title: Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking.
abstract: Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function.
authors.affiliation: Department of Molecular and Cellular Biochemistry and.
authors.forename: Yunjie
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Molecular and Cellular Biochemistry and.
authors.forename: Smita
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Division of Cardiovascular Medicine, Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY;
authors.forename: Binggang
authors.initials: B
authors.lastname: Xiang
authors.affiliation: Department of Physiological Chemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan; and.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kanaho
authors.affiliation: Division of Cardiovascular Medicine, Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY;
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Vermont, Burlington, VT.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Bouchard
authors.affiliation: Department of Molecular and Cellular Biochemistry and.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Moncman
authors.affiliation: Department of Molecular and Cellular Biochemistry and.
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D000818:Animals; D019921:Biotinylation; D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D048429:Cell Size; D003018:Clot Retraction; D003594:Cytoplasmic Granules; D004705:Endocytosis / Q000502:physiology*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D000089982:ADP-Ribosylation Factor 6; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse