Proximity proteomics identifies septin and PAK2 as decisive regulators of actomyosin expulsion of von Willebrand factor.

In response to tissue injury, within seconds the ultra large glycoprotein, von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring, that forms, contracts and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies though characterising proteins associated with such a dynamic process has been challenging. We have combined APEX-2 proximity labelling with an innovative dual loss of function screen to identify proteins associated with actomyosin ring function. We show p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events control actomyosin ring function aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.