Thrombosis Research 141 (2016) 11–16
Contents lists available at ScienceDirect
Thrombosis Research
journal homepage: www.elsevier.com/locate/thromres
Sepsis-associated thrombocytopenia
Caroline M. Larkin a,b,⁎, Maria-Jose Santos-Martinez b, Thomas Ryan a,c, Marek W. Radomski b
a
Department of Anaesthesia and Intensive Care Medicine, St James's Hospital, Dublin, Ireland
b
School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Ireland
c
Department of Clinical Medicine, Trinity College Dublin, Ireland
a r t i c l e i n f o
Article history:
Received 22 December 2015
Received in revised form 8 February 2016
Accepted 19 February 2016
Available online 2 March 2016
1. Introduction and background
Severe sepsis now accounts for one quarter of all admissions to
Intensive Care Units (ICUs) and has an incidence of 60/100,000 in
the United Kingdom [1]. Based on recent figures from the United
States (US), 19 million cases every year is a conservative estimate
of the worldwide incidence of sepsis [2]. This incidence is increasing,
resulting in rising costs to healthcare systems. In the US the annual
direct cost of sepsis is estimated to have increased from $15.4 billion
in 2003 to $24.3 billion in 2007 [3]. Apart from the enormous eco-
nomic cost of sepsis there is also the societal and individual impact
of the considerable mortality attributable to such a common disease
as sepsis. In developed countries the mortality rate from septic shock
appears to be decreasing. A study involving 171 ICUs emanating from
Australia and New Zealand and including over 100,000 patients
reported a decreasing mortality rate in severe sepsis from 35% in
2000 to 18.4% in 2012 [4]. A separate meta-analysis suggests that
sepsis mortality was still disconcertingly high at 29% in the late
2000s [5]. Despite a reported declining mortality rate there remains
significant morbidity for many sepsis survivors including cognitive
impairment, neuropathies, myopathies, respiratory problems and
immunological dysfunction. In addition there appears to be an in-
creased mortality rate for sepsis survivors compared to those who
have had non-septic critical illness [6] with studies reporting a 5-
year mortality rate ranging from 61% to 81.9% [7,8].
A decrease in platelet numbers is often seen in patients who develop
severe sepsis. The development of thrombocytopenia during a septic
episode is recognised as a significant event associated with increased
mortality and morbidity. For this reason, platelet count is included in
ICU severity of illness scoring systems such as the Sequential Organ Fail-
ure Assessment (SOFA) score and the Multiple Organ Dysfunction score
(MODS) [9,10].
http://dx.doi.org/10.1016/j.thromres.2016.02.022
0049-3848/© 2016 Elsevier Ltd. All rights reserved.
Why are platelet levels so important in sepsis? In sepsis platelets
may exhibit immunologic properties. Interestingly, invertebrate an-
imals, such as arthropods, have nucleated cells called haemocytes
which are primarily involved in immunity but that can also aggre-
gate together to form clots. Platelets may have evolved from such
haemocyte-like cells and this evolutionary origin may in part explain
platelets' apparent additional functionality as elements of the innate
immune system [11]. Indeed, the horse-shoe crabs Limulus polyphe-
mus, an evolutionary relic, have haemocytes that serve both as
haemostatic and immune elements, generating nitric oxide (NO) to
support these functions [12]. Platelets mediate phagocytic-type
functions and can internalise both bacteria and viruses, although
whether this is a true phagocytosis or the internalisation of pathogens
into an extracellular space contained within platelets remains contro-
versial [13]. While it may appear that platelet phagocytosis is a benefi-
cial function, this may not necessarily be the case as platelets may, in
fact, contribute to the spread of infection [11]. Platelets are, therefore,
recognised as key players in the innate immune system and their critical
role in inflammation means that platelets are significantly involved in
the pathogenesis of sepsis [14]. In a murine model severe thrombocyto-
penia has been associated with a strongly impaired host defence [15].
Thus, it is plausible that sepsis-associated thrombocytopenia (SAT)
may not just represent a marker of disease severity but also that throm-
bocytopenia itself can contribute to increased mortality in sepsis.
In this review we will discuss the incidence and prognostic signifi-
cance of SAT. The numerous studies that have examined possible mech-
anisms underlying SAT will be summarised. In addition, the other
causes of thrombocytopenia seen in the setting of critical illness will
be discussed with an outline of a paradigm to distinguish between
them clinically.
2. Incidence and implications
The incidence of SAT varies based on the agreed definition, however,
broadly speaking a platelet count of below 150,000/μl is accepted as
thrombocytopenia. In one study of nosocomial blood stream infections
an incidence of thrombocytopenia of 43.2% was observed [16]. In
another study of 214 patients with nosocomial sepsis, 70.6% of patients
developed a platelet count below 150,000/μl. In a large Canadian study,
involving 12 ICUs with 1238 patients with severe sepsis, the overall
incidence of thrombocytopenia was 14.5%; 9.9% in survivors versus
22.5% in non-survivors, giving a mortality odds ratio (OR) of 2.12 [17].
Among ICU patients, sepsis appears to be the predominant cause of
12 C.M. Larkin et al. / Thrombosis Research 141 (2016) 11–16
thrombocytopenia with Baughman et al. reporting an OR of 16.2 for the
development of severe thrombocytopenia in sepsis [18].
Studies on organism specific development of SAT are conflicting.
Some studies have reported a higher incidence of SAT with fungal and
gram negative infections [19,20], while others have reported no differ-
ence in the incidence between gram positive, gram negative and fungal
infections [21]. When thrombocytopenia occurs during an episode of
sepsis, the mortality rate significantly increases and thus SAT has long
been considered as a poor prognostic indicator [22]. With platelet
counts of b 150,000/μl defined as mild and b 20,000/μl considered as
very severe thrombocytopenia, one study found a mortality rate of
49.3% in the “mild” group and 77.8% in the “very severe” group [16].
Reporting an incidence of SAT of 55%, Sharma et al. found that SAT
was associated with 1.4 times increased risk of mortality [23]. Defining
thrombocytopenia as a platelet count below 100,000/μl, Horino et al. re-
ported in a study of patients with Pseudomonas aeruginosa sepsis and
without haematologic disease an incidence of thrombocytopenia of
14.9% in survivors and 61.5% in non-survivors [24]. In a single centre
study involving over 1100 patients over 6 years, Azkarate et al. reported
an OR for mortality in sepsis of 2.7 with the development of thrombocy-
topenia [25]. In addition to an increased mortality rate, SAT is also
associated with increased major bleeding events and blood product
transfusion [26]. Although there is limited published research on the
time course of thrombocytopenia in sepsis, one study published in
1999 suggested an onset within 24 h of septic shock [27]. A more recent
study reported an incidence of thrombocytopenia of 53% at admission to
ICU in patients with severe sepsis or septic shock, suggesting that SAT is
more often observed in the early stages of sepsis. Vandjick et al. report-
ed the nadir platelet count to occur on day 2 of admission [16]. Several
studies have reported that failure of the platelet count to recover to nor-
mal levels during critical illness is associated with a higher mortality
[28–30].
3. Proposed mechanisms of thrombocytopenia in sepsis
The studies described below illustrate the efforts that have been
undertaken to find a pathway that could explain the mechanism of
thrombocytopenia in sepsis. Broadly speaking, these studies have
examined – decreased platelet production, the role of platelet receptors,
immune-mediated thrombocytopenia, platelet sequestration and con-
sumptive coagulopathy.
3.1. Platelet production
Traditionally the thrombocytopenia that can occur in septic shock
was assumed to reflect a decreased production of platelets [31], so
called “marrow failure”. Indeed bone marrow suppression may be a
factor in some septic patients [14]. However, there is evidence that
platelet production (thrombopoiesis) may in fact be increased during
septic episodes [32,33]. Eissa et al. studied thrombopoesis in neonatal
sepsis using a serum thrombopoietin (TPO) assay, flow cytometry
analysis of reticulated platelet percentage (RP%) and calculation of ab-
solute RP counts. While platelet and RP counts were decreased, TPO
and RP% were increased in septic neonates compared to healthy con-
trols, suggesting that neonates respond to sepsis by up-regulating
thrombopoiesis and that thrombocytopenia ensues when the rate of
platelet consumption exceeds the rate of platelet production [34]. Ex-
amining bone marrow aspirates in thrombocytopenic ICU patients
Baughman et al. reported that the large majority had a normal megakar-
yocyte count [18]. In a landmark study, demonstrating that anucleate
platelets are in fact capable of generating their own progeny, Schwerz
et al. also found that this process is inhibited by E.Coli [35]. Given the
likelihood that this process contributes minimally to the circulating
platelet population this observation may or may not be clinically
significant.
3.2. The role of platelet receptors
The transmembrane proteins Toll-like Receptors (TLRs) are found
on various mammalian cells including neutrophils, dendritic cells,
keratinocytes and macrophages [36]. These non-catalytic receptors are
key players in the innate immune system and have been suggested as
a possible mediator of SAT [37]. Shiraki et al. were the first group to con-
firm the expression of TLRs by human platelets (TLR-1 and − 6) [38].
Subsequently, Andonegui et al. identified functional levels of TLR-4 on
murine platelets [36]. Andonegui et al. further demonstrated that
platelet accumulation in the lungs was neutrophil dependent and that
P-selection expression by platelets was not increased by bacterial
lipopolysaccharide (LPS) infusion. Subsequently, TLR-4, in addition to
TLR-2 and TLR-9, was identified in human platelets by Cognasse et al.
in 2005 [39]. Cognasse's group demonstrated that platelet TLR-4 expres-
sion was decreased in activated platelets, and using TLR-4 deficient
mice they demonstrated that LPS-induced thrombocytopenia was at-
tenuated in the absence of platelet TLR-4. Aslam et al. confirmed these
finding using a similar murine model and in a separate experiment
examining human platelets with flow cytometry [40]. Importantly, de-
creased expression of TLR-4 was demonstrated in both human and mu-
rine platelets following infusion of LPS. The same group also found that
in TLR-4 deficient mice there were decreased levels of tumour necrosis
factor α (TNF-α) production (TNF-α being one of the key cytokines in-
volved in the acute phase reaction of inflammation). More recently,
platelet-derived TLR-4 has been linked to the promotion of microvascu-
lar thrombosis in endotoxaemia, thereby providing a putative mecha-
nism to link TLR-4 activation and thrombocytopenia in sepsis [41].
This effect was found to be independent of TNF-α and Interleukin-1β
(IL-1β).
Focusing on the role of the platelet TLR-4/MYD88 complex, Zhang
et al. researched the effects of LPS on platelet activation in mice [37].
They demonstrated that LPS stimulates platelet secretion of dense and
α-granules, as indicated by ATP release and P-selectin expression, and
thus enhances platelet activation induced by low concentrations of
platelet agonists. In addition, the effect of LPS on platelet activation
was abolished by an anti-TLR-4 blocking antibody and in a TLR-4
knockout model, suggesting that the effect of LPS on platelet aggrega-
tion requires the TLR-4 pathway. LPS also caused cyclic guanosine
monophosphate (cGMP) elevation which was inhibited by an anti-
TLR-4 antibody or by TLR-4 deficiency, suggesting that activation of
the cGMP/protein kinase G pathway by LPS involves the TLR-4 pathway.
Zhang found that LPS enhanced aggregation and secretion of wild-type
mouse platelets, but not myeloid differentiation factor 88 (MYD88) de-
ficient platelets, concluding that LPS stimulates platelet secretion and
aggregation via the TLR-4/MyD88 receptor-signalling complex. This
study has some limitations as, although the mouse model of sepsis ap-
pears to be better than the rat model, a number of platelet receptors
in mice are not present in humans. These inter-species differences in
platelet receptors may limit the extrapolation of their results to the clin-
ical environment.
Protease-activated receptors (PARs) (the cellular mediators of
thrombin) were examined by Camerer et al. in mice with endotoxaemia
[42]. The PAR deficient mice when presented with an endotoxin chal-
lenge failed to show improved survival or decreased cytokine responses,
notably IL-6 and IL-10, compared with wild-type mice. Interestingly, the
PAR-4 deficient mice developed the same level of endotoxin-associated
thrombocytopenia as wild-type mice and it was concluded that “a
mechanism independent of platelet activation by thrombin was suffi-
cient to cause thrombocytopenia in our model”. Camerer postulated
therefore that SAT may not be caused by DIC but by another process.
The receptor complex GPIb-IX facilitates platelet adhesion to vascu-
lar endothelium by binding to von Willebrand factor (vWF). Yin et al. in-
vestigated the effects of LPS-induced endotoxaemia on the GPIb-IX
complex [43]. They also developed a micellar peptide inhibitor, MPαC,
which specifically inhibited the binding of GPIb-IX to vWF and thus
 C.M. Larkin et al. / Thrombosis Research 141 (2016) 11–16
prevented platelet adhesion via this mechanism. MPαC alleviated LPS-
induced thrombocytopenia, as well as providing a mortality benefit in
the mouse endotoxaemia model. This led Yin et al. to conclude that
the GPIb-IX complex played a significant role in the development of
SAT and would be a potential therapeutic target given their findings.
Salat et al. investigated changes in platelet receptor expression in pa-
tients with septic shock from intra-abdominal infection [27]. They
found increased levels of CD63, CD62P and CD31 and a significant
downregulation of CD36 in the platelets of septic patients as compared
to the healthy controls. CD63 (LIMP-1) and CD62P (P-selectin) are
expressed on the platelet surface and reflect platelet activation. CD36
(GP IV) is a binding site for thrombospondin and may be involved in
platelet signal transduction. CD31 (platelet endothelial cell adhesion
molecule 1) is a glycoprotein found on numerous cells, including plate-
lets, neutrophils, monocytes and some T-cells, and is likely involved in
leukocyte migration. Salat et al. concluded that these results demon-
strate platelet activation in septic shock and suggest increased platelet
consumption as a cause for SAT. It is worth noting that the measure-
ment of these platelet receptors, including even P-selectin, is indicative
but not conclusive evidence of platelet activation.
Regarding the role of platelet activating factor (PAF), Tsuchiyaet al.
used a canine model and showed a decrease in SAT in the group that
were treated with a PAF antagonist [44], an effect reported in earlier
studies of rats and rabbits [45,46]. Subsequently, a multicentre ICU
study of a PAF antagonist in the treatment of sepsis failed to show a sur-
vival benefit however [47]. Attempting to understand the mechanisms
underlying platelet dysfunction in sepsis, Hu et al. examined platelet
protein expression in a rat model of sepsis and found upregulation of
expression of 8 proteins including thrombospondin 1 and fibrinogen
gamma chain; both proteins involved in platelet activation [48]. This
rat sepsis model, however, has significant shortcomings and results
should be interpreted with caution [49].
3.3. Immune-mediated thrombocytopenia
Haemophagocytosis was described by Francois et al. as the “unre-
strained proliferation and activation of monocytes and macrophages
that actively ingest haemopoietic cells” and was proposed as the process
by which thrombocytopenia in sepsis occurs [50]. They described
phagocytosis as the presence of intact marrow cells within macro-
phages and considered haemophagocytosis as present when, firstly,
the percentage of histiocytes exceeded 2% of marrow nucleated cells
and, secondly, there was more than 1 haemophagocytic cell per field.
Taking bone marrow sternal samples from 50 septic patients Francois
found haemophagocytosis in 64% of patients, and in addition linked
haemophagocytosis to higher levels of macrophage colony-stimulating
factor (M-CSF). Francois suggested that high M-CSF levels promoted
haemophagocytosis and accounted for the occurrence of thrombocyto-
penia in sepsis. This study has some limitations in that a qualitative ap-
proach was adopted and samples from septic patients without
thrombocytopenia were not examined, which may undermine the clin-
ical relevance of these findings.
Stephan et al. examined platelet-associated IgG antibodies in criti-
cally ill patients with thrombocytopenia [51]. Of 61 patients, 18 (30%)
had platelet-associated IgG antibodies and this was strongly associated
with sepsis and cardiopulmonary bypass. Antiplatelet autoantibodies
were identified in 6 patients with elevated platelet-associated IgG. Of
these, 4 patients had autoantibodies directed against glycoprotein IIb/
IIIa, 2 against glycoprotein Ib/IX and 2 patients had circulating immune
complexes. As these are the same autoantibodies that have been impli-
cated in the pathogenesis of Idiopathic Thrombocytopenic Purpura
(ITP), Stephan et al. suggested an immune mechanism underlying
SAT. Semple et al. demonstrated an increase in platelet phagocytosis
by monocytes when LPS-bound platelets were opsonized with auto-
antibody positive sera from ITP patients [52]. However while this is an
interesting association it may not be causal, as antibodies that attach
13
to specific platelet receptors may stimulate, inhibit or exert no effect
on platelet function.
Supporting evidence for the role of phagocytosis as a mechanism of
thrombocytopenia emanates from a study of patients with malaria. Ex-
amining samples from thrombocytopenic patients with Plasmodium
vivax infections, Coehlo et al. found that platelet phagocytosis by mono-
cytes may be the significant reason for the prevalence of thrombocyto-
penia in patients with malaria [53]. While it is very plausible that this is
the mechanism for thrombocytopenia with malarial infections, it is un-
likely that this would be generally applicable to other non-parasitic
causes of sepsis.
3.4. Platelet sequestration
When investigating the utility of triflavin (an Arg-Gly-Asp-contain-
ing disintegrin) in preventing SAT, Sheu et al. used Chromium radio-
labelled platelets to study platelet homeostasis in a rat model of sepsis
challenged with LPS [54]. In this model significant accumulation of
platelets within the liver in sepsis was observed. Furthermore, Sheu
et al. reported that triflavin prevented thrombocytopenia in sepsis.
They proposed that either triflavin markedly inhibited platelet aggrega-
tion, resulting in decreased thromboxane A2 formation or, alternatively,
that triflavin inhibited the adhesion of platelets to subendothelial ma-
trixes, thereby leading to a reversal of the sequestration of platelets in
blood and liver in LPS-treated rats. This particular rat model of sepsis
however has significant shortcomings and these results are difficult to
extrapolate to the human setting [49].
3.5. Consumptive coagulopathy
There is good evidence that there is widespread platelet activation in
sepsis [55–57] and that as part of this platelet activation there is in-
creased formation of platelet-leukocyte aggregates (PLAs) [58,59].
There is evidence to suggest that SAT could in part be explained by con-
sumption of platelets into such aggregates [60]. Sepsis-induced platelet
activation is likely to lead to changes in the activation of the coagulation
system. However, the disordered coagulation seen in sepsis is incom-
pletely understood. It would appear that in all patients with septic
shock there is a degree of coagulation activation and clotting regardless
of the presence of overt features of disseminated intravascular coagula-
tion (DIC), with the degree of coagulation activation being related to the
severity of the illness [61]. In one study an incidence of DIC of 25% was
observed in the patients with sepsis who had developed thrombocyto-
penia [26]. Presumably, in cases of septic shock with overt DIC, the
thrombocytopenia that occurs will in part be explained by the con-
sumptive coagulopathy. It is plausible that if the platelet count falls
below 50,000/μl the likely cause is DIC [62]. However, in cases where
there is septic shock without overt DIC, the reason why SAT occurs is un-
clear and are possibly manifold.
4. Other causes of thrombocytopenia in ICU
Examining the various causes of thrombocytopenia in critically ill
patients, Vanderschueren et al. reported that the sepsis syndrome was
by far the most common cause of a decline in platelet count in the
ICU, accounting for over half of the cases in their study [63]. However
when diagnosing SAT it is important to consider and outrule the numer-
ous other causes of a low platelet count that can be seen in the critically
ill. Table 1 lists some of these other causes that are more likely to
be found in the context of critical illness with comments on the
distinguishing features of the various causes. A thorough clinical history
is key to establishing the reason for a decline in platelet count in the
ICU. A patient with sepsis and thrombocytopenia cannot be assumed
to have SAT, as for instance the incidence of drug-induced thrombocyto-
penia in septic patients may be as high as 2.3% [64]. In some cases there
will be a degree of overlap. For example, in the immune-mediated
14 C.M. Larkin et al. / Thrombosis Research 141 (2016) 11–16
Table 1
Overview of other causes of thrombocytopenia in the ICU.
Cause Examples
Decreased Myelodysplastic syndromes
production Aplastic anaemia
Chemotherapy
Certain drugs e.g. thiazides
Thrombotic thrombocytopenic purpura
Increased destruction Thrombotic microangiopathies (e.g. TTP, HUS)
Disseminated Intravascular Coagulopathy (DIC)
Drug-induced thrombocytopenia e.g. linezolid,
ciprofloxacin, quinidine
Heparin-induced thrombocytopenia
Preeclampsia/HELLP syndrome
Platelet sequestration Splenic sequestration — liver cirrhosis
Haemodilution Massive crystalloid, colloid or blood product transfusion
Spurious EDTA sampling
thrombocytopenia of haemolytic-uraemic syndrome (HUS), this illness
will often be preceded by an infectious episode (typically from E.Coli)
which in rare cases may evolve into septic shock [65]. Furthermore,
sepsis in the context of a haematologic malignancy or myeloprolifera-
tive disorder is often accompanied by a profound and persistent throm-
bocytopenia that is more likely related to the underlying illness than
sepsis.
5. Future directions
IL-11 is a platelet growth factor and can be used to prevent
chemotherapy-induced thrombocytopenia [66]. Wan et al. recently
published a single centre case-control study [67] of the effects of the
pleiotropic cytokine recombinant human IL-11 in septic patients with
severe thrombocytopenia (platelet count b 50,000/μl) and found that it
significantly decreased the mortality rate as compared to a control
group. The levels of IL-6 were significantly lower in the treatment
group. In addition, there were lower levels of the inflammatory marker
procalcitonin. This is the first study that has highlighted the potential
therapeutic value of IL-11 in the setting of SAT. Our group is currently
investigating platelet aggregation patterns in sepsis using quartz
crystal microbalance with dissipation (QCM-D) technology and also
examining the role of the matrix metalloproteinases, MMP-2 and
MMP-9, in the development of SAT. Thrombopoietin, the key hor-
mone regulating the production of megakaryocytes [68], is currently
under investigation in a trial evaluating the efficacy of recombinant
thrombopoietin in the treatment of patients with severe sepsis
who develop thrombocytopenia, an initial single centre study hav-
ing suggested a mortality benefit [69]. There is continued interest
in the utility of anti-platelet drugs in the prevention and treatment
of sepsis with 2 large trials ongoing — the ANTISEPSIS trial (Aspirin
to Inhibit Sepsis, ACTRN12613000349741) in Australia and the
Aspirin for the Treatment of Sepsis trial (NCT01784159) in Brazil.
6. Conclusion
The development of thrombocytopenia, either a relative or an
absolute decrease, during a septic episode is a significant event,
associated with a doubling of the expected mortality rate from that
episode. The exact mechanism underlying sepsis-associated throm-
bocytopenia remains unclear and it may be that this is a multifacto-
rial phenomenon. Should a distinct or dominant pathway emerge
in this process it is probable that this would be a potential therapeu-
tic target.
Comment Ref
Clinical history, peripheral blood film, bone marrow aspirate with low [70]
immature platelet fraction.
Severe thrombocytopenia and anaemia, Coombs negative. [71]
DIC secondary to non-infectious causes (e.g. solid cancers, placental abruption) [72]
usually has less fibrinolysis – FDP elevated less than D-dimers.
Platelet counts falls in 2–3 days if drug taken previously, 7 days if not taken previously. [73]
Use 4 T scoring system then check for heparin-PF4 antibodies. [74]
Pregnant or post-partum patient. [75]
History of liver disease, splenomegaly. [76]
Clinical history, concomitant decrease in haemoglobin and haematocrit. [77]
Incidence 0.1% [78]
Conflicts of interest
None.
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