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THROMBOSIS AND HEMOSTASIS

Increasing concentrations of prothrombin complex concentrate induce

disseminated intravascular coagulation in a pig model of coagulopathy with
blunt liver injury
Oliver Grottke,1,2 Till Braunschweig,3 Henri M. H. Spronk,4 Stephanie Esch,1 Annette D. Rieg,1 Rene van Oerle,4
Hugo ten Cate,4 Christina Fitzner,5 Rene Tolba,2 and Rolf Rossaint1
1Department

of Anesthesiology, 2Institute for Laboratory Animal Science, and 3Department of Pathology, RWTH Aachen University Hospital, Aachen, Germany;
for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University
Medical Center, Maastricht, The Netherlands; and 5Department of Medical Statistics, RWTH Aachen University Hospital, Aachen, Germany
4Laboratory

Despite increasing use of prothrombin

complex concentrate (PCC) to treat
hemorrhage-associated coagulopathy,
few studies have investigated PCC in
trauma, and there is a particular lack of
safety data. This study was performed to
evaluate PCC therapy in a porcine model
of coagulopathy with blunt liver injury.
Coagulopathy was induced in 27 anesthetized pigs by replacing approximately 70%
blood volume with hydroxyethyl starch
130/0.4 and Ringers lactate solution;
erythrocytes were collected and retrans-

fused. Ten minutes after trauma, animals

randomly received PCC (35 or 50 IU/kg) or
saline. Coagulation parameters including
thromboelastometry, thrombin generation, and blood loss were monitored for
2 hours. Internal organs were examined
macroscopically and histologically to determine the presence of emboli and assess liver injury. Total blood loss was
significantly lower and survival was
higher in both PCC groups versus the
control group (P < .05). These outcomes
appeared to be dose-independent. Throm-

boembolism was found in all animals

treated with 50 IU/kg PCC; 44% also
showed signs of disseminated intravascular coagulation. Liver injury was similar in
all animals. In conclusion, 35 IU/kg PCC
safely improved coagulation and attenuated blood loss. However, the higher dose
of PCC (50 IU/kg) appeared to increase
the risk of thromboembolism and disseminated intravascular coagulation. (Blood.
2011;118(7):1943-1951)

Introduction
The complexity of trauma-related coagulopathy requires an early
and targeted strategy to protect traumatized patients from exsanguination.1 Most trauma centers use fresh frozen plasma (FFP) as part
of their massive transfusion protocol.2 To avoid the well-known
complications of treatment with FFP and red blood cells (eg,
volume overload and transfusion-related lung injury),3,4 the use of
coagulation factor concentrates has been investigated in animal
models.5-10
Prothrombin complex concentrates (PCCs) are derived from
human plasma. Most of these products contain the vitamin K
dependent coagulation factors II, VII, IX, and X. PCCs with low
levels of factor VII are available (3-factor PCCs), but products with
higher levels of factor VII (4-factor PCCs) are used more commonly. PCCs are standardized and administered according to the
quantity of factor IX, and there are considerable differences
between PCCs in the quantities of coagulation factors II, VII, and
X, relative to factor IX.11 PCCs may contain coagulation inhibitors
(protein C, protein S, protein Z, and antithrombin), but the levels of
these constituents differ among PCCs.11 The primary safety concerns with PCCs are thromboembolic complications.12-17 The
propensity of PCCs to increase thrombin generation is the key to
their efficacy, but it may also be the key to the risk of thromboembolic events. Excessive concentrations of prothrombin (factor II) in
relation to levels of antithrombin have been suggested as the cause
of thromboembolic complications.18,19 The quantity of factor

II administered varies among PCCs, and it is not usually monitored.

Moreover, the ratio of antithrombin to thrombin is low in all
preparations (range 0-1:50),11 meaning that there is always a risk of
factor II overload.
Currently, there are 2 major indications for PCCs: rapid reversal
of oral anticoagulation (vitamin K antagonists) and deficiency of
vitamin Kdependent coagulation factors in life-threatening bleeding.20,21 PCCs have been well studied for the first of these
indications, and their safety in that setting seems to be generally
acceptable.22 However, no prospective studies have been performed to investigate the use of PCC as first-line monosubstance
therapy for trauma-related coagulopathy. All evidence supporting
the use of PCCs in trauma-related coagulopathy is from observational and animal studies.5-7,23-25 Although these data show the
potential for PCCs to be effective in correcting coagulopathy
associated with bleeding,26 there is no clinical evidence demonstrating their safety in this setting. The potential risk is illustrated by a
recent animal study, which reported a fatal thromboembolic
complication after treatment of dilutional coagulopathy with fibrinogen concentrate and PCC.27
Using a pig model of blunt liver injury, we attempted to investigate
the safety and efficacy of 2 concentrations of PCC in trauma-related
coagulopathy. The primary endpoint of the study was blood loss;
secondary endpoints including thrombin generation were chosen to
show the impact of PCC therapy on overall coagulation status.

Submitted March 15, 2011; accepted May 27, 2011. Prepublished online as
Blood First Edition paper, June 13, 2011; DOI 10.1182/blood-2011-03-343046.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 USC section 1734.

The online version of this article contains a data supplement.

2011 by The American Society of Hematology

BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

1943

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1944

BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

GROTTKE et al

Methods

clamped cranial to the liver, and the intraperitoneal blood was collected to
determine the total blood loss postinjury. The lungs, heart, liver, and kidneys were
removed and prepared for histologic examination.

Ethics and anesthesia

Blood sampling and analytical methods
This study was performed at the RWTH Aachen University Hospital,
Aachen, Germany. All experiments were performed in accordance with
German legislation governing animal studies, which follows the Guide for
the Care and Use of Laboratory Animals.28 Official permission was granted
from the appropriate government office for animal care and use (Landesamt
fur Natur, Umwelt und Verbraucherschutz, Recklinghausen; No. 8.8750.10.35.09.019). Before surgery, pigs were housed in ventilated rooms and
allowed to acclimatize to their surroundings for a minimum of 5 days.
Animals were fasted overnight before the surgical procedure; water was
provided.
Twenty-seven male German landrace pigs (body weight 30-36 kg) received
an intramuscular injection of 4 mg/kg azaperone (Stresnil; Janssen Pharmaceuticals) and 0.1 mg/kg IM atropine (Atropinsulfate; B. Braun) as premedication.
Anesthesia was induced by intravenous injection of 3 mg/kg propofol (Disoprivan; AstraZeneca) followed by orotracheal intubation. The animals were
ventilated in a closed circuit (Physioflex; Draeger) with 16-20 breaths/min and a
tidal volume of 10 mL/kg to keep the end-tidal partial pressure of carbon dioxide
between 36 and 42 mmHg. Anesthesia was maintained with isoflurane (Forane;
Abbott Laboratories) at end-tidal concentrations of 1% and a continuous infusion
of fentanyl (Janssen Pharmaceuticals) at 3-4 g/kg/h at an inspiratory oxygen
fraction of 1.0. Ringers lactate solution (RL) was infused, initially at 4 mL/kg/h
at first and increased to 10 mL/kg/h after laparotomy until infliction of trauma.
Temperature was maintained at 36.5-37.0C throughout the experiment by using
a warming blanket.
The animals were monitored by electrocardiogram, tail pulse oximetry,
temperature, and arterial as well as central venous pressure by femorally
introduced catheters connected to a standard anesthesia monitor
(AS/3; Datex Ohmeda).
Surgical preparation and hemodilution
Two 8.5-French catheters were surgically implanted in the right and left
jugular veins for volume substitution and insertion of a pulmonary artery
catheter. After line placement, a midline laparotomy with splenectomy and
cystostomy was performed. Intravascular volume was diluted by replacing
approximately 70% of the estimated blood volume29 with hydroxyethylstarch 130/0.4 (Voluven; Fresenius) and RL in a ratio of 1:1. Blood was
withdrawn at a rate of 100 mL/min. Volume was substituted as soon as the
blood pressure had dropped to 40%-50% of the baseline value and
continued until it had returned to 80% of the baseline value. The collected
blood was processed (Cell Saver 5; Haemonetics), and the red cells were
retransfused (20 mL/kg) over 10 minutes to avoid early death from anemia.
Liver injury and PCC substitution
A reproducible grade 3 blunt liver injury30 was induced with a force of
219-289N, using a custom-made instrument described elsewhere.31 Five
minutes after injury, all animals received RL as a fluid bolus of 35 mL/kg,
followed by continuous infusion at 40 mL/kg/h until the end of the
experiment. Using field envelopes, the 27 animals were randomized 1:1:1 to
receive normal saline solution (controls), 35 IU/kg PCC (Cofact; Sanquin;
PCC35), or 50 IU/kg PCC (PCC50). PCC was prepared according to the
manufacturers instructions and infused at a rate of 4 mL/min. Based on a
biochemical analysis of 10 consecutive lots performed by the manufacturer,
reconstituted Cofact contains a mean of 26.8 IU/mL factor II, 12.1 IU/mL
factor VII, 25.4 IU/mL factor IX, and 26.2 IU/mL factor X. In the same
biochemical analysis, Cofact was found to contain the following mean
levels of anticoagulants: 24.3 IU/mL protein C and 3.1 IU/mL protein S.
Conversely, Cofact contains only a low level of antithrombin ( 0.6 IU/
mL) and no heparin. The doses of PCC were chosen according to the results
of studies published previously.5,8,32,33
The observation period ended 120 minutes after injury. Animals surviving for
the whole of this period were euthanized with fentanyl, propofol, and potassium
chloride. Immediately after death, the abdomen was reopened, the vena cava was

Blood was collected and arterial blood gas analysis was performed after
splenectomy (baseline), at the end of hemodilution (hemodilution), and
30, 60, and 120 minutes after liver injury. For animals dying before
120 minutes postinjury, the last assessment was performed immediately
after death. Hemoglobin concentration, pH, partial pressure of oxygen, and
partial pressure of carbon dioxide were measured with a blood gas analyzer
(ABL500; Radiometer). Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, D-dimers, and antithrombin
were determined by standard laboratory methods using the appropriate tests
from Dade Behring on an MC 4 plus steel-ball coagulometer (Merlin
Medical); fibrinogen concentration was measured using the Clauss assay.
Thrombinantithrombin (TAT) complexes were quantified by ELISAs
(Enzygnost; Dade Behring).
Thromboelastometry and thrombin generation
A coagulation analyzer (ROTEM; Tem International GmbH) was used to
perform the EXTEM assay. The following parameters were measured:
clotting time (CT; seconds), clot formation time (CFT; seconds), and
maximum clot firmness (MCF; millimeters).
Thrombin generation in plasma was measured using the Calibrated
Automated Thrombogram (CAT; Thrombinoscope BV) as described previously, using final concentrations of 1pM tissue factor and 4M phospholipids.34 To correct for inner filter effects and substrate consumption, each
thrombin generation analysis was calibrated against the fluorescence curve
obtained in the same plasma with a fixed amount of calibrator (Thrombin
Calibrator; Thrombinoscope BV). Fluorescence was read in an Ascent
Reader (Thermolabsystems OY) equipped with a 390/460 nm filter set, and
thrombin generation curves were calculated using Thrombinoscope Version
4 software (Thrombinoscope BV). All measurements were performed in
duplicate.
Pathologic examination
After death, internal organs (heart, lungs, liver, and kidneys) were removed
immediately and fixed in 10% buffered formalin. Injured parts of the liver
were cut into 3-mm-thick slices and examined macroscopically and
microscopically by a pathologist blinded to therapy to assess the degree of
injury. In addition, representative tissue sections of all 4 organs were
processed to determine the occurrence of thromboembolic events. All
samples were embedded in paraffin and stained, both by H&E and by a
standard elastica van Gieson protocol, for histologic examination under
light microscopy (Microscope: Nikon, Eclipse 50i; Camera: Nikon, DS2Mv; Hardware-Camera-Adaptor with Software: Nikon, DS-L1 [Softwareversion; Firmware Ver3.22], recording on CF card; Mode: TIF, 2 MP;
Objectives: Nikon, Plan Achromat, 2, NA 0.06 [used for images with 20
magnification]; Nikon, Plan Achromat 4, NA 0.1 [used for images with
40 magnification]; and Nikon, Plan Fluor 40, NA 0.75 [used for images
with 400 magnification]). Both staining methods were applied to sections
from all of the tissues. Sections of lung and liver tissue from regions with a
high likelihood of thrombus formation were immunostained to test for
fibrinogen and von Willebrand factor (both antibodies and detection kit
from Dako) as described elsewhere.9,10
Statistics
All statistical analyses were conducted using SAS (Version 9.1.3; SAS Institute
Inc). For graphical purposes, GraphPad Prism (Version 5.01; GraphPad Software
Inc) was used. Continuous variables were calculated as mean values SD.
Initially, the experiment was designed to compare 3 groups (control,
PCC35, and PCC50). Because of the occurrence of disseminated intravascular coagulation (DIC), the PCC50 group was divided into 2 groups: those
who developed DIC (PCC50DIC) and those who did not (PCC50DIC).
Determination of whether DIC had occurred in each animal was based on

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BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

PCC SUBSTITUTION AFTER LIVER INJURY

1945

Table 1. Laboratory parameters

Baseline

Hemodilution

30 min after
trauma

60 min after
trauma

120 min after

trauma

Hemoglobin, g/L
Control

8.0 0.5

7.5 0.3*

4.6 1.3

4.0 0.9

PCC35

8.0 0.4

7.4 0.4*

5.8 0.7

5.9 0.6

2.5 0.3
5.5 0.8

PCC50DIC

8.2 0.4

7.5 0.2*

5.5 0.7

5.5 0.5

5.6 0.6

PCC50DIC

8.3 0.5

7.3 0.4*

3.6 0.4

3.5 1.1

2.8 0.9

Platelet count, 103/L

Control

304 32

110 18*

100 25

87 28

71 21

PCC35

280 33

119 10*

118 19

114 11

106 11

PCC50DIC

290 36

120 8*

99 14

86 29

97 22

PCC50DIC

325 44

115 20*

81 18

75 23

40 8

Control

322 29

106 15*

61 24

55 18

38 18

PCC35

308 32

110 14*

87 13

87 12

79 10

PCC50DIC

323 25

120 22*

98 17

88 15

92 12

PCC50DIC

343 24

114 19*

43 23

32 13

20, ND

Control

12 1

19 2*

23 4

21 3

25 4

PCC35

12 1

19 2*

22 2

21 1

22 2

PCC50DIC

11 1

18 1*

21 2

21 2

24 2

PCC50DIC

11 1

20 2*

26 2

43 14

300, ND

Control

79 6

36 5*

29 9

33 6

25 8

PCC35

88 9

39 7*

38 3

39 4

41 6

PCC50DIC

79 5

34 3*

32 6

38 5

40 6

PCC50DIC

93 16

41 8*

23 7

19 4

21 12

Fibrinogen, mg/dL

aPTT, s

Antithrombin, %

Data are mean SD.

ND indicates not detectable.
*P .05 versus each group over time.
P .05 versus control.
P .05 versus PCC35.
P .05 versus PCC50DIC.

clinical judgment, with due consideration of the International Society on

Thrombosis and Hemostasis criteria.35 Differences in total blood loss
among groups (control, PCC35, PCC50DIC, and PCC50DIC) were analyzed using analysis of variance, with Tukey post hoc adjustment. Pairwise
log-rank tests were used for survival analysis. For coagulation parameters,
blood cell count, and hemodynamic variables, a repeated-measures model
was conducted with group and time as factors; the interaction between time
and group was included. Post hoc tests for subgroup analysis were
calculated; because of a statistically significant interaction term for each
parameter analyzed, these results (except for those appearing in the main
text) are provided in supplemental Methods (available on the Blood Web
site; see the Supplemental Materials link at the top of the online article).
Because of the exponential distribution of TAT and D-dimers, these data
were log-transformed for the model. Statistical tests were performed
2-tailed, and P .05 was considered a significant result.

Results
Baseline conditions

At baseline and after hemodilution, there were no clinically

significant differences among the study groups (Tables 1 and 2).
The hemodilution resulted in significant prolongation of PT and
aPTT (Figure 1; Table 1). The concentrations of fibrinogen and
antithrombin, as well as the platelet count, decreased significantly
as expected (Table 1). All parameters of ROTEM assessment using
the EXTEM assay indicated coagulopathy (Figure 2), although
TAT complexes and D-dimers were not yet elevated (Figure 3). The
hemodilution decreased maximum thrombin generation (peak

height decreased from 95 9 to 52 4nM; P .001), whereas

overall thrombin generation (endogenous thrombin potential [ETP])
remained constant (521 48nM min before hemodilution vs
532 44nM min after hemodilution; Figure 4).
Measurements after standardized liver trauma

Control group. The control animals developed severe coagulopathy with persistent prolongation of PT, CT, and CFT (Figures
1-2). These parameters continued to deteriorate over time, resulting
in significant prolongation compared with that in all other study
groups. Platelet count, MCF, and the concentrations of fibrinogen
and hemoglobin were also significantly lower in this group than in
the PCC35 and PCC50DIC groups (Table 1; Figure 2). Compared
with the PCC35 and PCC50DIC groups, controls had a lower mean
arterial pressure and a higher heart rate (Table 2). Correspondingly,
blood loss was highest among controls (2033 363 mL), lowest in
the PCC35 (941 199 mL) and PCC50DIC (964 215 mL)
groups, and intermediate in the PCC50DIC group (1624 115 mL;
Figure 5). Four animals in the control group (44%) died before the
end of the observation period, a significantly higher percentage
than that in the PCC35 group (P .010).
Substitution with 35 IU/kg PCC. PCC35 animals recovered
from trauma after PCC administration, and this was reflected by
significant decreases in PT, CT, and CFT (Table 1; Figure 2).
Directly after PCC application (30 minutes after trauma), thrombin
generation increased: ETP, 2726 531nM min; peak height;
168 10nM (P .001 vs control for both parameters; Figure 4).

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1946

BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

GROTTKE et al

Table 2. Hemodynamic variables

Baseline

Hemodilution

30 min
after
trauma

60 min
after
trauma

120 12

140 23

120 min
after
trauma

HR, beats/min
Control

86 8

80 6*

PCC35

82 11

83 8

94 10

98 10

155 26
93 13

PCC50DIC

91 6

85 5

99 17

94 19

108 18

PCC50DIC

85 9

76 4*

133 22

132 24

165 21

Control

96 11

86 7*

44 13

40 11

30 8

PCC35

94 10

87 10*

56 18

66 13

65 12

PCC50DIC

88 9

84 5

61 9

69 9

68 5

PCC50DIC

87 11

78 4

35 10

31 14

26 13

Control

71

71

41

31

21

PCC35

72

81

41

42

5 1

PCC50DIC

62

81

5 1

5 1

6 1

PCC50DIC

61

8 1*

41

51

5 1

MAP, mmHg

CVP, mmHg

MPAP, mmHg
Control

18 2

18 2

11 3

10 2

92

PCC35

17 3

20 3

12 4

13 3

13 5

PCC50DIC

17 2

18 2*

15 4

15 1

16 2

PCC50DIC

18 5

17 2

13 4

11 2

11 1

CO, L/min
Control

4.2 0.5

4.3 0.4

3.0 1.2

2.4 1.3

1.3 0.5

PCC35

4.3 0.4

4.4 0.5

3.5 0.4

3.3 0.4

3.3 0.5

PCC50DIC

3.9 0.4

4.3 0.6

3.7 0.7

3.7 0.6

3.2 0.6

PCC50DIC

4.1 0.5

3.9 0.2

2.6 0.9

2.4 0.9

1.7 0.2

Data are mean SD.

CO indicates cardiac output; CVP, central venous pressure; HR, heart rate; MAP, mean arterial pressure; and MPAP, mean pulmonary pressure
*P .05 versus each group over time.
P .05 versus control.
P .05 versus PCC35.
P .05 versus PCC50DIC.

Although both parameters gradually declined during the observation period, thrombin generation remained higher than baseline at
120 minutes after trauma (ETP, 1525 38nM min; peak height,
128 4nM). Levels of TAT complexes were significantly higher
than those in controls at all time points after PCC administration
(P .001; Figure 5), whereas significantly higher levels of Ddimers were only observed 120 minutes after trauma (P .001 vs
control). All animals in the PCC35 group survived until the end of
the experiment. No significant difference in survival (P .145)
was observed between the PCC35 and PCC50groups.
Substitution with 50 IU/kg PCC. In animals not developing
DIC (5 of 9 [56%] in the original PCC50 group), the coagulation
parameters, blood loss and hemodynamic stability were compa-

rable with those of animals in the PCC35 group (Tables 1-2;

Figures 1-2 and 5). Animals developing DIC (n 4, 44% of the
original PCC50 group) exhibited significantly prolonged PT, CT,
and CFT (Figures 1-2). This was accompanied by significant but
temporary decreases in the concentrations of fibrinogen and clot
strength (MCF; Table 1; Figure 2). Levels of platelets, hemoglobin,
and antithrombin decreased over time in the PCC50DIC group,
such that they were lower in this group than in all other groups at
120 minutes after trauma (Table 1).
Immediately after PCC infusion, TAT complexes and D-dimers
had increased in the PCC50DIC group, and these levels continued
to increase throughout the observation period. Conversely, in the
PCC50DIC group, there was no increase 60 minutes after trauma in

Figure 1. Prothrombin time. Prothrombin time was measured on

a coagulometer to determine the effect of PCC therapy. The data
are mean values. *P .05 vs control. P .05 vs PCC35;
P .05 vs PCC50DIC. , not detectable.

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BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

PCC SUBSTITUTION AFTER LIVER INJURY

1947

Figure 2. Thromboelastometry (ROTEM) parameters. The

ROTEM coagulation analyzer was used to assess blood clotting.
The EXTEM assay was used according to the manufacturers
instructions. Clotting was activated by the EXTEM reagent, which
contains tissue factor as the starting reagent. (A) CT, representing
the initiation of clot formation; this corresponds to the reaction
time. (B) CFT, the time between clot initiation and attainment of an
amplitude of 20 mm. (C) MCF, reflecting the strength of the
resulting clot. *P .05 vs control; P .05 vs PCC35; P .05
vs PCC50DIC. , not detectable.

levels of TAT complexes, although D-dimers started to increase

60 minutes after trauma (Figure 3). Overall trends in levels of TAT
complexes and D-dimers were similar in the PCC50DIC and
PCC35 groups.

Thrombin generation increased immediately after PCC administration in all animals receiving the 50 IU/kg dose. At 30 minutes
after trauma, ETP was 3455 494nM min, and peak height was
253 4nM (pooled data from PCC50DIC and PCC50DIC

Figure 3. TAT complexes and D-dimers. Markers of

coagulation activation at baseline (after splenectomy),
after hemodilution, and at 30, 60, and 120 minutes after
trauma. (A) TAT complexes. (B) D-dimers. Data are
shown as box plots (minimum, first quartile, median,
third quartile, and maximum). *P .05 vs control;
P .05 vs PCC35; P .05 vs PCC50DIC.

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1948

GROTTKE et al

BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

Figure 4. Representative curves from the thrombin generation assays. (A) Plasma thrombin generation, for assessment of hypercoagulability after PCC administration.
Thrombin generation in platelet-poor plasma was assessed by means of the CAT according to standard in-house procedures, using 1pM tissue factor and 4M phospholipids
to trigger the assay. Representative curves are shown for baseline, hemodilution, and 30, 60, and 120 minutes after trauma, following the administration of saline (top row: open
curves); 35 IU/kg PCC (middle row: light gray curves); and 50 IU/kg PCC (bottom row: dark gray curves). Independent of the dose of PCC, plasma thrombin generation was
characterized by a prolonged time to tail, indicating attenuated inhibition of coagulation. (B) 50 IU/kg PCC can induce a hypercoagulable state associated with DIC. Top row:
thrombin generation curves in plasma from an animal receiving 50 IU/kg PCC and not developing DIC. Bottom row: thrombin generation curves in plasma from an animal
receiving 50 IU/kg PCC and developing DIC (PCC50DIC). Remarkably, no difference was observed in thrombin generation before and after hemodilution.

animals; P .001 vs PCC35 for both parameters; Figure 4). Both

of these parameters remained higher than baseline at 120 minutes
after trauma (ETP, 2143 982nM min; peak height, 196 15nM).
Thrombin generation curves from animals in the PCC50DIC group
showed a lack of reduction in thrombin generation after hemodilution. One animal in both in the PCC50DIC and PCC50DIC groups

Figure 5. Total blood loss, measured 120 minutes after liver injury. Horizontal
lines depict the means. *P .05 vs control; P .05 vs PCC35; P .05 vs
PCC50DIC.

died before the end of the observation period. No significant

difference in survival was observed between the PCC50DIC and
PCC50DIC group compared with that of the control group
(P .937).
Histopathologic analysis

The histopathologic examination of injured liver sections revealed

homogeneous tissue damage and comparable lacerations of venous
vessels up to 4 mm in diameter (Figure 6A-B). No thromboemboli
or other pathologic changes were present in kidneys or nontraumatized liver tissue. In the myocardium, there were no thromboemboli, but signs of premortal hypoxia (waving) were observed.
Two control animals had singular thromboemboli, 1-2 mm in
diameter, inside the lung arterioles. In 3 animals from the PCC35
group, several thromboemboli 1-2 mm in diameter were found. All
animals from the PCC50 group had disseminated thromboemboli
in lung arterioles and arteries up to 4 mm in diameter (Figure
6C-E). Three of 4 animals from the PCC50DIC group also showed
signs of a reticular (net-like) pattern of fibrinogen deposits within
lung capillaries (Figure 6H). In contrast, fibrinogen deposits in lung
capillaries of all other animals were small in both size and number
(Figure 6F-G).

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BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

Figure 6. Macroscopic liver injury and microscopic histology of lung tissue:

representative samples. Postmortem internal organs were removed to evaluate the
degree of liver injury and to assess the occurrence of any thromboembolic events.
(A-B) Macroscopic and histologic images of liver trauma, with laceration of venous
vessels up to 4 mm in diameter. (B) H&E stain. (C-E) Multiple thromboemboli in the
PCC50 group (up to 4 mm in panel E). (C,E) H&E stain. (D) Immunohistochemical
staining against fibrinogen. (F-G) Samples from the control group and PCC50 without
DIC with rare, irregular, small deposits of fibrinogen in lung capillaries. (H) Net-like
staining pattern for fibrinogen in lung capillaries of animals of the PCC50 group wth
DIC. (F-H) Immunohistochemical stain against fibrinogen. Original magnification:
panels B and E, 20; panels C-D, 40; panels F-H, 400.

Discussion
This experimental animal study is the first to examine the effects of
2 doses of PCC on blood loss and coagulation parameters after
trauma. PCC at a dose of 35 IU/kg was effective in reversing
coagulopathy related to hemodilution and trauma, reducing blood
loss compared with that in controls. At 50 IU/kg, PCC treatment
was followed by DIC in approximately half of the animals.
Histopathologic assessment showed thromboemboli in all animals
receiving the higher dose, including those not developing DIC.
This was accompanied by a net-like pattern of pulmonary fibrinogen deposits in 3 of 4 animals developing DIC.
Our finding that PCC can, at an appropriate dose, be effective in
treating coagulopathy is in agreement with several animal studies
of PCC in dilutional coagulopathy.5-7 However, we also found that
50 IU/kg of PCC had no greater impact on blood loss than
35 IU/kg. This could be related to the fact that all thrombin-

PCC SUBSTITUTION AFTER LIVER INJURY

1949

generating drugs are dependent on an adequate level of the

substrate fibrinogen to be effective. Accordingly, it has been shown
that treatment with both recombinant factor VIIa and fibrinogen is
more effective in correcting coagulopathy than treatment with
either agent alone.10,36 Likewise, results from 2 different studies
performed in a porcine model of trauma indicated considerably
greater reduction in blood loss after coadministration of fibrinogen
plus PCC,8 compared with administration of fibrinogen alone.37
Thus, combined treatment with fibrinogen and PCC has been
advocated in trauma, with fibrinogen supplementation as the first
priority on the basis that critically low levels of fibrinogen are
encountered early in massive bleeding.8,23,38 It would certainly be
of interest to repeat our study with this treatment regimen. Studies
have shown that coagulopathy-related bleeding may be independently associated with low concentrations of either thrombin or
fibrinogen.39 For targeted therapy with coagulation factor concentrates, it is necessary to identify the cause of coagulation deficiency.
This approach allows therapy to be tailored to each patients
specific needs.23 In contrast, formula-driven general treatment with
allogeneic blood products (eg, administration of red blood cells/FFP/
platelets in a 1:1:1 ratio) may lead to unnecessary use of blood
products.
In our experimental animal model, the EXTEM assay did not
point to a hypercoagulable state after PCC infusion: CT was not
shortened (relative to baseline) in any of the treatment groups. In
addition, only small differences were observed between the 35 and
50 IU/kg doses of PCC in all of the EXTEM parameters (considering animals not developing DIC). Likewise, PT showed little
difference between the PCC35 and PCC50DIC groups, with little
change after hemodilution in these animals. In contrast, the
thrombin generation assay showed a clear, dose-dependent increase
in thrombin generation after PCC administration. Several different
factors may have contributed to these findings. First, the end point
of the CT and PT parameters is formation of fibrin, meaning that
they depend on an adequate level of fibrinogen. Second, the
relationship between coagulation factor levels and CT/PT values is
not linear: in hemodilution, the concentration of coagulation factors
needs to drop to 40% before prolongation of CT or PT is
observed.40,41 On the other hand, there is a linear relationship
between thrombin generation and levels of coagulation factors,
making this assay more sensitive to changes in these proteins.
Third, neither EXTEM CT nor PT is influenced by antithrombin,
whereas thrombin generation is strongly influenced by antithrombin. Fourth, much higher levels of tissue factor are used in the PT
and EXTEM assays than in the thrombin generation assay.
Prolongation of EXTEM CT has already been used as a trigger for
the administration of PCC in a goal-directed algorithm of trauma
bleeding management.23,38 The question of whether EXTEM CT
may be used to guide the dose of PCC has not yet been investigated,
but the present data suggest that it might not be appropriate for this
purpose.
The present study suggests that a clinical trial program (phases
II and III) is needed to establish the safety and efficacy of PCC in
patients with trauma-induced or dilutional coagulopathy and different underlying risk factors for thromboembolic events. In accordance with our expectations for clinical practice, such a program
would ideally investigate PCC therapy in combination with fibrinogen supplementation (with PCC being administered as the second
of these 2 interventions). Establishing the optimal PCC dose
would, of course, be a key reason for pursuing the clinical trial
program. The choice of 50 IU/kg as the highest dose in our study

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1950

BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

GROTTKE et al

was in accordance with previous studies. A clinical study of

individualized PCC dosing for the reversal of vitamin K antagonists included 50 IU/kg as the highest dose,33 whereas in another
publication 50 IU/kg PCC was recommended for vitamin K antagonist patients with an international normalized ratio 6.0.42 Furthermore, a pharmacokinetic study of PCC conducted in healthy
volunteers used a dose of 50 IU/kg.32 The lower dose (35 IU/kg)
was chosen on the basis that this dose was shown in previous
porcine studies to be effective in the treatment of dilutional
coagulopathy.5,8 The median PCC dose administered in a trial of
combined administration of fibrinogen concentrate and PCC in
trauma patients was approximately 22 IU/kg.23 Clinical doses of
PCC for trauma-induced coagulopathy have not yet been established; therefore, the optimal doses to include in investigation of
the safety or efficacy of PCC in this setting are poorly defined.
In the present study, protracted elevation of TAT and D-dimers
accompanied by fibrin precipitation in lung capillaries in a subset
of animals indicated an excessive activation of coagulation. The
increase in TAT immediately after the application of PCC was
expected. However, the continuous increase throughout the observation period is surprising and indicates ongoing activation of the
coagulation system. In a study of 50 IU/kg PCC for reversal of
rivaroxaban, thrombin generation (ETP) was measured for 24 hours
after PCC administration.43 ETP increased within 15 minutes of
PCC administration and continued to increase over the observation
period to reach a level 50% higher than the pre-rivaroxaban
baseline level. It remains unclear whether ETP would have returned
to the pre-rivaroxaban level. The protracted activation of coagulation after PCC administration may be attributable to an imbalance
of prohemostatic factors and inhibitors, specifically an insufficient
level of antithrombin compared with the potential for thrombin
generation. This hypothesis corresponds with in vitro and in vivo
data that identified prothrombin overload (and insufficient inhibition of newly generated thrombin by antithrombin) as the most
likely cause of thrombosis.18,44 It is also supported by our
observations of prolonged tails as well as large amplitudes in the
thrombin generation curves, indicating a lack of thrombin inhibition. In a biochemical comparison of 7 commercially available
PCCs, none of the products contained antithrombin at a sufficient
level to balance the thrombin-generating potential of prothrombin.11 Thus, the application of antithrombin might be considered
before PCC infusion for trauma-induced coagulopathy to protect
against excessive systemic thrombin levels.
The apparent potential for increased risk of thromboembolism
and DIC when PCC is used to treat trauma-induced coagulopathy is
of major clinical significance. The European Medicines Agency
core summary of product characteristics for PCCs gives treatment
of bleeding and perioperative prophylaxis of bleeding in acquired
deficiency of the prothrombin complex coagulation factors as an
indication.45 This provides authorization for the use of PCC in
trauma-induced coagulopathy. Physicians may be assured by
statements in the literature that PCCs are balanced products
because of the presence of anticoagulant proteins.11,46,47 However,
we believe that PCCs cannot be considered as balanced unless they
include antithrombin at an quantity equivalent to that of
prothrombin.
Several limitations of our study need to be acknowledged.
Hemodilution had to be induced before the infliction of injury to
standardize the degree of coagulopathy. Clinically, hemodilution
results from blood loss (caused by, eg, major trauma). This is

followed by the infusion of large volumes, and hemostatic agents

are administered as coagulopathy occurs. In addition, in the present
study, liver injury was induced in anesthetized healthy pigs.
Physiologic responses to factors such as pain and inflammation
may have additional effects on hemostasis, and these are not
reflected in our model. It is possible that fibrinogen levels were
overestimated in the present study, because hydroxyethyl starch
had been administered.48-50 The method used to determine the
incidence of DIC in this animal study was based on clinical
judgment, with consideration of the International Society on
Thrombosis and Hemostasis criteria, which were designed for
humans.35 There are no established diagnostic criteria for porcine
DIC, but the clinical signs of DIC were clear in this study.
Coagulation profiles differ between species; therefore, the extent to
which the present results can be extrapolated to humans needs to be
confirmed.
In conclusion, coagulopathy associated with hemodilution and
trauma was reversed in this study by early administration of
exogenous PCC at a dose of 35 IU/kg. Animals receiving this dose
showed lower blood loss and a higher survival rate. However, data
from this study show that PCCs can also induce a prothrombotic
state. Given the lack of clinical safety data for PCCs in this setting,
the present data provide the best available insight. Clinical studies,
particularly in patients with comorbidities, are needed to optimize
dosing and ascertain the safety of PCCs for coagulopathy associated with hemodilution and trauma.

Acknowledgments
The authors thank Thaddaus Stopinski (Institute of Laboratory
Animal Science, RWTH Aachen University Hospital) for animal
experiments and Professor Dietrich Henzler (Department of Anesthesia and Division of Critical Care, Dalhousie University Halifax)
for his critical reading.
This research was supported by the START program of the
Faculty of Medicine, RWTH Aachen, Biotest (Dreieich, Germany),
and Sanquin (Amsterdam, The Netherlands).

Authorship
Contribution: O.G. conceived and conducted the experimental
laboratory work and drafted the manuscript; T.B. performed the
pathologic analyzes and drafted the manuscript; H.M.H.S., R.v.O.,
and H.t.C. performed the CAT assays and helped to write the
manuscript; S.E. and A.D.R. helped to perform the study; C.F.
performed the statistical analyses; R.T. helped to prepare the
manuscript; and R.R. participated in the study design and helped to
write the manuscript. All authors read and approved the final
manuscript.
Conflict-of-interest disclosure: O.G. has received research funding from Novo Nordisk (Denmark) and R.R. has received honoraria for lectures and consultancy from CSL Behring (Germany),
Novo Nordisk (Germany), Bayer Healthcare (Germany) and Air
Liquide (France). The remaining authors declare no competing
financial interests. The sponsors had no influence on the design of
the study or the interpretation of results.
Correspondence: Oliver Grottke, Department of Anesthesiology, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074
Aachen, Germany; e-mail: ogrottke@ukaachen.de.

From www.bloodjournal.org by guest on October 19, 2016. For personal use only.
BLOOD, 18 AUGUST 2011 VOLUME 118, NUMBER 7

PCC SUBSTITUTION AFTER LIVER INJURY

1951

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From www.bloodjournal.org by guest on October 19, 2016. For personal use only.

2011 118: 1943-1951

doi:10.1182/blood-2011-03-343046 originally published
online June 13, 2011

Increasing concentrations of prothrombin complex concentrate induce

disseminated intravascular coagulation in a pig model of coagulopathy
with blunt liver injury
Oliver Grottke, Till Braunschweig, Henri M. H. Spronk, Stephanie Esch, Annette D. Rieg, Rene van
Oerle, Hugo ten Cate, Christina Fitzner, Rene Tolba and Rolf Rossaint

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