Rab27b regulates number and secretion of platelet dense granules - PubMed (original) (raw)

Rab27b regulates number and secretion of platelet dense granules

Tanya Tolmachova et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation of mice carrying the conditional and KO Rab27b alleles. (A) Targeting vector pTT29 carrying three loxP sites, a neomycin-resistance gene (_Neo_r), and two homology arms were used to generate the Rab27b3lox allele in GSI-1 ES cells by homologous recombination. Diagnostic HindIII and XbaI restriction sites and corresponding 5′ and 3′ probes were used to identify correctly targeted ES clones. _Cre_-mediated recombination among the three loxP sites within the Rab27b3lox allele results in three possible alleles: Rab27bflox, Rab27bnull, and Rab27bnull+Neo, which were distinguished by Southern blot analysis by using EcoRI digestion and probe A. (B) Results of Southern blot analysis by using HindIII digestion and 5′ probe and XbaI digestion and 3′ probe are shown for five correctly targeted clones (clones 33, 99, 131, 181, and 200) and a wild-type (wt) ES clone. (C) Results of Southern blot analysis by using EcoRI digestion and probe A are shown for Rab27bflox/WT, Rab27bnull/WT, and Rab27bnull/null (Rab27b KO) mice. (D) Western blotting by using platelet lysates from Rab27b KO and wild-type mice and anti-Rab27a antibody, 4B12, and anti-calnexin antibody as a control. GST-Rab27b protein was used as positive control.

Fig. 2.

Fig. 2.

Platelet aggregation studies. Washed platelets were prewarmed at 37°C for 3 min, and an agonist, either collagen (A) or thrombin (B), was added (marked by an arrow). The aggregation reaction was recorded at 37°C for 5 min by using a dual aggregometer (Chrono-log).

Fig. 3.

Fig. 3.

In vitro secretion assays. (A) Secretion of dense granules. PRP was isolated from the blood of wild-type (black bars), Rab27b KO (gray bars), Rab27a ash/+_Rab27b_−/− (white bars), and double KO (hatched bars) mice and incubated with 14C-5-HT at 37°C for 1 h. Platelets were sedimented, washed, and challenged with different doses of thrombin (0.25, 0.5, 1, 2 units/ml) for 5 min at 37°C after prewarming for 3 min. Platelets were fixed, and release of 5-HT was measured by using a scintillation counter. The formula used to determine the percentage of 5-HT release was as follows: (release−background) × 100/(total − background) (%). The assay was repeated three times; each time, blood from three mice of the same genotype was pooled. Data are presented as averages ± SD. The initial uptake of 14C-5-HT (dpm per 107 platelets) was as follows: wild type, 2,311 ± 79; Rab27b KO, 2,562 ± 301; double KO, 1,957 ± 167. (B) Secretion of α-granules. Whole blood from double KO mice (hatched bars) and double heterozygous control mice, Rab27a ash/+_Rab27b_−/+ (black bars), was stained with biotin-conjugated anti-CD62P antibody and phycoerythrin-conjugated streptavidin in the absence or presence of phorbol-12-myristate-13-acetate (0, 0.06, 0.125, 0.25, 0.5, and 1 μM) and measured by FACS. For each genotype, blood from three mice was used and counted individually. The data presented are geometric means. (P = 0.9498 as calculated by Student's t test for two populations.) The assay was repeated two times.

Fig. 4.

Fig. 4.

Transmission electron microscopy of wild-type (A), Rab27b KO (B), double KO (C), and _Rab27aash/ashRab27b_−/+ (D). Insets show dense granules in higher-magnification views of the boxed areas. (Scale bars, 500 nm.)

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