Vimentin Is a Novel Molecule Required for the Formation of Von Willebrand Factor Strings from the Vascular Endothelium

Background: Von Willebrand factor (VWF) is a multimeric plasma and subendothelial glycoprotein which is produced and secreted by endothelial cells. With intense stimulation (e.g. after vascular injury), endothelial cells secrete unusually large multimers of VWF in a hyper-adhesive string arrangement. Upon secretion, these long multimers or strings remain anchored to the cell surface and are capable of quickly attracting circulating platelets through interaction with the receptor GPIbα. In the absence of the VWF-protease, the VWF strings attached to the endothelium mediate spontaneous platelet adhesion that leads to the formation of microthrombi on the endothelial surface, resulting in vessel occlusion. To date, it is not clear which molecules allow VWF strings to remain docked on the surface of the endothelium once secreted. Vimentin is a cytoskeletal molecule and its extracellular form has been shown to be expressed on the surface of various cell types, including endothelial cells. Recent work from our lab has highlighted the role of extracellular vimentin in mediating platelet adhesion to VWF and that anti-vimentin antibodies inhibit this interaction. We have also found that vimentin binds the A2 domain of VWF, which is exposed on the newly secreted VWF strings. Therefore, we hypothesize that vimentin mediates the anchorage of VWF strings to the vascular endothelium. Understanding these interactions is important as VWF strings have been implicated in the pathophysiology of several disease states, such as sickle cell disease and malaria. Methods: Commercial human umbilical vein endothelial cells (HUVECs) were used. Cells were stimulated with histamine and analyzed under flow conditions to assess the quantity of VWF strings in the presence of soluble recombinant A2 domain, soluble recombinant vimentin, or anti-vimentin antibodies versus control buffer. VWF strings were visualized by tagging with commercial fluorescent-conjugated antibody. We also evaluated VWF string adherence to the endothelium of intact pressurized cerebral arteries from vimentin knockout mice versus wild-type (WT) mice ex vivo. Cerebral middle cerebral artery and parenchymal arterioles from mice were isolated, pressurized, and luminally perfused in a perfusion chamber. Histamine was applied to activate the endothelium and elicit VWF string formation. The negative control was an irrelevant isotype antibody. After histamine treatment, the arteries/arterioles were processed for VWF immunofluorescence to assess VWF string formation. VWF strings were quantified as length normalized to endothelial surface area. Results: As expected, HUVECs expressed surface vimentin as determined using flow cytometry and confocal microscopy. The presence of either soluble A2 or soluble vimentin significantly reduced the amount of VWF string formation from histamine-stimulated HUVECs in comparison to control. In some experiments, anti-vimentin antibodies decreased VWF string formation but findings were not significant. Vascular endothelial cells from vimentin knockout mice failed to form VWF strings after histamine stimulation in comparison to vimentin WT mice. Conclusions: These novel findings show that extracellular vimentin appears to play a role in VWF string formation likely via A2 domain binding. Further studies are necessary to shed light on the intricate pathways regulating VWF-mediated platelet adhesion. Our long term goals are to understand the novel interactions between vimentin and VWF strings in governing hemostasis and thrombosis. Disclosures No relevant conflicts of interest to declare.