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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, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls 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.

•Proximity proteomics and siRNA subscreens in endothelial cells reveal proteins associated with regulated actomyosin expulsion of VWF.•PAK2 and septin hetero-oligomeric complexes are required for actomyosin ring function and effective VWF release.

Cancelas: Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Hemanext: Consultancy, Research Funding.

Lifelong maintenance of the blood system requires the preservation of a healthy hematopoietic stem cell (HSC) pool. Integrity of the HSC compartment is disrupted by severe homeostatic perturbations following acute infection or irradiation, which result in HSC loss and lead to dramatic hematological dysfunctions. HSC fitness is also affected by organismal patho-physiological conditions, which are associated with chronic low-grade stress. These chronic conditions present, over time, a unique challenge to the maintenance of the long-lived HSCs. However, the mechanisms by which the HSC pool adapts to chronic, low-grade stress conditions remain largely unknown.

No relevant conflicts of interest to declare.

Fitzgibbon: Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

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Introduction