Introduction Von Willebrand factor (VWF) is a large multimeric glycoprotein that contributes to platelet recruitment at sites of vascular injury. VWF is mainly produced in endothelial cells from where it is secreted directly into the circulation or stored in the rod-shaped organelles called Weibel-Palade bodies. VWF present in the circulation does not bind to platelets. Stimulated endothelial cells secrete VWF that has the capacity to spontaneously interact with platelets. Conversion of the platelet-binding conformation of secreted VWF into the non-binding conformation of plasma VWF involves proteolytic processing by the metalloprotease ADAMTS13. At sites of vascular injury, binding of VWF to the exposed subendothelial collagen induces a conformational change in VWF allowing a strong interaction with the platelet receptor glycoprotein (Gp)IbĪ±. Undesired secretion of active VWF may also occur in several pathological conditions. One example is von Willebrand disease type 2B (VWD2B), where a gain of function mutation in the VWF/A1 domain induces a permanent platelet-binding state in the VWF molecule. As a consequence, VWF can spontaneously interact with platelets in the circulation, leading to thrombocytopenia, a hallmark of VWD2B.

Objective The aim of this study was to investigate whether VWF present in the Weibel-Palade bodies of endothelial cells is stored in a platelet-binding conformation.

Methods Immunofluorescence experiments were performed on wildtype and VWD2B endothelial cells. Monoclonal antibody AU/VWF-a11 is directed against the VWF/A1 domain and recognizes VWF only when it is in its GpIb-binding conformation. Monoclonal antibody AU/VWF-C37H is directed against the VWF/A3 domain and recognizes both the platelet-binding and the non-platelet- binding conformation. Experiments were performed on cultured endothelial cells to study the conformation of VWF in the Weibel-Palade bodies. To study the conformation of secreted VWF, stimulated endothelial cells were perfused with washed platelets.

Results AU/VWF-C37H fluorescence was observed in the Weibel-Palade bodies of both wildtype and VWD2B endothelial cells, whereas AU/VWF-a11 fluorescence was only detected in the Weibel-Palade bodies of the VWD2B cells. Perfusion of washed platelets over wildtype and VWD2B endothelial cells resulted in adhesion of platelets to thin strings of secreted VWF. These strings stained positive for both AU/VWF-C37H and AU/VWF-a11. Of note, significantly more platelets adhered to VWF secreted from VWD2B than from wildtype endothelial cells. This hyperactive VWD2B-like platelet adhesion pattern could be mimicked by wildtype endothelial cells upon perfusion with platelets that were mixed with ristocetin.

Conclusions VWF stored within the Weibel-Palade bodies of endothelial cells does not possess platelet-binding capacities. Upon secretion, VWF undergoes a conformational change that allows the adhesion of platelets. The presence of ADAMTS13 is necessary to prevent the release of platelet-binding VWF in the circulation. In the absence of ADAMTS13, secreted VWF does not expose all its GpIb-binding sites as more platelets adhere to ristocetin activated VWF-strings or strings released from VWD2B endothelial cells.

Disclosures: No relevant conflicts of interest to declare.

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