Rab27b regulates number and secretion of plateletdense granulesTanya Tolmachova*, Magnus Åbrink†, Clare E. Futter‡, Kalwant S. Authi§, and Miguel C. Seabra*¶

*Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom; †Department of MedicalBiochemistry and Microbiology, Uppsala University, 751 05 Uppsala, Sweden; ‡Institute of Ophthalmology, University College London, London EC1V 9EL,United Kingdom; and §Cardiovascular Division, King’s College London, London SE1 9NH, United Kingdom

Edited by David D. Sabatini, New York University School of Medicine, New York, NY, and approved February 9, 2007 (received for review November 7, 2006)

The Rab27 GTPase subfamily consists of two closely related ho-mologs, Rab27a and Rab27b. Rab27a has been shown previously toregulate organelle movement and regulated exocytosis in a widevariety of secretory cells. However, the role of the more restrictedlyexpressed Rab27b remains unclear. Here we describe the creation ofRab27b knockout (KO) strain that was subsequently crossed with thenaturally occurring Rab27a KO line, ashen, to produce double KO(Rab27aash/ash Rab27b�/�) mice. Rab27b KO (and double KO) exhibitsignificant hemorrhagic disease in contrast to ashen mice. In vitroassays demonstrated impaired aggregation with collagen andU46619 and reduced secretion of dense granules in both Rab27b anddouble KO strains. Additionally, we detected a 50% reduction in thenumber of dense granules per platelet and diminished platelet sero-tonin content, possibly due to a dense granule packaging defect intoproplatelets during megakaryocyte maturation. The presence ofRab27a partially compensated for the secretory defect but not thereduced granule number. The morphology and function of platelet�-granules were unaffected. Our data suggest that Rab27b is a keyregulator of dense granule secretion in platelets and thus a candidategene for �-storage pool deficiency in humans.

membrane traffic � Rab GTPase

Rab proteins constitute a large group within the Ras superfamilyof monomeric GTP-binding proteins (1, 2). Rab GTPases are

critical regulators of vesicular transport steps in endocytic andexocytic pathways, acting as molecular switches oscillating betweenactive GTP-bound and inactive GDP-bound states (3–5). Rab27aand Rab27b constitute the Rab27 subfamily and share 71% identity(2). Much recent interest has focused on Rab27a (6–8). Defects inRAB27A are responsible for Griscelli Syndrome in humans, a rareautosomal disorder characterized by partial albinism, variable cel-lular immunodeficiency, and an acute phase of uncontrolled Tlymphocyte and macrophage activation (9). A spontaneous mouseknockout (KO) of Rab27a, designated ashen, possesses similarcharacteristics, with coat color dilution and defective cytotoxic Tlymphocyte function (10, 11). Rab27a is expressed in a wide varietyof secretory cell types, where it localizes to mature secretory vesiclesand lysosome-related organelles (12). Proposed functions forRab27a include regulation of organelle motility and promotion ofthe tethering/fusion of vesicles in regulated secretion (6–8). Forexample, in pigmented skin melanocytes, Rab27a mediates theinteraction of melanosomes with the actin-based motor, myosin Va(8); when in cytotoxic T lymphocytes, it recruits Munc13-4, whichis necessary for the release of lytic granules (13).

In contrast to Rab27a, Rab27b expression is much more re-stricted. Recent studies suggest that Rab27b is expressed mainly inplatelets, stomach, large intestine, pancreas, pituitary, and bladder(14–17). The role of Rab27b remains unclear, although several factssuggest that Rab27a and Rab27b could be functionally redundant.First, Rab27a and Rab27b interact with the same family of effec-tors, including melanophilin, Myrip, the synaptotagmin-like pro-teins (Slp1–5), and Munc13-4 (6). Secondly, transgenic expressionof Rab27b cDNA in melanocytes, where it is not normally ex-pressed, rescues the coat color defect of ashen mice (14).

Rab27b (formerly named c25KG) was originally purified as anabundant GTP-binding protein in platelets (18), and our subse-quent studies have confirmed the high level of Rab27b expressionin platelets (14). We previously suggested a specific role for Rab27bin platelet function based on the finding that the expression ofRab27b, but not that of Rab27a or five other Rabs also expressedin platelets, appears to be regulated directly by the transcriptionfactor nuclear factor-erythroid 2 (NF-E2) (19). NF-E2 is a criticalcontroller of platelet biogenesis within megakaryocytes. NF-E2 KOmice lack circulating platelets (20), and these mice show reducedmRNA and undetectable protein levels of Rab27b (19). Here wegenerated Rab27b KO and double Rab27 KO (Rab27aash/ash

Rab27b�/�) mice, which exhibit defects in platelet function.

ResultsGeneration of Rab27b KO Mice. To generate Rab27b KO mice, weused the conditional Cre–loxP system of site-specific recombination(Fig. 1A). Initially, we obtained two 125-kb mouse BACs containingthe mouse Rab27b gene and cloned an 11.5-kb DNA fragmentcontaining exons 2 and 3. In the 5� end of intron 3 (700 bpdownstream of exon 3), we inserted a cassette containing two loxPsites flanking a neomycin resistance gene (Neor) under control ofthe PGK promoter, followed by a polyA sequence (Fig. 1A).Another loxP site was inserted 400 bp upstream of the 5� end of exon2 in the same orientation as the other two loxP sites, enablingdeletion of exon 2 and 3 after introduction of Cre recombinase.Deletion of exons 2 and 3 leads to the loss of the first 79 aa, withretention of only one methionine residue in exon 4. However, theresulting severely truncated protein product is predicted to be veryunstable and quickly degraded.

Mouse GSI-1 ES cells isogenic with the Rab27b BAC clone wereelectroporated with linearized targeting vector, and clones wereselected with G418. Initial screening of 336 G418-resistant clonesby Southern blotting with XbaI digestion and a 3�-end proberesulted in seven positive clones, all of which had correct integrationat the 5�-end confirmed by Southern blot with HindIII digestion anda 5�-end probe (Fig. 1B). Blots were stripped and reprobed with theneo probe to confirm single integration of the construct. To confirmthe presence of the loxP site in intron 1, we used a PCR approachand identified five correctly targeted clones.

Rab27b3lox/� mice were crossed with a transgenic mouse lineexpressing Cre–recombinase under the control of the ubiquitous

Author contributions: T.T., K.S.A., and M.C.S. designed research; T.T., M.Å., and C.E.F.performed research; T.T. and M.Å. contributed new reagents/analytic tools; T.T., K.S.A., andM.C.S. analyzed data; and T.T. and M.C.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Abbreviations: 5-HT, 5-hydroxytryptamine; 14C-5-HT, 2-(5-hydroxy-3-indolyl)[2-14C]ethyl-amine creatinine sulfate complex; KO, knockout; PRP, platelet-rich plasma.

¶To whom correspondence should be addressed at: Molecular and Cellular Medicine,National Heart and Lung Institute, Faculty of Medicine, Imperial College London, LondonSW7 2AZ, United Kingdom. E-mail: m.seabra@imperial.ac.uk.

© 2007 by The National Academy of Sciences of the USA

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PGK promoter to produce Rab27b KO alleles. Both males andfemales were used as a PGK-Cre-positive parent. In crosses withPGK-Cre-positive males, deletion occurred strictly with inheritanceof the transgene, whereas in crosses with PGK-Cre females deletionoccurred even without inheritance of the Cre transgene, confirmingprevious reports that the cytoplasmic pool of Cre protein within theoocyte is sufficient to promote recombination (21). Animals car-rying a Rab27bnull allele were identified by PCR, and Southernblotting and homozygous Rab27bnul/nulll (Rab27b�/�) mice weregenerated (Fig. 1C). We confirmed the absence of Rab27b proteinexpression in Rab27b�/� mice by immunoblotting with the use of aspecific anti-Rab27b antibody (Fig. 1D).

Rab27b�/� mice were carefully inspected at regular times insearch of any general phenotype, such as developmental abnor-malities, feeding, and other behavioral changes (e.g., deathrate and so on). No noticeable abnormalities were detected,including no evidence of coat color dilution. Rab27b�/� micewere crossed with ashen mice to produce heterozygous animals(Rab27aash/�Rab27b�/�), which were crossed to generate doubleKO animals (Rab27aash/ash Rab27b�/�). Double KO animalswere viable and similar to ashen mice in coat color.

Analysis of Platelet Function in Rab27 KO and Double KO Mice. Wehave previously described that ashen mice do not exhibit plateletabnormalities (14). Our findings were contrary to results producedby Wilson et al. (11), but the same authors recently described thatthe bleeding phenotype observed in their ashen strain was in factdue to a defect in a second gene, Slc35d3 (22), thus supporting ourconclusion that Rab27a defect does not affect platelet function.

As described above, Rab27b is more likely to be functionallyimportant in platelets, and the availability of the new strains allowedan examination of platelet morphology and function. We startedwith standard hematological analysis. First, we measured plateletnumbers in wild-type, Rab27b KO, and double KO mice by usinga ZM counter (Beckman Coulter, Fullerton, CA). We found nodifferences among wild-type (5.8 � 1.0 � 108 platelets per milliliterof blood), Rab27b KO (5.6 � 0.9 � 108), and double KO (5.7 �0.5 � 108) blood samples. Cell parameters also were within thenormal range according to FACS measurements that were per-formed with FITC-conjugated anti-CD41 antibody. The followingvalues were obtained for wild type, Rab27b KO, and double KO,respectively: forward scatter (arbitrary units): 15.14 � 1.00, 14.89 �0.52, and 16.28 � 0.64; side scatter (arbitrary units): 204.11 � 10.6,206.86 � 3.02, and 197.01 � 4.81; platelets in relation to totalnumber of cells (%): 8.29 � 0.96, 9.54 � 1.39, and 8.87 � 1.63.These results suggest that neither Rab27b KO nor double KOexhibits reduced platelet number or size.

Next, we performed bleeding tests. Initially, we started with acommon method of immersing the tail of a deeply anesthetizedmouse in saline and measuring the time needed for the bleeding tostop. However, we found this method unreliable, because bleedingwould frequently never stop completely, or could stop and restart.Hence, we developed a semiquantitative bleeding test based onmeasuring the weight loss after 10-min bleeding from the tail tip,which is a reflection of the blood loss during the test (see Materialsand Methods). We observed a significant bleeding defect in allmouse strains homozygous for the Rab27b KO allele (Table 1).These mice lost significantly more weight (0.34–0.39 g) than miceheterozygous for Rab27b� allele or wild-type mice (0.13–0.16 g).The presence or absence of Rab27a had no noticeable effect onweight loss (bleeding). As a positive control, we performed similarbleeding test for RabGGTase-deficient gunmetal mice, previouslyshown to exhibit a bleeding defect (23). We found that the bleedingdefect caused by Rab27b KO is more severe than that of gunmetalmice, which lost, on average, 0.18 � 0.1 g per mouse. These resultsindicate that Rab27b deletion causes a severe defect in plateletfunction in vivo.

Next, we tested the aggregation response in vitro to various

Fig. 1. Generation of mice carrying the conditional and KO Rab27b alleles.(A) Targeting vector pTT29 carrying three loxP sites, a neomycin-resistancegene (Neor), and two homology arms were used to generate the Rab27b3lox

allele in GSI-1 ES cells by homologous recombination. Diagnostic HindIII andXbaI restriction sites and corresponding 5� and 3� probes were used to identifycorrectly targeted ES clones. Cre-mediated recombination among the threeloxP sites within the Rab27b3lox allele results in three possible alleles:Rab27bflox, Rab27bnull, and Rab27bnull�Neo, which were distinguished bySouthern blot analysis by using EcoRI digestion and probe A. (B) Results ofSouthern blot analysis by using HindIII digestion and 5� probe and XbaIdigestion 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 Southernblot analysis by using EcoRI digestion and probe A are shown for Rab27bflox/WT,Rab27bnull/WT, and Rab27bnull/null (Rab27b KO) mice. (D) Western blotting byusing platelet lysates from Rab27b KO and wild-type mice and anti-Rab27aantibody, 4B12, and anti-calnexin antibody as a control. GST-Rab27b proteinwas used as positive control.

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agonists. Aggregation in response to collagen was severely com-promised in Rab27b KO and double KO. At a high dose of collagen(20 �g/ml), aggregation responses in Rab27b KO and double KOwere reduced in comparison with wild-type mice (49 � 8% and45 � 9% vs. 62 � 5%) (Fig. 2A). At a lower dose of collagen (5�g/ml), aggregation responses in Rab27b KO and double KO wereseverely reduced in comparison with wild-type mice (9 � 6% and

7 � 4% vs. 46 � 7%). Consistently, aggregation responses to thethromboxane A2 mimetic U46619 in Rab27b KO and double KOwere noticeably reduced in comparison with control. At high dose(10 �M) of U46619, aggregation responses of Rab27b KO anddouble KO mice were 48 � 9% and 47 � 6% vs. 62 � 7% for thewild type. At a low dose (1 �M) of U46619, aggregation responseswere further reduced for Rab27b KO and double KO platelets(41 � 7% and 34 � 10%), whereas wild type remained unchanged(65 � 8%). As low dose of collagen and U46619 require generationand release of secondary agonists for complete aggregation re-sponses (e.g., secretion of ADP, serotonin from dense granules, andformation of thromboxane A2), these results suggest that Rab27bmay play a role in these amplification cascades. Conversely, theplatelet aggregation responses to thrombin in mutant platelets werestrong and similar to control, although the shape of the curveshowed a reduced rate of primary aggregation (Fig. 2B).

In vitro secretion of platelet dense granules was assessed by a5-hydroxytryptamine (5-HT) release assay (Fig. 3A). The uptake of2-(5-hydroxy-3-indolyl)[2-14C]ethylamine creatinine sulfate com-plex (14C-5-HT) by platelets in all strains during 1 h of incubationat 37°C was similar. Rab27b KO and double KO platelets exhibit a

Table 1. Bleeding test in Rab27-deficient mice

Mouse genotype

No. of micetested

Blood lossper mouse, g PRab 27a Rab27b

�/� �/� 15 0.13 � 0.08 n/a�/� �/� 26 0.13 � 0.08 0.9920ash/� �/� 7 0.14 � 0.11 0.7102ash/ash �/� 9 0.14 � 0.1 0.6377�/� �/� 10 0.34 � 0.2 0.0011ash/� �/� 9 0.37 � 0.2 0.0004ash/ash �/� 7 0.39 � 0.17 0.0001

Values for blood loss are averages � SD. P values are according to Student’st test for two populations; each mouse strain was compared to Rab27a�/�

Rab27b�/�. n/a, not applicable.

Fig. 2. Platelet aggregation studies. Washed platelets were prewarmed at37°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 5min by using a dual aggregometer (Chrono-log).

Fig. 3. In vitro secretion assays. (A) Secretion of dense granules. PRP wasisolated from the blood of wild-type (black bars), Rab27b KO (gray bars),Rab27aash/�Rab27b�/� (white bars), and double KO (hatched bars) mice andincubated 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) for5 min at 37°C after prewarming for 3 min. Platelets were fixed, and release of5-HT was measured by using a scintillation counter. The formula used todetermine the percentage of 5-HT release was as follows: (release�background) � 100/(total � background) (%). The assay was repeated threetimes; 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 per107 platelets) was as follows: wild type, 2,311 � 79; Rab27b KO, 2,562 � 301;double KO, 1,957 � 167. (B) Secretion of �-granules. Whole blood from doubleKO mice (hatched bars) and double heterozygous control mice, Rab27aash/�

Rab27b�/� (black bars), was stained with biotin-conjugated anti-CD62P anti-body and phycoerythrin-conjugated streptavidin in the absence or presenceof phorbol-12-myristate-13-acetate (0, 0.06, 0.125, 0.25, 0.5, and 1 �M) andmeasured by FACS. For each genotype, blood from three mice was used andcounted individually. The data presented are geometric means. (P � 0.9498 ascalculated by Student’s t test for two populations.) The assay was repeatedtwo times.

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significant defect in 14C-5-HT release in response to stimulus. Incontrast to nearly 100% release of the 14C-5-HT taken up by thewild-type platelets, maximal release for Rab27b KO was only 76.1 �7.5% and was even lower for the double KO (46.4 � 6.5%). For thelatter, the maximal release remained similar when the concentra-tion of thrombin was increased further (up to 4 units/ml). Inter-estingly, we observed that the defect in 14C-5-HT secretion inRab27aash/�Rab27b�/� mice was as severe as in double KO mice(Fig. 3A). These data further suggest that the absence of Rab27bleads to a dense granule secretory defect. Furthermore, it suggeststhat Rab27a also participates in secretion of dense granules asdouble KO platelets exhibit a more severe defect than singleRab27b KO platelets. Consistently, a reduced level of Rab27a inplatelets seems to limit the ability of Rab27a to functionallycompensate for Rab27b.

In the 5-HT release test, we noticed that background levels ofradioactivity in the absence of agonist were consistently higher inRab27b KO and double KO mice (12% of total loaded radioactivitycompared with 5% for the wild type). These data suggested thepossibility that Rab27b KO might lead to increase in constitutivesecretion (unstimulated release) of dense granules. To address thisquestion, we performed similar 14C-5-HT loading experiments andincubating platelets for up to 40 min at 37°C without agoniststimulation. The amount of released 14C-5-HT did not increase withtime (wild type, 5.6 � 0.8%; Rab27b KO, 12.7 � 1.6%; and doubleKO, 12.5 � 1.8%). These data suggest that Rab27b loss does notlead to constitutive secretion of dense granules.

Next, we investigated secretion of �-granules by measuringexposure of P-selectin on the platelet surface by FACS afteractivation with phorbol-12-myristate-13-acetate (Fig. 3B). Plateletswere identified by staining with FITC-conjugated anti-CD41 anti-body, whereas P-selectin was detected by using biotinylated anti-P-selectin antibody and phycoerythrin-conjugated streptavidin. Weobserved no significant differences in P-selectin levels on thesurface of activated platelets from Rab27b KO (data not shown)and double KO mice, suggesting that Rab27b does not play a majorrole in secretion of platelet �-granules.

Morphologic Analysis of Dense Granules in Rab27 KO Mice. Plateletmorphology was studied by conventional EM, and no gross abnor-malities were detected (Fig. 4). However, quantitative analysis ofgranule numbers revealed that Rab27b KO and double KO plateletsexhibited 50% reduction in the number of dense granules perplatelet (Table 2). Consistently, the total amount of platelet en-dogenous 5-HT in Rab27b KO lines was reduced �2-fold (Table 2).Both observations are consistent with the compromised plateletresponses in vivo (tail bleeding) and in vitro (aggregation) in thesemice. Moreover, the total platelet serotonin content was lower inRab27b�/� mice irrespective of Rab27a (Table 2). These datasuggest that Rab27b, not Rab27a, is implicated in controllingplatelet dense granule number.

DiscussionThe aim of our study was to unravel a biological role for Rab27b.For this purpose, we created a Rab27b gene deletion in mice andsubsequently a double KO by crossing with the natural Rab27a KOstrain, ashen (Rab27aash/ash). Rab27b KO and double KO miceexhibit a serious bleeding defect, and aggregation studies in vitroshowed reduced responses to several agonists, such as collagen andU46619. In vitro secretion of 5-HT but not P-selectin was compro-mised in both Rab27b KO and double KO mice. Finally, absence ofRab27b leads to a 50% reduction in the number of dense granulesper platelet. Our data suggest that Rab27b is critical for thepackaging and secretion of platelet dense granules, resulting in thebleeding diathesis observed in vivo.

The function of Rab27b in platelet dense granule secretion isconsistent with a flurry of recent evidence linking Rab27 proteinswith regulated exocytosis in nonneuronal cells (6, 7, 12). Rab27a has

been more thoroughly investigated to date, presumably because itis widely expressed in secretory cells (12). However, defects inRAB27A in humans result in Griscelli syndrome affecting primarilyonly two cell types, melanocytes and cytotoxic T lymphocytes (9).To explain this intriguing disparity, we have previously suggestedthat Rab27a activity can be compensated in other cell types byRab27b and possibly other related proteins, such as the Rab3isoforms (14). The study of the newly generated Rab27b KO miceand double Rab27 KO mice allowed us to further address this issue,and the results presented here suggest several novel ideas. First, thedouble KO mice are viable and have no gross organ/tissue abnor-mality, suggesting that potential widespread defects in regulatedsecretion due to loss of Rab27a and Rab27b could be compensatedfor by other Rab proteins, at least partially. Second, our studiessuggest that specific cell types rely differentially on a single Rab27isoform, such as melanocytes and cytotoxic T lymphocytes forRab27a, and platelets for Rab27b. Third, the present study suggests

Fig. 4. Transmission electron microscopy of wild-type (A), Rab27b KO (B),double KO (C), and Rab27aash/ashRab27b�/� (D). Insets show dense granules inhigher-magnification views of the boxed areas. (Scale bars, 500 nm.)

Table 2. Number and content of dense granules

Mouse genotype

Total serotonin content,�g per 109 platelets

Dense granulesper cellRab27a Rab27b

�/� �/� 5.76 � 0.73 0.33 � 0.07�/� �/� 5.58 � 1.31 NDash/� �/� 5.37 � 1.00 0.33 � 0.02ash/ash �/� 4.88 � 0.78 0.34 � 0.04�/� �/� 2.64 � 0.56 0.16 � 0.02ash/� �/� 2.36 � 0.59 NDash/ash �/� 2.21 � 0.31 0.19 � 0.01

Data presented are averages � SD. To calculate the dense granules per cell,the total number of granules visible was divided by the total number of cellsin the same field. For each genotype, three of four fields were quantitatedcorresponding to 200–300 cells examined. ND, not determined.

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that Rab27 isoforms may perform similar (redundant) but alsononredundant functions in the same cell type. Our present resultssuggest that both Rab27a and Rab27b participate in secretion ofplatelet dense granules. Rab27a can indeed partially compensatefor the absence of Rab27b in dense granule secretion as the in vitrosecretion tests demonstrated a more severe defect in the double KOvs. the single Rab27b KO. This partial compensation could bebecause of lower Rab27a expression in platelets compared withRab27b (14), a hypothesis supported by the fact that we observeda sharp decline in functional compensation in Rab27b KO mice thatwere heterozygous for the Rab27aash allele (Fig. 3). Neverthelessother Rab27b functions appeared nonredundant, such as a possiblerole in dense granule formation or ‘‘packaging’’ (see below). Finally,we detected no structural or functional impairment in �-granules,despite the localization of (a minor pool of) Rab27 proteins to thesegranules observed previously (14). These findings suggest that theRab27 proteins perform nonessential functions on these organelles.

Rab27b function in dense granule secretion may be mediated byMunc13-4, a member of a protein family implicated in regulatedexocytosis (24). Munc13-4 binds directly to both Rab27a andRab27b and has been implicated in regulated secretion of plateletdense granules (25). A physiological role for Munc13-4 in hema-topoietic cell secretion has been confirmed recently in a study ofpatients with familial hemophagocytic lymphohistiocytosis causedby mutations in this gene (26). Munc13-4-deficient familial he-mophagocytic lymphohistiocytosis patients exhibit low plateletcount, together with other characteristics of the disease, such asfever, hepatosplenomegaly, and hemophagocytosis (27). In addi-tion to Munc13-4, Rab27 proteins bind numerous other effectors(19), which also could be important for platelet function. Futurework should help elucidate this issue.

It is now accepted that complete aggregation response to manyagonists requires an amplification cascade that involves the releaseof ADP and other mediators from dense granules followed bystimulation of P2Y12 receptors enhancing Gi-mediated effects (28).The reduced ability of Rab27b KO and double KO mouse plateletsto secrete dense granules explains the markedly reduced aggrega-tion responses to U46619 and especially collagen. Our data showingthat aggregation is unaffected for thrombin also are consistent withthe above model, because thrombin itself can activate the guaninenucleotide inhibitor factor-mediated pathway. Still, much remainsto be established regarding the signaling mechanism associated withgranule secretion post-surface-receptor activation. PKC activity haslong been established as being required, including pharmacologicalevidence for PKC�-dependent PAR1-mediated dense granule se-cretion (29). However, little is known linking post-PKC events togranule movement and fusion to the plasma membrane events thatinvolve Rab27b, its effectors, and SNARE members.

In addition to defective secretion, we observed a significantreduction in the number of dense granules by using EM quantitativeanalysis and measurement of total platelet 5-HT. Interestingly, thisdefect appears to be independent of Rab27a because the resultswere similar whether none, one, or two copies of Rab27a werepresent (Table 2 and Fig. 4). The reduction in dense granulenumber could reflect a defect in organelle biogenesis. Rab27b maybe controlling a post-Golgi trafficking pathway necessary for thematuration of dense granules in megakaryocytes, as recently shownfor Rab38 and Rab32 in melanosome biogenesis, a related lyso-some-related organelle (30). However, it is more likely that thereduction in dense granule number in mature platelets is due to adense granule packaging defect. Platelets derive from megakaryo-cytes during a complex differentiation process that involves thepackaging of cytoplasmic components onto discrete areas ofmegakaryocyte dendritic projections, called proplatelets (31).Rab27b may control the cytoskeletal-mediated transport and dis-tribution of dense granules during proplatelet formation inmegakaryocytes, as described for Rab27a in melanosomes periph-eral distribution (8, 19). The absence of Rab27b may lead to

reduced or absent transition from microtubule-mediated to actin-mediated transport and hence to reduced numbers of dense gran-ules retained in proplatelets.

The morphology and function of �-granules was not affected inany of the KO strains, in agreement with only minor association ofRab27a and Rab27b with �-granule membranes (14). This suggeststhat Rab27 proteins are not important for secretion of �-granulesor that their loss could be compensated by other Rabs. One possiblecandidate would be Rab4, which was shown to be an essentialregulator of Ca2�-induced exocytosis of �-granules (32). Specificdense granules defects have been observed in a rare geneticcondition called �-storage pool disease (33). The similarities be-tween the patients reported and the mice in this study led us tospeculate that RAB27B is a candidate gene for �-storage pooldisease.

Materials and MethodsGeneration of Rab27b KO Mice. To generate targeting vector pTT29,a 11.5-kb ClaI–KpnI DNA fragment containing exons 2 and 3 ofmouse Rab27b gene was subcloned into pBlueScript vector. Thirtymicrograms of targeting vector pTT29 were linearized with NotIendonuclease, purified by ethanol precipitation, and used forelectroporation of 3 � 107 GSI-1 cells, which were then cultured inthe presence of 0.4 mg/ml G418. To test for correct integration, thefollowing probes were generated by PCR with a 5�-end probe(forward primer, 5�-agcctttactagcagggcaaaccaagatgc; reverseprimer, 5�-ttaaggaagggatttggtagagacccaccg; size, 1 kb) and a 3�-endprobe (forward primer, 5�-accactgccttcttcagagatgccatgggc; reverseprimer, 5�-agctacagacatgaagtccaggcagagggg; size, 1.1 kb). To con-firm single integration of the targeting construct, a 500-bp neoprobe was generated by using forward primer 5�-ccgcgctgttctcctct-tcc and reverse primer 5�-gtacgtgctcgctcgatgcg. To confirm thepresence of the most 5� loxP site, PCR was performed with forwardprimer 5�-ttggatccaggaaacataggtactgaaatgg and reverse primer 5�-ttcagctggcagaatgaagaagttggagat. This PCR amplifies the wild-typeallele (product size, 520 bp) and modified allele (570 bp). Two malechimeras were generated by blastocyst injections of targeted ESclone 181 into C57BL/6 embryos. Both chimeras were 80–90%chimeric and were transmitting the targeted allele.

Mouse Strains. Mice were bred and maintained in the ImperialCollege animal facility in accordance with the rules and regulationsof the Home Office on project license 70/6176. The transgenic lineof PGK-Cre mice was obtained from Ian Roswell (Cancer ResearchUK, London, U.K.) and out-crossed for three generations withC57BL/6J mice. Probe A is a 1.2-kb PCR fragment generated withforward primer 5�-tccacataaatcgtgtgtgtcctcttctcc and reverse primer5�-gtgcagaatgaagaagttggagagttttgc. Rab27bnull allele was identifiedwith forward primer B19 (5�-ctgctgcaggatctcacatcagtg) and reverseprimer B21 (5�-gaaatgggacattgggacaggagg); the size of the PCRproduct was 250 bp. Rab27bWT and Rab27b3lox alleles were identi-fied with forward primer B19 and reverse primer B20 (5�-agcatctgtaacctagacattggc); the sizes of the PCR products were 320and 370 bp, respectively. Routine genotyping was performed byusing three primers (B19, B20, and B21) in the same PCR. Thediagnostic PCR assay for Rab27bflox allele was preformed withforward primer B5 (5�-aacactgttgcatgagctgatcgc) and reverseprimer B21; the size of a product was 350 bp, whereas, for the wildtype, the allele size of the product was 440 bp. The PCR cyclingconditions were as follows: 94°C for 3 min, followed by 32 cycles of94°C for 40 sec, 62°C for 40 sec, 72°C for 1 min, and, finally, 72°Cfor 10 min.

Immunoblotting. Blood was drawn by cardiac puncture under ter-minal anesthesia in 0.4 M sodium citrate (ratio 1:9; Sigma, St. Louis,MO) or ACD (20 mM citric acid/110 mM sodium citrate/5 mMdextrose; ratio 2:8). Blood was diluted with a half volume of EHS(150 mM NaCl/1 mM EDTA/10 mM Hepes, pH 7.6) and spun at

5876 � www.pnas.org�cgi�doi�10.1073�pnas.0609879104 Tolmachova et al.

125 � g for 15 min. Platelet-rich plasma (PRP) was collected andcentrifuged at 1,000 � g for 15 min. The pellet was resuspended inlysis buffer (50 mM Hepes/10 mM NaCl/1 mM DTT, pH 7.2)supplemented with Complete protease inhibitor mixture (RocheDiagnostics, Uppsala, Sweden). Protein extracts were subjected toSDS/PAGE and transferred to PVDF membranes. The antibodiesused were polyclonal anti-Rab27b antibody S086 at a 1:400 dilution(13) and polyclonal anti-calnexin antibody at a 1:5,000 dilution(Stressgen Bioreagents, Ann Arbor, MI).

Determination of Platelet Counts and Size. To obtain platelet counts,50 �l of a 1:2,000 dilution of the PRP was counted in a ZM counter.To assess platelet size, 50 �l of whole blood was stained withFITC-conjugated rat anti-mouse CD41 (integrin �IIb chain) at afinal concentration or 10 �g/ml (BD Pharmingen, Franklin Lakes,NJ) for 30 min. FITC-conjugated rat IgG1, � monoclonal Ig isotypecontrol was used at similar concentration. The analysis was per-formed by using FACS Calibur (BD Immunocytochemistry Sys-tems) by using CellQuest version 3.1f software (BD Biosciences).Measurements were made on pools of blood from three mice of thesame genotype and performed four to five times for each genotype.Results are presented as averages � SD.

Bleeding Test. Eight-week-old mice (not previously genotyped)were anesthetized by i.p. injections of 30 �l of a 2:1 ketamine/xylazine mixture. Mouse body weight was measured, a 5-mm tail tipwas cut off, and tail was immersed into saline prewarmed to 37°C.Bleeding was allowed for 10 min, and then the tail was cauterizedand mouse weight was determined.

Aggregation. PRP was spun at 1,000 � g for 15 min, resuspendedin Tyrode’s buffer (138 mM NaCl/2.9 mM KCl/12 mM NaHCO3/0.36 mM NaH2PO4/5.5 mM glucose/10 mM Hepes/0.4 mM MgCl2,pH 7.4), spun at 1,000 � g for 15 min, and resuspended in Tyrode’sbuffer at a final concentration of 2.5 � 108 platelets per milliliter.The aggregation traces were registered by using a dual-channelaggregometer (Chrono-log, Havertown, PA) at 37°C, with stirringat 1,000 rpm. Platelets were prewarmed at 37°C for 3 min, and thenagonist was added and traces were recorded for 5 min. The agonistsused were thrombin (Sigma), U46619 (Sigma), and Horm collagen(Nycomed, Roskilde, Denmark).

5-HT Release Assay. PRP was incubated with 0.1 �Ci/ml of 14C-5-HTfor 1 h at 37°C. Platelets were spun at 1,000 � g for 15 min and

washed and resuspended in Tyrode’s buffer at 2.5 � 108 plateletsper milliliter; 200 �l of washed platelets was used per reaction. 5-HTrelease assay was performed in a shaking water bath at 37°C witha 3-min warming-up period before the addition of thrombin(Sigma). Reaction was stopped after 5 min by addition of 1/3volume of ice-cold 4% paraformaldehyde in 50 mM EDTA, spunat 9,300 � g for 5 min. Supernatant was collected and radioactivitywas counted by using a liquid scintillation analyzer (model 1900TR;Packard Instrument Co., Meridan, CT). ‘‘Total’’ radioactivity val-ues were obtained by counting 200 �l of labeled, washed, unstimu-lated platelets. To obtain a ‘‘background’’ value, 200 �l of labeledand washed platelets was kept at 37°C for 8 min, fixed, and spun,and radioactivity of the supernatant was measured. To measureplatelet uptake, platelets were labeled with 14C-5-HT for 1 h at 37°Cand washed, and incorporated radioactivity was counted. Theuptake per 107 platelets is calculated as (uptake � 107)/(totalnumber of platelets).

Determination of Endogenous 5-HT Levels. The method used was amodified procedure of Drummond and Gordon (34) that has beendescribed previously in detail (14).

Detection of P-Selectin at the Platelet Surface. Blood was drawn with4% sodium citrate as above and diluted 1:6 with Tyrode’s buffer.Biotinylated anti-P-selectin antibody (5 �g/ml), FITC-conjugatedanti-CD41 antibody (10 �g/ml), and phycoerythrin-conjugatedstreptavidin (10 �g/ml) were added to 50 �l of diluted blood,together with different concentrations of phorbol-12-myristate-13-acetate, and incubated for 30 min at room temperature. Sampleswere fixed with 1 volume of 1% paraformaldehyde in Tyrode’sbuffer and analyzed by FACS.

Electron Microscopy. PRP was collected and spun at 1,000 � g for10 min. Pellet was washed twice with 0.4% sodium citrate in 1� PBScontaining 8 �M prostaglandin E1. Pellet was resuspended in 2%paraformaldehyde and 1.5% glutaraldehyde and fixed for 1–2 h.Platelets were then osmicated, stained with tannic acid, and em-bedded in Epon. Ultrathin sections stained with lead citrate wereviewed on a Joel 1010 transmission electron microscope (Joel,Tokyo, Japan).

We thank Ross Anders for mouse breeding; Holly Graham for help withPCR analysis; Duarte Barral for support, expertise, and advice; and IanRoswell for providing the PGK-Cre strain. This work was supported by theWellcome Trust. K.S.A. was supported by the British Heart Foundation.

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