Chemico-Biological Interactions 284 (2018) 1–11

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Drug-induced thrombocytopenia: Focus on platelet apoptosis T

a,b a,b,c,∗
Enoli De Silva , Hugh Kim
a
Centre for Blood Research, University of British Columbia, Vancouver, Canada
b
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada
c
Faculty of Dentistry, University of British Columbia, Vancouver, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Thrombocytopenia is a serious and potentially fatal complication of drug therapy that results either from a
Drugs decrease in bone marrow platelet production or the excessive destruction of circulating platelets. Although
Thrombocytopenia multiple mechanisms are responsible for deregulated platelet clearance, the role of programmed platelet death
Platelets (apoptosis) in drug-induced thrombocytopenia has been relatively under-investigated until recently. Here we
Apoptosis
review apoptotic signaling pathways in platelets, with a focus on current data that provide mechanistic insights
into drug-induced apoptosis and thrombocytopenia.

1. Introduction configurations of the drug and antibody molecules interacting at the

Platelets are anucleate blood cells essential for hemostasis and
wound healing; tight regulation of platelet numbers is crucial for
human health. Platelets are synthesized and released from bone marrow
megakaryocytes into the circulation where they remain for 7–10 days
[1]. In humans, the normal circulating platelet count ranges from
150,000–450,000 platelets/μl of whole blood. A reduction in platelet
count below 150,000 platelets/μl is termed thrombocytopenia, a con-
dition that increases the risk of potentially life-threatening hemorrhage
[2]. Drug-induced thrombocytopenia (DIT) can occur following ad-
ministration of a wide range of medications, including antibiotics,
cardiac drugs and anti-neoplastic agents [3]. DIT-inducing agents can
perturb platelet counts in one of 2 ways: centrally, by exerting cytotoxic
effects on bone marrow megakaryocytes, thus reducing platelet synth-
esis (Fig. 1A); or peripherally, by enhancing clearance of platelets al-
ready in circulation (Fig. 1B). Drugs can accelerate the destruction of
circulating platelets through both immune- and nonimmune-mediated
mechanisms.
2. Immune-mediated platelet destruction
In immune-mediated platelet destruction, drug-dependent anti-
bodies bind to platelets, either directly or alongside accessory proteins
[4]. Platelets are then phagocytosed by macrophages that recognize the
drug-dependent antibody on the platelet surface. The subject of im-
mune-mediated DIT is covered in-depth by several excellent review
articles [3,5–7] and as such is only briefly mentioned here. There are 6
subtypes of immune-mediated DIT that reflect different spatial
platelet surface. These subtypes of immune-mediated platelet destruc-
tion are summarized in Table 1.
3. Nonimmune-mediated platelet destruction
In contrast to immune-mediated DIT, nonimmune-mediated platelet
destruction is described as a direct cytotoxic effect of the drug mole-
cules on the platelets. For example, thrombocytopenia was previously
reported to result from the chemotherapeutic administration of inter-
feron-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). The
purported mechanism was attributed to altered platelet-endothelial cell
interactions, and therefore, antibody-independent [8,9]. However, over
the past 7 years, considerable evidence has emerged in support of an-
other form of nonimmune-mediated platelet destruction: platelet
apoptosis, or programmed cell death. Published evidence from multiple
groups clearly indicate that thrombocytopenia-inducing drugs at-
tenuate platelet numbers by triggering pro-apoptotic signaling.
4. Pro-apoptotic signaling in platelets
Apoptosis is a programmed form of cell death that results in a
controlled clearance of cells by macrophages, without eliciting an in-
flammatory response. Apoptosis is essential for the removal of damaged
cells from the body and maintaining appropriate cell populations in
developing tissues [10]. This form of cell death contrasts with necrosis,
which is characterized by the uncontrolled rupture and release of cel-
lular contents [11], resulting in tissue damage. The recognition that
platelets undergo apoptosis is a relatively new concept that has been

∗
Corresponding author. UBC Centre for Blood Research, 2350 Health Sciences Mall, 4th Floor, Vancouver, BC, V6T 1Z3, Canada.
E-mail address: hughkim@dentistry.ubc.ca (H. Kim).

https://doi.org/10.1016/j.cbi.2018.01.015
Received 31 July 2017; Received in revised form 23 December 2017; Accepted 18 January 2018
Available online 02 February 2018
0009-2797/ © 2018 Elsevier B.V. All rights reserved.
E. De Silva, H. Kim Chemico-Biological Interactions 284 (2018) 1–11

Fig. 1. Platelet production and clearance. A. Decreased platelet production by megakaryocytes (MK) in the bone marrow can result from impaired megakaryopoiesis, thrombopoietin
(TPO) signaling defects and drug-induced myelosuppression. B. Circulating platelets may be prematurely cleared due to sequestration in the spleen, autoantibodies against platelet
glycoproteins and drug-induced platelet destruction via both immune- and non-immune mediated mechanisms. De-regulation of platelet production and/or clearance can lead to
thrombocytopenia.

Table 1
Mechanisms of immune-mediated platelet destruction.

Name Mechanism Typical drug Refs

Hapten-induced antibody • Drug (hapten) covalently binds to the amine groups of proteins, which elicits an immune response Antibiotics [3,5]
Drug-dependent antibody • Drug associates with platelet glycoproteins that is then targeted by antibodies Quinine [3,5]
• Drug exposes glycoprotein sites that are readily recognized by the immune system
• Immune response requires a soluble drug
Drug-induced autoantibodies • Autoantibody directly targets platelets Procainamide [5,9]
• Drug exposes glycoprotein sites that are readily recognized by the immune system, which triggers production
of autoantibodies
Immune complex • complex
Heparin binds and structurally modifies platelet factor 4 causing the formation of antibodies against this Heparin [3,5,97]

Drug-specific antibody • Aoccurring

chimeric (human/mouse) Fab domain that binds βA domain of GPIIIa, which is recognized by a naturally
antibody or drug-sensitive antibody
Abciximab [3,5,97]

Fiban-induced thrombocytopenia • Drug binds to the arginine-glycine-aspartic acid (RGD) recognition site on platelet GPIIb/IIIa receptor, which is
recognized by a naturally occurring antibody
Tirofiban [3,5]

increasingly documented over the past 10–15 years [12–14].
As is observed in nucleated cells, apoptotic platelets typically ex-
hibit cytoplasmic shrinkage, membrane protrusions known as “blebs”
and increased cell surface exposure of phosphatidylserine (PS) [10,15]
which denotes a cell's slated clearance by phagocytes [16]. Pro-apop-
totic signaling pathways are classified as extrinsic or intrinsic (Fig. 2).
Extrinsic apoptosis is specifically mediated by extracellular ligand
binding to “death” receptors, however, both pathways culminate in the
activation of caspases 3 and 7, cysteine proteases that orchestrate cell
death by degrading cellular proteins. Therefore, the de-regulation of
normal apoptotic signaling mechanisms would plausibly explain am-
plified platelet destruction observed in drug-induced thrombocyto-
penia.
signaling cascade leading to caspase-8 activation, followed by caspase-3
activation and apoptosis [10]. There is relatively little evidence to
suggest that platelets undergo extrinsic apoptosis as they do not express
the prototypical Fas death receptor [13]. However, this contention is
mitigated by the finding that platelets do express caspase-8, a marker of
extrinsic apoptosis [17]. Moreover, thrombin, a platelet agonist and
ligand for the protease-activated receptor-1 (PAR-1) [18], reportedly
induces caspase-3 activation and cell death [19]. This suggests the ex-
istence in platelets of an extrinsic apoptotic pathway that operates in-
dependently of the classical death receptors. Consequently, the precise
determinants of receptor-mediated platelet apoptosis remain undefined
at this time.

4.2. Intrinsic apoptosis

4.1. Extrinsic (receptor-mediated) apoptosis
The extrinsic apoptosis pathway is initiated by the binding of ex-
tracellular ligands with cell surface receptors. For example, members of
the tumor necrosis factor (TNF) receptor superfamily (FasR, TNFR1,
DR3, DR4 and DR5) are activated by their respective ligands (FasL,
TNF-α, Apo3L, Apo2L, Apo2L) [10]. Death receptor ligation triggers a
In contrast to the extrinsic apoptosis pathway, intrinsic apoptosis is
a receptor-independent process initiated by stimuli such as radiation,
toxins, free radicals or an increase in intracellular calcium concentra-
tion ([Ca2+]i) [10]. A central feature of intrinsic apoptosis is depolar-
ization of the mitochondria, and this is regulated by several pro-apop-
totic and anti-apoptotic accessory proteins (Fig. 2).

2
E. De Silva, H. Kim Chemico-Biological Interactions 284 (2018) 1–11

Fig. 2. The intrinsic apoptosis pathway. Schematic diagram depicts the steps in the intrinsic apoptosis pathway. BH3-only domain pro-death proteins are induced by cellular stress and/or
pro-apoptotic chemical stimuli (1). In the resting cell, multi-domain pro-survival proteins suppress activation of multi-domain pro-apoptotic proteins, Bax/Bak (depicted in red).
Activated BH3-only domain pro-death proteins can activate Bax/Bak via two mechanisms: by inhibiting Bcl-2 pro-survival proteins thereby removing their suppression of Bax/Bak (2a);
or by directly activating Bax/Bak (2b). Activated Bax translocates to the mitochondria and oligomerizes on the mitochondrial membrane (3). Bax/Bak mediate mitochondrial outer
membrane permeabilization (MOMP) (4). The loss of mitochondrial potential and release of cytochrome c into the cytosol (5). Cytosolic Apaf-1 binds cytochrome c, and recruits and binds
pro-caspase-9; this assembly is known as the apoptosome (6). The apoptosome activates caspase-9 (7). Activated caspase-9 subsequently activates caspase-3/ −7 (8), leading to cell death.
(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4.2.1. Bak/Bax and mitochondrial depolarization and outer membranes is termed the intermembrane space (IMS)
The mitochondrion is essential for regulating cell survival. Its ul- (Fig. 3A) [20]. The OM allows passage of specific cytosolic molecules
trastructure consists of a central matrix surrounded by an inner mem- through the voltage-dependent anion channel (VDAC). The IM contains
brane (IM) and an outer membrane (OM). The space between the inner a complex of proteins, called the electron transport chain (ETC), that

Fig. 3. Mitochondria-mediated apoptosis. A. The mitochondrial ultrastructure consists of the matrix, the inner membrane (IM), outer membrane (OM) and intermembrane space (IMS). B.
The mitochondrial permeability transition pore (MPTP) consists of the Voltage Dependent Anion Channel (VDAC), Adenine Nucleotide Translocator (ANT) and Cyclophilin D (CypD)
components. Under apoptotic conditions, the pore complex transports water and large molecules from the cytosol into the matrix. Consequently, the matrix swells and ruptures the IM and
OM. This results in the release of cytochrome c located in the intermembrane space (IMS). The MPTP can also lead to the dissipation of the H+ gradient that leads to mitochondrial
depolarization. C. Bak/Bax localized to the mitochondrial outer membrane (OM) can homo-oligomerize thus facilitating cytochrome c release.

3
E. De Silva, H. Kim
donate/accept electrons to build a proton gradient between the matrix
and IMS [20]. This proton gradient maintains the mitochondrial elec-
trical potential characteristic of cells in their resting state.
Intrinsic apoptosis is associated with a marked increase in mi-
tochondrial membrane permeability, due to the formation of a mi-
tochondrial permeability transition pore (MPTP) complex (Fig. 3B). The
MPTP permeabilizes the IM to the larger solutes of the IMS, which
eventually leads to the swelling of the matrix and loss of mitochondrial
potential (depolarization) [20,21]. This increased permeability also
releases pro-apoptotic factors, namely cytochrome c, into the cytosol
[20,21]. Cytochrome c then binds to apoptotic protease-activating
factor (Apaf-1) to form a complex called the ‘apoptosome’ [20]. The
apoptosome recruits and autoactivates caspase-9, which subsequently
activates caspase-3 to execute cell death.
The MPTP complex is regulated by pro-apoptotic proteins, notably,
activated Bax and Bak [22]. Activated Bax and Bak may homo-oligo-
merize into pore complexes on the OM facilitating the release of mi-
tochondrial cytochrome c [21,23] (Fig. 3C). Consequently, intrinsic
apoptosis is also marked by MPTP formation, mitochondrial translo-
cation of Bax, mitochondrial depolarization and cytochrome c release
[15]. A second group of inhibitory proteins, termed Bcl-2 proteins, bind
Bax and Bak to prevent their translocation to the mitochondria [14,24],
thus exerting an anti-apoptotic effect (Fig. 2).
4.2.2. Calcium (Ca2+) signaling
Mitochondrial depolarization is also triggered by excessive accu-
mulation of intracellular Ca2+ that is transferred to the mitochondria
[25,26]. Under apoptotic conditions, Ca2+ is released by the en-
doplasmic reticulum (ER). Importantly, platelets contain an ER-like
dense tubular system (DTS) that serves as an intracellular Ca2+ store
[27]. Uptake of excess intracellular Ca2+ (Ca2+
i ) by the mitochondria
leads to mitochondrial swelling and depolarization. In addition to an-
tagonizing the pro-apoptotic Bak and Bax proteins, the pro-survival Bcl-
2 proteins also prevent apoptosis by reducing the amount of releasable
Ca2+ from the ER and mitochondria [28]. A decreased Bcl-2/Bax (pro-
survival/pro-apoptotic) protein ratio was noted in platelets treated with
calcium ionophore [29], consistent with notion that Ca2+ influxes
trigger apoptosis.
4.2.3. Reactive oxygen species (ROS)
ROS are a family of oxygen-containing chemically reactive mole-
cules. This family includes short-lived free radicals such as hydroxyl
(•OH), alkoxyl (RO•) or peroxyl (ROO•), and medium lifetime radicals
such as superoxide (O2•) or nitroxyl radical (NO•). Other ROS include
hydrogen peroxide (H2O2), organic hydroperoxides (ROOH) and hy-
pochlorous acid (HOCl) [30]. The accumulation of ROS in cells can
promote excessive apoptosis. For example, ROS can target the mi-
tochondria by oxidizing lipids on the mitochrondrial IM thereby en-
hancing cytochrome c release [31] and mitochondrial depolarization
[32]. Platelet apoptosis can be triggered both by platelet-derived or
vasculature-derived ROS [33]. Treating platelets with H2O2 evokes the
major hallmarks of apoptosis [27,34]. Conversely, inhibition of H2O2
by catalase precludes PS exposure and caspase activation [27]. H2O2
treatment reportedly promotes the mitochondrial translocation of the
pro-apoptotic proteins Bid and Bax [34]. ROS can also activate other
signaling pathways, such as the mitogen activated protein kinase
(MAPK) pathways, which also have documented pro-apoptotic effects
[35].
4.2.4. MAP kinase (MAPK) signaling
Mitogen-activated protein kinases (MAPK) signaling controls cell
proliferation and survival [36]. MAPK signaling entails the sequential
phosphorylation and activation of downstream target proteins. There
are 3 MAPK modules associated with apoptosis: the c-Jun NH2 terminal
kinase (JNK) pathway; the p38 MAPK pathway; and the extracellular
signal regulated kinase (ERK) pathway. In the JNK pathway, pro-
4
Chemico-Biological Interactions 284 (2018) 1–11
apoptotic signals such as DNA damage or ROS, activate MKK4/7 kinase,
which then activates JNK. Activated JNK upregulates pro-apoptotic
proteins such as Bad thus promoting mitochondrial depolarization and
apoptosis [37]. Similarly, p38 MAPK is activated by the MKK3/6 ki-
nase, whose purported downstream effects include cell cycle arrest and
apoptosis [36]. Finally, ERKs are activated upstream by phosphoryla-
tion by MEK1/2. ERK activation can directly stimulate the mitochon-
drial pathway of apoptosis, including cytochrome c release and caspase
activation [38].
4.2.5. Phosphoinositide 3-kinase (PI3K) signaling
PI3K phosphorylates the 3′ position of the inositol ring of phos-
phoinositides, which activates phosphoinositide-dependent kinase 1
(PDK1) kinase and protein kinase B (PKB) [39]. In nucleated cells, ac-
tivated PDK1 phosphorylates and activates PKB, which promotes cell
survival by phosphorylating and inactivating the pro-apoptotic protein
Bad [39].
4.3. Caspase-independent apoptosis
While caspase activation is a well-documented endpoint of pro-
apoptotic signaling, it should be noted that programmed platelet death
can also occur in a caspase-independent manner. Wolf and coworkers
noted that calcium ionophore treatment of platelets induces the cell
shrinkage, plasma membrane microvesiculation and PS externalization
that are characteristic of apoptosis [40]. However, the authors also
noted that this Ca2+-driven apoptosis was mediated by the protease
calpain and was not contingent on caspase activation [40]. In an in vitro
study of co-cultured platelets and macrophages, Brown et al docu-
mented that aging platelets expressed increased Bak/Bax and PS ex-
posure but that the clearance of platelets by macrophages did not re-
quire caspase-3 activity [41]. Collectively, these findings indicate that
Ca2+ signaling fluxes can induce platelet apoptosis and clearance in-
dependently of caspase activity.
Key points: apoptotic signaling in platelets (Fig. 2).
• During platelet apoptosis, the pro-apoptotic proteins Bak and Bax
bind and permeabilize the mitochondrial membrane. The resultant
leaching of cytochrome c forms an Apaf-1/caspase-9 protein com-
plex (apoptosome) that activates caspase-3, causing cell death.
• The pro-apoptotic activity of Bak and Bax are constrained by a group
of proteins, termed “pro-survival” Bcl-2 proteins (Bcl-2, Bcl-xL).
• Another group of proteins known as BH3-only proteins (e.g. Bad,
Bim, Bik, Puma), displace Bcl-2 proteins from Bak/Bax, thereby
promoting apoptosis.
• Apoptotic platelets typically exhibit mitochondrial depolarization,
increased intracellular Ca2+ ([Ca2+]i) and surface PS exposure.
5. Drugs with documented pro-apoptotic effects on platelets
More than 200 drugs are reported to cause thrombocytopenia
[5,42]. The concept of platelet apoptosis is relatively new and was only
recently evaluated in the context of drug-induced thrombocytopenia
[43]. Table 2 lists the drugs presently known to induce apoptosis in
platelets. Below we provide a detailed review of the drugs' putative pro-
apoptotic mechanisms, based on currently available experimental evi-
dence.
5.1. Acetylsalicylic acid (aspirin)
Aspirin is a common analgesic and anti-inflammatory drug used for
thrombosis prevention by 24–35% of adults over the age of 50 [44].
The side effect of aspirin-induced thrombocytopenia was documented
as early as 1974 [45]. Recently published data by Zhao and colleagues
suggest that this may occur via apoptosis [46]. Using fluorescent dyes
to measure mitochondrial potential and PS exposure in washed human
 Table 2
Thrombocytopenia-inducing drugs with documented pro-apoptotic effects on platelets.

Drug class Drug name Clinical Indication Pro-apoptotic mechanism(s) of action Refs
E. De Silva, H. Kim

NSAID Aspirin Analgesia, anti-inflammatory, thrombosis prevention • Induction of mitochondrial depolarization, ROS generation and caspase-3 activation [46,47]
• Conformational modification of Bax that promotes Bax insertion into the outer mitochondrial
membrane
2+ 2+
Antibiotic Balhimycin Infection Increased intracellular Ca
• caspase-3 levels ([Ca ] ), induction of mitochondrial depolarization and
i [51]
activation
of pro-apoptotic ceramides
2+
Antibiotic Vancomycin Infection
• Formation
Increased [Ca ] , induction of mitochondrial depolarization, ceramide formation, increased
i [50]
• caspase-3 activity
2+ 2+
Antipsychotic Trifluoperazine Schizophrenia • Inhibition of calmodulin, thus inducing apoptosis by increasing [Ca ] and mitochondrial Ca
i [25]
overload
• Induction of mitochondrial depolarization, caspase-3 activation
Anti-neoplastic Carmustine Brain tumors • Induction of mitochondrial depolarization and caspase-3 activation [62]
• Upregulation of pro-apoptotic Bax; downregulation of anti-apoptotic Bcl-2
• c-Jun NH2-terminal kinase (JNK)-mediated mitochondrial depolarization
Anti-neoplastic ABT-737 (in development) • Main target Bcl-x < SUB > L < /SUB > [64,98–100]
• Induction of mitochondrial depolarization and release of cytochrome c, caspase-9, -8, -3 activation
Anti-neoplastic Cisplatin Various forms of cancer • Increased expression of pro-apoptotic Bax/Bak, decreased expression of anti-apoptotic Bcl-2/Bcl- [57,101]
X < SUB > L < /SUB >
• Increased binding of active Bax to mitochondrial membrane
of mitochondrial depolarization, caspase-3 activation, ROS production, increased
2+
• Induction
[Ca ] i
by MEK/ERK signaling but not p38 MAPK or JNK signaling
2+
Anti-neoplastic Tamoxifen Breast cancer
• Mediated of mitochondrial depolarization, caspase-3 activation, PS exposure, increased [Ca ]i [25]
Anti-neoplastic Navitoclax (ABT-263) (in development)
• Induction
with high affinity to Bcl2, Bcl-xL, Bcl-w, thus precluding their anti-apoptotic functions. [64,100]
2+
• Bind intracellular Ca by depletion of intracellular calcium stores (ER)

5
2+
Anti-neoplastic, immunosuppressant Methotrexate Hematologic cancers; rheumatoid arthritis
• Increased
generation, Ca release from ER, peroxidation of cardiolipin [59]
• ROS of mitochondrial depolarization, cytochrome c release, active caspase-9/ −3
• Induction expression of Bad, Bax, tBid; suppression of Bcl-2
• Increased
induces increased expression of Bad, Bax, suppression of anti-apoptotic Bcl-2 proteins,
• JNK
caspase-3 activation
2+
Anti-neoplastic NF-κB inhibitors Adenocarcinoma • ER stress, Ca release from ER, eIF2-α phosphorylation [67]
• MPTP formation, mitochondrial depolarization, cytochrome c release, caspase-3 activation, PS
exposure
• Decreased Bcl-2 levels, increased Bax levels
Anti-neoplastic Lovastatin Malignant glioma • Mitochondrial depolarization, upregulation of Bak, downregulation of Bcl-xL, caspase-3/-9 [71]
activation
• Integrin IIb 3
α β -mediated caspase-8 activation
Anti-neoplastic Doxorubicin Various forms of cancer • Mitochondrial translocation of Bax and mitochondrial depolarization, cytochrome c release, [76]
caspase-3 activation, PS exposure
• Augmented levels of mitochondrial ROS and cardiolipin peroxidation
Anti-neoplastic Bexarotene Colon, breast and lung cancer • Augmented thrombin and/or CRP stimulated intracellular calcium concentration [73]
• Caspase-3 IIb 3
activity, PS exposure and α β integrin activity
Anti-neoplastic Arsenic trioxide (ATO) Acute promyelocytic leukemia • Mitochondrial depolarization, upregulation of Bax, downregulation of Bcl-X < SUB > L < / [63]
SUB > , caspase-3 activation, PS exposure
• Induces apoptosis in a JNK-dependent manner
Food/consumable Ethanol Alcoholic beverages • Induction of mitochondrial depolarization and caspase-3 activation [83]
• Upregulation of Bax; downregulation of Bcl-2
of JNK
2+
Hormonal supplement Melatonin Purported benefits in regulating immunological,
• Activation ROS and H O levels, increase intracellular Ca
2 2 by depletion of intracellular stores [85]
cellular and endocrine responses
• Increased of mitochondrial depolarization, cytochrome c release, caspase-9 and -3 activity
2+
Natural derivative (black seed oil) Thymoquinone Purported benefits for multiple conditions, including
• Induction caspase-3 activity and [Ca ] < SUB > I < /SUB > [88]
cancer
• Increased of mitochondrial depolarization; increased pro-apoptotic ceramide formation
2+
• Induction i
• PI3K-mediated caspase-3 activation, increase in [Ca ] and mitochondrial depolarization (continued on next page)
Chemico-Biological Interactions 284 (2018) 1–11
E. De Silva, H. Kim Chemico-Biological Interactions 284 (2018) 1–11

platelets, the authors determined that aspirin induced platelet apoptosis

in a dose-dependent manner. Increased caspase-3 activation was also
[84,102]
noted in aspirin-treated platelets relative to controls. A separate study

[91]

[17]
Refs

documented that aspirin-treated platelets exhibit increased ROS gen-

caspase-3 and caspase-8 activity (not via classical death receptors), cleavage of Bid into eration and higher levels of the active form of the pro-apoptotic protein

of mitochondrial depolarization via both intrinsic and extrinsic pathways, caspase-9 and
Bax [47]. While aspirin's anti-thrombotic activity is attributed to the
caspase-9/-3 activity, induction of mitochondrial depolarization and cytochrome c

inhibition of cyclooxygenase (COX) synthesis [48], recent data raise the

possibility that aspirin's antithrombotic effects may be achieved by re-
• Bax translocation from cytosol to mitochondrial membrane, cytochrome c release ducing platelet numbers via apoptosis.

5.2. Antibiotics: vancomycin and balhimycin

ROS and H2O2 levels, increased [Ca ] < SUB > I < /SUB >

Antibiotics are another class of drugs associated with thrombocy-

topenia [49] although this was previously attributed to an antibody
-3 activation via caspase-8 activation, cleavage of Bid into tBid,

(hapten)-dependent mechanism [6] (Table 1). Two recent studies

evaluated the pro-apoptotic effects of antibiotics on human platelets
[50,51]. In these studies, treatment of platelets with vancomycin [50]
and balhimycin [51] significantly induced mitochondrial depolariza-
2+

tion, caspase-3 activation and PS exposure. Moreover, both antibiotics

stimulated the production of ceramides, which are lipids with docu-
of mitochondrial depolarization

mented pro-apoptotic functions [52]. In addition, increases in in-

tracellular [Ca2+] ([Ca2+]i) were observed in vancomycin- and bal-
Pro-apoptotic mechanism(s) of action

himycin-treated platelets loaded with the calcium indicator dye Fluo 3/

AM [50,51]. Interestingly, antibiotic-induced PS exposure, the endpoint
marker for apoptosis, was abrogated following calcium chelation with
tBid (BH3-only protein)

EDTA. However, PS exposure was not affected by caspase-3 inhibition

with zVAD-FMK, suggesting that [Ca2+]i influx, and not caspase-3 ac-
tivation, is the critical signaling event during antibiotic-induced platelet
apoptosis. These data suggest that vancomycin induces apoptosis in
• Induction
• Induction
• Increased
• Increased
• Increased

platelets in a caspase-independent manner (see section 4.3 above).

release

Further research is required to determine whether other antibiotics

induce thrombocytopenia through a comparable mechanism.

5.3. Anti-neoplastic agents

More than 100 chemotherapeutic drugs are currently employed as

anti-cancer agents [53]. Thrombocytopenia is a common side effect to
chemotherapy, due to the drugs' suppression of bone marrow and
thrombopoiesis [54]. However, emerging evidence published over the
Purported anti-oxidant properties

Purported anti-oxidant properties

Chronic inflammatory diseases

past 5 years suggests that chemotherapeutic agents may target circu-

lating platelets and induce their death by apoptosis.

5.3.1. Tamoxifen
Clinical Indication

Tamoxifen is a well-known chemotherapeutic used in the treatment

of breast cancer [55]. Tamoxifen-treated human platelets reportedly
exhibit increased mitochondrial depolarization and PS exposure; the
drug's antagonism of calmodulin (CaM) was proposed as the mechanism
of action [25]. CaM is a Ca2+-binding protein that modulates Ca2+-
dependent signaling processes [56] and also regulates cytosolic calcium
concentration [25]. These data suggest that tamoxifen induces in-
Andrographolide

tracellular Ca2+ fluxes by antagonizing CaM, thus promoting platelet

apoptosis.
Resveratrol
Drug name

Sesamol

5.3.2. Cisplatin
A more complete mechanistic description of drug-induced apoptosis
was provided for the anti-cancer agent cisplatin [57]. Consistent with
Natural derivative (plant-derived)

Natural derivative (plant-derived)

the findings reported in other studies of washed human platelets, cis-

Natural derivative (sesame seeds)

platin treatment induced mitochondrial depolarization, caspase-3 acti-

vation, Ca2+
i influx and PS exposure [57]. Moreover, as was observed in
vancomycin-induced apoptosis [50], cisplatin-driven platelet apoptosis
was shown to be Ca2+-dependent. However, the authors reported four
Table 2 (continued)

(4) additional features of cisplatin-induced platelet apoptosis. Firstly,

immunoblots of cisplatin-treated platelets showed increased expression
Drug class

of the pro-apoptotic Bax and Bak proteins relative to untreated controls.

Conversely, levels of the anti-apoptotic proteins Bcl-2 and Bcl-XL are
attenuated following cisplatin exposure. Secondly, immunoblot analysis

6
E. De Silva, H. Kim
of mitochondrial fractions revealed increased mitochondria-associated
Bax in cisplatin-treated platelets, indicating that cisplatin drives the
translocation of Bax to the mitochondria. Thirdly, the authors identified
the MEK/ERK signaling pathway as the target for cisplatin-induced
apoptosis, since mitochondrial depolarization, caspase-3 activation and
PS exposure were abrogated in platelets pre-treated with the MEK in-
hibitor PD98059 [57]. Fourthly, cisplatin reportedly promotes ROS
generation by platelets, as quantified by a fluorescent ROS-labeling dye;
interestingly, quenching of ROS by a reducing agent (DTT) also nulli-
fied cisplatin-induced apoptosis [57]. Consequently, this study provides
a thorough description of a drug-induced apoptotic pathway that is
simultaneously dependent on ROS generation, as well as Ca2+ and
MEK/ERK signaling.
5.3.3. Methotrexate, carmustine and arsenic trioxide (ATO)
Methotrexate is an anti-cancer drug that is also used to treat rheu-
matoid arthritis [58] and whose role in platelet apoptosis was recently
documented [59]. Methotrexate treatment induced endogenous ROS
production in platelets, as previously reported with cisplatin [57]. Mi-
tochondrial depolarization, increased [Ca2+]i, caspase-3 activation and
PS exposure were all observed in methotrexate-treated platelets [59]. In
this study, methotrexate induced JNK phosphorylation; this effect was
nullified by the JNK inhibitor dicumarol as well as the ROS antagonist
Mito-TEMPO. Further, methotrexate treatment upregulated the ex-
pression of the pro-apoptotic Bad and Bax proteins while down-
regulating expression of the anti-apoptotic Bcl-2. These findings were
reversed following JNK inhibition, thus indicating that methotrexate
induces platelet apoptosis by specifically targeting the JNK pathway in
a ROS-dependent manner [59]. Similar pro-apoptotic mechanisms were
reported for carmustine, an alkylating chemotherapeutic used to treat
several different cancers, including brain tumors and lymphomas [60]
as well as for arsenic trioxide (ATO), a highly potent anti-neoplastic
drug used to treat acute promyelocytic leukemia [61]. As observed with
other drugs, carmustine and ATO induce mitochondrial depolarization,
upregulation of Bax, downregulation of Bcl-2 and caspase-3 activation
in human platelets [62,63]. Like methotrexate, carmustine- and ATO-
induced mitochondrial depolarization are reportedly nullified in the
presence of dicumarol, and thus appear to be JNK-dependent [62,63].
5.3.4. Bcl-2-inhibiting drugs
Another class of anti-cancer drugs target the anti-apoptotic Bcl-2
proteins, which bind and inhibit the pro-apoptotic proteins Bak and Bax
[24]. ABT-737, and its oral analogue, ABT-263 (Navitoclax), bind to
Bcl-2, thus nullifying its anti-apoptotic effect. Vogler et al observed that
ABT-737 and ABT-263 induced mitochondrial depolarization, increased
[Ca2+]i caspase-3 activation and PS exposure in platelets [64]. Another
group reported that ABT-737 activates the p38 MAP kinase pathway in
platelets, and that drug-induced mitochondrial depolarization, caspase-
3 activation and PS exposure were all attenuated by SB202190, a p38
MAPK inhibitor [65]. In addition, it was noted that ABT-737-induced
platelet apoptosis is caspase-3-dependent, in contrast with the caspase-
3-independent mechanism observed with vancomycin-induced platelet
apoptosis [50]. Collectively, these data underscore the heterogeneous,
multi-pronged (and possibly drug-specific) nature of pro-apoptotic
signaling pathways (Fig. 4).
5.3.5. Nuclear factor kappa B (NF-κB) inhibitors
The NF-κB family of proteins regulate gene transcription and pro-
mote cell survival; consequently, the NF-κB signaling pathway is a
target for cancer therapy [66]. Recent evidence suggests that NF-κB
inhibition via the drugs BAY11-7082 and MLN4924 induces platelet
apoptosis by promoting endoplasmic reticulum (ER) stress [67].
ER stress is characterized by the accumulation of unfolded proteins
in the ER whose downstream effects include apoptosis [68] and phos-
phorylation of eukaryotic initiation factor 2 alpha (eIF2α). Paul et al
evaluated platelets treated with NF-κB inhibitors and found that these
7
Chemico-Biological Interactions 284 (2018) 1–11
drugs promoted increased [Ca2+]i, and eIF2α phosphorylation [67].
Platelets treated with BAY11-7082 and MLN4924 also exhibited, in a
dose-dependent manner, decreased Bcl-2 expression, increased Bax
expression, MPTP formation, cytochrome c release and caspase-3 acti-
vation. Importantly, the mitochondrial depolarization and PS exposure
induced by the NF-κB inhibitors were rescued by incubating the pla-
telets with an ER stress inhibitor (Salubrinal), further supporting the
notion that NF-κB inhibition drives platelet apoptosis via ER stress [67].
5.3.6. Lovastatin
Lovastatin is a member of the statin family of drugs used to lower
blood cholesterol, with additional applications as an anti-cancer ther-
apeutic [69,70]. Recently published data indicate that washed human
platelets treated with lovastatin displayed dose-dependent increases in
mitochondrial depolarization, upregulation of Bak, downregulation of
Bcl-xL and activated caspase-3/-9, relative to controls [71]. A notable
finding from of this study was the abrogation of mitochondrial depo-
larization in the presence of an αIIbβ3 integrin-blocking peptide, sug-
gesting that lovastatin targets the αIIbβ3 integrin complex (also termed
glycoprotein IIbIIIa) to induce platelet apoptosis since [71]. These data
provide additional evidence in support of receptor-mediated platelet
apoptosis that is independent of the classical Fas death receptors.
5.3.7. Bexarotene
Bexarotene is a stimulator of retinoid X receptors that is used as a
chemotherapeutic agent against several different types of cancer [72].
The pro-apoptotic effects of bexarotene were recently documented in
mouse platelets stimulated concurrently with thrombin or collagen-re-
lated peptide (CRP) [73]. Cao et al found that thrombin and CRP in-
duced caspase-3 activity, [Ca2+]i and PS exposure, and that these ef-
fects were significantly enhanced in the presence of bexarotene [73].
Interestingly, these data also suggest that bexarotene, like lovastatin,
targets the αIIbβ3 integrin complex (GPIIbIIIa), since CRP-induced
αIIbβ3 activation was slightly increased in the presence of bexarotene
[73].
5.3.8. Doxorubicin
Doxorubicin (DOX) is another chemotherapeutic drug used for the
treatment of multiple forms of cancer [74]. As is observed with many
anti-neoplastic agents, DOX is associated with thrombocytopenia [75].
In a report by Wang and colleagues, DOX-treated platelets exhibited
increased mitochondrial translocation of Bax and mitochondrial depo-
larization, which was also associated with cytochrome c release, cas-
pase-3 activation, PS exposure and increased ROS generation [76].
Importantly, DOX-induced apoptotic markers in platelets were in-
hibited by Mito-TEMPO, a mitochondrial reactive oxygen species (ROS)
antagonist [76]. These data imply that mitochondrial ROS play a cen-
tral role in DOX-induced platelet apoptosis.
5.4. Food-derived, natural and herbal substances
Alternative medicines, including herbal medicines, are commonly
used to manage symptoms associated with chronic pain [77], ulcerative
colitis [78] and even menopause [79]. As is the case with medications
prescribed by health care providers, these natural remedies may confer
significant side effects, such as thrombocytopenia [80]. Published data
from the past 5 years demonstrate the ability of several naturally-de-
rived products to induce apoptosis in platelets.
5.4.1. Ethanol
Chronic alcohol use has long been associated with an increased
susceptibility for bleeding and thrombocytopenia [81,82]. Newly pub-
lished work identifies ethanol as a pro-apoptotic stimulus for platelets
[83]. Washed platelets treated with ethanol exhibit increases in mi-
tochondrial depolarization, caspase-3 activation, as well as up- and
down-regulation of Bax and Bcl-2, respectively [83]. The authors also
E. De Silva, H. Kim Chemico-Biological Interactions 284 (2018) 1–11

Fig. 4. An overview of known platelet apoptosis-inducing drugs and pertinent signaling pathways. The extrinsic pathway includes caspase-8 activation, which can be induced by ABT-
737, andrographolide, lovastatin and resveratrol. The intrinsic pathway is defined by several key features, which can be induced by the following drugs: up-regulation of Bax expression
induced by aspirin, doxorubicin, Navitoclax, ethanol, ABT-737 and resveratrol; ROS production induced by aspirin, doxorubicin, sesamol, melatonin and methotrexate. Increased
intracellular calcium concentration ([Ca2+]i) is induced by methotrexate, tamoxifen, balhimycin, vancomycin, Navitoclax, melatonin, sesamol and bexarotene. ER-stress mediated
apoptosis is induced by NF-κB inhibitors. Cisplatin targets the intrinsic pathway via ERK signaling; JNK signaling is induced by carmustine, methotrexate and arsenic trioxide; PI3K
signaling is induced by thymoquinone. Legend: Blue boxes highlight the key markers of the extrinsic and intrinsic apoptosis pathways; green boxes represent the major signaling pathways
(e.g. MAPK, PI3K and ER-stress) that promote platelet apoptosis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this
article.)

report an ethanol-induced increase in JNK activation in platelets, as was
previously reported for methotrexate [59] and carmustine [62].
Moreover, the administration of ethanol to mice reduced their circu-
lating platelet counts in vivo and also increased bleeding times [83].
These data provide a partial explanation for the thrombocytopenia
observed with chronic alcohol use.
5.4.2. Sesamol and melatonin
Sesamol is a phenolic compound contained in sesame seeds that
reportedly exhibits anti-neoplastic properties, but also promotes pla-
telet apoptosis [84]. Sesamol-treated platelets exhibited increased ROS
(H2O2) production, [Ca2+]i, mitochondrial depolarization, cytochrome
c release, caspase-3 and -9 activation and PS exposure [84]. The same
group examined the pro-apoptotic effects of melatonin, an en-
dogenously-produced hormone available as an over-the-counter dietary
supplement [85]. Practically identical results were noted in melatonin-
treated platelets [85]. In addition, the authors noted that melatonin
treatment induced the phosphorylation of PI3-K, Syk and Src kinases
[85] although inhibition of these signaling pathways were not tested in
this study.
5.4.3. Thymoquinone
Thymoquinone is a chemical derived from the plant Nigela sativa
[86] that reportedly exhibits anti-cancer properties [87]. Thymoqui-
none treatment reportedly increases in [Ca2+]i, mitochondrial depo-
larization, caspase-3 activity and PS exposure [88,89]. These increases
were abrogated in the presence of the PI-3-kinase inhibitor wortmannin
[88], suggesting that thymoquinone-driven apoptosis is dependent on
PI-3-kinase signaling. This finding seemingly contradicts the anti-
apoptotic effect of PI-3-kinase signaling reported in nucleated cells
[39], suggesting that the downstream effects of certain signaling
pathways are cell-type specific.
5.4.4. Andrographolide and resveratrol: evidence for extrinsic apoptosis in
platelets
Andrographolide is a compound derived from the plant
Andrographis paniculata that is employed as a herbal medicine for its
purported anti-inflammatory and anti-cancer properties [90]. Andro-
grapholide was reported to induce caspase-3 as well as caspase-8 acti-
vation in human platelets [91]. Similar data were obtained in a separate
study investigated the pro-apoptotic properties of resveratrol (3, 4′, 5-
trihydroxy-trans-stilbene), a polyphenol found in red wine [17]. In this
study, resveratrol induced platelet apoptosis while exhibiting caspase-8
activation, as was observed in andrographolide-treated platelets [17].
The activation of caspase-8 by these drugs raises the novel and inter-
esting concept that components of extrinsic apoptotic signaling may in
fact exist in platelets.
Key points: drug-induced platelet apoptosis.
• Multiple drugs, including herbal substances, induce apoptosis in
platelets.
• Most of the available evidence is limited to in vitro studies using
washed platelets.
• Apoptotic drug-treated platelets exhibit: (1) mitochondrial depo-
larization; (2) increased [Ca2+]i; (3) caspase-3 activation and (4) PS
exposure.
• However, different drugs appear to trigger apoptosis via different
mechanisms. These include: Bak activation, Bcl-2 inhibition, sti-
mulation of different MAPK signaling modules (Fig. 4).
• Drug-induced platelet apoptosis occurs mainly via receptor-in-
dependent (intrinsic) pathways, although some drugs induce cas-
pase-8 activation, suggesting possible involvement of the extrinsic
pathway.

8
E. De Silva, H. Kim
6. Thrombocytopenia-inducing drugs: indirect evidence for pro-
apoptotic activity
Research from the past decade has revealed the importance of
apoptosis in controlling platelet lifespan [13,92]. We have also gleaned
considerable insight into apoptosis as a previously unidentified me-
chanism for drug-induced thrombocytopenia. This encourages spec-
ulation that other drugs known to cause platelet destruction, may in
fact do so by promoting platelet apoptosis. For example, interleukin-2
(IL-2) therapy is associated with thrombocytopenia [93] and reportedly
induces apoptosis in thymocytes [94] but this has not been evaluated in
platelets. The platelet-destructive mechanisms of multiple drugs remain
unknown [9]. It would therefore be of considerable interest to evaluate
the effects of these drugs in the context of platelet apoptosis.
7. Perspectives
7.1. Conclusion
Platelet apoptosis is a relatively new and understudied concept and
there now exists convincing evidence that drugs cause thrombocyto-
penia via pro-apoptotic signaling. There is remarkable congruity across
all studies with regards to the drugs' endpoint effects on Ca2+ signaling,
mitochondrial depolarization and PS exposure in platelets. However,
the different drugs also target distinct signaling pathways (e.g. JNK vs.
ERK) (Fig. 4) and show other subtle differences in their modulation of
apoptosis. For example, vancomycin-induced apoptosis is Ca2+-de-
pendent but unaffected by caspase-3 blockade [50], whereas ABT-737
and thymoquinone induce apoptosis in a caspase-3-dependent manner
[65,89]. It is tempting to hypothesize that drug-induced thrombocyto-
penia could one day be mitigated by the concurrent administration of
targeted anti-apoptotic agents, but this would obviously require a
thorough and complete understanding of all drug-induced apoptotic
pathways.
7.2. Future directions
A major limitation of the available evidence is that nearly all of the
studies were conducted in vitro using washed platelets. Future studies
would need to directly evaluate the effect of drug administration on
circulating platelet counts in vivo. To thoroughly dissect the drug-in-
duced apoptotic signaling pathways in vivo, future studies could employ
knockout mice where selected elements of putative signaling pathways
such as JNK or ERK are specifically deleted from platelets using Cre/lox
technology [95]. These elegant approaches could also be combined
with advanced imaging methods [96] to detect and analyze drug-in-
duced apoptosis in animal models. Further studies in this area are thus
likely to yield valuable new knowledge regarding the molecular me-
chanisms of drug function.
Acknowledgements
The authors thank all members of the Kim laboratory and the UBC
Centre for Blood Research, for helpful discussions. This work was
supported, in part, by a Canadian Institutes of Health Research (CIHR)
(grant number MC2-127872) Clinician-Scientist Salary Award (to H·K.).
Transparency document
Transparency document related to this article can be found online at
http://dx.doi.org/10.1016/j.cbi.2018.01.015.
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