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

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ₃, α_vβ₃), and purinergic receptors (P2Y₁, P2Y₁₂), 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, solubleN-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 hadlessplatelet-associatedFg, indicatingadefect inFguptake/storage.Othergranulemarkers 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 colocalizationwithRab4, Rab11, orVWF.Loss ofVAMP-3 also affectedsome acute platelet functions, causing enhanced spreadingon Fg and fibronectin and faster clot retraction comparedwithwild-type. In addition, the rateof Janus kinase 2phosphorylation, initiatedthrough 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.