Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation

ORIGINAL ARTICLE

Elevated thrombopoietin in plasma of burned patientswithout and with sepsis enhances platelet activation

E . LUP IA ,* 1 O . B OS C O, * 1 F . MAR IANO,� A. E . DONDI ,* A . GOFF I , * T . SPATOLA ,*

A . CUCCURULLO,* P . T IZZANI , * G . BRONDINO,§ M. STELLA� and G . M O NT R UC C H IO **Department of Clinical Pathophysiology, University of Turin, Turin; �Department of Medicine Area, Nephrology and Dialysis Unit, and

�Department of Plastic Surgery and Burns Unit, CTO Hospital, Turin; and §Department of Housing and City, Polytechnic University of Turin,

Turin, Italy

To cite this article: Lupia E, Bosco O, Mariano F, Dondi AE, Goffi A, Spatola T, Cuccurullo A, Tizzani P, Brondino G, Stella M, Montrucchio G.

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation. J Thromb Haemost 2009; 7:

1000–8.

Summary. Background: Thrombopoietin (TPO) is a humoral

growth factor that does not induce platelet aggregation per se,

but enhances platelet activation in response to several agonists.

Circulating levels of TPO are increased in patients with sepsis

and are mainly related to sepsis severity. Objectives: To

investigate the potential contribution of elevated TPO levels

in platelet activation during burn injury complicated or not by

sepsis.Methods: Westudied 22 burned patients, 10without and

12 with sepsis, and 10 healthy subjects. We measured plasma

levels of TPO, as well as leukocyte-platelet binding and P-

selectin expression. The priming activity of plasma fromburned

patients or healthy subjects on platelet aggregation and

leukocyte-platelet binding, and the role of TPO in these effects

were also studied in vitro.Results: Burned patients without and

with sepsis showed higher circulating TPO levels and increased

monocyte-platelet binding compared with healthy subjects.

Moreover, TPO levels, monocyte-platelet binding and P-

selectin expression were significantly higher in burned patients

with sepsis than in burned patients without sepsis. In vitro,

plasma from burned patients without and with sepsis, but not

from healthy subjects, primed platelet aggregation, monocyte-

platelet binding and platelet P-selectin expression. The effect of

plasma fromburnedpatientswith sepsiswas significantly higher

than that of plasma from burned patients without sepsis. An

inhibitor of TPO prevented the priming effect of plasma from

burned patients. Conclusions: Increased TPO levels may

enhance platelet activation during burn injury and sepsis,

potentially participating in the pathogenesis of multi-organ

failure in these diseases.

Keywords: burn, leukocyte-platelet adhesion, platelet activation

markers, platelet aggregation, sepsis, thrombopoietin.

Introduction

Thrombopoietin (TPO) is a humoral growth factor originally

identified for its ability to stimulate the proliferation and

differentiation of megakaryocytes [1,2]. TPO is constitutively

produced by the liver and kidneys, and is then cleared from

circulation upon binding with its receptor, c-Mpl, expressed

mainly on platelets and megakaryocytes [1,2]. We and others

have shown that, in addition to its action on megakaryocytes

and bone marrow progenitor cells [1,2], TPO also directly

modulates the response of mature platelets to several stimuli

and thereby their homeostatic potential [3,4]. In particular

TPO, which does not induce platelet aggregation per se,

enhances platelet activation in response to different agonists

[3,4], and the subsequent leukocyte-platelet adhesion via P-

selectin [5].

Elevated TPO levels have been reported in different clinical

conditions, from acute coronary syndromes [6] to hematolog-

ical diseases, where increased circulating TPO may be a

response to altered bone marrow hematopoiesis [7–9]. Several

investigations show that dysregulation of the TPO/Mpl

receptor system is also present in sepsis. In particular, elevated

TPO levels have been reported in healthy volunteers after

endotoxin infusion [10], as well as in septic children and

neonates [11–15] and septic adult patients [16,17]. In particular,

Zakynthinos et al. showed that TPO levels were greatly

increased in patients with sepsis compared with control

subjects, and that sepsis severity is the major determinant of

elevated TPO levels in these patients [17].

The aim of the present study was to investigate the potential

role of TPO in sustaining platelet aggregation and leukocyte-

Correspondence: Giuseppe Montrucchio, Dipartimento di Fisiopatologia

Clinica, Universita degli Studi di Torino, Via Genova 3, 10126 Torino,

Italy.

Tel.: +39 011 6705386; Fax: +39 011 6705367.

E-mail: [email protected]

1These authors contributed equally.

Received 22 December 2008, accepted 12 March 2009

Journal of Thrombosis and Haemostasis, 7: 1000–1008 DOI: 10.1111/j.1538-7836.2009.03348.x

� 2009 International Society on Thrombosis and Haemostasis

platelet interaction in patients with burn injury complicated or

not by the occurrence of sepsis.

Materials and methods

Patients

We studied 12 burned patients with sepsis, 10 patients with

burns and no evidence of infection, and 10 healthy subjects

(Table 1). All patients had burns covering 15–40% of their

total body surface area.

The diagnosis of sepsis was confirmed before blood collec-

tion by an experienced intensivist, blinded to the goals of the

study, according to the modified criteria for the diagnosis of

sepsis proposed in burned patients [18]. During the first 24 h

after the diagnosis of sepsis, burned patients with sepsis were

enrolled in the study.

Patients with burns and no evidence of infection were

monitored after enrollment for the appearance of signs of

infection, and kept in the study only if they did not match the

criteria for sepsis in burned patients within the following 72 h.

Exclusion criteria included the following: (i) age <16 years;

(ii) presence of overt disseminated intravascular coagulation

(DIC) [19] or platelet count <100 · 109 L)1; (iii) presence of

chronic hepatic failure, chronic renal failure, or hematological

disease affecting coagulation, platelet, or TPO production

[1,7,8,16]; (iv) the presence of malignancies under chemother-

apy or malignancies related to coagulation or platelets; and (v)

assumption of medications affecting platelet behaviour.

Severity of organ dysfunction was estimated using the

sequential organ failure assessment (SOFA) score [20].

The control group consisted of healthy volunteers, receiving

nomedications. None had shown any evidence of febrile illness

during the previous 2 weeks. Their hematological indices, and

liver and kidney function tests were within normal ranges.

The study was conducted according to the Helsinki Decla-

ration and it was approved by the Institutional Ethical

Committee �A.O.U. San Giovanni Battista di Torino - A.O.

C.T.O./Maria Adelaide di Torino�, dossier n. CEI/221.

Informed consent was obtained from the patients or their

caring relatives.

Blood collection protocol

Blood collection was performed using a central venous catheter

or, for healthy subjects, by clean venipuncture using a 19-gauge

butterfly infusion set, without venous stasis. After discarding

the first 4 mL, blood entered Vacutainers containing EDTA or

3.8% trisodium citrate, as appropriate.

To obtain plasma samples, EDTA-anticoagulated tubes

were centrifuged at 1600 ·g for 10 min at 4 �C. Plasma was

then centrifuged again at 12 500 ·g for 10 min at 4 �C, andimmediately frozen and stored at )70 �C.

Platelet-rich plasma (PRP) was prepared by centrifugation

of 3.8% trisodium citrate-anticoagulated blood for 15 min at

180 ·g [3].

Thrombopoietin measurement

TPO levels in EDTA-anticoagulated plasma were measured

using an ELISA assay (R&D Systems Inc., Minneapolis, MN,

USA) according to the manufacturer�s instructions.

Flow cytometry

All experiments were conducted using 3.8% trisodium citrate-

anticoagulated blood. Leukocyte-platelet aggregates in vivo

were analyzed using three-colour staining of whole blood

samples, as previously described [6]. Briefly, blood was diluted

1:1 with Tyrodes�s HEPES buffered saline (pH 7.4), and added

to a mixture of FITC-conjugated anti-CD45 (Beckman Coul-

ter, Miami, FL, USA), ECD-conjugated anti-CD14 (Beckman

Coulter), and PE-conjugated anti-CD41 (Dako Cytomation,

Glostrup,Denmark)monoclonal antibodies, and incubated for

15 min at room temperature. Cells were then fixed with 1%

paraformaldehyde and resuspended in 0.5 mL of phosphate-

buffered saline (PBS), after removal of erythrocytes by

hypotonic lysis.

Table 1 Clinical characteristics

Characteristics

Healthy subjects

(n = 10)

Burned patients

(n = 10)

Septic burned

patients (n = 12) P-value

Age (years) 50.0 ± 4.0 54.4 ± 6.0 57.8 ± 6.4 0.631

Gender (Male/female) 5/5 6/4 10/2

Outcome (Dead/alive) NA 2/8 8/4�

SOFA score* NA 4.80 ± 0.29 9.50 ± 0.75� <0.0001

Platelets (109 L)1)* 216.9 ± 16.7 177.1 ± 26.2 218.1 ± 25.7 0.856

Leukocytes (106 L)1)* 6153 ± 589 9070 ± 1408 13058 ± 1566§,� 0.004

Monocytes (106 L)1)* 474 ± 49 870 ± 204 965 ± 173 0.122

Thrombopoietin (pg mL)1)* 68.90 ± 17.16 153.79 ± 33.07§ 374.93 ± 62.41**,– <0.0001

Data represent means ± standard error. The last right column reports the P-value obtained by comparing the three experimental groups using

one-way ANOVA.

NA, non applicable; SOFA, sequential organ failure assessment. *Data at enrollment. �P < 0.05 vs. burned patients. �P < 0.0001 vs. burned

patients. §P < 0.01 vs. healthy subjects. –P < 0.01 vs. burned patients. **P < 0.001 vs. healthy subjects.

TPO enhances platelet activation in burn injury 1001


Samples were analyzed on an EPICS–XL flow cytometer

(Coulter Corp, Hialeah, FL, USA) using adequate compensa-

tion for different fluorochromes. Total leukocytes were iden-

tified by their positive staining with anti-CD45, and

lymphocyte, polymorphonuclear and monocyte populations

were discriminated on the ground of CD45 vs. forward scatter.

The percentage of leukocyte subgroups co-expressing CD45-

CD41 (polymorphonuclear leukocytes-platelets) or CD14-

CD41 (monocytes-platelets) over the total population of

leukocytes expressing CD45 or CD14 was used as an index

of leukocyte-platelet adhesion [6].

P-selectin expression was evaluated in whole blood using a

mixture of FITC-conjugated anti-CD62P/P-selectin (Ancell

corporation, Bayport, MN, USA) or the appropriate isotypic

control, and PE-conjugated anti-CD41monoclonal antibodies.

Platelets were identified by their characteristic light scatter and

the positive signal provided by the platelet marker PE-anti-

CD41 monoclonal antibody.

For in vitro experiments, 100 lL of diluted blood from

healthy adult donors was pre-incubated at 37 �Cwith 25 lL of

plasma of patients or normal subjects for 5 min and then

stimulated with adenosine 5¢-diphosphate (ADP, 0.8 lM;Helena Laboratories, Beaumont, TX, USA) or epinephrine

(EPI, 3 lM; Helena Laboratories). Samples were then pro-

cessed and analyzed as described above.

In separate experiments, plasma was incubated with a

humanTPO receptor (TPOR)-Fc chimera (2.5 lg mL)1; R&D

Systems Inc.) for 5 min at 37 �C; the mixture of sample and

TPOR-Fc chimera was added to whole blood, further

incubated for 5 min at 37 �C and stimulated with ADP or

EPI. We have previously reported that the TPOR-Fc chimera

inhibited the priming effect exerted by recombinant human

TPO in PRP and whole blood, but did not affect the

aggregation induced by ADP or EPI [6].

Platelet aggregation

Platelet aggregation was evaluated in 3.8% trisodium citrate-

anticoagulated PRP and whole blood as previously described

[3,6]. PRPorwholebloodwasobtained fromhealthy subjects as

described above and incubated with 25 or 100 lL of plasma,

respectively, at 37 �C. In selected experiments, the effect of

tumor necrosis factor-a (TNF-a), interleukin (IL)-1, IL-6, IL-3,granulocyte colony stimulating factor (GCSF), or granulocyte

macrophage-colony stimulating factor (GMCSF) was evalu-

ated.When evaluating priming activity, ADPorEPIwas added

as secondary agonist. For each experiment, the ADP (0.8–

2 lmol L)1) or EPI (0.1–3 lmol L)1) concentration that

induced theminimummeasurable aggregationwas determined.

The priming index (PI)was calculated as the response to plasma

and agonist together, divided by the sum of the individual

responses elicited by plasma and the agonist separately [3,6].

Using this calculation, a PI > 1 indicated synergism, a PI = 1

indicated additive response, and PI < 1 indicated inhibition.

In separate experiments, plasma was incubated with the

human TPOR-Fc chimera (2.5 lg mL)1) for 5 min at 37 �C;

the mixture of sample and TPOR-Fc chimera was added to

PRP or whole blood, further incubated for 5 min at 37 �C and

stimulated with ADP or EPI.

Statistical analysis

Values are represented by means ± standard error (SE).

Comparisons between groups were carried out using ANOVA

followed by Newman–Keuls multicomparison test or Student�st-test where appropriate; categorical variables were compared

using two-way cross-tabulation with the chi-square test.

Normality was assessed using the Shapiro–Wilk analysis,

whereas the Bartlett test was used to test variance hetero-

geneity. When ANOVA assumptions were violated, data

were transformed using the Box–Cox power transformation

method.

The relationship between variables was investigated using

Pearson�s correlation test.

A P value <0.05 was considered significant.

All statistics were done using GRAPHPAD PRISM 4.00

(GraphPad Software, La Jolla, CA, USA) and R 2.7.1.

Results

Patient clinical characteristics and TPO levels

Table 1 gives demographic and clinical data for patients and

healthy subjects. Burned patients with sepsis did not differ

regarding demographic characteristics either from burned

patients without sepsis or from healthy subjects. All septic

patients required vasopressors, and the intra-hospital mortality

in this group of patients was about 66% (eight out of 12).

Platelet counts were not different between the groups. Leuko-

cyte counts were significantly higher in burned patients with

sepsis (13058 ± 1566 · 106 L)1) than in burned patients

without sepsis (9070 ± 1408 · 106 L)1) and healthy subjects

(6153 ± 589 · 106 L)1); in contrast, absolute monocyte

counts were not different between the groups (healthy subjects

474 ± 49, burned patients 870 ± 204, septic burned patients

965 ± 173 · 106 L)1) (Table 1). Plasma TPO concentrations

were significantly higher in burned patients, both without

(153.79 ± 33.07 pg mL)1) and with sepsis (374.93 ± 62.41

pg mL)1), than in healthy subjects (68.90 ± 17.16 pg mL)1);

moreover, burned patients who developed sepsis had higher

circulating levels of TPO than burned patients without sepsis

(Table 1). No significant correlation was found between

circulating TPO levels and platelet count (not shown).

In vivo platelet activation

A significantly higher percentage of monocyte-platelet binding

was found in burned patients, both without (36.25 ± 2.58%)

and with sepsis (57.61 ± 7.87%), compared with healthy

subjects (23.21 ± 2.72%); burned patients with sepsis had a

significantly higher percentage of monocyte-platelet binding

than burned patients without sepsis (Fig. 1A, B). On the

1002 E. Lupia et al


contrary, polymorphonuclear-platelet aggregates were not

significantly different between the three groups, although a

tendency towards an increase in polymorphonuclear-platelet

adhesionwas observed in burned patients who developed sepsis

(not shown).

The percentage of platelets expressing P-selectin was signif-

icantly higher in burned patients with sepsis (14.16 ± 2.63%)

than in burned patients without sepsis (2.78 ± 0.26%) or

healthy subjects (2.38 ± 0.18%) (Fig. 1C, D).

TPO levels in plasma significantly correlated withmonocyte-

platelet aggregation (r = 0.52; P < 0.005), and with platelet

P-selectin expression (r = 0.67; P < 0.0001).

Effect of plasma from burned patients on platelet activation

in vitro

The effect of plasma fromburnedpatients or normal subjects on

platelet aggregation was tested in vitro on PRP from healthy

donors. Plasma from burned patients, both without and with

sepsis, did not induce platelet aggregation per se, but it

significantly enhanced the aggregation induced by ADP

(PI = 1.57 ± 0.10 for burned patients without sepsis;

PI = 2.04 ± 0.20 for burned patients with sepsis) (Fig. 2A)

or EPI (PI = 1.84 ± 0.16 for burned patients without sepsis;

PI = 2.83 ± 0.25 for burned patients with sepsis) (Fig. 2B).

When EPI was used as a secondary agonist to trigger platelet

aggregation in PRP, the priming effect induced by plasma from

burned patients with sepsis was significantly higher than that

observed with plasma from burned patients without sepsis

(Fig. 2B), whereas no difference was seen when ADP was used

as a secondary agonist (Fig 2A). This priming effect on platelet

aggregation inPRPwas seenwith all plasma samples examined.

Also in whole blood, pre-incubation of samples with plasma

obtained from burned patients primed aggregation induced by

ADP(Fig. 3A)orEPI (Fig. 3B). In this experimental condition,

the priming effect induced in whole blood by plasma from

burned patients with sepsis was significantly higher

(PI = 2.23 ± 0.15 for ADP; PI = 2.49 ± 0.34 for EPI) than

that induced by plasma from burned patients without sepsis

(PI = 1.75 ± 0.08 forADP;PI = 1.81 ± 0.09 forEPI)when

both ADP and EPI were used as secondary agonists (Fig. 3).

Plasma from healthy subjects did neither prime platelet

aggregation in PRP nor in whole blood (Figs 2 and 3).

In parallel experiments, we tested the activity of several

cytokines known to be released during sepsis [21] to assess their

potential contribution to the priming effect exerted by plasma

samples of burned patients with sepsis. We did not detect any

priming activity on ADP- or EPI-induced platelet aggregation

using TNF-a, IL-1, IL-6, IL-3, GCSF or GMCSF (data not

shown).

Healthy subject Burned patient

Burned patient

Septic burned patient

aggr

egat

es (

%)

Healthy subject Septic burned patient

CD41 PE

CD41 PE

CD41 PE

CD41 PE

E1

E3 E4

E2

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E3 E4

E2

E1

E3 E4

E2

E1

E3 E4

E2

103

103

102

102

101

101

100

103

102

101

100

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102

101

100

103

102

101

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103102101100

103102101100

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CD

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CD

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62P

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CD

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CD

14 E

CD

CD41 PE

E1

E3 E4

E2

80P < 0.001

P < 0.001

P < 0.05

P < 0.001

P < 0.01

Health

y

Burne

d

burn

edSep

tic

Health

y

Burne

d

burn

edSep

tic

60

40

20

20

15

10

5

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0

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102

101

100

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100

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CD41 PE

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CD

14 E

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Mon

ocyt

e-pl

atel

etP

late

let P

-sel

ectin

(%

)

A B

C D

Fig. 1. Representative flow cytometry analysis (Panels A, C) and data quantification (Panels B, D) of the percentage of monocyte (CD14-positive)-platelet

(CD41-positive) aggregates and platelet CD62P/P-selectin expression detected in vivo in healthy subjects and in burned patients without and with sepsis.

Data represent means ± SE. One-way ANOVA with Newman–Keuls multicomparison test was performed.



Neither plasma from burned patients, both without andwith

sepsis, nor from healthy subjects increased monocyte-platelet

binding and platelet P-selectin expression in whole blood per se.

On the contrary, plasma from burned patients without and

with sepsis, but not from healthy subjects, significantly

enhanced monocyte-platelet binding and platelet P-selectin

expression induced byADP (Fig. 4A, B) or EPI (Fig. 4C,D) in

whole blood, as determined using flow-cytometric analysis. The

effect induced by plasma from burned patients with sepsis was

significantly higher than that induced by plasma from burned

patients without sepsis when EPI was used as secondary

agonist to trigger platelet activation, as evaluated in terms of

both monocyte-platelet binding (52.20 ± 3.06% vs. 36.63

± 2.10%) and platelet P-selectin expression (19.39 ± 1.81%

vs. 14.83 ± 0.84%) in whole blood (Fig. 4C, D). However,

whenADPwas used as a secondary agonist, only the difference

in monocyte-platelet binding was statistically significant

(41.67 ± 2.48% vs. 33.13 ± 2.52%) (Fig. 4A), whereas no

difference was seen in terms of P-selectin expression

(16.28 ± 1.52% vs. 13.28 ± 1.02%) (Fig. 4B).

Role of TPO in the priming activity of plasma from burned

patients

Circulating TPO levels measured in vivo correlated with the

in vitro priming activity exerted by plasma samples on platelet

aggregation both in PRP and in whole blood. In particular,

plasma TPO levels significantly correlated with the ADP-

induced priming index in PRP (r = 0.39;P < 0.05) andwhole

blood (r = 0.58; P < 0.02), as well as with the EPI-induced

priming index in PRP (r = 0.42; P < 0.02) and whole blood

(r = 0.52; P < 0.05).

Moreover, pre-incubation of plasma from burned patients

without and with sepsis with an inhibitor of TPO biological

activity, a human TPOR-Fc chimera, significantly reduced the

priming effect exerted on platelet aggregation in PRP by ADP

(from PI = 1.57 ± 0.10 to 1.29 ± 0.07 for burned patients

without sepsis; from PI = 2.04 ± 0.20 to 1.35 ± 0.06 for

burned patients with sepsis) (Fig. 5A, B) or EPI (from

PI = 1.84 ± 0.16 to 1.38 ± 0.09 for burned patients without

sepsis; from PI = 2.83 ± 0.25 to 1.44 ± 0.03 for burned

patients with sepsis) (Fig. 5C, D). A similar reduction in the

priming effect on platelet aggregation induced by ADP (from



patients with sepsis) (Fig. 6A, B) or EPI (from



patients with sepsis) (Fig. 6C, D) was also observed in whole

blood. Pre-treatment of plasma from burned patients without

and with sepsis with the TPOR-Fc chimera also significantly

decreased monocyte-platelet binding induced in whole blood

using ADP (from 33.13 ± 2.52% to 24.67 ± 2.46% for

Prim

ing

inde

x

Ligh

t tra

nsm

issi

on (

%)

Ligh

t tra

nsm

issi

on (

%)

5%5%

Septic burned

Burned

Healthy

patient

Septic burnedpatient

4

P < 0.001

P < 0.001

P < 0.01

P < 0.01

P < 0.01

Health

y

Burne

d

burn

edSep

tic

Health

y

Burne

d

burn

edSep

tic

3

2

0

1

Prim

ing

inde

x4

3

2

0

1

patient

Burnedpatient

subject

Healthysubject

1 min

1 min

Plasma

Plasma

ADP

EPI

A

B

Fig. 2. Representative aggregation traces and quantification of the in vitro

priming activity induced by plasma from healthy subjects and burned

patients without and with sepsis on ADP- (Panel A) or EPI- (Panel B)

induced platelet aggregation in platelet-rich plasma. Data represent

means ± SE. One-way ANOVA with Newman–Keuls multicomparison

test was performed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.

Impe

danc

e (Ω

)Im

peda

nce

(Ω)

Septic burned

Burned

Healthy

patient

patient

subject1 min

ADP

EPI

Plasma

2Ω

Septic burned

Burned

Healthy

patient

patient

subject1 min

Plasma

2Ω

P < 0.001

P < 0.05

P < 0.01

3

2

0

1

Prim

ing

inde

x

Health

y

Burne

d

burn

edSep

tic

P < 0.001

P < 0.05

P < 0.001

Health

y

Burne

d

burn

edSep

tic

Prim

ing

inde

x

3

2

0

1

A

B

Fig. 3. Representative aggregation traces and quantification of the in vitro

priming activity induced by plasma from healthy subjects and burned

patients without and with sepsis on ADP- (Panel A) or EPI- (Panel B)

induced platelet aggregation in whole blood. Data represent means ± SE.

One-way ANOVA with Newman–Keuls multicomparison test was per-

formed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.

1004 E. Lupia et al


burned patients without sepsis; from 41.67 ± 2.48% to

26.87 ± 1.99 for burned patients with sepsis) (Fig. 7A) or

EPI (from 36.63 ± 2.10% to 26.53 ± 1.18% for burned

patients without sepsis; from 52.20 ± 3.06 to 36.19 ± 2.79 for

burned patients with sepsis) (Fig. 7C), as evaluated by flow-

cytometric analysis. An analogous inhibitory effect of TPOR-

Fc chimera was seen on platelet P-selectin expression (ADP:

from 13.28 ± 1.02% to 10.23 ± 1.26% for burned patients

without sepsis; from 16.28 ± 1.52% to 10.62 ± 1.25% for

burned patients with sepsis; EPI: from 14.83 ± 0.44% to

12.18 ± 0.49% for burned patients without sepsis; from

19.39 ± 1.81% to 12.80 ± 1.54% for burned patients with

sepsis) (Fig. 7B, D). On the contrary, pre-incubation of plasma

from healthy subjects with the TPOR-Fc chimera had no

significant effect (data not shown).

Discussion

The first result of our study is that patients with a burn injury

have increased monocyte-platelet aggregates and platelet P-

selectin expression comparedwith healthy subjects. In addition,

monocyte-platelet aggregates were significantly higher in

burned patients with sepsis than burned patients without

sepsis. It is known that monocyte-platelet aggregation is not

only a sensitive measure of platelet activation, but also has

significant pro-inflammatory and pro-thrombotic conse-

quences, in particular in acute coronary syndrome [22]. Our

present findings show that increased platelet activation (i.e. P-

selectin expression) and eterotypic aggregation (i.e. monocyte-

platelet adhesion) also occur in burn injury, especially after

sepsis development, suggesting that activated platelets amplify

the inflammatory reactions and favor the insurgence of organ

damage in these pathological conditions.

Moreover, we found that burn injury is associated with a

significant increase in the circulating levels of TPO, about 2-

fold the levels measured in healthy subjects. TPO levels further

increase upon development of sepsis, suggesting that the

development of sepsis, in addition to burn injury, may

contribute to increase circulating TPO levels in these patients.

These results are substantially in agreement with those already

reported by Zakynthinos et al. [17] in a larger population of

patients with sepsis. However, our data are the first to evaluate

TPO levels in burned patients without and with sepsis.

Thrombocytopenia has been correlated with poor outcome

in extensive burns and may be considered a sensitive marker of

sepsis [18,23]. However, septic patients in our study had a

platelet count within the normal range. As thrombocytopenia

and DIC are common complications of sepsis, and TPO levels

are elevated during DIC [16], we excluded from the study those

patients whomatched the diagnostic criteria for overt DIC [19],

and in particular had a platelet count <100 · 109 L)1. The

application of this exclusion criterionmay explain our failure to

detect thrombocytopenia in burned and septic burned patients,

which may also be related to the small number of patients

studied and to the relatively early enrollment of septic patients.

The precise origin of the rise in TPO levels observed in

burned patients without and with sepsis remains unclear. TPO

levels are well known to be primarily regulated by platelet mass

[1,2], and yet we did not detect thrombocytopenia in septic

patients in our study. Burned patients without and with sepsis

also showed increased indices of in vivo platelet activation

compared with healthy subjects.Moreover, we found a positive

correlation between (i) TPO levels and monocyte-platelet

binding in vivo, and (ii) TPO levels and platelet P-selectin

expression in vivo. Therefore, platelets themselves may repre-

sent a major contributor to increased TPO levels, as they are

known to release full-length biological active TPO upon

stimulation [16]. Finally, as IL-6, themain acute-phase reactant

produced in the liver, enhances TPO synthesis [24], high TPO

levels in burned and septic patients may also depend on

increased hepatic synthesis.

The presence of TPO in the circulation precludes the

evaluation of its role on platelet aggregation directly on blood

samples obtained from patients. Therefore, we studied the

contribution of TPO to platelet aggregation by adding patient

plasma samples to platelets of healthy subjects in vitro and

inhibiting TPO biological activity using the TPOR-Fc chimera.

In these experimental conditions, plasma from burned patients

without and with sepsis, but not from healthy subjects,

Mon

ocyt

e-pl

atel

etag

greg

ates

(%

)M

onoc

yte-

plat

elet

aggr

egat

es (

%)

60

Health

y

Burne

d

Septic

burn

ed

Health

y

Burne

d

Septic

burn

ed

Health

y

Burne

d

Septic

burn

ed

P < 0.001

P < 0.001

P < 0.001

P < 0.05

P < 0.001

P < 0.001

P < 0.05

P < 0.05

P < 0.05

P < 0.001P < 0.05

0

20

20

25

15

10

5

0

25

40

20

0

40

Pla

tele

t P-s

elec

tin (

%)

Health

y

Burne

d

Septic

burn

ed

20

25

15

10

5

0

Pla

tele

t P-s

elec

tin (

%)

A B

C D

Fig. 4. In vitro effect of plasma from healthy subjects and burned patients

without and with sepsis on ADP- (Panels A, B) or EPI- (Panels C, D)

induced monocyte-platelet aggregation and platelet P-selectin expression,

analyzed by flow cytometry in whole blood. Data represent means ± SE.

One-way ANOVA with Newman–Keuls multicomparison test was

performed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.



enhances platelet aggregation as well as monocyte-platelet

binding and platelet P-selectin expression in blood samples

from healthy donors. The priming effect induced by plasma

from burned patients with sepsis was significantly higher than

that induced by plasma from burned patients without sepsis in

all the experimental conditions tested. However, when ADP

was used as a secondary agonist in PRP for platelet aggregation

studies or in whole blood for the evaluation of P-selectin

Plasma

Burned patient

Burned patient

Septic burned

Septic

burn

ed

4

3

2

1

0

Burne

d

Septic burned

patient


patientBurned patient+ TPORFc

Burned patient+ TPORFc

+ TPORFc

Burne

d

+ TPORFc

Septic

burn

ed

+ TPORFc

Septic

burn

ed

Burne

d

Burne

d

+ TPORFc

Septic

burn

ed

+ TPORFc


+ TPORFc

1 min

1 min

1 min

1 min

ADP

Plasma EPI

Plasma ADP

Plasma EPI

Ligh

t tra

nsm

issi

on (

%)

Ligh

t tra

nsm

issi

on (

%)

5%5%

5%

Prim

ing

inde

xP

rimin

g in

dex

5%

P < 0.001

P < 0.0001

P < 0.005

P < 0.005

4

3

2

1

0

A B

DC

Fig. 5. Representative aggregation traces and quantification of the effect of plasma from burned patients without andwith sepsis pre-incubated or not with

the TPOR-Fc chimera on ADP- (Panels A, B) or EPI- (Panels C, D) induced platelet aggregation in platelet-rich plasma. Data represent means ± SE.

Student�s t-test was performed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.

Impe

danc

e (Ω

)Im

peda

nce

(Ω)

Burned patient

Septic burned

Septic

burn

ed

3

2

1

0

Prim

ing

inde

x

2

1

0

Prim

ing

inde

x

Burne

d

patient



+ TPORFc



+ TPORFc

Burne

d

+ TPORFc

Septic

burn

ed

+ TPORFc

Septic

burn

ed

Burne

d

Burne

d

+ TPORFc

Septic

burn

ed

+ TPORFc

Burned patient


Plasma ADP Plasma ADP

Plasma EPI Plasma EPI

2Ω

1 min 1 min

1 min 1 min

2Ω

2Ω2Ω

2Ω

P < 0.005

P < 0.01

P < 0.013

P < 0.001BA

C D

Fig. 6. Representative aggregation traces and quantification of the effect of plasma from burned patients without andwith sepsis pre-incubated or not with

the TPOR-Fc chimera onADP- (Panels A, B) or EPI- (Panels C, D) induced platelet aggregation in whole blood. Data represent means ± SE. Student�s t-test was performed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.

1006 E. Lupia et al


expression by flow cytometry, the priming effect was not

different between burned patients without and with sepsis.

These results may be related to the higher variability of the

experimental data in these settings. The contribution of TPO to

the priming effect exerted by plasma samples from burned

patients without and with sepsis is suggested by: (i) the

correlation analysis showing that TPO levels and ADP- and

EPI-induced priming index in PRP and whole blood consen-

sually increased in the three groups; and (ii) the inhibitory effect

of the TPOR-Fc chimera. Taken together, our in vivo and in

vitro data support the hypothesis that TPO present in the

circulation of burned patients, especially those developing

sepsis, may facilitate platelet activation by sensitizing circulat-

ing platelets to the action of other agonists, thus precipitating

the occurrence of microvascular thrombosis and the clinical

onset of multi-organ failure. This model is in agreement with

the priming effect observed in vivo on platelet activation after

TPO infusion in non-human primates; platelets isolated from

TPO-treated animals showed indeed a heightened sensitivity to

substances that stimulate platelet aggregation during the first

few days of treatment [25].

Other humoral mediators may also be evoked as potential

candidates for the initiation of leukocyte-platelet activation and

microvascular damage during burn injury and/or sepsis

[21,22,26]. In our experimental model, no priming effect on

platelet aggregation in both PRP and whole blood was seen

using TNF-a, IL-1, IL-6, IL-3, GCSF, or GMCSF, as also

previously shown by others [27]. However, platelets express

Toll-like receptors, which, upon binding with bacterial lipo-

polysaccharides, are responsible for sepsis-induced thrombo-

cytopenia and microvascular injury [28–30]. Therefore,

endotoxins may also cooperate with TPO in sustaining

enhanced platelet activation in burned patients who develop

sepsis.

We studied burned patients, who may be considered a good

model to evaluate the pathogenesis of organ failure in sepsis.

They are a homogeneous population in which the initial insult

can be quantified, and sepsis often develops in severe burns [31].

However, cautionmust be taken when generalizing conclusions

drawn by studies in selected populations, as that considered in

our study, to all patients affected by sepsis, or to patients with

sepsis originated from other infectious sites.

In conclusion, increased TPO levels may enhance platelet

activation during burn injury and sepsis and have a role in the

pathogenesis of multi-organ failure in these pathological

conditions.

Mon

ocyt

e-pl

atel

etag

greg

ates

(%

)

Burne

d

Septic

burn

ed

Septic

burn

ed

+ TPORFc

Burne

d

+ TPORFc

Burne

d

Septic

burn

ed

Septic

burn

ed

+ TPORFc

Burne

d

+ TPORFc

Burne

d

Septic

burn

ed

Septic

burn

ed

+ TPORFc

Burne

d

+ TPORFc

60

40

20

0

P < 0.05

P < 0.0001 P < 0.001

P < 0.01

P < 0.005

P < 0.005

20

25

15

10

5

0

Pla

tele

t P-s

elec

tin (

%)

Burne

d

Septic

burn

ed

Septic

burn

ed

+ TPORFc

Burne

d

+ TPORFc

20

25

15

10

5

0

Pla

tele

t P-s

elec

tin (

%)

Mon

ocyt

e-pl

atel

etag

greg

ates

(%

)

60

P < 0.005

P < 0.0001

0

20

40

A

C D

B

Fig. 7. Effect of TPOR-Fc chimera on the priming activity induced by plasma from burned patients without and with sepsis on ADP- (Panels A, B) or

EPI- (Panels C, D) induced monocyte-platelet aggregation and platelet P-selectin expression, analyzed by flow cytometry in whole blood. Data represent

means ± SE. Student�s t-test was performed. ADP, adenosine 5¢-diphosphate; EPI, epinephrine.



Addendum

Conception and design: E. Lupia, G. Montrucchio, F.

Mariano. Patient recruitment: M. Stella, F. Mariano. Analysis

and interpretation of data: E. Lupia, O. Bosco, A. Cuccurullo,

T. Spatola, A.E. Dondi, A. Goffi, P. Tizzani, G. Montrucchio.

Statistical Analysis: G. Brondino. Drafting, critical revision

and final approval of the manuscript: E. Lupia, O. Bosco, G.

Montrucchio.

Acknowledgement

This work was supported by MURST ex-60% to GM, and

Progetto di Ricerca Sanitaria Finalizzata – Regione Piemonte

2006 to EL and GM.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation