Hypercoagulation and Hypermetabolism of Fibrinogen in Severely Burned Adults

Hypercoagulation and Hypermetabolism of Fibrinogen in Severely Burned Adults
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Wenjun Z Martini1, PhD, John B Holcomb2, MD, Yong-Ming Yu3, MD, PhD, Steven E Wolf4,
MD, Leopoldo C Cancio 1,MD1, Anthony E Pusateri1, PhD, and Michael A Dubick 1, PhD
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US Army Institute of Surgical Research, Ft Sam Houston, TX 78234
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University of Texas Health Science Center at Houston, TX 77030
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Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
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Shriner’s Burn Hospital for Children, Galveston, TX 77550
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Corresponding author:
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Wenjun Z. Martini, Ph.D.
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The US Army Institute of Surgical Research

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3650 Chambers Pass

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Ft. Sam Houston, TX 78234
210-539-4573 (Tel)
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210-539-6244 (Fax)
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wenjun.z.martini.civ@mail.mil
This work was funded by the U.S. Army Medical Research and Materiel Command
Published by Oxford University Press on behalf of the American Burn Association 2019. This work is
written by (a) US Government employee(s) and is in the public domain in the US.
ABSTRACT
Introduction: This study investigated changes in plasma fibrinogen metabolism and changes in
coagulation in severely burned adults.

Methods: Ten patients (27±3 years; 91± 6kg) with 51±3% TBSA were consented and enrolled
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into an institutional review board approved prospective study. On the study day, stable isotope
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infusion of 1-13C-phenylalanine and d5-phenylalanine was performed to quantify fibrinogen
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production and consumption. During the infusion, vital signs were recorded and blood samples
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were drawn every hour. Coagulation was measured by thromboelastograph (TEG). Ten normal
healthy volunteers (37±7years; 74±4 kg) were included as the control group.
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Results: Burned adults had elevated heart rates (120±2 vs. 73±5 (control) beats/min), respiration
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rates (23±2 vs. 15±1 breaths/min), plasma glucose (127±10 vs 89±2 mg/dL) and fibrinogen
levels (613±35 vs 239±17 mg/dL); and decreased albumin (1.3±0.2 vs. 3.7±0.1 g/dL) and total
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protein (4.4±0.2 vs. 6.8±0.1 g/dL, all p<0.05). Fibrinogen breakdown was elevated in the burn
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group (2.3±0.4 vs. 1.0±0.3 µmol/kg/min); and fibrinogen synthesis was further enhanced in the
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burn group (4.4±0.7 vs 0.7±0.2 µmol/kg/min, both p<0.05). Clotting speed (TEG-alpha) and clot
strength (TEG-MA) were increased in the burn group (62±4 vs. 50±4, and 76±2 vs. 56±2 mm,
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respectively, both p<0.05). Fibrinolysis of TEG-LY60 was accelerated in the burn group (16±6 vs.
3±1) and so was the increase in D-dimer level in the burn group (4.5±0.4 vs 1.9±0.3 mg/L, both
p<0.05).
Conclusions: The hypercoagulable state postburn is in part a result of increased fibrinogen synthesis,
over and above increased fibrinogen breakdown.
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Key words: burn injury, fibrinogen, protein catabolism, stable isotope infusion, and
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coagulation. Ac
INTRODUCTION
Burn injury causes profound hypermetabolic and catabolic responses, which may last months to
years1-4. These metabolic deteriorations induce severe muscle loss, prolonged ventilation days,

impaired immune responses, and eventual death1, 4-7. Meanwhile, burn injury is also known to
cause coagulation dysfunction, one of the important contributors to trauma mortality8-11.
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However, the comprehensive understanding of the dynamic change and interrelation between
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coagulation and metabolism in burn patients is indeed lacking.
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As the precursor for clot formation, fibrinogen availability is of vital importance for sufficient
and timely coagulation12. Following trauma and hemorrhagic shock, fibrinogen loss is associated
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with bleeding and mortality13-16, and supplementation of fibrinogen may improve outcomes17-19.
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In general, fibrinogen availability reflects the dynamic balance of fibrinogen synthesis and
breakdown. Thus, investigating changes of fibrinogen metabolism may provide mechanisms

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related to changes in coagulation function. In addition, as one the most abundant plasma
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proteins, fibrinogen metabolic changes may shed light on host metabolic responses to insults.
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Thus, investigating fibrinogen metabolism will provide insights connecting changes of
coagulation and metabolism after burn.

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In order to determine metabolic changes of fibrinogen, we have developed an in vivo technique
in a swine model20. Applying this technique, we have investigated fibrinogen metabolic changes
under traumatic hemorrhage, hypothermia and acidosis in experimental animals21-23. Although
the extent of changes in fibrinogen synthesis and breakdown varies under different trauma
conditions, fibrinogen deficiency was consistently demonstrated shortly after trauma 24. These
findings provide an explanation for the initial drop of fibrinogen concentration observed
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clinically after trauma13, 25, and support the approach of early supplementation of fibrinogen
concentrate to restore coagulation in trauma. In this study, we used the same technique to
investigate changes of fibrinogen metabolism and coagulation in severely burned adult patients,

in parallel with coagulation assessments.
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MATERIALS AND METHODS
Patient enrollment:
This study was reviewed and approved by the Institutional Review Board at the Brooke Army

Medical Center. Patients admitted to the Burn Center at the US Army Institute of Surgical
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Research with burns ≥ 20% total body surface area (TBSA) and age between 18 and 65 years old
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were screened. Those with pre-existing chronic diseases (i.e., liver, heart, and lung), acute life-
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threatening illness, pregnancy or nursing, prisoner status, and allergy to iodine or any dye were
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excluded. Following inclusion and exclusion criteria, 10 burned patients with 51 ± 3% total body
surface area (27 ± 4 years old, 91 ± 6 kg) were consented and enrolled in the study. Ten normal
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healthy volunteers (37 ± 5 years old, 74 ± 4kg) were included in the study as healthy controls.
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Isotope infusion study:
Stable isotope ring-d5-phenylalanine and 1-13C-phenylalanine were purchased from Cambridge

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Isotope Laboratories (Andover, MA). Sterile indocyanine green (ICG) dye and 0.45% normal
saline were purchased from Akorn, Inc. (Buffalo Grove, IL) and Baxter Healthcare Corporation,
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(Deerfield, IL), respectively. Stable isotope solutions of d 5-phenylalanine (80 µmol/ml) and 1-
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C-phenylalanine (100 µmol/ml) were prepared at the Pharmacy Department at Brooke Army
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Medical Center under sterile conditions and certified fume hoods on the day prior to infusion
study.
Upon enrollment, the timing of the isotope infusion study was selected based upon the physiological
stability of the patients and availability of patients with 10 hours free of clinical care (i.e., dressing
changes). The physiological steady state, as indicated by temperature and hemodynamics, was the
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assumption for valid quantification of protein metabolism using stable isotope infusion. This
assumption excluded initial resuscitation periods and days of surgery and frequent dressing
changes. Once the physiological steady state and the availability of 10 hours free of clinical care

were confirmed, the isotope infusion study was performed in each of the enrolled patients (Table
1). On average, the isotope infusion study was performed at 18±5 days after burn. On the study
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day, when patient’s vital signs were stable for 3 hours (defined as less than 20% of fluctuation
over the beginning values), baseline vital signs were recorded and blood samples were
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withdrawn as baseline measurements. Afterwards, a primed constant infusion of d5-
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phenylalanine (0.2 µmol/kg/min, for 4h) and 1-13C-phenylalanine (0.2 µmol/kg/min, for 8h) was
performed in each subject through 0.2 µm sterile and non-pyrogenic Tuffryn® membrane filters.
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Hourly arterial blood samples were taken during the infusion. ICG dye was injected at 6h to
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measure plasma volumes during the infusion. Vital signs were monitored and recorded hourly
during the study. The entire infusion study is depicted in Fig. 1. If the patient was treated with
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drugs such as propranolol, insulin or oxandrolone (Table 2), the treatment was not stopped for
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the isotope infusion period. In addition, the same isotope infusion and associated procedures
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were performed in 10 healthy subjects in an unoccupied ward in the Burn Center.

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Calculations of fibrinogen synthesis and breakdown: Fibrinogen synthesis and breakdown were
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calculated based on the labeling changes during the infusion, as described previously20. Briefly,
plasma fibrinogen fractional synthesis rate (FSR) was calculated based on the increase of
fibrinogen bound phenylalanine labeling over the 8 h infusion of 1-13C-phenylanaline. Plasma
fibrinogen fractional breakdown rate (FBR) was determined by the decrease of fibrinogen-bound
phenylalanine labeling after the infusion of d5-phenylalanine was stopped at 4 h. The plasma
fibrinogen absolute synthesis rate was calculated by multiplying FSR with plasma fibrinogen
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concentration and plasma volume (measured using ICG dye). Similarly, plasma fibrinogen
absolute breakdown rate was calculated by multiplying FBR with plasma fibrinogen
concentration and plasma volume.

Coagulation measurements: Plasma prothrombin time (PT) and activated partial thromboplastin
time (aPTT) were measured by standard clinical lab techniques using the BCS Coagulation
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System (Dade Behring, Deerfield, IL). The coagulation profile was assessed in fresh whole
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blood samples (0.35 ml) with tissue factor using thrombelastography (TEG, TEG 5000
Hemostasis Analyzer, Haemoscope Corp, Niles, IL) as described previously20 8. Tissue factor
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solution was prepared daily by 1:1000 dilution of recombinant human tissue factor (Dade
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Behring, Deerfield, IL) with saline. Heparinase cups were used in TEG analysis since all patients
were prescribed heparin for DVT prophylaxis. TEG measurements included R time (the time that
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the initial clot is formed), K time (the time when clot strength reaches 20 mm), maximum
amplitude (MA) (maximum clot strength),  angle (the rapidity of fibrin build-up and cross-
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linking), and LY60 (rate of amplitude reduction 60 min after MA to determine fibrinolysis). TEG
measurements were made in triplicate at every time point and performed for 1.5 h to ensure the
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completion of LY60.
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Analytical methods
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Blood chemistry was measured using a Dimension Clinical Chemistry System (Dade Behring
Inc., Newark, DE), which included plasma total protein content, plasma concentrations of
albumin and liver enzymes. Plasma fibrinogen concentration was measured using the BCS
Coagulation System (Dade Behring, Deerfield, IL).
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For assessment of plasma free amino acid enrichments, 0.5 ml of acidified plasma was loaded on
a cation exchange column (AG 50W-X8 resin, 200-400 mesh, H+ form, Bio-rad). Amino acids
were separated after elution with ammonic hydroxide. The extracts were dried under speed

vacuum and derivatized by N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide at 100 °C for
1 h. Plasma fibrinogen was isolated by adding 0.5M CaCl2 and thrombin to form fibrin clot,
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following the procedure described by Stein et al. 26. The clot was then washed and hydrolyzed in
6N HCl at 110 °C for 24 h and dried under speed vacuum. The released amino acids after
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hydrolysis were isolated, dried, and subjected to derivatization in the same manner as for plasma
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free amino acids. The enrichments of phenylalanine from plasma free amino acid pool and in
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fibrinogen protein were determined by gas chromatography-mass spectrometry (GC-MS, model
5973, Hewlett-Packard) in the electron impact ionization mode. A selective ion-monitoring

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method was used at nominal mass-to-charge ratio (m/z) of 336 (m+0), 337 (m+1), 338 (m+2),
339 (m+3), 340 (m+4), and 341(m+5) for phenylalanine. The enrichment was expressed as tracer
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(labeled amino acids) to tracee (unlabeled amino acids) ratio (TTR).

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Statistical analysis
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Comparisons in physiological measurements, liver enzyme activities, substrate concentrations,

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fibrinogen metabolism and coagulation between the burn and control groups were performed by
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one-way analysis of variance followed by the Tukey correction for post hoc comparisons. All
results are expressed as means ± SE. Statistical significance was set at the 0.05.
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RESULTS
The demographics and clinical characteristics of the 10 enrolled burn patients are described in
Table 1. All enrolled burn patients received deep venous thrombosis (DVT) prophylaxis within

24 hours after admission in accordance with our institution’s clinical practice guidelines. Prior to
study day, all of the enrolled patients were screened for evidence of hemorrhage before study and
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none of them was actively bleeding. The mean hospital stay of the enrolled patients was 87±19
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days (Table 1). The 10 normal healthy volunteers were studied during the same time period.
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Physiological and Biochemical Measurements
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A physiological steady state was observed in the control and burn groups during the study, with
no significant changes in vital signs during the 8 h stable isotope infusion in either group.
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Compared with the control group, burn patients had significant increases in respiratory rate and
heart rate (Fig 2). Burned patients had increased levels of plasma glucose and lactate, as well as
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reduced levels of total protein and albumin (Table 3). Fibrinogen concentration and platelet
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counts in burn patients were increased to 2.5 times and 1.8 times of the control values,
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respectively (Table 4). In addition, burn patients had elevated serum liver enzyme concentrations
of alanine aminotransferase, asparate amino transferease, -glutamyl transferase and lactate

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dehydrogenase (Table 5).
Fibrinogen Metabolism
Plasma phenylalanine TTR (m + 1) from 1-13C-phenylalanine infusion in both groups reached
plateau values (32.0±1.0% in control and 14.5±0.9% in burn ) after 1 h. The rate of appearance
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of free phenylalanine was 0.6±0.0 μmol/kg/min in control and 1.4±0.1 μmol/kg/min in burn
patients (p < 0.05).
Fibrinogen-bound phenylalanine TTR (m + 1) increased linearly during the infusion of 1-13C-

phenylalanine. Plasma fibrinogen fractional synthesis rate, based upon the increases of
fibrinogen-bound phenylalanine TTR, was 0.54±0.08%/h in control and 1.25±0.12%/h in burn
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patients (p < 0.05). Plasma volume was 50±2 ml/kg in the burn group and 35±2 ml/kg in the
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control group (p < 0.05). In burn patients, fibrinogen absolute synthesis rate was increased to 4
times of the control values in burn patients (Fig 3).
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Fibrinogen fractional breakdown rate was measured based upon changes in fibrinogen-bound
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phenylalanine TTR (m+5), after the infusion of d5-phenylalanine stopped at 4 h. Fibrinogen
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fractional breakdown rate was 0.84±0.16%/h in control and 0.72±0.14%/h in burn. Fibrinogen
absolute breakdown rate was accelerated to twice the control values (Fig 4).
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Coagulation Function
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Coagulation variables did not change significantly during the 8h isotope infusion study in either
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group. Thus, the averages of baseline values and measurements made at 8h were pooled for
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comparison.
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PT and aPTT were similar between the burn and control group, but D-dimer was elevated to 2.5
times of the control value (Table 4). The initial clotting time (R time) from TEG analysis was
similar in the control (4.4±0.4 min) and the burn (6.5±1.0 min) groups. However, clotting
rapidity (α angle), clot strength (MA), and fibrinolysis (LY60) were all increased in the burn
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group, as compared to the control values (Fig 5&6). Increased fibrinolysis observed in burn
patients is also supported by the higher D-dimer levels in these patients (Table 4).
DISCUSSION

Hypermetabolism and significant protein catabolism are consistent features of severe burn injury.
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The present study demonstrated a wide range of pathophysiological alterations in burn patients,
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including elevated respiratory rate, heart rate, glucose, and activities of serum markers of liver
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enzymes. Protein catabolic responses were also prominent, with accelerated breakdown rates of
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fibrinogen and whole-body protein, and decreased total serum protein content. Concurrently, a
hypercoagulable state was present with increased clotting speed and clot strength, together with
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enhanced fibrinolysis. All of these changes occurred during the flow phase of the burn,
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suggesting prolonged and sustained metabolic and coagulation disturbances from burn injury.
As an acute phase protein, fibrinogen has been shown to be increased in various stress
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circumstances, such as in sepsis, cancer, surgery, trauma injury27, 28. In the current study, plasma
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fibrinogen levels also increased several folds following burn injury. Although commonly
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observed, the underlying mechanisms regulating the increase in fibrinogen levels remain
unknown. The increased availability may be due to increased production, and/or decreased
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degradation. In this study, with direct measurements of synthesis and breakdown, we revealed
mechanisms contributing to the increase of fibrinogen levels. Our data showed that both
fibrinogen synthesis and breakdown were increased after burn. However, the magnitude of
increase in fibrinogen synthesis was larger than that of fibrinogen breakdown, resulting in the
increase of fibrinogen availability after burn injury. It should be emphasized that, like other
proteins, fibrinogen synthesis rate and breakdown rate undergo dynamical changes. Results from
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this study were only reflect fibrinogen metabolism at 18±5 days post burn injury. Fibrinogen
synthesis and breakdown at different time periods may be different from what is presented in this
study.

Metabolic responses to traumatic injury, infection, surgery, and sepsis are characterized by
increases in whole-body protein turnover and loss of lean-body-mass protein29, 30. Free amino
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acids are released from whole-body protein breakdown and amino acids are taken up in the
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splanchnic bed30, 31. In this study, whole-body protein breakdown in severely burn patients was
doubled that of control values and fibrinogen synthesis by the liver was increased four-fold. The
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flow of amino acids released by peripheral protein catabolism and the increase in acute phase
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protein synthesis by the liver were previously described by Clowes et al. in patients with sepsis
or trauma31, 32. Thus, this process may represent a generalized host response after injury.
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In addition to hemostasis, fibrinogen plays vital roles in many biological and physiological
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functions, including angiogenesis, wound healing, inflammation, infection, and neoplasia .
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During wound healing, fibrinogen plays an essential role by establishing initial matrix and
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primary wound-healing structure, strength and stability and facilitating later cellular migration
and remodeling of the healing process12. Delayed wound healing is associated with high risk of
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mortality in burn patients37. In host defense, fibrinogen functions as a barrier to prevent pathogen
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spread, as well as a modulator of host immune function, including activation of immune cells for
degranulation, oxidative response, and expression of cytokines36. Thus, it is possible that
increasing fibrinogen availability under various stressed circumstances reflects host responses to
promote recovery. Furthermore, the fact that increasing fibrinogen production at the cost of
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accelerating whole-body protein breakdown may indicate that it is a host priority for survival.
However, further efforts are needed to test this hypothesis.
This study is a continuation of our effort in coagulation research in injured patients. Previously

we investigated the acute changes of coagulation function following burn injury8. In that study,
we enrolled 25 burn patients (30±4 %TBSA) and measured coagulation profiles daily for 7 days
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after admission. A hypercoagulable state was observed in those patients, as indicated with
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elevated clotting speed and increased clot strength as compared to normal controls8. Fibrinogen
levels increased daily and plateaued at day 3. In the present study, during the flow phase of
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recovery, we also observed a hypercoagulable state, suggesting the sustained changes in
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coagulation profile after burn injury. Meanwhile, the extent of fibrinolysis was also enhanced. It
is unclear at what point the coagulation process may recover to normal values. In addition, we
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previously investigated coagulation changes and clinical outcomes in 25 burn patients and 33
non-burn trauma patients daily for 7 days after admission8. We observed that one burn patient
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and two non-burn trauma patients developed pulmonary embolism8, despite DVT prophylaxis. In
the present study, most patients enrolled had combined burn and traumatic injuries. With the
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small sample size and combined injuries, it was unclear whether burn patients had the same level
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of risk, morbidity, or mortality from thromboembolic complications as trauma patients.

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Prospective clinical trials with appropriate sample sizes are warranted to address the question.
Disseminated intravascular coagulation contributes to significant morbidity and mortality in
severe sepsis38-41. This response results from interconnected cellular and molecular reactions at
local and systemic levels and develops into inflammation-induced coagulation activation and
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subsequent fibrinolytic activation38, 39. Following consumption of pro-coagulant and anti-
coagulant and pro-fibrinolytic and anti-fibrinolytic factors, the response progresses to
hemorrhagic or thrombotic events, which implies the importance of timing in therapies 41. In

present study, several patients were receiving antibiotics, such as ciprofloxacin, vancomycin,
and/or imipenem-cilastatin, at the time of the infusion study, and 8 of 10 patients developed
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bacteremia several weeks after the study. Thus, it is possible that an underlying infection in some
patients might influence the coagulation changes observed in this study, in addition to that
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induced by the burn injury. Future effort is needed to differentiate the effects of burn injury and
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infection on coagulation.
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There are a few limitations worthy noting about the present study. First, there was a significant
difference in mean body weight between the burn and control groups. This may have represented
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fluid accumulation from resuscitation in the burn patients. We did not measure BMI in the study.
Since fibrinogen metabolic changes were expressed as mg fibrinogen per kg body weight per
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hour, the larger body weight in the burn group could potentially result in underestimation of
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accelerated fibrinogen synthesis and breakdown from burn injury. Second, wound healing status
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was not available during the study thus we are unable to assess the impact of wound healing on
metabolism and coagulation in analysis. Third, insulin administration in burn patients has been
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shown to improve skeletal muscle protein synthesis 42, 43. Since 90% of the enrolled patients
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received insulin during the infusion, the findings of this study might include the stimulatory
effect of insulin on protein metabolism. However, the effects of propranolol or oxandrolone
cannot be determined, as few patients received these medications during the study. Future effort
is needed to identify the effects of these drugs on fibrinogen metabolism.
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In addition, although hyperfibrinolysis was shown in the present study, it is important to note
that with the small sample size of 10 patients, it was not able to capture the variations of
coagulation responses to burn. Significant heterogeneities have been demonstrated in trauma and

burn patients 44, 45. In a multicenter cohort study of 795 trauma patients, Roberts et al. reported
that 44% of patients presented with fibrinolysis shutdown, 36% of patients had normal
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fibrinolysis, and 21% patients had hyperfibrinolysis45. The highest mortality within the first 24
hours was observed in hyperfibrinolytic patients. After 24 hours, the mortality increased with
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fibrinolyltic shut down patients45. These time-dependent fibrinolytic responses may have
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profound implications on the efficacy and safety of tranexamic acid therapy. Thus, fibrinolytic
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responses to burn and trauma are complex and findings from our study should interpreted with
caution.
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In conclusion, our study demonstrated sustained hypermetabolism and catabolism in severely
burned adults during the flow phase. We observed accelerated protein breakdown at the whole
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body level, as well as in the individual protein fibrinogen. However, fibrinogen availability was
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increased due to a higher increase in fibrinogen production as compared to consumption.

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Coagulation was enhanced together with increased fibrinolysis. In order to determine the best
treatment options and time windows in burn management, further effort is warranted to
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investigate the time course and implications of accelerated fluxes of coagulation and fibrinolysis
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as well as fibrinogen synthesis and breakdown.
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ACKNOWLEDGEMENTS
The authors would like to thank Annette McClinton for her regulatory assistance throughout the
study and Dr David Chinkes for his assistance in protein kinetics calculation. The authors

appreciate support received from research nurse Nancy Molter, Elisabeth Frail, Audrey Parker,
Peggy Bielke, Kari Williams, Chaya Galin and Michelle Leas at the US Army Institute of
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Surgical Research and the Pharmacy Department at Brooke Army Medical Center.
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The opinions or assertions contained herein are the private views of the authors and are not to be
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construed as official or as reflecting the views of the Department of the Army or the Department
of Defense.
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REFERENCES
1. Wolfe RR. Review: acute versus chronic response to burn injury. Circ Shock.
1981;8(1):105-115.

2. Herndon DN, Tompkins RG. Support of the metabolic response to burn injury. Lancet.
2004;363(9424):1895-1902.
t
ip
3. Jeschke MG, Gauglitz GG, Kulp GA, Finnerty CC, Williams FN, Kraft R, Suman OE,
cr
Mlcak RP, Herndon DN. Long-term persistance of the pathophysiologic response to severe burn
injury. PLoS One. 2011;6(7):e21245.
us
4. Porter C, Tompkins RG, Finnerty CC, Sidossis LS, Suman OE, Herndon DN. The
an
metabolic stress response to burn trauma: current understanding and therapies. Lancet.
2016;388(10052):1417-1426.
M
5. Jeschke MG, Chinkes DL, Finnerty CC, Kulp G, Suman OE, Norbury WB, Branski LK,
Gauglitz GG, Mlcak RP, Herndon DN. Pathophysiologic response to severe burn injury. Ann
d
te
Surg. 2008;248(3):387-401.
6. Newsome TW, Mason AD, Jr., Pruitt BA, Jr. Weight loss following thermal injury. Ann
ep
Surg. 1973;178(2):215-217.
c
7. Hart DW, Wolf SE, Mlcak R, Chinkes DL, Ramzy PI, Obeng MK, Ferrando AA, Wolfe
Ac
RR, Herndon DN. Persistence of muscle catabolism after severe burn. Surgery. 2000;128(2):312-
319.
8. Park MS, Martini WZ, Dubick MA, Salinas J, Butenas S, Kheirabadi BS, Pusateri AE,
Vos JA, Guymon CH, Wolf SE, et al. Thromboelastography as a better indicator of
hypercoagulable state after injury than prothrombin time or activated partial thromboplastin time.
J Trauma. 2009;67(2):266-275; discussion 275-266.

18
9. Park MS, Salinas J, Wade CE, Wang J, Martini W, Pusateri AE, Merrill GA, Chung K,
Wolf SE, Holcomb JB. Combining early coagulation and inflammatory status improves
prediction of mortality in burned and nonburned trauma patients. J Trauma. 2008;64(2

Suppl):S188-194.
10. Geerts WH, Code KI, Jay RM, Chen E, Szalai JP. A prospective study of venous
t
ip
thromboembolism after major trauma. N Engl J Med. 1994;331(24):1601-1606.
11. Schreiber MA. Coagulopathy in the trauma patient. Curr Opin Crit Care.
cr
2005;11(6):590-597.
us
12. Weisel JW, Litvinov RI. Fibrin Formation, Structure and Properties. Subcell Biochem.
2017;82:405-456.
13.
an
Armand R, Hess JR. Treating coagulopathy in trauma patients. Transfus Med Rev.
M
2003;17(3):223-231.
14. Hardy JF, De Moerloose P, Samama M. Massive transfusion and coagulopathy:

d
pathophysiology and implications for clinical management. Can J Anaesth. 2004;51(4):293-310.

te
15. Sherman LA. DIC in massive transfusion. Prog Clin Biol Res. 1982;108:171-189.
ep
16. Sherry S. Hemostatic mechanisms and proteolysis in shock. Fed Proc. 1961;20:209-218.
17. Stinger HK, Spinella PC, Perkins JG, Grathwohl KW, Salinas J, Martini WZ, Hess JR,
c
Dubick MA, Simon CD, Beekley AC, et al. The ratio of fibrinogen to red cells transfused affects
Ac
survival in casualties receiving massive transfusions at an army combat support hospital. J
Trauma. 2008;64(2 Suppl):S79-85; discussion S85.
18. Gonzalez EA, Moore FA, Holcomb JB, Miller CC, Kozar RA, Todd SR, Cocanour CS,
Balldin BC, McKinley BA. Fresh frozen plasma should be given earlier to patients requiring
massive transfusion. J Trauma. 2007;62(1):112-119.
19
19. Ketchum L, Hess JR, Hiippala S. Indications for early fresh frozen plasma,
cryoprecipitate, and platelet transfusion in trauma. J Trauma. 2006;60(6 Suppl):S51-58.
20. Martini WZ, Chinkes DL, Pusateri AE, Holcomb JB, Yu YM, Zhang XJ, Wolfe RR.

Acute changes in fibrinogen metabolism and coagulation after hemorrhage in pigs. Am J Physiol
Endocrinol Metab. 2005;289(5):E930-934.
t
ip
21. Martini WZ, Chinkes DL, Sondeen J, Dubick MA. Effects of hemorrhage and lactated
Ringer's resuscitation on coagulation and fibrinogen metabolism in swine. Shock.
cr
2006;26(4):396-401.
us
22. Martini WZ. The effects of hypothermia on fibrinogen metabolism and coagulation
23.
an
function in swine. Metabolism. 2007;56(2):214-221.
Martini WZ, Holcomb JB. Acidosis and coagulopathy: the differential effects on
M
fibrinogen synthesis and breakdown in pigs. Ann Surg. 2007;246(5):831-835.
24. Martini WZ. Fibrinogen metabolic responses to trauma. Scand J Trauma Resusc Emerg
d
Med. 2009;17(1):2.
te
25. Collins JA. The pathophysiology of hemorrhagic shock. Prog Clin Biol Res. 1982;108:5-
ep
29.
26. DeLoughery TG. Coagulation defects in trauma patients: etiology, recognition, and
c
therapy. Crit Care Clin. 2004;20(1):13-24.

Ac
27. Fleck A. Clinical and nutritional aspects of changes in acute-phase proteins during
inflammation. Proc Nutr Soc. 1989;48(3):347-354.
28. Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci.
1982;389:39-48.
20
29. Jahoor F, Desai M, Herndon DN, Wolfe RR. Dynamics of the protein metabolic response
to burn injury. Metabolism. 1988;37(4):330-337.
30. Clowes GH, Jr., Randall HT, Cha CJ. Amino acid and energy metabolism in septic and

traumatized patients. JPEN J Parenter Enteral Nutr. 1980;4(2):195-205.
31. Clowes GH, Jr., Heideman M, Lindberg B, Randall HT, Hirsch EF, Cha CJ, Martin H.
t
ip
Effects of parenteral alimentation on amino acid metabolism in septic patients. Surgery.
1980;88(4):531-543.
cr
32. Clowes GH, Jr., George BC, Villee CA, Jr., Saravis CA. Muscle proteolysis induced by a
us
circulating peptide in patients with sepsis or trauma. N Engl J Med. 1983;308(10):545-552.
33. an
Litvinov RI, Weisel JW. What Is the Biological and Clinical Relevance of Fibrin? Semin
Thromb Hemost. 2016;42(4):333-343.

M
34. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of
fibrinogen and fibrin. Ann N Y Acad Sci. 2001;936:11-30.

d
35. Zuliani-Alvarez L, Midwood KS. Fibrinogen-Related Proteins in Tissue Repair: How a

te
Unique Domain with a Common Structure Controls Diverse Aspects of Wound Healing. Adv
ep
Wound Care (New Rochelle). 2015;4(5):273-285.
36. Ko YP, Flick MJ. Fibrinogen Is at the Interface of Host Defense and Pathogen Virulence
c
in Staphylococcus aureus Infection. Semin Thromb Hemost. 2016;42(4):408-421.

Ac
37. Rowan MP, Cancio LC, Elster EA, Burmeister DM, Rose LF, Natesan S, Chan RK,
Christy RJ, Chung KK. Burn wound healing and treatment: review and advancements. Crit Care.
2015;19:243.
38. Iba T, Watanabe E, Umemura Y, Wada T, Hayashida K, Kushimoto S, Japanese
Surviving Sepsis Campaign Guideline Working Group for disseminated intravascular c, Wada H.
21
Sepsis-associated disseminated intravascular coagulation and its differential diagnoses. J
Intensive Care. 2019;7:32.
39. Samuels JM, Moore HB, Moore EE. Coagulopathy in Severe Sepsis: Interconnectivity of

Coagulation and the Immune System. Surg Infect (Larchmt). 2018;19(2):208-215.
40. Liu J, Liu Y, Liu S, Zhang Q, Zheng J, Niu Y, Wang X. Hypocoagulation induced by
t
ip
broad-spectrum antibiotics in extensive burn patients. Burns Trauma. 2019;7:13.
41. Scarlatescu E, Tomescu D, Arama SS. Anticoagulant Therapy in Sepsis. The Importance
cr
of Timing. J Crit Care Med (Targu Mures). 2017;3(2):63-69.
us
42. Sakurai Y, Aarsland A, Herndon DN, Chinkes DL, Pierre E, Nguyen TT, Patterson BW,
an
Wolfe RR. Stimulation of muscle protein synthesis by long-term insulin infusion in severely
burned patients. Ann Surg. 1995;222(3):283-294; 294-287.

M
43. Diaz EC, Herndon DN, Porter C, Sidossis LS, Suman OE, Borsheim E. Effects of
pharmacological interventions on muscle protein synthesis and breakdown in recovery from

d
burns. Burns. 2015;41(4):649-657.

te
44. Shupp JW, Prior SM, Jo DY, Moffatt LT, Mann KG, Butenas S. Analysis of factor XIa,
ep
factor IXa and tissue factor activity in burn patients. Burns. 2018;44(2):436-444.
45. Roberts DJ, Kalkwarf KJ, Moore HB, Cohen MJ, Fox EE, Wade CE, Cotton BA. Time
c
course and outcomes associated with transient versus persistent fibrinolytic phenotypes after
Ac
injury: A nested, prospective, multicenter cohort study. J Trauma Acute Care Surg.
2019;86(2):206-213.
22
TABLES
Table 1. Burn patient demographics and characteristics
Patient # Age (yr)/ Body TBSA Other Injury Infusion Hospital

Military (M) or Weight (%) study time Stay

Civilian (C) (kg) (Days after (Days)
burn)
P-01 26/M 88 50 Fracture 10 125
t
ip
P-02 20/M 100 59 Fracture 26 83
cr
P-03 31/M 93 75 Inhalation 58 228
P-04 46/C 62 57 Inhalation 14 53
us
P-05 32/C 88 37 None 6 17
P-06 25/M 135 an

53 Fracture 14 127
P-07 26/M 90 40 Fracture 9 58

M
P-08 23/C 88 41 None 22 86
P-09 18/M 78 56 None 12 49

d
P-10 24/M 102 48 None 13 46

te
Ave ± SE 27±3 91±6 52%±4% 18±5 87±19

c ep
Ac
23
Table 2. Burn patient medication records during the isotope infusion study
Patient # Insulin Propranolol Oxandrolone Blood products
P-01 Yes No No No

P-02 Yes Yes No No
P-03 Yes No No No
t
ip
P-04 Yes No No No
cr
P-05 Yes No No No
P-06 Yes No No No
us
P-07 No No No No
P-08 Yes Noan No No
P-09 Yes Yes Yes No

M
P-10 Yes Yes No No
d
te
c ep
Ac
24
Table 3. Plasma protein levels in the burn and control groups
Glucose Lactate Albumin Total protein

(mg/dL) (mg/dL) (g/dL) (g/dL)
t
ip
Control 89 ± 2 9.6 ± 0.7 3.7 ± 0.1 6.8 ± 0.1
cr
Burn 126 ± 11* 13.4 ± 1.0* 1.3 ± 0.2* 4.4 ± 0.2*
us
an
* p < 0.05 compared with control. N=10 in each group.
M
d
te
c ep
Ac
25
Table 4. Serum coagulation measurements in the burn and control groups
Fibrinogen Platelets PT aPTT D-dimer

(mg/dL) (109/L) (sec) (sec) (mg/L)
t
ip
Control 247±20 240±15 10.7±0.2 27.3±0.6 1.8±0.4
cr
Burn 612±35* 430±70* 11.1±0.2 28.2±1.0 4.5±0.4*
us
an
*p < 0.05 compared with control. N=10 in each group.
M
d
te
c ep
Ac
26
Table 5. Serum liver enzyme concentrations in the burn and control groups
Alanine Aspartate -glutamyl Lactate

aminotransferase aminotransferase transferase dehydrogenase
(U/L) (U/L) (U/L) (U/L)
t
ip
cr
Control 33±4 18±3 21±4 145±8
us
Burn 120±26* 99±28* 104±37* 273±35*
an
M
*p < 0.05 compared with control. N=10 in each group.
d
te
c ep
Ac
27
FIGURE LEGENDS
Figure 1 Stable isotope infusion for fibrinogen synthesis and breakdown rates in the burn

and control groups.
t
ip
Figure 2 Heart rate and respiration rate in the burn and control groups.
*p<0.05 compared to control
cr
us
Figure 3 Fibrinogen synthesis rate in the burn and control groups.
*p<0.05 compared to control an

M
Figure 4 Fibrinogen breakdown rate in the burn and control groups

d
te
Figure 5 Clotting initiation time (R time) and clotting speed (α angle) in the burn and
ep
control groups.

c
Ac
Figure 6 Clot strength (MA) and fibrinolysis (LY60) in the burn and control groups.
28
8
7
ICG
6
5
1-13C-Phenylalanine
4
3
2H5-Phenylalanine
2
1
Figure 1
Blood Sampling
0
Time (h)
*
*
*
*
*
Burn
Burn
*
*
*
*
Control
Control
Respiration rate
*
*
Heart rate
*
*
*
Figure 2
Beat/min Breath/min
*
Figure 3
mg/kg/h
*
Figure 4
mg/kg/h
*
Angle (α)
R time
Figure 5
Minutes Degrees
*
*
LY60
MA
Figure 6
mm %

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Hypercoagulation and Hypermetabolism of Fibrinogen in Severely Burned Adults

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Hypercoagulation and Hypermetabolism of Fibrinogen in Severely Burned Adults

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The US Army Institute of Surgical Research

3650 Chambers Pass

Ft. Sam Houston, TX 78234

coagulation in severely burned adults.

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over and above increased fibrinogen breakdown.

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cause coagulation dysfunction, one of the important contributors to trauma mortality8-11.

breakdown. Thus, investigating changes of fibrinogen metabolism may provide mechanisms

Thus, investigating fibrinogen metabolism will provide insights connecting changes of

coagulation and metabolism after burn.

In order to determine metabolic changes of fibrinogen, we have developed an in vivo technique

under traumatic hemorrhage, hypothermia and acidosis in experimental animals21-23. Although

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Stable isotope ring-d5-phenylalanine and 1-13C-phenylalanine were purchased from Cambridge

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were performed in 10 healthy subjects in an unoccupied ward in the Burn Center.

fibrinogen bound phenylalanine labeling over the 8 h infusion of 1-13C-phenylanaline. Plasma

concentration and plasma volume.

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Coagulation System (Dade Behring, Deerfield, IL).

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5973, Hewlett-Packard) in the electron impact ionization mode. A selective ion-monitoring

(labeled amino acids) to tracee (unlabeled amino acids) ratio (TTR).

Comparisons in physiological measurements, liver enzyme activities, substrate concentrations,

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of alanine aminotransferase, asparate amino transferease, -glutamyl transferase and lactate

dehydrogenase (Table 5).

Plasma phenylalanine TTR (m + 1) from 1-13C-phenylalanine infusion in both groups reached

patients (p < 0.05).

Fibrinogen-bound phenylalanine TTR (m + 1) increased linearly during the infusion of 1-13C-

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fibrinogen-bound phenylalanine TTR, was 0.54±0.08%/h in control and 1.25±0.12%/h in burn

times of the control values in burn patients (Fig 3).

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degranulation, oxidative response, and expression of cytokines36. Thus, it is possible that

However, further efforts are needed to test this hypothesis.

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of risk, morbidity, or mortality from thromboembolic complications as trauma patients.

Disseminated intravascular coagulation contributes to significant morbidity and mortality in

coagulant and pro-fibrinolytic and anti-fibrinolytic factors, the response progresses to

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effect of insulin on protein metabolism. However, the effects of propranolol or oxandrolone

is needed to identify the effects of these drugs on fibrinogen metabolism.

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increased due to a higher increase in fibrinogen production as compared to consumption.

as well as fibrinogen synthesis and breakdown.

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injury. PLoS One. 2011;6(7):e21245.

J Trauma. 2009;67(2):266-275; discussion 275-266.

prediction of mortality in burned and nonburned trauma patients. J Trauma. 2008;64(2

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14. Hardy JF, De Moerloose P, Samama M. Massive transfusion and coagulopathy:

pathophysiology and implications for clinical management. Can J Anaesth. 2004;51(4):293-310.

survival in casualties receiving massive transfusions at an army combat support hospital. J

Trauma. 2008;64(2 Suppl):S79-85; discussion S85.

massive transfusion. J Trauma. 2007;62(1):112-119.

cryoprecipitate, and platelet transfusion in trauma. J Trauma. 2006;60(6 Suppl):S51-58.

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