive. The aim of the present study was to analyze the relative contribution of EC-derived VWF versus platelet-derived VWF in the pathophysiology of cerebral malignancy.

Methodology: Fluorescence microscopy and ELISA technique was used to monitor tumor cell-induced EC activation in human primary microvascular ECs of the brain compared with macrovascular ECs (HUVECs). Intraluminal VWF string formation and platelet accumulation was verified in a mouse model for spontaneous melanoma development with metastasis to the brain. For evaluation of the clinical significance, plasma and tissue of patients was analyzed. To evaluate the relative contribution of endothelial- and platelet-derived VWF, microfluidic devices, impedance measurements and transwell assays were applied.

Findings: Our in vitro examinations demonstrated significant lower levels of VWF in brain microvascular ECs compared with HUVECs reflected by low numbers of luminal VWF networks upon tumor cell-induced endothelial stimulation. In line, cerebral microvessels of wild type mice exhibited low amounts of VWF stored in the vessel wall and few VWF-platelet aggregates. However, using a transgenic mouse model for brain metastasis and metastatic brain tissue of patients, we observed that intraluminal VWF networks and platelet aggregates were significantly elevated in brain metastases. Importantly, intracranial VWF-platelet thrombi were already increased in brains without visible metastatic foci, indicating that VWF fibers foster initial steps of the brain metastatic cascade. Implementation of high resolution microscopy identified activated thrombocytes followed by secretion of VWF fibers in the cerebral microvasculature. Importantly, platelets lacking VWF (VWF^−/−) showed reduced aggregation in a model to mimic the microcirculation within the brain. Finally, systemic anticoagulation using clinically approved low-molecular-weight heparins abrogated platelet accumulation, VWF network formation and reduced tumor cell-induced permeability and tumor cell transmigration.

Conclusion: Our findings provide a novel mechanism by which platelet-derived VWF promotes cerebral thrombosis and suggests platelet activation as therapeutic target for prevention of brain metastases.