3 years ago

Cellubrevin/Vesicle-Associated Membrane Protein-3 (VAMP-3)-mediated endocytosis and trafficking regulate platelet functions.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ3, αvβ3) and purinergic receptors (P2Y1, P2Y12), and thus for 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 N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called Cellubrevin/Vesicle-Associated Membrane Protein-3 (VAMP-3) in platelet function. While 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 3D-Structured Illumination Microscopy. Dextran uptake was rapid compared to 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 two 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 to wild-type. Additionally, the rate of JAK2 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.

Publisher URL: http://doi.org/10.1182/blood-2017-02-768176

DOI: 10.1182/blood-2017-02-768176

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