Cellubrevin/vesicle-associated membrane protein-3–mediated endocytosis and trafficking regulate platelet functions

Abstract

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ, αβ), and purinergic receptors (P2Y, P2Y), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble -ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3 mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional-structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4, Rab11, and von Willebrand factor (VWF) compartments in wild-type platelets in a time-dependent manner. In VAMP-3 platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3 platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

“…Very little is known about the difference between the two Rab8 isoforms, although it is postulated that they function in both distinct and overlapping compartments in various cell types (Chen, Liang, Chia, Ngsee, & Ting, 2001;Sato et al, 2014 (Figures 2c and 4c). We also found that depletion of VAMP3, a downstream regulator fusion event controlled by both Rab8b and Rab11b, could result in an increased number of intact vacuoles harbouring the ΔsdhA strain (Figure 5c) (Banerjee et al, 2017;Finetti et al, 2015;Wilcke et al, 2000). VAMP3 is SNARE protein that drives membrane fusion, thus pointing to a model in which disruption of membrane integrity is a consequence of fusion with a compartment that destabilises the LCV (Hu, Hardee, & Minnear, 2007).…”

“…We demonstrated that depletion of the Rab5 effector EEA1 and the Rab11b effector Rab11FIP1 partially restored integrity of the ΔsdhA‐ containing vacuole and increased intracellular growth of the mutant, generating phenotypes indistinguishable from depletion of the GTPases (Figures c and c). We also found that depletion of VAMP3, a downstream regulator fusion event controlled by both Rab8b and Rab11b, could result in an increased number of intact vacuoles harbouring the ΔsdhA strain (Figure c) (Banerjee et al, ; Finetti et al, ; Wilcke et al, ). VAMP3 is SNARE protein that drives membrane fusion, thus pointing to a model in which disruption of membrane integrity is a consequence of fusion with a compartment that destabilises the LCV (Hu, Hardee, & Minnear, ).…”

“…Of particular note regarding the vacuole integrity readouts, both shRNA (Figure d) and siRNA in primary macrophages (Figures and ) gave identical results for both the Rab8 and Rab11 isoforms, further supporting isoform specificity. Similar to knockdown of Rab5 and Rab8 effectors, siRNA directed against VAMP3, a SNARE protein that promotes vesicle fusion events and is a downstream effector of both Rab11 and Rab8, was sufficient to partially restore ΔsdhA vacuole integrity and intracellular growth (Banerjee et al, ; Christoforidis et al, ; Finetti et al, ; Lindsay & McCaffrey, ; Wilcke et al, ) (Figure c).…”

“…On the other hand, Rab11a has been connected to regulated disintegration of the Shigella vacuole (Mellouk et al, 2014;Weiner et al, 2016). The identification of knockdown candidates that were downstream effectors of Rab11 (Rab11Fip1; Figure 1d) and Rab8 (Vamp3; Figure 5) supports a role for these proteins in destabilising the LCV surrounding the ΔsdhA mutant (Banerjee et al, 2017;Christoforidis et al, 1999;Finetti et al, 2015;Lindsay & McCaffrey, 2004;Wilcke et al, 2000). LCVs, arguing that the effects are highly isoform-specific ( Figure 4b).…”

VAMP-3 mediates platelet endocytosis

“…1 P latelet activation depends upon exocytosis, a process that involves platelet granules releasing their prothrombotic contents into the blood. Banerjee and colleagues now show that platelets rely on endocytosis as well.…”