Small GTPase Rab4 regulates Ca2+-induced α-granule secretion in platelets

Abstract

Upon activation, platelets release many active substances stored in α- and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca2+-induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca2+-induced secretion of von Willebrand factor (vWF) stored in α-granules, but not that of [3H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 cofractionated with vWF and P-selectin (an α-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [3H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing α- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca2+-induced exocytosis of α-granules.