Programmed Anuclear Cell Death
Delimits Platelet Life Span
Kylie D. Mason,2,4Marina R. Carpinelli,1Jamie I. Fletcher,2Janelle E. Collinge,1Adrienne A. Hilton,1
Sarah Ellis,5Priscilla N. Kelly,2Paul G. Ekert,6Donald Metcalf,3Andrew W. Roberts,3David C.S. Huang,2,7,*
and Benjamin T. Kile1,4,7,*
1Molecular Medicine Division
2Molecular Genetics of Cancer Division
3Cancer and Hematology Division
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia
4Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
5Peter MacCallum Cancer Centre, Trescowthick Research Laboratories, St. Andrew’s Place, East Melbourne,
Victoria 3002, Australia
6Children’s Cancer Centre, Royal Children’s Hospital, Parkville, Victoria 3052, Australia
7These authors contributed equally to this work.
*Correspondence: firstname.lastname@example.org (B.T.K.), email@example.com (D.C.S.H.)
Platelets are anuclear cytoplasmic fragments
essential for blood clotting and wound healing.
Despite much speculation, the factors deter-
mining their life span in the circulation are un-
known. We show here that an intrinsic program
for apoptosis controls platelet survival and
dictates their life span. Pro-survival Bcl-xLcon-
strains the pro-apoptotic activity of Bak to
maintain platelet survival, but as Bcl-xL de-
grades, aged plateletsare primed forcell death.
Genetic ablation or pharmacological inactiva-
tion of Bcl-xL reduces platelet half-life and
causes thrombocytopenia in a dose-dependent
manner. Deletion of Bak corrects these defects,
and platelets from Bak-deficient mice live lon-
ger than normal. Thus, platelets are, by default,
genetically programmed to die by apoptosis.
The antagonistic balance between Bcl-xLand
Bak constitutes a molecular clock that deter-
mines platelet life span: this represents an
important paradigm for cellular homeostasis,
and has profound implications for the diagnosis
and treatment of disorders that affect platelet
number and function.
In metazoans, new cells are constantly generated to re-
place those that are aged, damaged, or functionally
expended. Such tissue homeostasis can be regulated
at multiple levels and in a variety of ways in diverse cell
for normal health is the hematopoietic system. Beginning
with hematopoietic stem cells, multiple rounds of prolifer-
ation, differentiation, and commitment give rise to the
various lineages of blood cells. Each mature cell has a
specialized function and a life span characteristic of its
lineage. For example, memory B lymphocytes can survive
for many years, erythrocytes for months, and platelets
quent stresses and insults to which they are exposed.
Persistence of cells that should normally be destroyed
and removed contributes to diseases such as cancer and
autoimmunity (Danial and Korsmeyer, 2004; Rudin and
Thompson, 1997; Strasser et al., 2000).
play an essential role in blood clotting and wound healing.
They are produced by megakaryocytes: large, polyploid
cells that develop in the bone marrow and spleen. Mega-
karyocytes shed platelets into the blood stream where, in
humans, they circulate for around 10 days (Leeksma and
Cohen, 1955) before being destroyed by the reticuloendo-
thelial system, primarily in the liver and spleen. Like all lin-
eages of blood cells, the steady state number of mature
platelets is the result of a balance between their produc-
tion and destruction. While some of the mechanisms reg-
ulating platelet biogenesis have been clarified in recent
years (Kaushansky, 2005; Patel et al., 2005), the factors
that control their life span, particularly at steady state—a
subject of speculation since the 1960s (Mustard et al.,
1966)—have remained elusive.
A number of recent studies have suggested that plate-
lets can undergo apoptosis (Brown et al., 2000; Pereira
et al., 2002; Rand et al., 2004; Vanags et al., 1997). Plate-
lets express several Bcl-2 family members, and, in re-
sponse to various stimuli, exhibit features characteristic
of cell death; however, the physiological significance of
this phenomenon remains to be established. Here we
report that platelets are intrinsically programmed to un-
dergo cell death in vivo, and that their life span in the
circulation is circumscribed by the initiation of apoptosis.
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1173
ber of the extended Bcl-2 protein family (Adams, 2003;
Danial and Korsmeyer, 2004), is the key mediator of plate-
ing Bcl-xLor its pharmacological inhibition by the BH3
mimetic compound ABT-737 (Oltersdorf et al., 2005) de-
crease platelet half-life (t1/2) and cause thrombocytopenia
in a dose-dependent manner. The major downstream
effector responsible for mediating platelet death is pro-
apoptotic Bak. Deletion of Bak, and to a lesser extent, its
relative Bax, can extend platelet life span and reverse
the effects of Bcl-xLantagonism both in vitro and in vivo.
Weshow thatBcl-xLandBak constitute themajor compo-
nents of a ‘‘molecular clock’’ that determines platelet life
span: as platelets age, degradation of Bcl-xLtriggers Bak-
mediated apoptosis and clearance from the circulation.
Mutations in Bcl-x Cause Thrombocytopenia
We conducted a genome-wide mutagenesis screen in
wild-type mice to identify mutations causing thrombocy-
topenia. Male BALB/c mice were treated with the chemi-
cal mutagen N-ethyl-N-nitrosourea (ENU) and mated to
untreated BALB/c females. First-generation (G1) offspring
were bled at 7 weeks of age, and mice exhibiting circulat-
ing platelet counts below 900 3 103/ml (lower end of the
normal range) were re-bled at 9 weeks. Several G1mice
exhibited persistent thrombocytopenia (Figure 1A), the
heritability of which was in each case tested by mating
to wild-type BALB/c mice. The G2offspring from these
matings were bled at 7 weeks of age, and the presence
of animals with low platelet counts confirmed that five
pedigrees were segregating heritable dominant mutations
Two of these mutations, denoted Plt20 and Plt16, were
both mapped via a standard positional cloning approach
to the distal end of chromosome 2. Fine mapping refined
the candidate regions for Plt20 (Figure 1B) and Plt16
(Figure 1C) to overlapping intervals of 16.3 and 1.9 Mb, re-
spectively. The exons and splice junctions of candidate
genes were directly sequenced, and mutations in the
Bcl-x gene were identified in affected animals from both
the Plt20 (Figure 1D) and Plt16 (Figure 1E) pedigrees. In
the case of Plt20, an A-to-G transition is predicted to
cause the substitution of cysteine for tyrosine at residue
In Plt16, the mutation is a T-to-A transversion predicted
to cause the substitution of asparagine for isoleucine at
Like Bcl-xPlt20and Bcl-xPlt16Mice, Bcl-xL-Deficient
Mice Are Also Thrombocytopenic
Bcl-xL(Boise et al., 1993) is a pro-survival member of the
Bcl-2 protein family (which includes Bcl-2, Bcl-w, Mcl-1,
and A1) that regulates developmentally programmed
and stress-induced cell death (Adams, 2003; Danial and
Korsmeyer, 2004). The thrombocytopenia exhibited by
mice carrying the Plt20 and Plt16 alleles of Bcl-x sug-
gested that Bcl-xL contributes to the maintenance of
platelet numbers. To verify the role of Bcl-xLand to ex-
clude linked ENU-induced mutations as the cause of the
thrombocytopenia, we examined animals that had been
specifically engineered to lack Bcl-xL(Motoyama et al.,
1995). Bcl-x+/?mice develop normally and are born at
the expected Mendelian frequency (Motoyama et al., 1995).
We found that, like Bcl-x+/Plt20and Bcl-x+/Plt16mice,
Bcl-x+/?animals exhibited platelet counts significantly
(?1100 3 103/ml) (Figure 2A), confirming that haploinsuffi-
ciency of Bcl-x results in thrombocytopenia.
Unlike Bcl-xPlt16/Plt16animals, of which only a few were
tation (Motoyama et al., 1995), Bcl-xPlt20/Plt20mice were
born at the expected Mendelian frequency and survived
to at least 6 months of age, indicating that this allele of
Bcl-x is hypomorphic, rather than a complete loss-of-
function. Aside from a mild increase in splenic erythropoi-
esis (data not shown), Bcl-xPlt20/Plt20mice did not display
any other gross abnormalities in the hematopoietic com-
partment (Table 1), and in contrast to animals carrying
indicating that spermatogenesis is not significantly com-
promised. Significantly, platelet counts in homozygous
Bcl-xPlt20/Plt20mice were further reduced to approximately
25% of that of wild-type counterparts (Table 1), demon-
strating that incremental reductions in Bcl-xLproduce a
phenotypic gradient with respect to platelet number.
Thrombocytopenia is not a general result of inactivating
Bcl-2-like pro-survival proteins: unlike Bcl-x mutant mice,
platelet counts in Bcl-2+/?, Bcl-w?/?, and Mcl-1+/?ani-
mals were normal (Figure 2A).
Increased Rates of Platelet Clearance
in Bcl-x Mutant Mice
Robust megakaryocytopoiesis in the bone marrow and
spleens of Bcl-x mutant mice argued against defective
topenia. Megakaryocyte progenitor numbers were normal
in Bcl-x+/Plt20mice and Bcl-xPlt20/Plt20littermates (see Ta-
were marginally increased in Bcl-x+/Plt20and Bcl-x+/Plt16
mice, and significantly elevated in Bcl-xPlt20/Plt20homozy-
gotes (see Figure S1 in the Supplemental Data and Table
S1). In all Bcl-x mutant mice, they were morphologically
normal and exhibited ploidy profiles similar to those of
wild-type counterparts (Figure S2A). Additionally, mega-
karyocyte progenitors from the mutant mice were not
prone to spontaneous apoptosis in vitro (Figure S2B), and
recovered as vigorously as wild-type counterparts from
stress-induced thrombocytopenia in vivo (Figure S2C).
We then examined whether increased splenic se-
questration might account for the reduction in platelet
counts. However, removing spleens from homozygous
Bcl-xPlt20/Plt20mice only partially corrected the thrombo-
cytopenia (from 367 ± 41 to 516 ± 63 3 103/ml, compared
1174 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
Figure 1. Isolation and Molecular Identification of Mutations in Bcl-x
(A) Peripheral blood platelet counts from 810 7-week-old G1offspring of ENU-mutagenized BALB/c males. Each circle represents an individual
mouse. Founder animals for the Plt20 and Plt16 pedigrees are indicated. The heritability of three additional thrombocytopenias (Plt17, Plt18, and
Plt21) was confirmed; these pedigrees are at various stages of the genetic mapping process. Plt17 was mapped to chromosome 11 and a mutation
in the gene encoding GpIba was identified. Plt18 maps to an interval on chromosome 16, while Plt21 is yet to be assigned a map location. No
additional heritable mutations causing thrombocytosis were identified.
(B and C) Mapping haplotypes for Bcl-xPlt20(B) and Bcl-xPlt16(C). Markers used and their positions on the April 2006 University of California, Santa
Cruz (UCSC) mouse genome are indicated. Defining recombinant events are shaded gray; those confirmed by heritability testing are shown in bold.
An interval of 16.3 Mb wasdefined for Bcl-xPlt20,betweenJCCA19 and D2Mit500.The candidate interval for Bcl-xPlt16wasrefined to1.9 Mb, between
JCCA9 and D2Mit139.
(D and E) DNA sequence electropherograms showing the nucleotide changes in animals heterozygous for the Bcl-xPlt20(D) or Bcl-xPlt16(E) mutations.
Further sequencing established that neither the Plt16 nor Plt20 mutation is present in the parental BALB/c strain or the C57BL/6 mapping strain.
Tyrosine 15 and isoleucine 182, the residues substituted in the Plt20 and Plt16 pedigrees, respectively, are conserved between mouse and human
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1175
with 1279 ± 200 to 1650 ± 136 3 103/ml in wild-type litter-
mates), indicating that abnormal splenic function is not
primarily responsible for low platelet counts.
Next, we considered whether Bcl-xL, in its capacity as
a pro-survival regulator, might directly influence the fate
of platelets, reasoning that platelets with reduced Bcl-xL
function might die prematurely. By tracking the survival
of biotin-labeled platelets in vivo (Ault and Knowles,
1995), we found that the Bcl-xPlt20mutation decreased
platelet t1/2 in a dose-dependent manner (Figure 2B).
One mutant allele of Bcl-x reduced the normal platelet t1/2
byapproximately 50%(?57hrto24hr),whereas mutating
both alleles triggered a further reduction in t1/2to less than
12 hr (Figure 2B). Similarly, platelets from Bcl-x+/Plt16and
Figure 2. Like Bcl-xPlt20and Bcl-xPlt16Mutant Mice, Mice Lacking One Bcl-x Allele Are Thrombocytopenic
a significant decrease in platelet number. Results were compared using two-tailed unpaired Student’s t test. *p < 0.05.
(B) Decreased life span of Bcl-xPlt20platelets. Peripheral blood samples were taken from Bcl-xPlt20mice 0, 4, 8, 12, 24, 28, 48, 72, and 96 hr after
injection with NHS-biotin. Whereas wild-type (Bcl-x+/+) platelets exhibited a t1/2of 57 hr, consistent with published observations (Berger et al.,
1998), the Bcl-xPlt20mutation caused a dose-dependent decrease to ?24 hr in heterozygotes and ?10 hr in Bcl-xPlt20/Plt20homozygous mice.
(C) Bcl-x mutations shorten platelet life span. Half-lives of platelets in mice of the indicated genotypes determined as in (B). Like the Bcl-xPlt20
mutation, Bcl-xPlt16or Bcl-x deletion (Bcl-x+/?) also decreased platelet half-lives relative to that of wild-type (Bcl-x+/+) littermate controls.yDetailed
information on the genetic background of the mice is provided in Experimental Procedures.
(D) Reduced platelet half-life (t1/2) in Bcl-x mutant mice is a platelet-intrinsic defect. Biotinylated platelets from mice of the indicated genotypes were
adoptively transferred into unmanipulated recipients of the indicated genotypes, and their clearance from the circulation was measured as in (B).
(E) Loss of Bcl-xLincreases platelet turnover, resulting in a proportionately younger platelet population. The percentage of reticulated platelets was
determined by staining with Thiazole orange (Kienast and Schmitz, 1990); each symbol represents an individual mouse.
(F) Platelet production is not impaired by mutations in Bcl-x. Absolute numbers of reticulated platelets were determined by Thiazole orange staining
and measuring platelet count at steady state.
Data in (B), (C), and (D) represent means ± SD of five to eight mice at each time point.
1176 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
Bcl-x+/?mice also exhibited shortened platelet life spans
To confirm that changes in circulating t1/2 reflected
properties intrinsic to platelets, we performed reciprocal
Bcl-x+/Plt20and Bcl-xPlt20/Plt20platelets were cleared more
quickly than wild-type platelets (Figure 2D), with half-lives
indistinguishable from those seen in unmanipulated mice.
Conversely, the clearance of wild-type platelets trans-
ferred into Bcl-x+/+, Bcl-x+/Plt20, or Bcl-xPlt20/Plt20mice
was identical regardless of the recipient’s genotype
by staining with Thiazole orange, a marker of young, RNA-
replete, ‘‘reticulated’’ platelets (Ault and Knowles, 1995;
Kienast and Schmitz, 1990). The Plt20 and Plt16 muta-
tions in Bcl-x caused dose-dependent increases in the
proportion of positive cells (Figure 2E), indicating that
platelet populations in mice carrying these mutations
life span being shortened. Absolute reticulated platelet
rable with those of wild-type mice (Figure 2F), again
supporting the conclusion that mutation of Bcl-x does
not significantly impair platelet production (Table 1 and
Tables S1 and S2; Figures S1 and S2).
Bcl-xPlt20and Bcl-xPlt16Mutations Destabilize Bcl-xL
Neither the Plt20 nor the Plt16 mutation is predicted to di-
rectly affect the BH3 binding groove of Bcl-xL(Figure 3A),
a region critical for its function (Adams, 2003; Liu et al.,
2003; Sattler et al., 1997). Consistent with this, the capac-
ity of the mutant Bcl-xLproteins to bind the essential
downstream mediators of apoptosis, Bax and Bak (Cheng
et al., 2001; Lindsten et al., 2000), appeared largely intact
(Figure 3B). When overexpressed in cell lines (includ-
ing immortalized Bcl-x?/?mouse embryo fibroblasts
[MEFs]), the mutantBcl-xLproteins werenot constitutively
cytotoxic (data not shown), but were less stable than
wild-type Bcl-xL(Figure S3A). Likewise, we found that
the stability of endogenous full-length Bcl-xL (normal
t1/2= ?18 hr) was moderately reduced in MEFs derived
from Bcl-xPlt20/Plt20mice (t1/2= ?12 hr) (Figure 3C). Even
the basal level of endogenous Bcl-xL was reduced in
Bcl-xPlt16/Plt16cells, consistent with the propensity of this
mutant form of Bcl-xLto be degraded (Figure 3C and
Interestingly, the destabilization of Bcl-xL selectively
sensitized MEFs to apoptosis when protein synthesis
was inhibited (Figure 3D and Figures S3B and S4B), prob-
apoptotic Bak (Willis et al., 2005), were both degraded
Bak was maintained even 24 hr after this treatment. Be-
cause both pro-survival proteins (Bcl-xLand Mcl-1) need
to be inactivated for Bak-mediated apoptosis (Willis et al.,
2005), the greater stability of wild-type Bcl-xL allowed
prolonged survival following cycloheximide treatment (Fig-
ure 3D and Figure S3B) even when Mcl-1 was degraded
(Figure 3C). In contrast, the mutations had no effect on the
sensitivity to other damaging signals that did not directly
affect protein synthesis, such as treatment with the broad-
spectrum kinase inhibitor staurosporine (Figure S4A).
Thus, we conclude that Bcl-xPlt20and Bcl-xPlt16are hypo-
morphic alleles of Bcl-x that encode labile proteins.
A BH3 Mimetic Compound Causes Acute
The small molecule ABT-737 is a BH3 mimetic drug that
antagonizes pro-survival Bcl-xL(Oltersdorf et al., 2005).
It selectively targets Bcl-2, Bcl-xL, and Bcl-w, but not
the other pro-survival proteins Mcl-1 or A1 (Oltersdorf
et al., 2005; van Delft et al., 2006). To date, it is reported
to be well-tolerated in mice and demonstrates single-
agent efficacy against certain tumor cell lines, particularly
those derived from small cell lung cancers or lymphomas
Table 1. Peripheral Blood Cell Values of Mice Carrying Mutant Alleles of Bcl-x
Erythrocytes (3 106/ml)10.6 ± 0.3b
10.7 ± 0.410.8 ± 0.510.2 ± 0.59.5 ± 0.4
Hematocrit (%) 51.5 ± 1.751.7 ± 2.352.4 ± 2.0 50.9 ± 1.649.6 ± 2.9
MCVc(femtoliters)48.5 ± 0.748.5 ± 1.148.6 ± 1.050.2 ± 1.452.1 ± 1.6
Leukocytes (3 103/ml)8.2 ± 1.58.2 ± 1.9 7.9 ± 1.7 8.4 ± 2.18.9 ± 0.8
Neutrophils (3 103/ml) 1.2 ± 0.21.2 ± 0.3 1.1 ± 0.21.4 ± 0.41.0 ± 0.3
Lymphocytes (3 103/ml) 7.1 ± 1.56.8 ± 1.76.7 ± 1.6 6.9 ± 1.6 7.3 ± 0.5
Monocytes (3 103/ml) 0.1 ± 0.00.1 ± 0.00.1 ± 0.00.1 ± 0.0 0.2 ± 0.0
Platelets (3 103/ml)1137 ± 82 598 ± 59 596 ± 65 265 ± 47 279 ± 48
MPVd(femtoliters) 7.1 ± 0.7 7.3 ± 0.7 6.7 ± 0.6 7.3 ± 0.67.3 ± 0.6
bData represent means ± SD for 10–12 mice per genotype.
cMCV, mean corpuscular volume.
dMPV, mean platelet volume.
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1177
(Oltersdorf et al., 2005). Notably, it was observed that the
drug reduced platelet counts in mice (Oltersdorf et al.,
2005), with features of apoptosis evident when platelets
were exposed to ABT-737 in vitro (Zhang et al., 2007).
Within 2 hr of injecting a single dose of ABT-737 (but not
the vehicle control) into wild-type C57BL/6 mice, platelet
counts dropped to less than 30% of normal, with the nadir
at4hr(Figure 4Aand FigureS5A).Thrombocytopenia was
dose dependent (data not shown) and platelet counts
treatment (Figure 4B). This recovery, associated with sus-
tained production of thrombopoietin (TPO) (Figure 4B),
was observed even with daily injections of ABT-737.
When ABT-737 was continued for 14 days, platelet levels
Figure 3. The Bcl-xPlt20and Bcl-xPlt16Mutations Destabilize the Bcl-xLProtein
(A) Location of the Plt20 and Plt16 mutations on Bcl-xL. The two mutations (in blue) are mapped on the 3D structure of mouse Bcl-xL(light gray) in
complex with a Bim BH3 peptide (red) (1PQ1) (Liu et al., 2003). Y15 (Plt20) is partially solvent exposed, while I182 (Plt16) is completely buried,
with neither contributing directly to the BH3 binding groove. The structural depiction was prepared using PyMOL (http://www.pymol.org).
(B) The mutant proteins encoded by the Plt20 and Plt16 alleles of Bcl-x can still bind Bax and Bak. FLAG-tagged wild-type Bcl-xL, or the Plt20 (Y15C)
and Plt16 (I182N) mutants were coexpressed in 293T cells with HA-tagged Bax (upper) or Bak (lower), and the Triton X-100 containing lysates immu-
noprecipitated with the mouse monoclonal anti-FLAG (FL; M2 clone), -HA (HA.11 clone), or an irrelevant control antibody (C; -GluGlu). The blot was
probed with rat monoclonal antibodies to FLAG (9H1) or HA (3F10).
(C) The Plt20 and Plt16 mutations destabilize Bcl-xL. (Upper panels) Decreased basal expression of Bcl-xLPlt16 protein. Immunoblotting for Bcl-xL,
alent lysates prepared from wild-type or Bcl-xPlt20/Plt20primary MEFs 0–24 hr after exposure to 50 mg/ml cycloheximide (protein synthesis inhibitor) in
the presence of the broad-spectrum caspase inhibitor qVD.OPh (50 mM) were probed for indicated proteins. Data shown is representative of at least
two cell lines of each genotype analyzed.
(D) Bcl-xPlt20or Bcl-xPlt16MEFs are susceptible to protein synthesis inhibition. The viability (determined by PI exclusion) of representative primary
MEFs derived from wild-type, Bcl-xPlt20/Plt20, or Bcl-xPlt16/Plt16mice after exposure to 50 mg/ml cycloheximide for 0-30 hr is shown. Data represent
means ± SD of representative cell lines. y, <1% viability.
1178 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
were maintained at ?60%–70% of normal until the ther-
apy was stopped (Figure S5B). In contrast, when ABT-737
was given weekly, acute thrombocytopenia ensued in
a cyclical manner (Figure 4C), with platelet counts recov-
ering in the intervening periods.
Interestingly, when the age profile of circulating plate-
lets post-ABT-737 was examined by Thiazole orange
staining, we noted a transient increase in the proportion
of reticulated platelets (Figure 4D). This suggested that
older platelets might be more susceptible to the effects
of the drug. To investigate this possibility, we treated
mice with anti-platelet serum (APS) in order to artificially
synchronize platelet production. Platelet counts post-
APS decreased rapidly to almost undetectable levels at
24 hr before recovering over the course of 7 days (Fig-
ure 4E). During this recovery, the Thiazole orange profile
changed dramatically, witha largely homogenous popula-
tion of new, reticulated platelets prominent on day 2 after
APS (Figure 4F). As the population aged, the proportion
of reticulated platelets dropped to near-normal levels at
7 days post-APS. Newly synthesized young platelets
(2 days post-APS) were highly resistant to the effects of
ABT-737 (Figure 4F). In sharp contrast, aged platelets
(7 days post-APS) were susceptible to ABT-737 in vivo,
confirming that the drug acts primarily on older platelets.
Drugs are a well-known cause of thrombocytopenia.
agents,thisisusuallyimmunemediated. Typically, insuch
cases, onset of thrombocytopenia occurs 7–10 days after
initial exposure to a drug and is inevitably exacerbated by
continued treatment (George et al., 1998). Conversely, the
effect of ABT-737 was extremely rapid (Figure 4A) and
platelet counts in the treated mice partially recovered
despite ongoing therapy (Figure S5B), suggesting that
a new rheostat for maintaining platelet levels had been
set. We observed that TPO levels (Figure 4B) and mega-
mice (Figure S5C). Furthermore, when ABT-737 was
tested in progenitor cell cultures in vitro, no effect on the
number of megakaryocyte colonies formed was evident
(Figure S5D). Thus, in contrast to the well-characterized
effects observed when drugs impair platelet production
or trigger immune-mediated destruction, our results point
toward a direct cytotoxic action of ABT-737 on platelets.
ABT-737 Induces Caspase-Dependent
Given the observation that Bcl-xLis likely to be the key
survival factor expressed in platelets (Figure 5A), we rea-
soned that ABT-737, which targets Bcl-2, Bcl-xL, and
Bcl-w (Oltersdorf et al., 2005), might kill these anuclear
cytoplasmic fragments by specifically inhibiting Bcl-xL,
rather than another pro-survival protein. As anticipated,
heterozygosity for a null allele of Bcl-xL, but not Bcl-2,
exacerbated ABT-737-induced thrombocytopenia (Fig-
ure 5B). Exposure to ABT-737 triggered cleavage and
full activation of caspase-3 as well as cleavage of gelsolin,
a known caspase substrate, in cultured platelets (Fig-
ure 5C). Furthermore, inhibiting caspases, the down-
stream effectors of apoptosis (Thornberry and Lazebnik,
1998), with the broad-spectrum inhibitor qVD.OPh (Case-
rta et al., 2003) partially ameliorated the cytotoxic effect of
platelets in culture. Of note, exposure to ABT-737 ex vivo
did nottriggerplatelet activation oradversely impact upon
the ability of platelets to aggregate in response to ADP or
collagen (Figure S6).
Loss of Bak Ameliorates Thrombocytopenia
Caused by ABT-737
destruction by caspases, we next considered the poten-
tial molecular target or targets of its activity. The likeliest
candidates are the multidomain pro-apoptotic family
members Bax and Bak; they have been shown to be es-
sential mediators of apoptotic cell death (Cheng et al.,
2001; Lindsten et al., 2000; Rathmell et al., 2002; Zong
et al., 2001) that act upstream of the caspases. Further-
more, Bcl-xLhas the capacity to keep Bak in check by di-
rectly binding this cell death mediator (Figure 3B; see dis-
cussion above) (Willis et al., 2005), thereby preventing its
downstream actions. We therefore examined the effect
of ABT-737 in mice deficient for either one or both of these
proteins. The absence of Bak markedly blunted the action
of ABT-737 on platelet viability in culture (Figure 5D) and
significantly buffered against the apoptotic effects of
ABT-737 in vivo (Figure 5F). While the loss of Bax alone
had little impact (data not shown), the complete absence
of Bak combined with the additional loss of one Bax allele
rendered platelets entirely refractory to ABT-737 (Fig-
the killing of platelets by neutralizing the pro-survival ac-
tion of Bcl-xL, thereby allowing the unrestrained action
of the key pro-apoptotic mediators Bak and, to a lesser
In Platelets, Pro-Apoptotic Bak Is the Critical Target
for Pro-Survival Bcl-xL
Since deletion of the downstream effectors Bak and Bax
protected platelets against ABT-737-induced killing (Fig-
ure 5), we examined the role of these molecules in normal
platelet homeostasis. At steady state, platelet counts in
Bax-deficient mice were indistinguishable from those of
wild-type counterparts (Figure 6A). In contrast, Bak?/?
ure 6A). Bak?/?platelets were morphologically normal
(Figure6B)andBak?/?micedidnothave defects(e.g., hy-
posplenism) that might account for the thrombocytosis. It
was therefore of interest to find that platelet t1/2in these
animals, as determined by in vivo labeling assays, was
increased by almost 50% (Figure 6C and Figure S7). We
next examinedplatelet counts andhalf-lives in micecarry-
ing combinations of mutant Bcl-x, Bak, and Bax alleles.
Strikingly, deletion of one Bak allele rescued the thrombo-
cytopenia in Bcl-x+/?mice (Figure 6D). Deletion of both
alleles caused a thrombocytosis similar to that seen in
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1179
Figure 4. The BH3 Mimetic ABT-737 Triggers Acute Thrombocytopenia
and platelet counts were determined. All injected mice exhibited a significant reduction in platelet counts, with the nadir (<30% normal) occurring
approximately 4 hr after injection; each symbol represents a mouse.
(B) Platelet recovery after a single dose of ABT-737. Platelet counts (blue symbols; left axis) were determined 2–96 hr after a single dose of ABT-737
(red arrow). Note full recovery by day 3, and rebound thrombocytosis by day 4. Orange symbols represent serum TPO levels (right axis).
(C) Cyclical acute thrombocytopenia triggered by ABT-737. Platelet counts were determined before and 8 hr after a single dose of ABT-737 (red
arrows) given at weekly intervals. The drug caused a comparable drop in platelets each time. During recovery, baseline counts drifted upwards.
1180 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
Bak-deficient animals (cf. Figure 6D with Figure 6A), with a
corresponding increase in platelet t1/2 observed (Fig-
ure 6C). Furthermore, the thrombocytopenia seen in het-
erozygous Bcl-xPlt20or Bcl-xPlt16mice was exacerbated
versed by loss of one Bak allele (Figure 6E). Thus, Bak lies
biochemically (Willis et al., 2005) (Figure 3 and Figure 5)
and genetically (Figure 6) downstream of Bcl-x.
Bcl-xLMaintains Platelet Survival by Restraining
These results presented here identify Bcl-xLas the major
homeostatic regulator of platelet survival. In contrast, the
netic mutation or pharmacological antagonism of Bcl-xL
in vivo or in vitro caused a dose-dependent diminution
of platelet survival and life span, but mutations in Bcl-2,
Bcl-w, or Mcl-1 did not. A central role for pro-survival
Mcl-1 or A1 also seems unlikely since the BH3 mimetic
compound ABT-737 does not target these members of
the Bcl-2 family (Oltersdorf et al., 2005; van Delft et al.,
2006).Although the precise contribution various apoptotic
regulators make to platelet production remains to be
determined (Clarke et al., 2003; De Botton et al., 2002;
Kaluzhny et al., 2002; Ogilvy et al., 1999), we conclude
that Bcl-xLis not absolutely required for megakaryocyte
proliferation and differentiation. Whether it plays a role in
theprocess ofplateletshedding remainsto beaddressed.
We propose that the amount of Bcl-xLa platelet inherits
determines its life span. As Bcl-xLis degraded over time,
a threshold is reached, upon which pro-apoptotic Bak
is freed and platelet apoptosis is induced. Inhibition of
Bcl-xL, either genetically or pharmacologically, speeds
up the molecular clock, bringing forward the point of entry
into cell death and subsequent platelet clearance from the
circulation. The model is supported by our observation
that Bcl-xLand Bak have different half-lives: in the ab-
sence of new protein synthesis, Bcl-xLdegrades more
rapidly than Bak. Given their limited synthetic capacity, it
might therefore be expected that all else being equal,
aging platelets would be unable to counter an inexorable
decline in Bcl-xLrelative to Bak. Indeed, previous reports
suggest that while Bak levels are stable in platelets stored
at 37?C (Brown et al., 2000), Bcl-xLlevels decrease over
time (Bertino et al., 2003). A corollary is that older circulat-
ing platelets harboring less Bcl-xLshould be more sus-
ceptible to the effects of ABT-737 than their younger
counterparts, and our experiments with APS followed
by ABT-737 treatment clearly demonstrate that this is
the case. This probably explains near-normal recovery of
platelet counts in mice treated daily with ABT-737,
whereas mice that received the drug weekly exhibited
acute-onset thrombocytopenia interspersed with com-
plete recovery between injections. In the face of sustained
Bcl-xLinhibition, the age profile of platelets changes such
that the circulating population comprises primarily youn-
ger cells that are more refractory to ABT-737. With weekly
injections, the age profile instead reverts to normal as the
drug is cleared and the circulating platelet population is
therefore normosensitive to ABT-737.
Thus, ourstudies demonstrate thatthe intrinsic machin-
ery for programmed cell death (apoptosis) regulates the
life span of the anucleate platelet. It has been reported
previously that enucleated cytoplasts can undergo apo-
ptosis (Jacobson etal.,1994). To ourknowledge, platelets
are the first example of an unmanipulated anucleate cell
that is not only capable of proceeding through pro-
grammed cell death, but whose physiological life span is
governed by the interplay between pro-survival and pro-
apoptotic factors. It will be interesting to examine whether
other cells lacking a nucleus, such as erythrocytes, are
controlled by similar mechanisms.
Pharmacological Inhibition of Bcl-xL: Implications
for Targeting Pro-Survival Proteins
in Cancer Therapy
As evasion of programmed cell death is a hallmark of can-
cer (Hanahan and Weinberg, 2000), there is currently
much interest in discovering and developing pharmaco-
logical inhibitors of anti-apoptotic Bcl-2 proteins to cir-
cumvent prolonged tumor cell survival (Fesik, 2005). In
contrast to most other drugs that cause thrombocytope-
nia, either by bone marrow suppression (e.g., cytotoxics)
or by immune-mediated platelet destruction (e.g., qui-
nine), our results and those from Abbott Laboratories
(Zhang et al., 2007) demonstrate that ABT-737 is directly
cytotoxic to platelets. This represents a novel mechanism
of drug-induced thrombocytopenia. However, this is likely
to compensate by increased platelet production. Other
agents antagonizing Bcl-xL will also be anticipated to
cause thrombocytopenia, a convenient surrogate bio-
marker for Bcl-xLinhibition in the clinic.
(D) ABT-737 acts selectively on aged platelets. Representative flow cytometric profiles of Thiazole-orange-stained platelets after a single dose of
ABT-737 (red arrow); note increased proportion (percentage indicated in blue) of younger (reticulated) platelets after ABT-737 treatment.
(E) Synchronizing platelets. A single dose of anti-platelet serum (APS; blue arrow) treatment provokes an acute, severe thrombocytopenia (platelet
(blue symbols; right axis).
(F) Young platelets are resistant to ABT-737. Wild-type C57BL/6 mice were treated with APS, and then injected with ABT-737 (red arrows) either
2 or 7 days afterwards. Absolute platelet counts and the percentage of reticulated platelets were measured. The top panels show representative
flow cytometric profiles following Thiazole orange staining before or after ABT-737 injections. The bottom panels show platelet counts prior to or
2 hr post ABT-737 injection.
Data in (B), (C), (E), and (F) represent means ± SD of three to six mice at each time point.
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1181
Figure 5. The BH3 Mimetic ABT-737 Triggers Platelet Apoptosis
(A) Expression of Bcl-2 family proteins inplatelets. Lysates prepared from 50mg or 5mg plasma enriched for mouseplatelets or MEFs were probed for
Bcl-xL, Mcl-1, Bcl-2, Bak, Bax, or Actin (loading control).
(B) Genetic ablation of Bcl-xLexacerbates ABT-737-induced thrombocytopenia. Wild-type C57BL/6, Bcl-x+/?, or Bcl-2+/?mice were treated with
a single dose of ABT-737 (75 mg/kg; red arrow) and the platelet counts were determined 2–24 hr afterwards.
(C) ABT-737 triggers caspase activation in platelets. Immunoblotting for full-length intact caspase-3 (p32; top panel), cleaved p17 fragment (middle),
or gelsolin (bottom) of cell lysates prepared from freshly isolated or cultured platelets that were left untreated or after exposure to ABT-737 (1 mM) with
or without the broad-spectrum caspase inhibitor qVD.OPh (50 mM), qVD.OPh alone, or Etoposide (10 mM) is shown. ABT-737 induced complete
caspase-3 activation and gelsolin cleavage that was partially blocked by qVD.OPh.
(D) ABT-737 triggers Bak-mediated caspase-dependent loss of platelets in culture. Wild-type C57BL/6 or Bak?/?platelets were counted 1 hr after
being left untreated, or after exposure to ABT-737 (1 mM), with or without qVD.OPh (50 mM), qVD.OPh alone, or Etoposide (10 mM). Data represent
means of normalized platelet counts (untreated = 100%) ± SD of four independent experiments, using platelets pooled from six mice of each
(E) Human platelets exhibit caspase-dependent susceptibility to ABT-737 (1 mM for 1 or 2 hr). Data representation as in (D).
(F) Absence of Bak protects platelets against ABT-737. Wild-type C57BL/6, Bak?/?, and Bak?/?Bax+/?mice were treated with a single dose of
ABT-737 (75 mg/kg; red arrow) and the platelet counts were determined 0–24 hr afterwards. Bak?/?mice were unaffected by ABT-737 at early
time points (up to 4 hr), whereas Bak?/?Bax+/?mice were completely protected.
Data in (B) and (F) represent means ± SD of at least six mice at each time point.
1182 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
The Bcl-xL:Bak Axis in Platelet Disorders
Our studies raise the possibility that mutations in the key
genes controlling platelet survival account for some
cases of inherited or acquired thrombocytopenias and
thrombocytoses. They also predict that strategies to pro-
mote platelet survival by inhibiting apoptosis could be
Figure 6. In Platelets, Bak Is the Major Target Of Pro-Survival Bcl-xL
(A) Deletion of the gene encoding Bak results in thrombocytosis. Automated analysis of platelet counts in wild-type C57BL/6, Bax?/?, Bak?/?, or
Bak?/?Bax+/?mice demonstrated that loss of Bak significantly elevated platelet numbers; Bax plays a less prominent role.
(B) Normal platelet ultrastructure in Bak?/?platelets. Transmission electron microscopic images of representative platelets from wild-type (upper
panel) or Bak?/?(lower) mice are shown.
(C) Bak?/?platelets have increased life spans. Half-lives of platelets in mice of the indicated genotypes determined as described (Figure 2B and
Figure S7) are shown. Data represent means ± SD from eight mice.
(D) Genetic ablation of Bak prevents the thrombocytopenia caused by loss-of-function mutations in Bcl-xL. Platelet counts of mice with the indicated
genotypes were compared. Deletion of one Bak allele prevented thrombocytopenia in Bcl-x+/?mice, whereas the loss of both alleles resulted in
thrombocytosis indistinguishable from that caused by deletion of Bak alone.
(E) Thrombocytopenia in heterozygous Bcl-xPlt20or Bcl-xPlt16mutant mice on a mixed genetic background was prevented by loss of Bak and exac-
erbated by constitutive absence of Bcl-x.
*p < 0.05; statistical analyses in (A), (D), and (E) were performed using two-tailed unpaired Student’s t test.
Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc. 1183
advantageous in some patients with thrombocytopenia.
Conversely, patients suffering from thrombotic conditions
or thrombocytosis may well benefit from treatment with
BH3 mimetics like ABT-737 to promote platelet destruc-
tion and thus prevent the sequelae associated with path-
Our results also have potential implications for the han-
dling and storage of platelets prior to transfusion. Apopto-
tic processes have been implicated in the rapid decline in
platelet viability observed ex vivo—the platelet storage
lesion (Li et al., 2000). Indeed, while Bcl-xLlevels decline
in human platelets stored at 37?C, they do not appear to
do so in platelets subjected to routine blood bank storage
procedures at 22?C (Bertino et al., 2003). In light of our
be a key mechanism by which platelet viability is main-
tained at the lower temperature. Thus, valuable improve-
ments in platelet viability duringstorage and perhaps even
post-transfusion might be possible by stabilization of
Bcl-xLor inhibition of Bak. Several groups have examined
whether inhibition of caspases, the downstream demoli-
tion enzymes, can effectively delay storage-associated
bition was achieved, there was little impact on platelet
viability (Bertino et al., 2003; Brown et al., 2000; Cohen
et al., 2004; Li et al., 2000). This may reflect the short
half-lives of some inhibitors or their failure to completely
abolish caspase activation, since low-level activity suf-
fices for apoptosis (Methot et al., 2004). More likely,
even complete caspase inhibition may not prevent the
organellar (particularly mitochondrial) damage directly in-
iting the apoptotic cascade at the level of the Bcl-xL:Bak
axis may potentially overcome this important limitation.
Whether inhibiting platelet apoptosis and prolonging
let functions (hemostasis, clot formation, vascular remod-
eling, wound healing) remains to be addressed.
Control of Cellular Life Span: Death
as the Default State
Our results support the model that cellular life span is ge-
netically predetermined and that certain cells are intrinsi-
cally programmed to die by default in the absence of
external influences (Raff, 1992). Without biosynthetic ma-
chinery that might allow platelets to respond vigorously to
such extrinsic signals, their survival and life span is gov-
erned primarily by the stability of the apoptotic regulators
(Bcl-xLversus Bak). Although the precise molecules will
differ, this paradigm for the control of cellular life span im-
posed by the molecular regulators of the cell death ma-
chinery may well operate fordiverse cell types. Inmanyin-
stances, extrinsic survival signals, such as those provided
by growth factors, contact with neighboring cells, or stro-
mal support, may prolong cellular life spans by enhancing
the production or stability of key pro-survival Bcl-2-like
proteins to contain the pro-apoptotic activity of the long-
lived mediators Bax and Bak.
Generation and Isolation of Bcl-xPlt20and Bcl-xPlt16
Male BALB/c mice were injected intraperitoneally with three weekly
100 mg/kg doses of ENU (Sigma N3385 1g isopac). Treated mice
were mated with untreated BALB/c females to yield first-generation
(G1) progeny. At 7 weeks of age, blood from G1mice was collected
from the retro-orbital plexus into tubes containing potassium EDTA
(Sarstedt), and the number of platelets in the peripheral blood was de-
termined using an Advia 120 automated hematological analyzer
The Plt20 and Plt16 mutations were mapped by outcrossing affected
animals to wild-type C57BL/6 mice. Affected F1mice were outcrossed
to wild-type C57BL/6 mice to produce the F2generation. Genomic
DNA was collected from 40 F2animals in each case and a genome-
wide scan was performed with a panel of 80 simple sequence length
polymorphism (SSLP) markers. Candidate intervals were refined by
analyzing the products of additional meioses with MIT and in-house
CA repeat markers at increasing density.
Platelet Clearance Analysis
Mice were injected intravenously with 600 mg N-hydroxysuccinimido-
biotin (NHS-biotin) (Sigma) in buffer containing 140 mM NaCl and
10% DMSO. At various time points whole tail blood was isolated and
mixed with BSGC buffer (116 mM NaCl, 13.6 mM tri-sodium citrate,
8.6 mM Na2HPO4, 1.6 mM KH2PO4, 0.9 mM EDTA, 11.1 mM glucose).
The equivalent of 1 ml blood was washed in balanced salt solution
(BSS: 149 mM NaCl, 3.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4,
7.4 mM HEPES, 1.2 mM KH2PO4, 0.8 mM K2HPO4, 3% bovine calf se-
rum), pelleted at 1210 g for 10 min, and stained with FITC-conjugated
rat anti-CD41 (BD) and phycoerythrin-conjugated streptavidin (BD) for
1 hr on ice. Samples were washed again in BSS and flow cytometry
was performed on an LSR flow cytometer (BD).
Reticulated Platelet Labeling
Staining reactions contained 1 ml blood, 50 ml Thiazole orange
(0.1 mg/ml in PBS), 0.25 ml phycoerythrin-conjugated CD41 antibody,
and 9 ml PBS. Reactions were incubated in the dark at room tempera-
ture for 15 min before fixation by the addition of 1 ml of PBS 1% PFA.
Adoptive Platelet Transfer
Mice were injected intravenously with 600 mg NHS-biotin (Sigma) in
tion, mice were heart-bled using a heparinized syringe and two millili-
ters blood mixed with five milliliters BSGC buffer. Blood was centri-
fuged twice at 600 g for 3 min and 5 ml platelet-rich plasma was
removed each time. Pooled platelet-rich plasma was pelleted at
injection into recipient mice.
All animal experiments conformed to the regulatory standards of, and
were approved by, the Melbourne Health Research Directorate Animal
Miscellaneous Experimental Procedures
Detailsforthefollowing experimentalprocedures areprovidedwiththe
Supplemental Data: platelet aggregometry, hematological analyses,
thrombopoietin analysis, nucleic acid sequencing, flow cytometric
analysis of megakaryocytes, expression constructs, tissue culture,
cell death induction, retroviral infections and apoptosis assays, immu-
noprecipitation, immunoblotting and immunostaining, ABT-737 ad-
ministration, and ex vivo platelet assays.
1184 Cell 128, 1173–1186, March 23, 2007 ª2007 Elsevier Inc.
The Supplemental Data for this article can be found online at http://
The authors wish to thank D. Hilton and W. Alexander for generously
sharing resources and insights; Abbott Laboratories (S. Rosenberg,
unpublished data,and insights;J.Adams,P. Bouillet, S.Cory, S.Jack-
son, N. Nicola, H. Puthalakath, A. Strasser, C. Thompson, W. Steven-
son, S. Watowich, and A. Wei for comments and discussions; F. Bat-
tye, J. Corbin, L. DiRago, B. Helbert, K. Henley, J. Heraud, C. Hyland,
H. Ierino, S. Meusburger, S. Mifsud, S. Mihajlovic, M. Robati, L. Tai,
E. Tsui, and M. White for excellent technical assistance; S. Juneja
and colleagues at Royal Melbourne Hospital for platelet aggregation
assays; E. Salt, M. Sacco, T. Carle, D. Cooper, N. Iannarella, K. Pioch,
and G. Siciliano for outstanding animal husbandry; and P. Bouillet,
S. Korsmeyer, D. Kwiatkowski, Y. Lazebnik, T. Lindsten, D. Loh, N.
Motoyama, A. Strasser, and C. Thompson for mouse strains and
reagents. Our work is supported by the Cancer Council of Victoria
(K.D.M., Post-Graduate Research Scholarship; D.M., Carden Fellow-
ship), the Australian Research Council (B.T.K., Queen Elizabeth II
Fellowship); Australian NHMRC (Program Grants 257502, 257500;
(CA80188, CA43540); and Leukemia & Lymphoma Society (SCOR
7015-02). B.T.K. is a consultant to, and this work was supported in
part by, Murigen Pty Ltd.
Received: October 4, 2006
Revised: December 4, 2006
Accepted: January 6, 2007
Published: March 22, 2007
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