Upon activation, platelets release many active substances stored in ␣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 Ca 2؉ -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

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... rong inhibition of the Ca 2؉induced secretion of von Willebrand factor (vWF) stored in ␣-granules, but not that of [ 3 H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 cofractionated with vWF and P-selectin (an ␣-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 [ 3 H]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 ␣and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca 2؉ -induced exocytosis of ␣-granules. -associated protein of 23 kDa; GDI, GDP disso-ciation inhibitor; HSP60, heat shock protein 60; SLO, streptolysin O; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; wt, wild type.