Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

Abstract

• Arf6 selectively regulates endocytic trafficking of platelet a IIb b 3 .• Endocytosis contributes to acute platelet function.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 a IIb b 3 -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 a IIb b 3 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 a IIb b 3 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 FeCl 3 -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. (Blood. 2016;127(11):1459-1467 IntroductionIt is increasingly clear that platelets are capable of different types of intracellular membrane trafficking. Platelet exocytosis and autophagy are critical for hemostasis. [1][2][3][4] Platelet endocytosis is important for granule cargo loading. Platelets selectively take up certain cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor) from plasma and package it into a-granules. [5][6][7][8] However, the molecular mechanisms of platelet endocytosis are largely unknown. It is also unclear whether platelet endocytosis contributes to acute platelet functions beyond granule cargo loading. The goal of this study is to begin to address these fundamental questions.The Ras-like, small guanosine triphosphate (GTP)-binding proteins called adenosine 5'-diphosphate-ribosylation factors (Arf's), are key intracellular trafficking regulators. They cycle between inactive guanosine diphosphate (GDP)-bound and active GTP-bound states and are important for multiple functions (eg, actin cytoskeleton remodeling, lipid metabolism, and vesicle traffickin...

Platelet secretion in inflammatory and infectious diseases

“…These findings are consistent with Kanamarlapudi and colleagues who showed that Arf6 regulates P2Y trafficking in platelets as well as dynamin-dependent fission of coated vesicles during endocytosis[50]. Additionally, two Rab proteins (Rab4 and Rab11) are also involved in integrin-mediated fibrinogen uptake and trafficking in platelets[49, 51]. …”

“…Endocytosis is an important process in platelets and megakaryocytes for loading certain granule cargo, such as fibrinogen (Fg) and vascular endothelial growth factor (VEGF)[46–48]. While the mechanisms of platelet endocytosis remain incompletely understood, recent studies demonstrate that adenosine 5′-diphosphate–ribosylation factor 6 (Arf6), a small guanosine triphosphate-binding protein, regulates integrin α2bβ3 trafficking and fibrinogen uptake[49]. Genetic ablation of Arf6 in platelets enhanced clot retraction and spreading, indicating that endocytosis contributes to rapid platelet functions.…”

Integrin αIIbβ3 outside-in signaling

“…141 Integrins can also crosstalk with other cell-surface proteins/receptors to mediate outside-in signaling, and a IIb b 3 makes functionally important interactions with tetraspanins such as CD151 and tumor-suppressing subchromosomal transferable fragment cDNA 6 (TSSC6). 23,142,143 The small GTPase Arf6 can regulate a IIb b 3 trafficking, 144 and many further proteins are likely to be involved in this important, yet understudied, aspect of a IIb b 3 biology.…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…ADP‐ribosylation factor 6 (Arf6) belongs to the Arf family of small G‐proteins and is involved in receptor trafficking in platelets 97. In contrast to Rap1, Rac1, and RhoA, Arf6 is inhibited during platelet activation, whereas cyclic nucleotide pathways elevate Arf6‐GTP levels 98.…”