Background: Von Willebrand factor (VWF) is a multimeric protein that plays a crucial role in hemostasis. Endothelial cells synthesize and process VWF and store mature VWF multimers in specialized organelles called Weibel-Palade bodies (WPBs). In steady-state VWF from WPBs is continuously secreted into the vascular lumen through the basal pathway to maintain circulating VWF plasma levels. Upon vascular injury rapid stimulus-induced WPB exocytosis occurs via the regulated pathway to enable formation of VWF strings, which promote platelet adhesion at the damaged site. Rab GTPases (Rabs) belong to a family of proteins involved in vesicle trafficking. The secretory Rabs Rab3B, Rab3D, and Rab27A have been implicated in WPB trafficking, but how these Rabs are activated and recruited to WPBs remains unknown. Rabs require a guanine exchange factor (GEF) for their activation and recruitment to specific membranes. Previously, MAP-kinase activating death domain (MADD) has been identified as a GEF for Rab27A and Rab3 isoforms in melanocytes and neuroendocrine cells, respectively.

Aims: We investigated if and how MADD is involved in the regulation of VWF secretion.

Methods: We performed shRNA-based knockdown in primary endothelial cells and determined intracellular localization of WPBs and Rabs by immunofluorescence microscopy. Rab activity was assessed using GST-tagged Rab-specific effectors as pulldown bait and readout by Western blotting. VWF secretion was measured by ELISA.

Results: MADD knockdown resulted in loss of WPBs at the cell periphery resembling the accumulation of WPBs in the perinuclear region previously observed in Rab27A depleted cells. Recruitment of Rab27A and Rab3D to WPBs was decreased upon MADD knockdown, suggesting that MADD activates these Rabs. Indeed, Rab activity assays showed reduced active Rab27A, Rab3D, and Rab3B upon MADD silencing. MADD depleted cells exhibited decreased histamine-evoked VWF release, but increased basal release.

Conclusions: MADD acts as a master regulator in VWF secretion through activation and recruitment of secretory Rabs to WPBs.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/madd-regulates-von-willebrand-factor-trafficking-in-endothelial-cells-via-activation-of-weibel-palade-body-associated-rab-gtpases/