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 flowcytometry, were unchanged; yet, Arf6KOplatelets exhibited enhanced spreading on Fg and faster clot retraction. Thiswas not the result of alterations in αIIbβ3 signaling, becausemyosin 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.
|Number of pages||9|
|State||Published - Mar 17 2016|
Bibliographical noteFunding Information:
The authors are immensely grateful to members of the Whiteheart laboratory for their careful reading of this manuscript and their helpful comments. The authors thank the staff of the University Imaging Facility for help with the electron microscopy analysis, Greg Bauman at the University Flow Cytometry Core Facility for help with the FACS analysis, and the staff of Department of Laboratory Animal Resources for their assistance. The authors also thank Dr Richard T. Premont at Duke University for generously providing us GIT1 and GIT2 brain tissue for western blots. The Departmental Imaging Facility, where all the fluorescence imaging was done, is supported by a grant from the National Institutes of Health National Center for Research Resources (P20 RR020171). This work was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL56652 and HL082193 [S.W.W.] and HL68819 [Z.L.]), an American Heart Association (AHA) predoctoral fellowship (11PRE7500051) (Y.H.), a Grant-in-Aid from AHA Great Rivers Affiliate (Z.L.), and a Scientist Development Grant from AHA Great Rivers Affiliate (B.X.).
© 2016 by The American Society of Hematology.
ASJC Scopus subject areas
- Cell Biology