Small GTPase Rab4 regulates Ca2+-induced alpha-granule secretion in platelets.
Upon activation, platelets release many active substances stored in alpha- 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 strong inhibition of the Ca(2+)-induced secretion of von Willebrand factor (vWF) stored in alpha-granules, but not that of [(3)H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 co-fractionated with vWF and P-selectin (an alpha-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 alpha- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca(2+)-induced exocytosis of alpha-granules.
Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
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R
Shirakawa
A
A
Yoshioka
H
H
Horiuchi
H
H
Nishioka
A
A
Tabuchi
T
T
Kita
D001426:Bacterial Proteins; D001792:Blood Platelets / Q000648:ultrastructure*; D002118:Calcium / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000502:physiology*; D005089:Exocytosis; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000502:physiology; D006801:Humans; D013301:Streptolysins / Q000494:pharmacology; D020695:rab4 GTP-Binding Proteins / Q000032:analysis / Q000502:physiology*; D014841:von Willebrand Factor / Q000032:analysis
D001426:Bacterial Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D013301:Streptolysins; C025964:streptolysin O; D014841:von Willebrand Factor; D020695:rab4 GTP-Binding Proteins; D002118:Calcium