JBUR 5837 No.

of Pages 14

burns xxx (2019) xxx –xxx

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier.com/locate/burns

Plasma protein C levels are directly associated with

$
better outcomes in patients with severe burns

Thomas Charles Lang a,c, * , Ruilong Zhao a, Albert Kim b,

Aruna Wijewardena b, John Vandervord b , Rachel McGrath b ,
Siobhan Fitzpatrick b , Gregory Fulcher b, Christopher John Jackson a
a
Sutton Laboratories Level 10, The Kolling Institute, The University of Sydney, Northern Clinical School, Royal North
Shore Hospital, Reserve Rd, St. Leonards, 2065, NSW, Australia
b
Royal North Shore Hospital, Reserve Rd St., Leonards, 2065, NSW, Australia
c
Department of Anaesthesia, Prince of Wales and Sydney Children’s Hospitals, Barker St, Randwick, 2031, NSW, Australia

article info abstract

Article history: Protein C circulates in human plasma to regulate inflammation and coagulation. It has
Accepted 1 May 2019 shown a crucial role in wound healing in animals, and low plasma levels predict the presence
Available online xxx of a wound in diabetic patients. However, no detailed study has measured protein C levels in
patients with severe burns over the course of a hospital admission. A severe burn is
associated with dysfunction of inflammation and coagulation as well as a significant risk of
Keywords:
morbidity and mortality. The current methods of burn assessment have shortcomings in
Protein C
reliability and have limited prognostic value. The discovery of a biomarker that estimates
Severe burns
burn severity and predicts clinical events with greater accuracy than current methods may
Surgery
improve management, resource allocation and patient counseling. This is the first study to
Clinical outcomes
assess the potential role of protein C as a biomarker of burn severity.
Intensive care
We measured the plasma protein C levels of 86 patients immediately following a severe
Fluid resuscitation
burn, then every three days over the first three weeks of a hospital admission. We also
analysed the relationships between burn characteristics, blood test results including
plasma protein C levels and clinical events. We used a primary composite outcome of
increased support utilisation defined as: a mean intravenous fluid administration volume
of five litres or more per day over the first 72 h of admission, a length of stay in the
intensive care unit of more than four days, or greater than four surgical procedures during
admission. The hypothesis was that low protein C levels would be negatively associated
with increased support utilisation.
At presentation to hospital after a severe burn, the mean plasma protein C level was 76  20%
with a range of 34–130% compared to the normal range of 70–180%. The initial low can be
plausibly explained by impaired synthesis, increased degradation and excessive consump-
tion of protein C following a burn. Levels increased gradually over six days then remained at a
steady-state until the end of the inpatient study period, day 21. A multivariable regression
model (Nagelkerke’s R2 = 0.83) showed that the plasma protein C level on admission
contributed the most to the ability of the model to predict increased support utilisation

$
The Department or Institution to which this work should be attributed is The Kolling Institute of The University of Sydney, associated
with Royal North Shore Hospital.
* Corresponding author at: Department of Anaesthesia, Prince of Wales and Sydney Children’s Hospitals, Barker St, Randwick 2031, NSW
Australia.
E-mail address: thomas.lang@sydney.edu.au (T.C. Lang).
https://doi.org/10.1016/j.burns.2019.05.001
0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14

2 burns xxx (2019) xxx –xxx

(OR = 0.825 (95% CI = 0.698-0.977), P = 0.025), followed by burn size (OR = 1.252 (95% CI = 1.025–
1.530), P = 0.027), burn depth (partial thickness was used as the reference, full thickness
OR = 80.499 (1.569–4129.248), P = 0.029), and neutrophil count on admission (OR = 1.532 (95%
CI = 0.950–2.473), P = 0.08). Together, these four variables predicted increased support
utilisation with 93.2% accuracy, 83.3% sensitivity and 97.6% specificity. However if protein C
values were disregarded, only 49.5% of the variance was explained, with 82% accuracy, 63%
sensitivity and 91.5% specificity. Thus, protein C may be a useful biomarker of burn severity
and study replication will enable validation of these novel findings.
© 2019 Elsevier Ltd and ISBI. All rights reserved.

sepsis after a burn, [17,18] and inflammatory cytokine

1. Background
A severe burn is associated with dysfunction of both
inflammation and coagulation and a significant risk of
morbidity and mortality. Uncontrolled inflammation follow-
ing burns leads to the systemic inflammatory response
syndrome [1] and a paradoxically increased risk of infection
and sepsis [2]. Disseminated intravascular coagulation, mi-
crovascular thrombosis, haemoconcentration [3], ineffective
fibrin aggregation, platelet dysfunction [4] and aggressive fluid
resuscitation primarily with crystalloid solution [5] all con-
tribute to widespread disruption to coagulation processes and
poor tissue perfusion. Both inflammatory and coagulation
dysfunction pathways converge to produce, in many cases,
multi-organ dysfunction syndrome [6] These complications
benefit from intensive and coordinated treatment to prevent
morbidity and mortality.
Adjusted inpatient mortality incidence is approximately
4%. [7] This is similar to the inpatient mortality incidence
among patients with acute myocardial infarction [8] or
pulmonary embolus [9]. The adjusted all-cause mortality rate
ratio is 1.8 times higher among adolescent, adult and middle-
aged patients with burns compared to an index hospital
population without burns [10]. Among older patients, long-
term mortality attributable to burn injury is 29% [11]. Morbidity
is prevalent but more difficult to quantify given the heteroge-
nous nature of the condition and the variety of methods used
to report outcomes after a burn [12–14].
Given this risk of mortality and morbidity, prognostic
models are used in the care of patients with burns. These
models are conventionally derived from regression analyses
of clinical data [15]. Such data includes findings from the
clinical examination like burn size and burn depth. Prognos-
tic models are often referred to as burn severity scores.
However, many scores are not properly validated, do not
provide odds ratios and cannot quantify the risk of a given
outcome [16]. Therefore, other data should be used in
conjunction with the clinical examination to accurately
predict clinical events.
The discovery of a biomarker, which provides clinicians
with an adjunct to the clinical examination in order to
estimate burn severity, quantify risk and predict clinical
events with greater accuracy than current methods, may
improve management decisions, resource allocation and
patient counseling. Indeed, studies have shown an associ-
ation between clinical events following a sever burn and the
results of blood tests. For example, procalcitonin predicts
levels such as IL-8, [19,20], IL-4, GM-CSF, MCP-1 are
associated with mortality [20] as are enzymes TIMP-1 [21]
and gelsolin [22].
Protein C is a serine protease synthesised largely by the
liver [23], as well as vascular endothelial cells and epidermal
keratinocytes [24], which circulates in plasma as a zymogen
of activated protein C (APC) [23]. It has a molecular weight of
62 kD and consists of 419 amino acids: three major moieties
comprise the light chain, which connects to the catalytic
heavy chain by a disulphide bridge [23]. The protein C system
is best known for its function of regulating coagulation [25].
In addition, APC exerts potent anti-inflammatory and
cytoprotective functions [23]. Protein C circulates in normal
plasma at an average concentration of 4 mg/mL [26]. The
plasma concentration of APC is 2000 times lower than the
concentration of protein C [23] and measurement is not
feasible in clinical practice as the half-life of APC is only
15 min [27]. Protein C levels are far more stable and are easily
measured by analysing plasma with an assay which
determines functional protein C levels, and reporting protein
C levels as a percentage compared to normal levels [26]. The
current World Health Organisation (WHO) standardised
units for reporting protein C are IU/mL [28] although it is
also reported with units of mg/mL [26], % [29] and U/dL [30].
We have shown previously that protein C levels are an
independent predictor of the presence of a wound in age, sex
and type of diabetes-matched subjects, to a greater extent
than HbA1c% or C-reactive protein (CRP) levels [31]. However,
diabetic wounds are typically chronic and there is limited
data about plasma protein C levels in patients with acute
wound states, such as a burn injury. Protein C levels in sheep
with experimentally induced burns decreased over the first
24 h after injury [32]. A small study of five patients with burns
of 20–70% total body surface area (TBSA) found that protein C
levels ranged from 26 to 89% of normal [33]. Another more
recent small study of nine patients with burns of 25–95%
TBSA found that protein C levels taken at hospital admission
ranged from 30 to 95% of normal [29]. Interestingly, this in-
depth study of dozens of clotting factors and proteins
showed that protein C was the only component of the
coagulation system that was significantly different between
survivors and non-survivors at time of admission (82  9% vs.
56  18%, P = 0.04) [29].
There is physiological congruity between the role of
protein C and the pathophysiology of burns, and there is the
potential for improvement of methods estimation of severity
of injury and prognostication in patients with burns, and

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14
burns xxx (2019) xxx –xxx
there is a growing body of evidence showing prognostic
utility of several blood markers of inflammation in burns.
Therefore, we measured protein C levels of patients with
severe burns on admission and serially throughout their
hospital stay and then investigated the relationship between
patient characteristics, burn characteristics, several blood
test results including protein C levels, and clinical outcomes,
in order to test our hypothesis. Our hypothesis was that
protein C levels would be negatively associated with support
utilisation in patients with severe burns. Other aims of the
study included the quantification of protein C levels over
time in patients with burns, as well as comparison of protein
C levels between groups stratified by burn size, burn depth
and survival, as well as the establishment of predictors of
outcome following a severe burn based on a multivariable
regression model of the collected data.
2. Methods
This was a single-centre prospective observational cohort
study undertaken in Royal North Shore Hospital, a large
teaching hospital in New South Wales, Australia, which also
functions as a Level 1 Trauma Centre and Burns Referral
centre. Participating departments included the Severe Burns
Unit (SBU), the Intensive Care Unit (ICU) and the Emergency
Department (ED). The SBU provides ward-level care for
patients with severe burns admitted under the Burns surgical
team. It is staffed with specialist burns staff, who also see
patients in the SBU outpatient clinic for follow up. The study
was designed and completed in accordance with the
Strengthening Reporting of Observational Studies in Epide-
miology (STROBE) statement [34] and approved by the
Northern Sydney Local Health District Human Research
Ethics Committee.
Patients with severe burns were admitted to hospital and
after providing written informed consent were enrolled as
participants according to the following inclusion and exclu-
sion criteria: participants were above eighteen years of age and
had burns between 10–80% TBSA involved, with some area of
the burn as partial thickness. These burn sizes were selected
on the rationale that burns below 10% TBSA would not lead to
an appreciable change in protein C levels, while the cut-off of
80% was decided by our ethics committee as a suitable upper
limit for patient inclusion. Burn depth was determined
clinically by consensus among senior burns surgeons at the
time of presentation and was defined as the most prevalent
depth of burn. If there was conjecture about burn size or depth,
or difficulty with clinical examination in the ED, the burn size
and depth were clarified when the patient was under general
anaesthesia prior to excision and grafting. This practice is part
of usual care at this centre.
Patients were not eligible to be enrolled if they were
under eighteen years of age, pregnant or lactating, had
clinically significant clotting or bleeding disorders (includ-
ing any pre-existing condition requiring anticoagulation
with warfarin or other oral anticoagulants) or had active
local or systemic infection. The age cut-off was determined
by the ethics committee while pregnancy, infection and
clotting disorders can all affect baseline protein C levels
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
3
therefore patients were excluded if any were present, in
order to maximize the likelihood of including patients with
normal baseline protein C levels. Participants were deemed
to have severe burns by definition of transfer criteria to this
facility in accordance with the New South Wales Burns
Transfer Guidelines [35].
Ethics committee approval was gained for the recruitment
of 86 patients into this study and was adequately powered for
data analysis of approximately 60 patients, based on results
from a similar study, using an a prioi analysis [31]. Selection
bias was minimised by the prospective nature of enrolment
and strict adherence to inclusion and exclusion criteria, and
loss-to-follow up bias was minimised by active retention and
regular follow up in our SBU outpatient clinic. Potential
confounders were minimised by recording all exposures
during the patient’s admission and the cohort underwent
stratification and regression.
Patients were assessed immediately on arrival to the ED
by senior burns surgeons as part of usual care, which
included visual assessment of the burn with formal quanti-
fication of burn size and depth. All patients received usual
care throughout and after the inpatient study period of
21 days. Blood samples were collected on the day of
admission (day 0) and subsequently every three days for
three weeks, until day 21 or discharge. Time of hospital
presentation, rather than time of injury, was selected as time
point zero for several reasons. Firstly, we lacked accurate
data for all patients about the time of injury. Secondly, the
risk of data collection and data entry error was felt to be
unacceptably high if time of injury was selected as time point
zero, because all electronic medical record data used time of
presentation as time point zero. Thirdly, the estimated time
of injury occurred within 24 h to presentation to hospital for
all patients. Based on the understanding of human protein C
production and consumption, this was felt to be an
acceptable time-window before the first blood sample was
obtained. Blood samples were taken third-daily, rather than
another frequency, based on the rationale that more
frequent collection would be unlikely to yield more valuable
data for the purposes of the study, whilst also being unlikely
to yield clinically valuable data and unacceptably increasing
patient discomfort. Blood samples were also taken at follow-
up at 12 months after discharge.
Blood samples were obtained and analysed for the total
amount of functional protein C by trained pathologists in the
Royal North Shore Hospital Pacific Laboratory Medicine
Services using a Diagnostica Stago STA-R Evolution (France)
coagulation analyser (Series Number CA81044175). Blood
samples were included for analysis if they were collected
within 24 h of each designated time point. Clinical data were
also collected as part of routine care and included demograph-
ic details, intravenous fluid, details of surgery undertaken,
length of stay (LOS) within the hospital and within the ICU and
several blood markers of inflammation and organ function.
1 1
Data was analysed with IBM SPSS Statistics v20 and
1
GraphPad Prism v7. A statistician not involved in data
collection independently oversaw analysis of the data.
Statistical tests were undertaken to assess the relationships
between patient characteristics, laboratory data and clinical
events.
JBUR 5837 No. of Pages 14
4 burns xxx (2019) xxx –xxx
2.1. Primary outcome: increased support utilisation
throughout admission
We used a binary composite outcome for this study, termed
increased support utilisation. This outcome identified
patients who received large amounts of intravenous fluid,
and/or had a prolonged LOS in the ICU, and/or had numerous
surgical procedures during their admission. Each of these
components of care is considered standard treatment but
each carries risks, which contribute to patient morbidity and
therefore excess exposure to these treatments comprises a
complicated admission utilising increased support. It is
important to identify these clinical events in order to
carefully consider exactly how best to select the best
treatment for these patients.
2.1.1. Volume of intravenous fluid administered
In patients with severe burns, complications of high-volume
fluid resuscitation are well described and include pulmonary
oedema [36], intraabdominal hypertension (IAH) and abdomi-
nal compartment syndrome (ACS) [37–42]. Fluid infusion
regimes such as the Parkland formula provide high volumes
of fluid in the first 8 h, with the volume administered over the
subsequent 16 h guided by urine output [43]. Most burns
centres, including ours, utilise the Parkland protocol [44,45].
However, the majority do not adhere to the original protocol
and subsequently, patients are administered too much fluid
[46–48]. There is clear evidence of a relationship between the
volume of intravenous fluid administered, particularly crys-
talloid solutions, and the incidence of adverse events. A lack of
validated protocols for fluid administration in patients with
severe burns beyond the first 24 h after injury may contribute
to this phenomenon.
Klein et al. found a total volume of 250 mL/kg of crystalloid
fluid led to patients having a higher risk of death, acute
respiratory distress syndrome, pneumonia, and other com-
plications [49]. Ivy et al. found a threshold of 250 mL/kg of fluid
led to ACS at 48 h after burn [50]. Oda et al. found a fluid
threshold of 300 mL/kg at any time point in admission to
produce IAH, which in their cohort was approximately 21 L of
intravenous fluid administered throughout admission. In that
study, patients received intravenous fluid infusions for at least
4 days which is equivalent to 5.25 L per day for four days in a
70 kg patient [51]. In a randomised trial, surgical patients who
were administered higher volumes of intravenous fluids had
higher rates of complications compared to patients who were
administered restricted volumes of fluid and this was most
marked for patients who gained >5.51 kg of body weight with
the amount of weight gain in kg closely related to the volume of
intravenous fluid administered in L, with the highest risks of
complications occurring in the first 72 h [52]. Given that the
usual duration of fluid resuscitation in patients with burns is at
least three days [51], the average ideal body weight in a patient
with burns is approximately 70 kg [53], and the total volume of
intravenous fluid which is associated with morbidity is
approximately 250 mL/kg, the approximate time point at
which a high-risk volume of fluid of is administered is three
days. Therefore, we instituted part of our primary outcome to
include a mean intravenous fluid administration volume of 5 L
or more per day over the first 72 h of admission.
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
2.1.2. ICU LOS
Mortality rates remain steady [7] therefore studies of pre-
dictors of patient outcomes focus on morbidity. Measures of
morbidity are not equal. Some are easy to measure, like
hospital LOS. However, hospital LOS alone does not accurately
capture the degree of support during an admission and does
not impact the cost of a hospital admission, whereas ICU LOS
does [54]. LOS in the ICU provides a clearer picture of a patient
who utilises significant support during their admission and it
also predicts mortality after discharge [55,56]. More recent
studies of patients with burns have used ICU LOS as their
primary outcome [57] or as part of a composite primary
outcome [58–61]. For example, one study of 250 patients with a
mean TBSA of 32.2% showed that patients spent a mean
duration in ICU of 0.23 days per 1% TBSA [54]. ICU LOS is
important to determine the amount of support a patient needs,
the cost of admission, and predicting mortality after discharge.
Furthermore, longer stays in ICU are associated with adverse
outcomes. In 821 adult patients with respiratory failure or
sepsis without known underlying cognitive deficit and a
median ICU LOS of five days, 34% of patients exhibited signs of
a moderate traumatic brain injury and 24% exhibited signs of
Alzheimer’s disease at one year follow up [62]. In 34,696 elderly
ICU patients with a mean LOS in ICU of 3.4 days, each day
beyond seven days in ICU increased the odds of death at one
year by 104% [63]. A systematic review of sequential organ
failure assessment (SOFA) models in patients in ICU showed
that severity of illness measured over five days correlated
strongly with mortality [64]. Thus we included patients in the
outcome of increased support if they had an ICU LOS of five or
more days.
2.1.3. Number of surgical procedures undertaken
Practices vary between centres regarding the timing and
amount of surgery undertaken [65]. In our centre, almost all
patients with severe burns undergo excision and grafting.
Graft loss is a complication of excision and grafting following
a burn. Among patients with graft loss, re-operation is
common [66]. Most of our procedures are performed under
general anaesthesia. It is known that perioperative compli-
cations increase with the number of surgeries during
admission [67–69] such as pathological scarring [70] and
general anaesthesia associated post-operative cognitive
dysfunction syndrome [71–73]. There is limited data to
suggest a threshold number of surgeries that predisposes
someone to increased risk of adverse outcomes and further
studies are required. So in order to identify patients who may
have graft failure and who may potentially be at risk of
complications from multiple general anaesthetics, we de-
fined a group of patients who had a higher than expected
number of surgeries. The mean number of surgeries each
patient underwent in our study was four, so we included
patients in this outcome who underwent more than the
mean of four surgical procedures.
Thus, we instituted a composite binary classification to
identify patients who used increased support throughout
admission. This included patients with any of the following: a
mean intravenous fluid administration of 5 L or more per day
over the first 72 h of admission, a LOS in the ICU of five or more
days, or five or more surgeries during the admission.
JBUR 5837 No. of Pages 14

burns xxx (2019) xxx –xxx 5

2.2. Statistics Table 2 – Clinical overview of patient treatment

throughout their admission.
Descriptive statistics are expressed with the mean followed by All patients
standard deviation, or by n followed by %, unless otherwise
Intubated, n (%)
stated. Statistical tests used are described when relevant.
Yes 23 (27)
No 63 (73)
Admitted to, n (%)
3. Results SBU 49 (57)
ICU 37 (43)
3.1. Patient and burn characteristics LOS (days), mean  SD
Total 25  31
ICU 6  14
All 86 enrolled patients who met inclusion and exclusion
Duration of intubation in days, mean  SD 88
criteria were included in the prospective analysis. Patient and Intravenous fluid administration (L), mean  SD
burn characteristics are shown in Table 1. One quarter of Day 0 3.64  3.09
patients were female and the cohort had a mean age of Day 1 4.14  2.94
44 years. The most common type of burn was thermal (90%) Day 2 3.23  1.93
and the mean burn size was 21% of TBSA. Two-thirds of Surgical management, n (%)
Yes 76 (88)
patients had primarily partial thickness burns (66%) and
No 10 (12)
almost a third of patients had full thickness burns (32%).
Number of surgeries, mean  SD 43
Table 3 summarises the results of commonly performed blood Mortality, n (%) 3 (3)
tests.

3.2. Patient treatment

Table 3 – Baseline laboratory results for blood tests on
Table 2 summarises some key features of patient treatment presentation to hospital.
through the phases of initial resuscitation, intensive care All patients
management, and surgical management. Evidence of an 9
Neutrophils 10
airway burn was found in 25 patients (29%) on presentation
Mean  SD 10.7  6.1
and endotracheal intubation was performed in 23 of those Normal range 2–8  109
patients (27%). Over half of the patients (57%) were admitted to Lymphocytes 109
the ICU and the remainder were admitted to the SBU. Of Mean  SD 2.1  1.1
patients admitted to the ICU, the mean LOS was six days. The Normal range 1–4  109
overall mean hospital LOS for the entire cohort was 25 days. Platelets 109
Mean  SD 240  84
Among patients who were intubated, the mean duration of
Normal range 150–400  109
intubation and ventilation was 8 days. The mean volume of
Albumin (g/L)
intravenous fluid provided to patients during resuscitation Mean  SD 37  6
and their first day in hospital was 3.64 L, with a mean of 4.14 L Normal range 35–52 g/L
then 3.23 L on subsequent days. Surgical intervention was Creatinine (mmol/L)
Mean  SD 80  31
Normal 45–90 umol/L
C-reactive protein (mg/L)
Mean  SD 39  52
Table 1 – Clinical overview of patient and burn Normal <5 mg/L
characteristics on admission. INR (International normalized ratio)
All patients Mean  SD 1.1  0.2
APTT seconds
Total patients, n 86 Mean  SD 28.9  4.1
Female, n (%) 22 (26) Normal range 24–36 s
Male, n (%) 64 (74) Lactate (mmol/L)
Age (years), mean  SD 44  19 Mean  SD 2.0  1.4
Mechanism of injury, n (%) Normal 0.5–1.6 mmol/L
Thermal — flame 65 (76)
Thermal — scald 9 (10)
Thermal — other 3 (3)
Chemical 7 (8)
undertaken in 76 patients (88%) and of these patients the mean
Electrical 1 (1)
number of surgeries was four. Three patients did not survive
Friction 1 (1)
Burn size (% TBSA), mean  SD 21  13 admission.
Primary depth of burn, n (%)
Full thickness 27 (31) 3.3. Protein C levels on admission
Partial deep 31 (36)
Partial superficial 26 (30) On admission, the mean protein C level for all patients was
Superficial 1 (1)
76  21% with a range of 34–130% compared to the normal

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14
6 burns xxx (2019) xxx –xxx
range of 70–180% [74]. Values were normally distributed.
Twenty-one of the 59 patients (35%) who provided admission
blood samples had lower than normal levels of protein C
(<70%). These patients had a mean protein C level of 56  12%
on admission. Eleven patients had full thickness burns, ten
had burns of partial thickness, and there was a mean burn size
of 27  18% TBSA. The mean daily intravenous fluid adminis-
tration volume over the first 72 h of admission was 4.3  2.3 L/
day, and the mean number of surgeries was 6  5 surgeries.
These patients had a mean ICU LOS of 16  24 days and a mean
total hospital LOS of 41  44 days. All three non-survivors had
admission protein C levels <70% on admission.
3.4. Protein C levels throughout admission
Fig. 1 and Table 4 outline plasma protein C levels in this cohort
throughout the 3-week study period of their admission.
Regarding the change in protein C values among the entire
cohort over the 21 day study period, an ordinary one-way
ANOVA analysis (which was significant, P = 0.0003) followed by
Dunnett’s multiple comparisons showed a statistically signif-
icant increase in mean protein C level compared to admission
by day 6 and this difference remained significant until day 21.
Fig. 1 – Plasma protein C levels in patients with severe burns.
In a cohort of 86 patients with severe burns with a mean TBSA
% of 2113, plasma protein C levels were measured every
three days over 21 days of a hospital admission. Asterixes
represent a statistically significant increase in mean protein
C level from admission. ****P <0.001. Bars show SEM.
Table 4 – Protein C levels in patients with severe burns throughout hospital admission.
Day n Mean  SD (%)
Normal = 70–180%
0 59 76  20
3 65 89  25
6 69 106  30
9 56 116  33
12 47 117  33
15 34 116  39
18 30 121  36
21 23 114  36
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
We explored the relationship between plasma protein C
and burn depth and size. Protein C levels were lower in full
thickness burns than partial thickness burns on admission
(64  18% vs. 82  19%, P = 0.0008) and this difference remained
statistically significant up to and including day 6. Patients with
larger burns (defined as TBSA  30% [37,75]) had lower protein
C levels than patients with smaller burns (TBSA < 30%) on
admission (55  17% vs. 80  19%, P = 0.0003), and this differ-
ence remained statistically significant up to and including day
12. These results are summarised in Fig. 2a and b.
Table 5 and Fig. 2d outline the protein C levels of three
patients (3%) who died during their hospital admission. Two of
these patients had protein C levels taken on presentation to
the hospital (49% and 47% respectively). Their protein C levels
remained low throughout admission. Statistical tests were not
undertaken due to the low number of values for each time
point but it is clear that these patients’ protein C levels were
lower than those of survivors, and remained very low
throughout admission.
3.5. The association between protein C levels on admission
and increased support use
Patients requiring increased support throughout admission
had significantly lower levels of protein C on admission than
those who did not (55  17% vs. 80  18%, P < 0.0001, Fig. 2c) and
this difference remained statistically significant up to and
including day 12. Table 6 shows the patient characteristics of
those who underwent increased support, and those who had a
standard admission. There was a statistically significant
difference between these two groups with regards to patient
and burn characteristics, pathology results, and treatment
details; mean burn size and depth were significantly different
between the two groups, as were the proportion of patients
who were intubated and sent immediately to ICU from the ED
(each group P < 0.0001).
Table 7 shows blood test results on admission which
showed a significant difference between patients who went on
to use increased support, and patients who did not, were
as follows: protein C level (P < 0.0001); neutrophil count
(P < 0.0001); creatinine (P = 0.012) and lactate (P = 0.0231). The
following blood test results were not significantly different
between the two groups on presentation to hospital: CRP
(P = 0.0938); platelet count (P = 0.3997); INR (P = 0.3842); lym-
phocyte count (P = 0.4899); albumin level (P = 0.8054) and APTT
(P = 0.7602).
Range (%) Adjusted P value (comparison to day 0 value)
34–130 –
45–135 0.1197
41–183 <0.0001
40–185 <0.0001
52–200 <0.0001
32–185 <0.0001
31–193 <0.0001
31–170 <0.0001
JBUR 5837 No. of Pages 14

burns xxx (2019) xxx –xxx 7

Fig. 2 – The cohort was stratified according to: burn depth; burn size; the need for increased support throughout hospital
admission; and mortality. The plasma protein C level is shown among each of these groups. Increased support was defined as
any of a mean intravenous fluid administration volume of >5L/day over the first 72h of hospital admission, five or more days
spent in ICU, or five or more surgical procedures undertaken during admission. Groups were analysed for statistically
significant differences in protein C levels at each time point. Asterixes represent a statistically signifiicant differece in mean
between groups at each time point. ****P <0.001; ***P <0.01; **P <0.02; and *P <0.05. Bars show SEM. (a) Plasma protein C levels in
patients with full thickness burns compared to patients with partial thickness burns. Plasma protein C levels were lower in
patients with full thickness burns (black line) compared to patients with partial thickness burns (grey line) until day 9. (b) Plasma
protein C levels in patients with large burns compared to patients with small burns. Plasma protein C levels were lower in
patients with large (30% TBSA) burns (black line) compared to patients with small (<30% TBSA) burns (grey line) until day 12. (c)
Plasma protein C levels in patients with severe burns requiring increased support compared to those who did not. Plasma
protein C levels were lower in patients who had increased support throughout admission (black line) compared to patients who
did not require increased support (grey line) until day 12. (d) Plasma protein C levels in patients with severe burns who died
during admission compared to patients who survived. Plasma protein C levels appeared lower in patients who died during their
hospital admission than those who survived. Statistical tests were not performed due to the small sample size of patients who
died.

Table 8 shows the clinical events that occurred throughout
admission among the two groups, and unsurprisingly, because
the events that occurred during the admission defined each
group, there were multiple statistically significant differences
in the occurrence of events such as amount of fluid adminis-
tered, number of surgeries, LOS and LOS in ICU, and mortality
incidence.
Regression analysis (Table 9) was performed to ascertain
the extent to which patient characteristics, burn character-
istics and blood test results on admission determined a
patient’s outcome after adjustment for the influence of other
variables. Out of the total 86 patients, 57 patients could be
included in the stepwise analysis due to missing data, and of
these, 19 were identified as using increased support. We
identified, with a comprehensive and systematic method
exploring plausible factors which may influence the outcome,
the following variables that significantly increased the risk of
patients with increased support: burn size (n = 86), burn depth
(n = 86), presence of inhalational injury (n = 86), day 0 protein C
(n = 57), day 0 neutrophil count (n = 88), day 0 lactate (n = 55) and
day 0 creatinine (n = 87).
Table 10 shows the final four variables on admission that
contributed most to influencing increased support throughout
admission based on multiple imputations of several regres-
sion model combinations. These variables are: protein C level
on admission, burn size, burn depth, and neutrophil count on
admission. Of these, protein C levels contributed the greatest
log likelihood change for the regression modelling. This
model’s performance was statistically significant, P < 0.0005.
It was then evaluated by several methods. Firstly, Nagelkerke’s

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14

8 burns xxx (2019) xxx –xxx

Table 5 – Protein C levels in patients with severe burns Table 7 – Baseline blood test results on admission.
who died during hospital admission.
Standard Increased P value
Day n Mean  SD (%) Range (%) admission support
0 2 48  1 47–49 Protein C (%)
3 3 56  10 45–62 Mean  SD 85  16 57  17 <0.0001
6 2 69  9 62–75 Normal range
9 2 68  40 40–96 70–180
12 0 – – Neutrophils 109
15 2 81  18 68-94 Mean  SD 8.9  4 14.7  7.9 <0.0001
18 1 81  0 81 Normal range 2–8
21 2 71  25 53–88 Lymphocytes 109
Mean  SD 2.1  1.2 1.9  0.9 0.4899
Normal range 1–4
Platelets 109
R [2] (0.83) showed that the model explained 83.1% of the Mean  SD 235  71 252  108 0.3997
variance in outcome, and correctly classified 93.2% of cases of Normal range
increased support. Sensitivity was 83.3% and specificity was 150–400
97.6%. Secondly, the Hosmer-Lemeshow goodness-of-fit test Creatinine (mmol/L)
Mean  SD 74  15 92  45 0.012
for calibration was appropriately not significant, P = 0.907.
Normal range
Thirdly, the area under the receiver-operator characteristic
45–90
(ROC) curve (AUC) for discrimination was 0.974 (95% CI, 0.940– Albumin (g/L)
1.000), P < 0.0005; and the optimism-corrected ROC AUC was Mean  SD 37  7 37  6 0.8054
0.937. These are outstanding levels of discrimination [76]. Normal range
Internal validation was achieved by calculating the optimism- 35–52
corrected ROC AUC from 200 bootstrapped samples. Further C-reactive protein (mg/L)
Mean  SD 52  70 24  32 0.0938
consideration of the Youden index from protein C’s ROC curve
Normal <5
suggested that an optimal protein C cut-off level of 70% leads to
INR
the ideal sensitivity and specificity values for the model in Mean  SD 1.1  0.1 1.1  0.2 0.3842
predicting the risk of increased support. This coincidentally is APTT (seconds)
the lower limit in normal in adults provided by our laboratory, Mean  SD 28.8  2.7 29  6.1 0.7602
when testing patients with venous thrombosis for inherited Normal range
protein C deficiency. When considering the performance of the 24–36
Lactate (mmol/L)
multivariate regression model in the absence of protein C, the
Mean  SD 1.6  0.7 2.5  1.9 0.0231
model’s performance deteriorated. It remained statistically Normal range
significant (P < 0.0005) however the goodness-of-fit test for 0.5–1.6

P values formatted to bold font are all values <0.05.

Table 6 – Clinical overview of patients on admission. P

value represents comparison of increased support vs.
standard admission. calibration trended towards significance (P = 0.115), Nagel-
Standard Increased P value kerke’s R [2] reduced to 0.495, the classification accuracy
admission support reduced to 82.6%, the sensitivity reduced to 63.0% and the
Total patients, n 59 27 –
specificity reduced to 91.5%.
Female, n (% of group) 14 (24) 8 (30) 0.6003
Male, n (% of group) 45 (76) 19 (70)
Age (years), 43  19 47  17 0.4233 4. Discussion
mean  SD
Mechanism of injury, n (%)
This is the first detailed study to measure protein C levels
Thermal (flame) 43 (72) 24 (86) 0.1965
over an extended period of time in patients with severe
Thermal (scald) 9 (15) 0 (0)
Thermal (other) 2 (3) 1 (4) burns. Assuming that this cohort had normal levels of
Chemical 5 (8) 2 (7) protein C before injury (which is likely because no patients
Electrical 0 (0) 1 (4) had an inherited thrombotic condition, infection, pregnan-
Friction 1 (2) 0 (0) cy or condition requiring anticoagulation) the results
Burn size (% TBSA), 17  8 31  19 <0.0001 suggest that a severe burn directly leads to a decrease in
mean  SD
plasma protein C. Patients who suffered large burns or deep
Primary depth of burn
burns or patients who did not survive admission had the
Full thickness 10 (17) 18 (64) <0.0001
Partial deep 25 (42) 8 (26) lowest levels of protein C on admission. We used a
Partial superficial 24 (40) 2 (7) composite primary outcome called increased support
Superficial 1 (1) 0 (0) utilisation to identify patients who went on to receive
P values formatted to bold font are all values <0.05. significant emergency, critical care or surgical intervention.
Of all the clinical and laboratory data collected, plasma

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14

burns xxx (2019) xxx –xxx 9

Table 8 – Clinical overview of patients throughout their Table 10 – Factors on admission which predict increased
admission. support in patients with severe burns (multivariable
regression).
Standard Increased P value
admission support Odds ratio (95% CI) P value
Admitted to, n (%) Burn size (% TBSA) 1.252 (1.025–1.530) 0.027
ICU 13 (22) 23 (85) 0.0001 Burn depth
SBU 46 (78) 4 (15) Partial thickness Reference 0.029
LOS (days) Full thickness 80.499 (1.569–4129.248)
SBU, mean  SD 16  21 44  40 <0.0001 Protein C (%) 0.825 (0.698–0.977) 0.025
ICU, mean  SD 01 17  21 <0.0001 Neutrophil count 109 1.532 (0.950–2.473) 0.080
Intubated, n (%)
Yes 8 (14) 15 (55) 0.0001
No 51 (86) 12 (45)
Duration of intubation 21 11  9 0.0147
showed that normal levels of protein C (70%) optimally
(days), mean  SD improved the sensitivity and specificity of the multivariable
Intravenous fluid administration regression model. This suggests that a patient with normal
Day 0, mean  SD 2.79  1.81 L 5.11  4.16 L 0.004 protein C levels may be protected from using increased
Day 1, mean  SD 2.52  1.35 L 6.86  2.89 L <0.0001 support. Burn size, burn depth, neutrophil count on admission
Day 2, mean  SD 2.31  1.33 L 4.39  1.98 L <0.0001
and protein C level on admission contributed together to
Surgical management, n (%)
predict increased support utilisation with high sensitivity,
Yes 51 (86) 25 (93) 0.4949
No 8 (14) 2 (7) specificity and accuracy. Including protein C level in the
Surgeries, mean  SD 21 65 <0.0001 multivariable regression predicted increased support utilisa-
Mortality, n (%) tion with an additional 20.3% sensitivity, 6.1% specificity,
Died 0 (0) 3 (11) 0.0286 10.6% accuracy, and almost doubled Nagelkerke’s R2 value
Survived 59 (100) 24 (89) explaining 33.8% more variance, compared to when protein C
P values formatted to bold font are all values <0.05. levels were not considered. Other studies have shown that
inhalational injury influences patient outcomes [77–79] how-
ever when we included it in the stepwise analysis, it lost
protein C level on admission was most strongly associated with significance and did not contribute to the multivariable
increased support. Twenty-seven patients (31%) had increased regression model. This may be due to the relatively low
support utilisation throughout admission and among these sample size or the strength of the model.
patients, the initial magnitude of loss of functional protein C The findings of this study may be explained by the
was proportionate to the risk of subsequently using increased excessive inflammation and coagulation that occurs after a
support throughout admission. burn. Based on other studies of protein C in patients and
In order to quantify a protein C cut-off value that was animals with an excessive systemic inflammatory response,
associated with a reduced risk of using increased support, we and an understanding of burn pathophysiology, we propose
applied Youden’s index from protein C’s ROC curve. This three main factors which could contribute to low protein C in
burns: impaired synthesis, increased degradation and exces-
sive consumption. The first proposed mechanism is impaired
synthesis. Protein C is synthesised largely by the liver, and a
Table 9 – Exploratory univariable regression for possible
burn acutely blunts synthesis of the hepatically produced
predictors of increased support in patients with burns on
admission to hospital. proteins a-1 acid glycoprotein, C-reactive protein and hapto-
globin [80]. The synthesis of protein C may also be suppressed
Covariate Odds ratio (95% CI) P value in the same manner after a burn. Protein C is also synthesised
Age (years) 1.010 (0.986–1.035) 0.419 by epidermal keratinocytes and vascular endothelium [24], so
Sex (female) 1.353 (0.488–3.756) 0.561 large burns with significant epidermal and endothelial
Burn size (TBSA %) 1.100 (1.042–1.160) 0.001 damage causes direct traumatic loss of functioning protein
Burn depth
C-producing cells. Furthermore, pro-inflammatory cytokines
Partial thickness Reference <0.0005
such as TNF-a and IL-1 effectively shut off the protein C system
Full thickness 8.330 (2.957–23.466)
Presence of inhalational injury 8.081 (2.841–22.987) <0.0005 [81]. In non-survivors after severe burns, at 24 h after
Protein C (%) 0.899 (0.849–0.953) <0.0005 presentation to hospital, IL-1 and TNF-a concentrations are
CRP (mg/L) 0.986 (0.971–1.003) 0.098 >1000% higher than normal levels [29]. TNF-a suppresses
Neutrophils 109 1.230 (1.105–1.368) <0.0005 protein C activation [82] while IL-1 suppresses protein C
Lymphocytes 109 0.893 (0.580–1.374) 0.606 expression [83] and because both cytokines are abundant
Platelets x109 1.003 (0.998–1.008) 0.285
immediately after a burn, the protein C system is shut off. The
INR 2.471 (0.136–44.988) 0.541
second proposed mechanism is increased degradation. Our
APTT (seconds) 1.020 (0.916–1.134) 0.723
Lactate (mmol/L) 1.964 (1.135–3.399) 0.016 results show that the neutrophil count is elevated immedi-
Creatinine (umol/L) 1.031 (1.003–1.059) 0.028 ately after a burn (mean neutrophil count on admission
Albumin (g/L) 0.989 (0.922–1.062) 0.769 10.7  6.1  10 [9], normal range 2–8  10 [9]), and protein C is
P values formatted to bold font are all values <0.05. very sensitive to complete degradation by brief exposures to
low concentrations of the proteolytic enzyme neutrophil

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
JBUR 5837 No. of Pages 14
10 burns xxx (2019) xxx –xxx
elastase [84]. This is secreted by neutrophils in response to
sepsis and burns [85]. The third proposed mechanism is
excessive consumption due to increased thrombin generation
following a burn. Thrombin generation is significantly accel-
erated, and to a greater than normal magnitude, following a
burn [86]. Thrombin adherence to the protein C complex is the
trigger for the activation of protein C to APC [23]. APC is then
rapidly depleted through neutralisation or adherence to
plasminogen activator inhibitor 1 [87] and it is plausible that
protein C levels decrease when APC is continuously exhausted
on a large scale in response to the widespread thrombin
generation seen following a burn.
The slow return to baseline levels can be explained by the
brief half-life of protein C, relative to the slow nature of protein
C synthesis. A study of protein C deficient patients who
administered a protein C rich substrate of either fresh frozen
plasma (FFP) or prothrombin concentrate (PCC), showed that
protein C levels declined steadily to baseline deficient levels
after approximately 12 and 48 h respectively, with half-lives of
7.8 and 7.4 h respectively [30]. Another study of protein C
deficient patients found the half-life of protein C following PCC
administration to be 7.9  1.6 h [88].
The effect of reduced protein C system activity is to lose
appropriate modulation of inflammation and coagulation.
Regarding inflammation, protein C modulates the inflamma-
tory response caused by tissue necrosis and apoptosis [89].
After tissue injury, protein C, which is circulating freely in
plasma, moves to bind to the vascular endothelial protein C
receptor (EPCR) where it is cleaved by thrombin to form APC
[90] and this activates nearby protease-activated receptors
(PARs) and the tyrosine kinase-associated receptor, Tie 2
[91,92]. PAR-1, PAR-2 and Tie2 activation [92] leads to a
reduction in NF-kB synthesis and nuclear translocation
followed by an overall reduction in production of inflammato-
ry mediators [89]. Chemotaxis and cytokine production are
then suppressed leading to both decreased leukocyte adhesion
and activation and decreased capillary permeability [89].
Multiple studies show the association between excessive
circulating pro-inflammatory cytokine levels, pro-inflamma-
tory enzyme levels and poor outcomes after burns [19–22].
Additionally, excessive capillary permeability following a burn
[93,94] can lead to catastrophic outcomes such as abdominal
compartment syndrome and acute pulmonary oedema [95]. In
contrast, a properly functioning protein C system effectively
modulates this pro-inflammatory response [23,89,96] and
stabilises the endothelium preventing capillary permeability
[31,90,97,98]. In unwell patients with normal protein C levels, a
drop in protein C levels precedes significant clinical deteriora-
tion. In patients who are febrile after chemotherapy-induced
neutropaenia, a significant decrease in protein C levels reliably
precedes the onset of sepsis [99]. Similarly, in patients who are
critically ill from various causes, a significant decrease in
protein C levels precedes the onset of severe pancreatitis and
multi-organ failure [100]. This suggests that protein C provides
a crucial amount of support for immune-mediated processes,
and if it is consumed or depleted, unregulated inflammatory
processes can take hold.
Regarding coagulation, protein C appropriately inhibits
factor Va and factor VIIIa, two cofactors which respectively
inhibit factor X and prothrombin so that fibrin clots form
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
appropriately [25]. A detailed study of nine patients with
severe burns (four were non-survivors) measured multiple
components of the coagulation system including coagulation
factors, anticoagulant proteins, the fibrinolytic markers and
inflammatory cytokines regularly over the first 96 h of a
hospital admission. The only components showing a signifi-
cant difference between non-survivors and survivors on
admission were protein C activity levels (56  18% for non-
survivors vs. 82  9% for survivors, P = 0.04) and factor VIII
activity levels (518  182% for non-survivors vs. 305  148% for
survivors, P < 0.05) [29]. Given that protein C inhibits factor
VIIIa, this fits with our findings. Given that severe burns lead to
an early hypercoagulable state [101] this may be mediated by
loss of activity of the protein C system. Given this initial loss of
anticoagulant activity, plus the fact that burns are initially
treated with high volumes of intravenous crystalloid solutions
[43–45] (which further exacerbates coagulopathy) [102–105], the
late increases in procoagulant components D-dimer and fibrin
[29] and the devastating effects of disseminated intravascular
coagulation (DIC) seen after a burn [106], significant attention
should be paid to the early reduction of functional protein C
levels in burns.
The findings of this study give rise to two new concepts
about the potential clinical utility of protein C in burns. Firstly,
measuring functional protein C levels provides useful prog-
nostic information in patients with burns, such that it may
become an adjunct to the clinical examination. Burns
clinicians rely heavily on the initial clinical examination to
determine early treatment decisions however this method
yields unreliable prognostic information [16]. Size estimations
[107], depth estimations [108–110] and the use of features on
history and examination to diagnose inhalational injury [111]
each have shortcomings in reliability and accuracy. Yet they
determine treatment that carries an appreciable risk of harm.
Further analysis of the role of protein C in burns may allow for
the development of superior predictive scores to those
currently used.
The second concept is that protein C deficiency caused by a
burn is harmful and this deficiency could be corrected by
replacing it. FFP contains a physiological amount [112] (87  15
units/dL) of protein C [30] and is used by several resuscitation
protocols. A well-designed randomised trial compared the use
of the Slater formula, which predominantly uses FFP, to the
Parkland formula, which predominantly uses Hartmann’s
solution. The FFP group achieved the desired urine output with
nearly half the fluid of the Hartmann’s group (140 mL/kg/day
vs. 260 mL/kg/day, P = 0.005), with significant and clinically
important differences favouring FFP with regards to weight
gain, peak intra-abdominal pressures, peak inspiratory pres-
sures and base deficit clearance [113]. These findings are
consistent with other studies [114,115]. Although there are rare
reports of lung injury following FFP transfusion [116], and FFP
is more costly than crystalloids or albumin [117], a retrospec-
tive review of 5 years’ experience with FFP-based resuscitation
deemed FFP to be safe and effective for fluid resuscitation [118].
Future studies of fluid administration in patients with severe
burns could help clarify the potential superiority of FFP over
other fluid types during acute resuscitation, and whether this
potential superiority is due to the influence of protein C or to
another property of colloids.
JBUR 5837 No. of Pages 14
burns xxx (2019) xxx –xxx
5. Conclusion
In summary, in patients with severe burns, protein C is low
on admission to hospital and returns gradually to a steady-
state after six days. The initial low measurement can be
plausibly explained by impaired synthesis, increased degra-
dation and excessive consumption of protein C following a
burn. A plasma protein C level on the day of admission,
together with burn size, burn depth, and neutrophil count on
the day of admission predicted with high accuracy, sensitiv-
ity and specificity the need for increased support throughout
admission after a severe burn. It may be a useful biomarker
of burn severity.
Funding
Funding for this study was supported by the Ramsay Research
and Teaching Fund.
Conflict of interest
Christopher Jackson, John Vandervord, Gregory Fulcher and
Aruna Wijewardena are shareholders in a company undertak-
ing a trial of a 3K3A-APC in diabetic ulcers. For other authors,
no competing financial interests exist. The content of this
article was expressly written by the authors listed. No
ghostwriters were used to write this article.
Acknowledgements
We thank Dr. Nancy Huang and Mr. Andrew Zimmerman for
their assistance with data collection, and to the staff of the
Emergency Department, Intensive Care Department and
the Severe Burns Unit at Royal North Shore Hospital for their
assistance in this project. We also thank Associate Professor
Meilang Xue for technical support and the biostatisticians at
The Kolling Institute, in particular Dr. Danny Kim for his advice
on regression models. We also thank David Bellingham for his
advice on data management and Dr. Boris Waldman for his
advice on testing outcome validity.
REFERENCES
[1] Girardot T, Rimmelé T, Venet F, Monneret G. Apoptosis-
induced lymphopenia in sepsis and other severe injuries.
Apoptosis 2017;22:295–305.
[2] Lopez ON, Cambiaso-Daniel J, Branski LK, Norbury WB,
Herndon DN. Predicting and managing sepsis in burn
patients: current perspectives. Ther Clin Risk Manag
2017;13:1107.
[3] Heideman M. The effect of thermal injury on hemodynamic,
respiratory, and hematologic variables in relation to
complement activation. J Trauma 1979;19:239–47.
[4] Bartlett RH, Fong SW, Marrujo G, Hardeman J, Anderson W.
Coagulation and platelet changes after thermal injury in
man. Burns 1981;7:370–7.
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
11
[5] Smith CA, Gosselin RC, Utter GH, Galante JM, Young JB,
Scherer LA, et al. Does saline resuscitation affect
mechanisms of coagulopathy in critically ill trauma patients?
An exploratory analysis. Blood Coagul Fibrinolysis
2015;26:250–4.
[6] Butt MA, Coulson A, Hull J, Ho T. Early treatment strategies in
sepsis. Br Med J 2008;336:521.
[7] Strassle PD, Williams FN, Napravnik S, et al. Improved
survival of patients with extensive burns: trends in patient
characteristics and mortality among burn patients in a
tertiary care burn facility, 2004-2013. J Burn Care Res
2017;38:187–93.
[8] Asaria P, Elliott P, Douglass M, et al. Acute myocardial
infarction hospital admissions and deaths in England: a
national follow-back and follow-forward record-linkage
study. Lancet Public Health 2017;2:e191–201.
[9] Giri S, Pathak R, Aryal MR, Karmacharya P, Bhatt VR, Martin
MG. Lack of “Weekend effect” on mortality for pulmonary
embolism admissions in 2011: data from nationwide
inpatient sample. Int J Cardiol 2015;180:151–3.
[10] Duke JM, Boyd JH, Randall SM, Wood FM. Long term mortality
in a population-based cohort of adolescents, and young and
middle-aged adults with burn injury in Western Australia: a
33-year study. Accid Anal Prev 2015;85:118–24.
[11] Duke JM, Boyd JH, Rea S, Randall SM, Wood FM. Long-term
mortality among older adults with burn injury: a population-
based study in Australia. Bull World Health Organ
2015;93:400–6.
[12] Griffiths C, Guest E, White P, et al. A systematic review of
patient-reported outcome measures used in adult burn
research. J Burn Care Res 2017;38:e521–45.
[13] Friedstat JS, Ryan CM, Gibran N. Outcome metrics after burn
injury: from patient-reported outcome measures to value-
based health care. Clin Plast Surg 2017;44:911–5.
[14] Osborne CL, Petersson C, Graham JE, et al. The Burn Model
Systems outcome measures: a content analysis using the
international classification of functioning, disability, and
health. Disabil Rehabil 2017;39:2584–93.
[15] Saly D, Yang A, Triebwasser C, et al. Approaches to predicting
outcomes in patients with acute kidney injury. PLoS One
2017;12:e0169305.
[16] Hussain A, Dunn KW. Predicting length of stay in thermal
burns: a systematic review of prognostic factors. Burns
2013;39:1331–40.
[17] Cabral L, Afreixo V, Almeida L, Paiva JA. The use of
procalcitonin (PCT) for diagnosis of Sepsis in burn patients: a
meta-analysis. PLoS One 2016;11:e0168475.
[18] Cabral L, Afreixo V, Meireles R, et al. Checking procalcitonin
suitability for prognosis and antimicrobial therapy
monitoring in burn patients. Burns Trauma 2018;6:10.
[19] Shelhamer MC, Rowan MP, Cancio LC, et al. Elevations in
inflammatory cytokines are associated with poor outcomes
in mechanically ventilated burn patients. J Trauma Acute
Care Surg 2015;79:431–6.
[20] Finnerty CC, Jeschke MG, Qian WJ, et al. Determination of
burn patient outcome by large-scale quantitative discovery
proteomics. Crit Care Med 2013;41:1421–34.
[21] Hastbacka J, Freden F, Hult M, et al. Matrix
metalloproteinases -8 and -9 and tissue inhibitor of
metalloproteinase-1 in burn patients. A prospective
observational study. PLoS One 2015;10:e0125918.
[22] Huang L, Yao Y, Dong N, et al. Prognostic significance of
plasma gelsolin in severe burn patients with sepsis.
Zhonghua Shao Shang Za Zhi 2016;32:77–81.
[23] Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective
protein C pathway. Blood 2007;109:3161–72.
[24] Xue M, Campbell D, Jackson CJ. Protein C is an autocrine
growth factor for human skin keratinocytes. J Biol Chem
2007;282:13610–6.
JBUR 5837 No. of Pages 14
12 burns xxx (2019) xxx –xxx
[25] Dahlbäck B, Villoutreix BO. Regulation of blood coagulation
by the protein C anticoagulant pathway: novel insights into
structure-function relationships and molecular recognition.
Arterioscler Thromb Vasc Biol 2005;25:1311–20.
[26] Miletich J, Sherman L, Broze G. Absence of thrombosis in
subjects with heterozygous protein C deficiency. N Engl J Med
1987;317:991–6.
[27] Fernandez JA, Petaja J, Gruber A, Griffin JH. Activated protein
C correlates inversely with thrombin levels in resting healthy
individuals. Am J Hematol 1997;56:29–31.
[28] Marlar RA, Gausman JN. Laboratory testing issues for protein C,
protein S, and antithrombin. Int J Lab Hematol 2014;36:289–95.
[29] Tejiram S, Brummel-Ziedins KE, Orfeo T, et al. In-depth
analysis of clotting dynamics in burn patients. J Surg Res
2016;202:341–51.
[30] Marlar RA, Sills RH, Groncy PK, Montgomery RR, Madden RM.
Protein C survival during replacement therapy in
homozygous protein C deficiency. Am J Hematol 1992;41:24–
31.
[31] Whitmont K, Fulcher G, Reid I, et al. Low circulating protein C
levels are associated with lower leg ulcers in patients with
diabetes. BioMed Res Int 20132013:.
[32] Esechie A, Enkhbaatar P, Nakano YY, Traber L, Herndon D,
Traber DL. Comparison of plasma concentrations of
antithrombin (AT) and protein c (PC) in burn and smoke
inhalation injury and sepsis. FASEB J 2006;20:A286–7.
[33] Lo S, Lai W, Kwok F. Protein C and Protein S levels in some
burns patients. Burns 1994;20:186–7.
[34] Von Elm E, Altman DG, Egger M, et al. The strengthening the
Reporting of Observational Studies in Epidemiology (STROBE)
statement: guidelines for reporting observational studies.
PLoS Med 2007;4:e296.
[35] Innovation AfC. NSW burn transfer guidelines. In: NGDo
Health, editor. Statewide burn injury service. .
[36] Demling RH, Wong C, Jin L, Hechtman H, Lalonde C, West K.
Early lung dysfunction after major burns: role of edema and
vasoactive mediators. J Trauma 1985;25:959–66.
[37] Ennis JL, Chung KK, Renz EM, et al. Joint Theater Trauma
System implementation of burn resuscitation guidelines
improves outcomes in severely burned military casualties. J
Trauma 2008;64:S146–51 Discussion S51–52.
[38] Malbrain ML, Cheatham ML, Kirkpatrick A, et al. Results from
the international conference of experts on intra-abdominal
hypertension and abdominal compartment syndrome. I.
Definitions. Intensive Care Med 2006;32:1722–32.
[39] Ivy ME, Atweh NA, Palmer J, Possenti PP, Pineau M, D’aiuto M.
Intra-abdominal hypertension and abdominal compartment
syndrome in burn patients. J Trauma Acute Care Surg
2000;49:387–91.
[40] Parra MW, Al-Khayat H, Smith HG, Cheatham ML.
Paracentesis for resuscitation-induced abdominal
compartment syndrome: an alternative to decompressive
laparotomy in the burn patient. J Trauma Acute Care Surg
2006;60:1119–21.
[41] Dulhunty JM, Boots RJ, Rudd MJ, Muller MJ, Lipman J.
Increased fluid resuscitation can lead to adverse outcomes in
major-burn injured patients, but low mortality is achievable.
Burns 2008;34:1090–7.
[42] Lund T, Onarheim H, Reed RK. Pathogenesis of edema
formation in burn injuries. World J Surg 1992;16:2–9.
[43] Baxter CR, Shires T. Physiological response to crystalloid
resuscitation of severe burns. Ann N Y Acad Sci
1968;150:874–94.
[44] Baker RHJ, Akhavani MA, Jallali N. Resuscitation of thermal
injuries in the United Kingdom and Ireland. J Plast Reconstr
Aesthetic Surg 2007;60:682–5.
[45] Gordon M, Marvin J, Greenfield E, et al. Regional and
institutional variation in burn care. J Burn Care Rehabil
1995;16:85–90.
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
[46] Engrav LH, Colescott PL, Kemalyan N, et al. A biopsy of the use
of the Baxter formula to resuscitate burns or do we do it like
Charlie did it? J Burn Care Rehabil 2000;21:91–5.
[47] Cartotto RC, Innes M, Musgrave MA, Gomez M, Cooper AB.
How well does the Parkland formula estimate actual fluid
resuscitation volumes? J Burn Care Rehabil 2002;23:258–65.
[48] Friedrich JB, Sullivan SR, Engrav LH, et al. Is supra-Baxter
resuscitation in burn patients a new phenomenon? Burns
2004;30:464–6.
[49] Klein MB, Hayden D, Elson C, et al. The association between
fluid administration and outcome following major burn: a
multicenter study. Ann Surg 2007;245:622–8.
[50] Ivy ME, Atweh NA, Palmer J, Possenti PP, Pineau M, D’Aiuto M.
Intra-abdominal hypertension and abdominal compartment
syndrome in burn patients. J Trauma Inj Infect Crit Care
2000;49:387–91.
[51] Oda J, Yamashita K, Inoue T, et al. Resuscitation fluid volume
and abdominal compartment syndrome in patients with
major burns. Burns 2006;32:151–4.
[52] Brandstrup B, Tønnesen H, Beier-Holgersen R, et al. Effects of
intravenous fluid restriction on postoperative complications:
comparison of two perioperative fluid regimens: a
randomized assessor-blinded multicenter trial. Ann Surg
2003;238:641.
[53] Liu NT, Fenrich CA, Serio-Melvin ML, Peterson WC, Cancio LC,
Salinas J. The impact of patient weight on burn resuscitation.
J Trauma Acute Care Surg 2017;83:S112–9.
[54] Maan ZN, Frew Q, Din AH, et al. Burns ITU admissions: length
of stay in specific levels of care for adult and paediatric
patients. Burns 2014;40:1458–62.
[55] Peck MD, Mantelle L, Ward CG. Comparison of length of
hospital stay to mortality rate in a regional burn center. J Burn
Care Rehabil 1996;17:39–44.
[56] Lemeshow S, Klar J, Teres D, et al. Mortality probability
models for patients in the intensive care unit for 48 or
72 hours: a prospective, multicenter study. Crit Care Med
1994;22:1351–8.
[57] Garside TL, Lee RP, Delaney A, Milliss D. Clinical practice
variation in acute severe burn injury. Anaesth Intensive Care
2018;46:321–5.
[58] Hodgman EI, Subramanian M, Wolf SE, et al. The effect of
illicit drug use on outcomes following burn injury. J Burn Care
Res 2017;38:e89–94.
[59] Blay B, Thomas S, Coffey R, Jones L, Murphy CV. Low vitamin d
level on admission for burn injury is associated with
increased length of stay. J Burn Care Res 2017;38:e8–e13.
[60] Issler-Fisher AC, Fakin RM, Fisher OM, et al. Microbiological
findings in burn patients treated in a general versus a
designated intensive care unit: effect on length of stay. Burns
2016;42:1805–18.
[61] van Langeveld I, Gagnon RC, Conrad PF, et al. Multiple-drug
resistance in burn patients: a retrospective study on the
impact of antibiotic resistance on survival and length of stay.
J Burn Care Res 2017;38:99–105.
[62] Pandharipande PP, Girard TD, Jackson JC, et al. Long-term
cognitive impairment after critical illness. N Engl J Med
2013;369:1306–16.
[63] Moitra VK, Guerra C, Linde-Zwirble WT, Wunsch H.
Relationship between ICU length of stay and long-term
mortality for elderly ICU survivors. Crit Care Med 2016;44:655.
[64] Minne L, Abu-Hanna A, de Jonge E. Evaluation of SOFA-based
models for predicting mortality in the ICU: a systematic
review. Crit Care 2008;12:R161.
[65] Israel JS, Greenhalgh DG, Gibson AL. Variations in burn
excision and grafting: a survey of the american burn
association. J Burn Care Res 2017;38:e125–32.
[66] Nosanov LB, McLawhorn MM, Hassan L, et al. Graft loss:
review of a single burn center’s experience and proposal of a
graft loss grading scale. J Surg Res 2017;216:185–90.
JBUR 5837 No. of Pages 14
burns xxx (2019) xxx –xxx
[67] Abbott TEF, Ahmad T, Phull MK, et al. The surgical safety
checklist and patient outcomes after surgery: a prospective
observational cohort study, systematic review and meta-
analysis. Br J Anaesth 2017;120:146–55.
[68] Urbach DR, Govindarajan A, Saskin R, Wilton AS, Baxter NN.
Introduction of surgical safety checklists in Ontario, Canada.
N Engl J Med 2014;370:1029–38.
[69] Li H, Yao Z, Tan J, et al. Epidemiology and outcome analysis of
6325 burn patients: a five-year retrospective study in a major
burn center in Southwest China. Sci Rep 2017;7:46066.
[70] Gangemi EN, Gregori D, Berchialla P, et al. Epidemiology and
risk factors for pathologic scarring after burn wounds. Arch
Facial Plast Surg 2008;10:93–102.
[71] Mason SE, Noel-Storr A, Ritchie CW. The impact of general
and regional anesthesia on the incidence of post-operative
cognitive dysfunction and post-operative delirium: a
systematic review with meta-analysis. J Alzheimers Dis
2010;22:S67–79.
[72] Desborough J. The stress response to trauma and surgery. Br J
Anaesth 2000;85:109–17.
[73] Guo Z, Liu J, Li J, et al. Postoperative delirium in severely
burned patients undergoing early escharotomy: incidence,
risk factors, and outcomes. J Burn Care Res 2017;38:e370–6.
[74] Royal North Shore Hospital LabInfo Test Directory. 2017
[Accessed 2 December 2017, at http://www.palms.com.au/
php/labinfo/info_index.php?tab=4].
[75] Rizzo JA, Rowan MP, Driscoll IR, Chan RK, Chung KK.
Perioperative temperature management during burn care. J
Burn Care Res 2017;38:e277–83.
[76] Hosmer [455_TD$DIFF]Jr D, Lemeshow S, Sturdivant R.
Applied logistic regression. 3rd ed. New York: John Wiley &
Sons. Inc; 2013.
[77] Shirani KZ, Pruitt [456_TD$DIFF]Jr BA, Mason Jr AD. The
influence of inhalation injury and pneumonia on burn
mortality. Ann Surg 1987;205:82.
[78] Smith DL, Cairns BA, Ramadan F, et al. Effect of inhalation
injury, burn size, and age on mortality: a study of
1447 consecutive burn patients. J Trauma 1994;37:655–9.
[79] Blot S. Development and validation of a model for prediction
of mortality in patients with acute burn injury: the Belgian
Outcome in Burn Injury Study Group. Br J Surg 2008;96:111–7.
[80] Thomas S, Wolf SE, Chinkes DL, Herndon DN. Recovery from
the hepatic acute phase response in the severely burned and
the effects of long-term growth hormone treatment. Burns
2004;30:675–9.
[81] Levi M, van der Poll T. Inflammation and coagulation. Crit
Care Med 2010;38:S26–34.
[82] Nawroth PP, Stern DM. Modulation of endothelial cell
hemostatic properties by tumor necrosis factor. J Exp Med
1986;163:740–5.
[83] Yamamoto K, Shimokawa T, Kojima T, Loskutoff DJ, Saito H.
Regulation of murine protein C gene expression in vivo:
effects of tumor necrosis factor-alpha, interleukin-1, and
transforming growth factor-beta. Thromb Haemost
1999;82:1297–301.
[84] Eckle I, Seitz R, Egbring R, Kolb G, Havemann K. Protein C
degradation in vitro by neutrophil elastase. Biol Chem Hoppe
Seyler 1991;372:1007–13.
[85] Kaufman T, Magosevich D, Moreno MC, et al. Nucleosomes
and neutrophil extracellular traps in septic and burn
patients. Clin Immunol 2017;183:254–62.
[86] Wiegele M, Schaden E, Koch S, Bauer D, Krall C, Adelmann D.
Thrombin generation in patients with severe thermal injury.
Burns 2019;45:54–62.
[87] Hazelzet JA. Levels of protein C and activated protein C: what
do they mean? Crit Care 2006;10:126.
[88] Riess H, Binsack T, Hiller E. Protein C antigen in prothrombin
complex concentrates: content, recovery and half life. Blut
1985;50:303–6.
Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001
13
[89] Esmon CT. Protein C anticoagulant system—anti-
inflammatory effects. Semin Immunopathol 2012;34:127–32.
[90] Jackson CJ, Xue M. Activated protein C: an anticoagulant that
does more than stop clots. Int J Biochem Cell Biol
2008;40:2692–7.
[91] Julovi SM, Xue M, Dervish S, Sambrook PN, March L, Jackson
CJ. Protease activated receptor-2 mediates activated protein
C-induced cutaneous wound healing via inhibition of p38.
Am J Pathol 2011;179:2233–42.
[92] Xue M, Chow SO, Dervish S, Chan YKA, Julovi SM, Jackson CJ.
Activated protein C enhances human keratinocyte barrier
integrity via sequential activation of epidermal growth factor
receptor and tie2. J Biol Chem 2011;286:6742–50.
[93] Pitt RM, Parker JC, Jurkovich GJ, Taylor AE, Curreri PW.
Analysis of altered capillary pressure and permeability after
thermal injury. J Surg Res 1987;42:693–702.
[94] Harms BA, Bodai BI, Kramer GC, Demling RH. Microvascular
fluid and protein flux in pulmonary and systemic circulations
after thermal injury. Microvasc Res 1982;23:77–86.
[95] Demling RH. The burn edema process: current concepts. J
Burn Care Rehabil 2005;26:207–27.
[96] Pina-Canseco MS, Páez-Arenas A, Massó F, et al. Protein C
activation peptide inhibits the expression of ICAM-1, VAM-1,
and interleukin-8 induced by TNF-a in human dermal
microvascular endothelial cells. Folia Histochem Cytobiol
2012;50:407–13.
[97] Whitmont K, Reid I, Tritton S, et al. Treatment of chronic leg
ulcers with topical activated protein C. Arch Dermatol
2008;144:1479–83.
[98] Jackson C, Whitmont K, Tritton S, March L, Sambrook P, Xue
M. New therapeutic applications for the anticoagulant,
activated protein C. Expert Opin Biol Ther 2008;8:1109–22.
[99] Mesters RM, Helterbrand J, Utterback BG, et al. Prognostic
value of protein C concentrations in neutropenic patients at
high risk of severe septic complications. Crit Care Med
2000;28:2209–16.
[100] Lindstrom O, Kylanpaa L, Mentula P, et al. Upregulated but
insufficient generation of activated protein C is associated
with development of multiorgan failure in severe acute
pancreatitis. Crit Care 2006;10:R16.
[101] Pruitt Jr. BA, Wolf SE. An historical perspective on
advances in burn care over the past 100 years. Clin Plast
Surg 2009;36:527–45.
[102] Ruttmann TG, James MFM, Finlayson J. Effects on coagulation of
intravenous crystalloid or colloid in patients undergoing
peripheral vascular surgery. Br J Anaesth 2002;89:226–30.
[103] Ruttmann TG, James MFM, Lombard EH. Haemodilution-
induced enhancement of coagulation is attenuated in vitro
by restoring antithrombin III to pre-dilution concentrations.
Anaesth Intensive Care 2001;29:489–93.
[104] Ng FJ, Lam CCK, Chan LC. In vivo effect of haemodilution with
saline on coagulation: a randomized controlled trial. Br J
Anaesth 2002;88:475–80.
[105] Boldt J, Haisch G, Suttner S, Kumle B, Schellhase F. Are
lactated Ringer’s solution and normal saline solution equal
with regard to coagulation? Anesth Analg 2002;94:378–84.
[106] Boral BM, Williams DJ, Boral LI. Disseminated intravascular
coagulation. Am J Clin Pathol 2016;146:670–80.
[107] Tocco-Tussardi I, Presman B, Huss F. Want correct
percentage of TBSA burned? Let a layman do the assessment.
J Burn Care Res 2018;39:295–301.
[108] Monstrey S, Hoeksema H, Verbelen J, Pirayesh A, Blondeel P.
Assessment of burn depth and burn wound healing potential.
Burns 2008;34:761–9.
[109] Watts AM, Tyler MP, Perry ME, Roberts AH, McGrouther DA.
Burn depth and its histological measurement. Burns
2001;27:154–60.
[110] Devgan L, Bhat S, Aylward S, Spence RJ. Modalities for the
assessment of burn wound depth. J Burns Wounds 2006;5:e2.
JBUR 5837 No. of Pages 14

14 burns xxx (2019) xxx –xxx

[111] Ching JA, Shah JL, Doran CJ, Chen H, Payne WG, Smith Jr. DJ.
The evaluation of physical exam findings in patients
assessed for suspected burn inhalation injury. J Burn Care Res
2015;36:197–202.
[112] Tait RC, Walker ID, Islam SI, et al. Protein C activity in healthy
volunteers—influence of age, sex, smoking and oral
contraceptives. Thromb Haemost 1993;70:281–5.
[113] O’Mara MS, Slater H, Goldfarb IW, Caushaj PF. A prospective,
randomized evaluation of intra-abdominal pressures with
crystalloid and colloid resuscitation in burn patients. J
Trauma 2005;58:1011–8.
[114] Du G-B, Slater H, Goldfarb IW. Influences of different
resuscitation regimens on acute early weight gain in
extensively burned patients. Burns 1991;17:147–50.
[115] Hoelscher V, Harvin J, Cotton B, Wade C, Huzar T. 218
impact of fresh frozen plasma infusions during
resuscitation in thermally-injured patients. J Burn Care Res
201839: S76-S.
[116] Jones LM, Deluga N, Bhatti P, Scrape SR, Bailey JK, Coffey RA.
TRALI following fresh frozen plasma resuscitation from burn
shock. Burns 2017;43:397–402.
[117] Cartotto R, Greenhalgh D. Colloids in acute burn
resuscitation. Crit Care Clin 2016;32:507–23.
[118] Jones L, Brown N, Philips G, et al. Burn resuscitation with
fresh frozen plasma: 5 years of experience with the west
Penn formula. Austin J Emerg Crit Care Med 2015;2:1018.

Please cite this article in press as: T.C. Lang, et al., Plasma protein C levels are directly associated with better outcomes in patients with
severe burns, Burns (2019), https://doi.org/10.1016/j.burns.2019.05.001