Background: Von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. 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: Rab activity assays revealed a reduction in Rab27A, Rab3D, and Rab3B activation upon MADD silencing (Figure 1). Rab activation was dependent on the DENN domain of MADD. Furthermore, immunofluorescent analysis showed that Rab27A and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting (Figure 2). Artificial mistargeting of MADD using a TOMM70-tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. MADD silencing did not have an impact on functional WPB biogenesis, MADD-depleted cells however exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through prior activation of Rab27A by MADD. 
MADD is a guanine nucleotide exchange factor for secretory Rabs Rab recruitment to WPBs is decreased upon MADD silencing

Conclusions: MADD acts as a master regulator in VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/gdp-gtp-exchange-factor-madd-drives-activation-and-recruitment-of-secretory-rab-gtpases-to-weibel-palade-bodies/