Page 254 - Haematologica May 2020

Ferrata Storti Foundation
Haematologica 2020 Volume 105(5):1414-1423
Platelet Biology & its Disorders
Dynamin 2 is required for GPVI signaling and platelet hemostatic function in mice
Nathan Eaton,1,2 Caleb Drew,1 Jon Wieser,1 Adam D. Munday,3,4 and Hervé Falet1,2
1Blood Research Institute, Versiti, Milwaukee, WI; 2Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI; 3Bloodworks Northwest Research Institute, Seattle, WA and 4Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA
ABSTRACT
Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemo- static function using both pharmacological and genetic approaches. Dnm2fl/fl Pf4-Cre (Dnm2Plt–/–) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2Plt–/– platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2Plt–/– platelets, which were dysfunctional in response to stim- ulation via GPVI, and to a lesser extent to thrombin. Dnm2Plt–/– platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.
Introduction
Receptor-mediated endocytosis (RME), the process by which cells internalize and sort specific extracellular material, plasma membrane proteins, and lipids, con- tributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration.1 RME requires membrane fission by the large and ubiq- uitous GTPase dynamin 2 (DNM2), which polymerizes at the neck of budding endocytic vesicles to mediate the GTP-dependent membrane fission required for their release into the cytosol prior to their incorporation into the endosomal com- partment.2 Consistent with its indispensable role in cellular homeostasis, DNM2 mutations have been associated with Charcot-Marie-Tooth disease, centronuclear myopathy, and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL),3-5 and Dnm2 deletion results in early embryonic lethality in mice.6 While other clas- sical dynamins (DNM1 and DNM3) are critical for activity-dependent vesicle recy- cling in presynaptic neurons,7,8 their functions in other cells remain less clear.
The most well-characterized physiological roles of RME are to regulate uptake of nutrients such as cholesterol and iron and to down-modulate cytokine receptor sig- naling.1 Lack of DNM2-dependent RME enhances responses to thrombopoietin in platelets and megakaryocytes (MK),9 and to epidermal growth factor and inter-
Correspondence:
HERVÉ FALET
hfalet@versiti.org
Received: February 19, 2019. Accepted: July 9, 2019. Pre-published: July 11, 2019.
doi:10.3324/haematol.2019.218644
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/5/1414
©2020 Ferrata Storti Foundation
Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- mercial purposes is not allowed without permission in writing from the publisher.
1414
haematologica | 2020; 105(5)
ARTICLE