An overview of the role of platelets in angiogenesis, apoptosis and autophagy in chronic myeloid leukaemia
Repsold et al. Cancer Cell Int
An overview of the role of platelets in angiogenesis, apoptosis and autophagy in chronic myeloid leukaemia
Lisa Repsold 0
Roger Pool 2
Mohammed Karodia 2
Gregory Tintinger 1
Annie Margaretha Joubert 0
0 Department of Physiology, Faculty of Health Sciences, School of Medicine, University of Pretoria , Pretoria, Gauteng , South Africa
1 Department of Internal Medicine, Faculty of Health Sciences, School of Medicine, University of Pretoria , Pretoria, Gauteng , South Africa
2 Department of Haematology, Faculty of Health Sciences, School of Medicine, University of Pretoria , Pretoria, Gauteng , South Africa
Amongst males, leukaemia is the most common cause of cancer-related death in individuals younger than 40 years of age whereas in female children and adolescents, leukaemia is the most common cause of cancer-related death. Chronic myeloid leukaemia (CML) is a chronic leukaemia of the haematopoietic stem cells affecting mostly adults. The disease results from a translocation of the Philadelphia chromosome in stem cells of the bone marrow. CML patients usually present with mild to moderate anaemia and with decreased, normal, or increased platelet counts. CML represents 0.5% of all new cancer cases in the United States (2016). In 2016, an estimated 1070 people would die of this disease in the United States. Platelets serve as a means for tumours to increase growth and to provide physical- and mechanical support to elude the immune system and to metastasize. Currently there is no literature available on the role that platelets play in CML progression, despite literature reporting the fact that platelet count and size are affected. Resistance to CML treatment with tyrosine kinase inhibitors can be as a result of acquired resistance ensuing from mutations in the tyrosine kinase domains, loss of response or poor tolerance. In CML this resistance has recently become linked to bone marrow (BM) angiogenesis which aids in the growth and survival of leukaemia cells. The discovery of the lungs as a site of haematopoietic progenitors, suggests that CML resistance is not localized to the bone marrow and that the mutations leading to the disease and resistance to treatment may also occur in the haematopoietic progenitors in the lungs. In conclusion, platelets are significantly affected during CML progression and treatment. Investigation into the role that platelets play in CML progression is vital including how treatment affects the cell death mechanisms of platelets.
Platelets; Chronic myeloid leukaemia; Angiogenesis; Apoptosis; Autophagy
Amongst males, leukaemia is the most common cause of
cancer-related death in individuals younger than 40 years
of age whereas leukaemia is the most common cause of
cancer-related death in female children and adolescents
]. One-third of all types of cancer identified in
children (1 month to 14 years) are attributed to leukaemia of
which 78% are acute lymphoblastic leukaemia (Table 1
and 2) [
Leukaemia results from the abnormal formation of
white blood cells during the process of haematopoiesis
]. Leukaemia can be divided into acute- or chronic
leukaemia and is further subdivided as either myeloid (from
myeloid cells) or lymphoid (from lymphocytes)
The most common types of leukaemia are acute
myeloid leukaemia (AML), acute lymphoblastic leukaemia
(ALL), chronic myeloid leukaemia (CML) and chronic
lymphocytic leukaemia (CLL) [
]. Acute leukaemia
refers to the rate at which the disease progresses which
is in acute cases is rapid development; without treatment
the disease would be fatal within a few months of disease
onset. The time of disease progression varies according to
the type of leukaemia, with accumulation of blood cells
that do not mature during haematopoiesis, referred to as
Chronic leukaemia is characterised by a long
subclinical period ranging from 3 to 5 years, where there is a
delayed build-up of abnormal lymphocytes or myeloid
cells. The abnormality differs for each type of leukaemia
depending on the genetic mutation present, and results
in the lymphocytes or myeloid cells not being able to
perform their functions. The latter may not be symptomatic
for a prolonged period ranging from months to years [
]. Leukaemia can also arise from erythrocytes or
platelets resulting in myeloid leukaemia or from the bone
marrow, lymph nodes and spleen [
As previously mentioned, the development of the
disease is a result of genetic mutation [
]. Genes involved in
the regulation of haematopoiesis are commonly mutated
in leukaemia, resulting in differentiation defects of
haematopoietic cells. Distinctive mutations are implicated
in each type of leukaemia [
]. Recurrent cytogenetic
abnormalities occur in 50% of AML patients and 80% of
ALL patients [
]. The rat sarcoma mitogen-activated
protein kinase (RAS-MAPK) signalling or
phosphatidylinositol 3-kinase (PI3k)/protein kinase B (AKT) signalling
allow for proliferation and survival of mutated cells of a
haematopoietic origin [
Treatment and survival rates of leukaemia depend
on the type of genetic mutation responsible and stage
at time of diagnosis (which varies per leukaemia type).
These include radiation therapy, chemotherapy,
targeted therapy and combinations of the three treatments
(Table 3) [
Stem cell transplantation may also be used as
treatment in cases of leukaemia and lymphoma [
cell transplantation consists of patients receiving initial
high dosages of chemotherapy and/or radiation therapy
eliminating the bulk of the patient’s stem cells, the dosage
hereof depends on the type of drug administered.
Following this therapy, patients receive a transplant of
compatible donor stem cells by infusion, replacing the lost stem
cells and producing new, unmutated stem cells [
Development of innovative targeted-molecular therapy
which comprises of drugs that target molecules including
those involved in cell growth signalling, tumour blood
vessel development and general markers of apoptosis
has transformed treatment of leukaemia and specifically
CML through the development of tyrosine kinase
Chronic myeloid leukaemia
CML is a chronic leukaemia of the haematopoietic stem
cells affecting mostly adults. In 2016 it was estimated that
there would be 8220 new cases of chronic myeloid
leukaemia and an estimated 1070 people would die of this
disease in the United States (Figs. 1, 2) [
]. Chronic myeloid
leukaemia represents 0.5% of all new cancer cases in the
United States [
]. CML results from a translocation of the
Philadelphia (Ph) chromosome in stem cells of the bone
marrow. This, in turn, leads to the collocation of the
Abelson murine leukaemia viral oncogene homolog 1 (ABL1)
gene from chromosome 9 and the breakpoint cluster
region protein (BCR) gene from chromosome 22 [
The latter causes the fusion of a BCR-ABL gene encoding
for the aforementioned transcripts and fusion proteins of
the BCR-ABL protein including tyrosine-kinase activity
involving the phosphorylation of several substrates
activating multiple signal-transduction cascades involved in cell
proliferation and differentiation [
Additional genetic events include mutations or
deletions of genes namely p53 and the retinoblastoma protein
(Rb) following the translocation of the Ph chromosome
resulting in the fusion of the BCR and ABL1 genes which
allow for the progression of disease [
BCRABL1 gene may function by hindering apoptosis in
targeted stem cells [
]. Inhibition of apoptosis in
hematopoietic progenitor cells expressing the fused BCR-ABL
gene is thought to occur through the phosphotyrosine
kinase activity of the BCR-ABL gene. This results in these
cells being able to evade dependency on growth factors
and resistance to harmful effects of drugs and irradiation
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CML can be divided into various phases of disease
progression; these are the initial chronic stable phase
and advanced phase which are partitioned into the
earlier accelerated phase and later acute- or blastic phase [
]. The chronic phase is usually the phase in which
most patients are diagnosed and is characterized by an
increased spleen size, while also being able to maintain a
normal range of blood counts as established by
comparing to reference levels, on therapy with tyrosine kinase
Diagnosis of CML includes the presence of
splenomegaly, leukocytosis and the incidence of the
BCRABL1 fusion gene present in leukaemia cells [
Diagnostic tests include cytogenetic analysis to detect
the Ph chromosome, fluorescence in situ hybridization
detecting the presence of the mutated BCR-ABL gene by
using fluorescent dyes and the polymerase chain reaction
which detects and measures these mutated BCR-ABL
Progression of CML from the initial, chronic stable
phase to the advanced, accelerated and blastic phases is
not well understood. Pathways implicated include the
atypical phosphorylation of intracellular proteins such as
Crk-like protein (Crkl), mitogen-activated protein kinase
1/2 (Mek 1/2), Rac and c-Jun N-terminal kinase (Jnk) [
]. The activation of signal transduction pathways rat
sarcoma (RAS) or signal transducer and activator of
transcription (STAT) may occur [
]. The potential
activation of the phosphatidylinositol 3-kinase/AKT pathway
that enables apoptosis is also implicated [
Research conducted to determine the cause of
increased bleeding in patients on TKI treatment showed
that Dasatinib and, to a lesser degree Imatinib, inhibit
platelet function by impairing arachidonic acid- and
epinephrine-induced aggregation. The exact mechanism by
which this platelet dysfunction is caused is not known; it
was shown not to be related to thrombocytopenia or the
presence of clonal haematopoiesis [
The most widely used treatment for CML is Imatinib,
an inhibitor of BCR-ABL tyrosine kinase, a specific
inhibitor of the BCR-ABL fusion protein, commonly
referred to as a tyrosine kinase inhibitor (TKI) [
If patients are receptive and responsive to TKI
treatment they are likely to survive in excess of 20 years after
diagnosis and patients may have an average lifespan
of 67 years of age [
]. In the case of patients who
don’t respond to TKI’s (which is usually about 20% of
patients) the disease progresses rapidly in 50% of these
patients into the more aggressive acute or blastic phase.
In these cases second, third and fourth generation TKI’s
are used for treatment as well as haematopoietic stem
cell transplants [
]. In the other 50% of patients,
CML progressively advances to the accelerated phase
which may last for months or even years before
progressing to the blastic phase [
]. Once the blastic
transformation has occurred in patients their survival may only be
3–9 months [
Platelets are known to serve as a means for tumours to
increase growth and provide physical- and mechanical
support to elude the immune system and metastasize
]. There is, however, no literature available on the
role that platelets play in CML progression. Due to the
fact that platelets fulfil an important role in cancer- and
tumour development, their role in CML and potential
influence in CML progression are of clinical significance.
Cancer metastasis is directly linked to platelet activity
and, in particular, the ability of cancer cells to elude the
immune system by formation of platelet-tumour
]. The latter takes place through the
binding of cancer cells (lung-, bone- and breast cancer) to
P-selectin and integrins expressed on the membrane of
platelets, thus activating the platelets [
Binding of cancer cells to platelets via P-selectin
consequently results in attraction of platelets to areas of
neovascularization and tumour growth by the release of
serotonin and thromboxane from platelets [
Serotonin is known to have a tumour-stimulatory role and also
contributes to cancer-related fatigue, while thromboxane
stimulates proliferation and prevents apoptosis of
cancer cells [
]. Mitogens including vascular endothelial
growth factor (VEGF), platelet derived growth factor
(PDGF) and transforming growth factor (TGF) are
subsequently released thereby increasing vascularization and
growth of the tumour [
Further activation of platelets ensues from the original
tumour; triggering enhanced growth of the tumour as a
result of the release of platelets granules [
]. Release of
the contents of the granules from platelets hinders the
ability of the immune surveillance system against
malignancy through cloaking tumour cells and protecting the
tumour cells from natural killer (NK) cells by providing a
physical barrier and also placing major
histocompatibility complex (MHC) class I antigen into the vicinity of the
tumour cell surface [
]. This process is referred to as
the platelet–cancer loop (Fig. 3) .
In a recent publication it was shown that platelets are
not solely produced in the bone marrow as
conventionally thought, but that platelet biogenesis is predominantly
located in the lungs, producing approximately 50% of the
total platelets in the circulation [
substantial populations of haematopoietic progenitors were
found to be produced in the lungs. These progenitors
could repopulate the bone marrow in cases of
thrombocytopenia and stem cell deficiency [
]. These significant
findings demonstrate that there are uncertainties
concerning the process of haematopoiesis and specifically
how this new source of haematopoiesis may affect our
understanding of the aetiology of leukaemia [
Platelets play an important role in cancer and tumour
development, in particular their direct involvement in
the process of angiogenesis in tumours. Therapy directed
at specifically targeting angiogenesis is a recognized
method of treatment, however, it is not well researched
in haematological malignancies [
]. The importance
of angiogenesis-targeted therapies in CML has recently
become clear as the occurrence of TKI resistance and
specifically Imatinib resistance increases (Fig. 4).
Failure of patients to respond to Imatinib treatment
can be a result of acquired resistance ensuing from
mutations in the BCR-ABL 1 tyrosine kinase domain, loss of
response and poor tolerance [
]. In CML, this resistance
has recently become linked to bone marrow (BM)
angiogenesis which aids in the growth and survival of
leukaemia cells [
]. However, with the discovery of the lungs
as a site of haematopoietic progenitors, this may indicate
that CML resistance is not localized to the bone marrow
and that the mutations leading to the disease and
resistance to treatment may also occur in the haematopoietic
progenitors in the lungs [
Angiogenesis is a well-known contributor to cancer
progression and is defined as a closely-controlled biological
process which takes place during foetal development of
blood vessels and wound healing [
]. Angiogenesis is a
process associated with the formation of new vascular
sections onto a pre-existing vascular system [
Tumour angiogenesis, the process leading to the
formation of new blood vessels within the tumour mass,
provides cancer cells with oxygen and nutrition and plays
a central role in cancer cell survival [
]. It also
promotes tumour growth and possible development of
distant metastases [
The angiogenesis-related proteins released during
angiogenesis can be differentiated into the angiogenic
activators and the angiogenic inhibitors [
Angiogenesis-activating proteins include VEGF, PDGF and
matrix metallopeptidase-9 (MMP-9), while the
angiogenic inhibitors include transforming growth factor β
30, 37, 38
]. These angiogenesis-regulatory
factors are released from activated platelets in
circulating blood of patients with cancer or the development of
VEGF is a dimeric glycoprotein and a member of the
PDGF family which contributes to angiogenesis by
promoting endothelial cell growth, maturation and survival,
enhancing vascular permeability and inhibiting
]. A wide variety of human tissues express low
levels of VEGF (around 108 pg/ml) [
]. High levels
(around 238 pg/ml) are produced where angiogenesis is
required such as in foetal tissue, the placenta, the corpus
luteum, as well as in the vast majority of human tumours
including breast, colorectal, bladder and ovarian cancers
]. Studies have shown that prostate- and colorectal
cancer patients have increased serum VEGF levels when
compared to healthy individuals [
36, 46, 47
It was reported that TGFβ, another
angiogenic-regulating factor released by platelets, also plays a role in the
inhibition of the antitumour activity of T-cells, NK cells,
neutrophils, monocytes and macrophages involved in
regulating cancer progression [
PDGF is present in a number of cells including
platelets, fibroblasts, keratinocytes, myoblasts, astrocytes,
epithelial cells and macrophages [
]. Expression of PDGF
and platelet derived growth factor receptors (PDGFRs)
are dynamic and characterized by a constant change in
levels; their biosynthesis and processing are controlled
at various levels where increased expression or levels are
indicative of several diseases and pathological conditions
which are categorized into three causative disease groups
namely tumours, vascular diseases and fibrosis [
Levels of PDGF, however, can be upregulated by a
variety of stimuli including hypoxia, thrombin, cytokines and
growth factors such as TGFβ [
]. Studies have shown
increased PDGF signalling in epithelial types of
cancer which affected tumour growth, angiogenesis,
invasion and metastasis [
]. This may be explained by the
fact that both PDGFR-α and PDGFR-β engage in
signaling pathways namely RAS-MAPK and PI3K known
to be involved in cellular- and developmental responses
including stimulation of cell growth, differentiation and
The process of angiogenesis (Fig. 5) is thought to be
primarily caused by hypoxia in tumours which activate
hypoxia-inducible factor-1 (HIF-1) [
]. HIF-1 is
responsible for increased expression of pro-angiogenic genes
including VEGF [
]. VEGF mediates the process of
angiogenesis through vasodilation of pre-existing blood
vessels via generation of nitric oxide [
]. VEGF is a main
contributor to angiogenesis by promoting endothelial cell
growth, maturation and survival, enhances vascular
permeability and inhibits apoptosis [
36, 43, 44
Nutrient deprivation within a tumour mass also signals
the release of various angiogenic molecules [
16, 17, 45–
]. The release of VEGF, epidermal growth factor (EGF),
angiopoietin 1 (Ang1) and basic fibroblastic growth
factor (bFGF) stimulates proliferation, migration and
assembly of the endothelium, while integrins αvβ3 and α5
mediate cell migration and spread [
]. Formation of
a new basement membrane is essential in maturation of
newly formed vessels which takes place through
recruitment of smooth muscle cells via PDGF .
Most of the above-mentioned angiogenesis-regulatory
factors are released specifically from the α-granules of
platelets, which also play a role in vascular repair and
cell to cell interactions [
]. Platelets contain 3 types of
secretory granules including α-granules, dense granules
and lysosomes. It has, however, been shown that
platelets can release either pro-angiogenic factors or
antiangiogenic factors differentially in response to various
tissue stimuli [
]. This suggests that platelets may hold
clinical implications once the mechanism of
differentiating release of pro- and anti-angiogenic factors is
elucidated to target specific release of antiangiogenic factors
at tumour sites [
Cell death: apoptosis
Many endogenous angiogenesis inhibitors have been
shown to induce apoptosis in vivo [
]. Apoptosis is
characterised by membrane blebbing, cell shrinkage,
hypercondensation of chromatin and formation of apoptotic
bodies, activated by either the intrinsic and/or the
extrinsic pathways [
]. Both of these pathways include the
interaction of death receptors with death ligands and
activation of caspases (Fig. 6). These can be divided into
two classes namely the initiator caspases (caspase 8 or 9)
and the executioner caspases (caspase 3, 6, and 7) [
In nucleated cells, the intrinsic apoptosis pathway
is initiated by stimuli which trigger cytochrome c to be
released from the mitochondria, and, in turn recruit’s
initiator caspase 9, thereby activating executioner caspase 3
resulting in apoptosis [
]. During the extrinsic apoptotic
pathway in nucleated cells, death receptors (DRs)
including DR5 bind to death ligands which employ the initiator
caspases 8 and 9, forming the death-inducing signalling
complex (DISC) and activation of the effector caspase 3,
ensuing in apoptosis [
52, 57, 58
The removal of apoptotic cells is a result of
phosphatidylserine (PS) collecting on the external layer of the cell
membrane which is initiated by activation of the
calcium-dependent phospholipid scramblase and signals
macrophages to stimulate the removal of the apoptotic
]. Once the PS has been externalized, a distinct
characteristic of apoptosis, it is possible to quantify the
extent of the PS-flip as binding sites are revealed during
the flip [
Apoptosis is closely associated with occurrences
within the nucleus and is consequently questioned in
platelets since they lack this cellular component [
]. Platelets display characteristic signs of nucleated
apoptosis including membrane blebbing, loss of the
integrity of the platelet membrane and
microparticle release [
]. The ability of platelets to undergo
apoptosis is a result of mitochondrial presence which
contributes to mitochondrial deoxyribonucleic acid
(DNA) and messenger ribonucleic acid (mRNA).
Mitochondrial DNA and mRNA aid in the platelets’ ability
to synthesise proteins contained within platelet
Thus, even though platelets do not possess a nucleus,
they exhibit biological apoptotic signals during stressed
conditions including activation of caspase 3 and
exposure/externalisation of phosphatidylserine [
 showed that platelets do undergo apoptosis via the
intrinsic apoptotic pathway that also regulates the
The intrinsic apoptotic pathway in platelets,
comparable to the process in nucleated cells, is characterised
by activation of Bak and Bax, members of the B-cell
lymphoma 2 (Bcl-2) protein family which promote
apoptosis, triggering damage of the mitochondria and
releasing cytochrome c and other apoptotic proteins
from the mitochondrial intermembrane space. The
release of cytochrome c allows for the formation of the
Apaf-1 apoptosome and subsequent recruitment of
initiator procaspase 9. Binding to the apoptosome activates
caspase 9 and leads to the activation of effector caspase
3, culminating in the execution phase of apoptosis [
Upstream of caspase 3 activation and PS exposure, the
mitochondrial inner transmembrane potential is
depolarized in platelets, similar to the mechanism of nucleate
cellular apoptosis (which is the programmed process of
apoptosis in nucleated cells) [
The resulting externalisation of PS then allows for
removal of apoptotic platelets. In platelets, PS is also
expressed on the cell surface, however, it can only be
recognized by macrophages for phagocytosis by recognition
via human cluster of differentiation 36 (CD36) present
on the membrane of human platelets [
externalisation of PS in platelets seems to also occur
independently of the intrinsic apoptotic pathway playing an
important role in formation of thrombin by assembling
the pro-thrombinase complex [
Cell death: autophagy
In addition to apoptosis in platelets, the role of autophagy
and the biological markers, including autophagy-related
proteins (Atg) and quantification of the conversion of
light chain 3-I (LC3-I) to LC3-II have not been researched
extensively in platelets. Since platelets do contain small
amounts of functional mitochondria, it has been
proposed to share characteristics of nucleated autophagy
mechanisms and markers (Fig. 7) [
ability to maintain cellular homeostasis and adjustment to
starvation is of importance in platelets as their lifespan
is only about 10 days in humans [
]. Autophagy can
also be triggered continuously under certain stressed
conditions such as starvation, cellular injury and contact
with certain chemicals such as lithium, which leads the
cell to progressively degrade vital cytoplasmic
components, essentially digesting itself [
The occurrence of autophagy in platelets is essential
in maintaining homeostasis within platelets and in the
number of platelet populations [
]. The incidence of
autophagy is not well documented in platelets. Literature
has shown that platelets do express Atg proteins and the
process is also activated by the inhibition of mTOR [
]. A defect in platelet autophagy may result in
compromised platelet adhesion and aggregation impacting on
coagulation and the resulting formation of a platelet plug
during damage to blood vessels .
CML patients have abnormal megakaryocytes that can
deliver unusual blast fragments to the peripheral blood
and patients are frequently found to have large and
heterogeneous platelets. Additionally, TKI treatment has been
shown to induce platelet dysfunction and may result in
coagulation abnormalities and an increased incidence of
]. Since platelets are significantly affected
during CML progression and treatment, investigation
into the role that platelets play in CML progression is
of importance, including how treatment effects the cell
death mechanisms of platelets. In light of new research
implicating the lungs as an additional production site not
only for platelets, but also haematopoietic progenitors,
research into platelet involvement in CML is of critical
ABL1: Abelson murine leukaemia viral oncogene homolog 1; AKT: protein
kinase B; ALL: acute lymphoblastic leukaemia; AML: acute myeloid leukaemia;
Ang1: angiopoietin 1; Apaf-1: apoptotic protease activating factor 1; Atg:
autophagy-related genes; Bcl-2: B cell lymphoma 2; BCR: breakpoint cluster
region protein; bFGF: basic fibroblastic growth factor; BM: bone marrow; CD:
cluster of differentiation; CLL: chronic lymphocytic leukaemia; CML: chronic
myeloid leukaemia; Crkl: Crk-like protein; DISC: death-inducing signaling
complex; DNA: deoxyribonucleic acid; DRs: death receptors; EGF: epidermal
growth factor; FADD: Fas-associated death domain; HIF-1: hypoxia-inducible
factor-1; Jnk: c-Jun N-terminal kinase; LC3: light chain-3; MAPK: mitogen
activated protein kinases; Mek 1/2: mitogen-activated protein kinase 1/2; MHC:
major histocompatibility complex; MMP-9: matrix metallopeptidase-9; mRNA:
messenger ribonucleic acid; mTOR: mammalian target of rapamycin; NK:
natural killer; PDGF: platelet derived growth factor; PDGFRs: platelet derived
growth factor receptors; PE: phosphatidylethanolamine; Ph: Philadelphia; PI3K:
phosphatidylinositol 3-kinase; PS: phosphatidylserine; RAS: rat sarcoma;
RASMAPK: rat sarcoma mitogen-activated protein kinase; STAT: signal transducer
and activator of transcription; TGFβ: transforming growth factor β; TKI: tyrosine
kinase inhibitor; ULK: uncoordinated 51-like kinase; VEGF: vascular endothelial
LR was responsible for literature review and the main contributor to drafting
of the manuscript. RP, MK, GT and AMJ assisted in drafting of the manuscript.
All authors read and approved the final manuscript.
Funding and Acknowledgements
The financial assistance of the National Research Foundation,
Struwig-Germeshuysen Research Trust, Medical Research Council of South Africa, the Cancer
Association of South Africa and the School of Medicine Research Committee
of the University of Pretoria is hereby acknowledged.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or
analysed during the current study.
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Springer Nature remains neutral with regard to jurisdictional claims in
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