DTIC ADA627892: The Effects of Hypothermia on Fibrinogen Metabolism and Coagulation Function in Swine

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MetabolismClinicalandExperimental56(2007)214–221
www.elsevier.com/locate/metabol
The effects of hypothermia on fibrinogen metabolism and
coagulation function in swine
Wenjun Zhou Martini
TheUSArmyInstituteofSurgicalResearch,Ft.SamHouston,TX78234,USA
Received17April2006;accepted10September2006
Abstract
Clinicalcoagulopathyfrequentlyoccursinthepresenceofhypothermia.Theprimarypurposeofthisstudywastoinvestigatetheeffects
of hypothermia on clotting protein fibrinogen metabolism and on coagulation function in a swine model. Twelve pigs were randomly
allocatedintocontrolandhypothermiagroups.Hypothermiaof328Cwasinducedusingablanketwithcirculatingwaterat48C.Fibrinogen
synthesisandbreakdownwerequantifiedusinga6-hourstableisotopeinfusionwithsubsequentgaschromatographandmassspectrometry
analysis. Clotting enzyme thrombin generation kinetics was quantified at baseline and at the end of the infusion. Changes in fibrinogen
metabolism and thrombin generation were correlated with coagulation function assessed by thromboelastography (TEG). Hypothermia
decreased fibrinogen synthesis from the control value of 2.6 F 0.4 to 1.2 F 0.2 mg kg(cid:2)1 h(cid:2)1 (P b .05), with no effect on fibrinogen
breakdown.Thrombingenerationattheinitiationphasewasdelayedbyhypothermia,buttherewerenochangesatthepropagationphase.
In thromboelastography measurements,theinitial clottingtime(R time)wasprolongedfrom thebaseline valueof 3.01F 0.13to4.30F
0.24minutes(P b.05)andclottingrapidity(anglea)wasdecreasedfromthebaselinevalueof72.30F0.90to65.34F1.07(P b.05).
Hypothermia caused no significant changes in clot strength (maximum amplitude) and clot lysis (LY ). We concluded that hypothermia
60
causedapotentialdeficitinfibrinogenavailabilityandadelayinthrombingeneration,consequentlyinhibitingcoagulationfunction.Ourdata
support the current practices of rewarming andprescribing recombinant factor VIIa forhypothermic patients with coagulation defects.
Published byElsevier Inc.
1. Introduction temperaturesdeclinedfrom358Ctolessthan328C[7].The
complexity of clinical settings, such as tissue injury, blood
Hypothermia, clinically defined as body temperature of
loss, resuscitation, and hypothermia, makes it difficult to
348C or less, is commonly observed in severely injured
clarify the mechanisms related to coagulation defects in
trauma patients. As much as 66% of trauma patients arrive
hypothermic patients. Therefore, we used a swine model to
in emergency departments with temperatures of less than
definetheeffectsofhypothermiaonthecoagulationprocess
368C [1]. Among nonsurviving trauma patients, approxi-
in this study.
mately80%havebodytemperaturesoflessthan348Catthe
The coagulation process involves fibrin clot formation
timeofdeath[2].Oneofthemostdistressingcomplications
from the precursor fibrinogen. Thrombin, catalyzing the
of hypothermia is the disruption of hemostasis. Compared
conversionoffibrinogentofibrinclot,playsanimportantrole
with patients with body temperatures of 36.18C F 0.78C,
inthisprocess[10].Thrombinisgeneratedfromprothrombin
there was a 2.4-fold increase of blood loss in postlapar-
duringtheinitiationandpropagationphases.Intheinitiation
otomy patients whose body temperature was 33.88C F
phase,asmallamountofthrombinisproducedasaresultof
0.58C [3]. The association of hypothermic coagulopathy
the formation of the activated tissue factor and factor VIIa
with high mortality has been well described [3-9]. In a
complex. In the propagation phase, a large amount of
group of patients with injury severity scores of more than
thrombin is generated upon the activation of a series of
25, the mortality increased from 10% to 100% when body
procoagulant enzymes (ie, factors V, VIII, and IX) and
platelets. Blood hemostasis requires rapid thrombingenera-
tionandsufficientfibrinogeninthecirculation.Theeffectsof
E-mailaddress: [email protected]. hypothermiaonthecoagulationprocesshavebeenestimated
0026-0495/$–seefrontmatter.PublishedbyElsevierInc.
doi:10.1016/j.metabol.2006.09.015
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The effects of hypothermia on fibrinogen metabolism and coagulation
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W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221 215
bycold-inducedchangesinstandardclinicallaboratorytests with recirculating water at 48C until the animal’s body
ofprothrombintime(PT)andactivatedpartialthromboplas- temperature reached 32.08C (blankets were not used in the
tin time (PTT) [11-16]. Prolonged PT and PTT have been control group). Afterward, pig body temperature was
showninhypothermicpatientsandexperimentalanimals,as maintained at 328C F 0.58C throughout the remainder of
wellasplasmasamplescooledinvitro[11-16].BecausePT the study. The Paratrend sensor for temperature measure-
andPTTprovideestimatesoftheextrinsic-commonandthe ment was placed into the right carotid artery and the
intrinsic-common pathways, respectively, those measure- recirculated-water blanket (48C) was placed on the pig
ments could not reflect the effects of hypothermia on a (fromnecktogroin).Becausethetemperaturegradientfrom
specificenzyme,suchasthrombin.Inaddition,theeffectsof the sensor (328C) to blanket (48C) remained unchanged
hypothermia on dynamic aspects of fibrinogen metabolism during the 6-hour isotope infusion, the likelihood of
havenotbeenidentified. artificially cooling the sensor by the blanket was minimal.
Thisstudywasdesignedtoinvestigatetheeffectsofhypo- Pig body temperatures in the control group (without
thermiaonfibrinogenmetabolism,thrombingeneration,and blankets) were maintained at 39.08C F 0.28C throughout
coagulation function in vivo. After the induction of hypo- the study. No heparin was used in this study.
thermia in a swine model, fibrinogen synthesis and break-
2.2. Stable isotope infusion for fibrinogen metabolism
down were quantified using our newly developed stable
isotopeinfusiontechnique[17],togetherwithquantifications The effects of hypothermia on fibrinogen metabolism
inthrombingenerationkinetics.Changesinfibrinogenmeta- were investigated using our newly developed stable isotope
bolismandthrombingenerationwerecorrelatedwithcoagu- technique [17]. Upon the completion of hypothermia
lationfunctionalassessmentbythromboelastography(TEG). induction, priming doses of stable isotopes containing
1-13C-phenylalanine (18 lmol/kg) and d -phenylalanine
5
(18 lmol/kg) were given, followed immediately by a
2. Methods constant infusion of tracer 1-13C-phenylalanine (0.3 lmol
kg(cid:2)1 min(cid:2)1) and d -phenylalanine (0.3 lmol kg(cid:2)1 min(cid:2)1).
2.1. Experimental design 5
The infusion of 1-13C-phenylalanine was maintained for
This study was approved by the US Army Institute of 6 hours and the d -phenylalanine infusion was maintained
5
Surgical Research Institutional Animal Care and Use for 4 hours (Fig. 1). Blood samples (10 mL each) were
Committee (A-03-003).Atotalof12crossbredcommercial
juvenileYorkshireswine(40.9F0.8kg,JohnAlbertSwine
Farm, Cibolo, TX) were randomized into 2 experimental
groups: normal control (n = 6) and hypothermia (n = 6).
After an overnight fast, animals were preanesthetized with
glycopyrrolate (0.1 mg/kg, IM) and Telazol (6 mg/kg, IM;
FortDodgeAnimalHealth,FortDodge,IA).Thepigswere
then intubated and anesthesia maintained by 1.5% to 2.5%
isoflurane in 100% oxygen by mask for the surgical
procedures. A multiple sensor (Paratrend Diametrics Med-
ical, High Wycombe, UK) was inserted in the right carotid
artery for measuring mean arterial pressure, temperature,
pH, and heart rate. A Swan-Ganz thermodilution catheter
was inserted in the left jugular vein to measure cardiac
output. The right femoral artery was cannulated for blood
sampling and the left femoral vein was cannulated for
infusionsofstableisotopesandmaintenancefluid.Theright
femoral vein was cannulated for intravenous anesthesia of
ketamine during the study.
Upon completion of surgical procedures, lactated Ring-
er’s solution was infused at 0.04 mL kg(cid:2)1 min(cid:2)1 as a
maintenance fluid in both control and hypothermia groups.
Anesthesia was switched to a combination of isoflurane
(0.5%) and continuous intravenous drip of ketamine (0.15
mLkg(cid:2)1h(cid:2)1of100mg/mL)inallpigsforthereminderof
the study period. After a 10-minute stabilization period,
blood samples were taken from the femoral artery for
baseline measurements. Hypothermia was induced in the
Fig.1.Measurementoftotalplasmaproteinandfibrinogenconcentrations
hypothermia group by means of a water-pumped blanket andbloodplateletlevelsduringhypothermia(328C)inpigs.
216 W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221
collected from the femoral artery cannula at 0, 1, 2, 3, 4, 5, measurement. Specifically in this study, d -phenylalanine
5
5.5,6,6.5,and7hours.Ofthe10-mLblood,3mLwasused wasinfusedfrom1to5hoursandstoppedat5hours.EB ,
pre
for measurements of blood chemistry (green-top vacutainer calculated using Eq. (3), is the predicted enrichment of
tube, Fisher, Houston, TX) and platelet (purple-top vacu- fibrinogen-bound amino acid if d -phenylalanine infusion
5
tainer EDTA tubes, Fisher); 2 mL for fibrinogen concentra- had not stopped at 5 hours but had continued to the end of
tion analysis (blue-top vacutainer tubes, Fisher); and 5 mL the study. EB was the actual measured fibrinogen
act
for fibrinogen kinetics analysis (purple-top vacutainer phenylalanine enrichment with d -phenylalanine infusion
5
EDTA tubes, Fisher). A bolus injection of sterile indoc- stopped at 5 hours. The values of EF, EB , and EB
pre act
yanine green was given and blood samples (2 mL each, in varied with time during the infusion study. For example, in
gold-top vacutainer tubes, Fisher) were collected before the one representative pig, EF, EB , and EB were 7.54%,
pre act
injectionandat5,10,and15minutespostinjectiontoassess 2.04%, and 1.95%, respectively, at 5 hours, and 5.02%,
pig plasma volume. Additional blood samples (3 mL each) 2.13%, and 2.06% at 5.5 hours. The changing patterns
were collected at 0 and 5 hours to assess changes in of these parameters between 5 and 7 hours allowed
coagulation function. Thus, a total of 114-mL blood was the calculation of the fibrinogen breakdown rate using
collected duringthe7-hour study,andthisvolumeofblood Eqs. (2) and (3).
wasadequatelycompensatedbythevolumeofmaintenance Fibrinogen FBRs represent the fraction (%) of total
fluid (0.04 mL kg(cid:2)1 min(cid:2)1). At the end of the isotope fibrinogen breakdown (or loss) during a unit of time;
infusion, animals were euthanized with sodium pentobarbi- absolutebreakdownratesrepresenttheamountoffibrinogen
tal (100 mg/kg, IV). (mg) breakdown per kilogram of body weight during a unit
Mean arterial pressure, temperature, and heart rate were oftime.Forexample,FBRof5%/hmeansa5%breakdown
recordedcontinuouslyduringthestudy.Cardiacoutputwas of the total fibrinogen pool in 1 hour; ABR of 3 mg kg(cid:2)1
determined by thermodilution in triplicate at baseline and h(cid:2)1meansa3-mgbreakdownoffibrinogenperkilogramof
hourly throughout the remainder of the study. body weight in 1 hour. Absolute breakdown rate was
calculated by multiplying FBR times pig total plasma
2.3. Calculations for fibrinogen synthesis and breakdown
volume and fibrinogen concentration (ABR = FBR (cid:3)
Fibrinogensynthesisandbreakdownrateswerequantified plasma volume (cid:3) fibrinogen concentration). Similarly,
using a primed constant infusion of stable isotope 1-13C- fibrinogen absolute synthesis rate was calculated by
phenylalanine and d -phenylalanine (Cambridge Isotope multiplying FSR times pig total plasma volume and
5
Laboratories, Andover, MA). In stable isotope d -phenylal- fibrinogen concentration.
5
anine,5hydrogenatoms(1H,massnumberof1)innaturally In stable isotope tracer methodology, protein synthesis
present phenylalanine C H CH CH(NH )COOH (FW 165) andbreakdownratesareindependentofthetracersusedfor
6 5 2 2
are replaced by 5 deuterium (2H, mass number of 2) to the quantifications. Specifically in this study, fibrinogen
become C d CH CH(NH )COOH (FW 170). Calculations incorporation rate calculated from d -phenylalanine is the
6 5 2 2 5
forfibrinogensynthesisandbreakdownhavebeendescribed same as that from 1-13C-phenylalanine. Likewise, fibrino-
previously [17]. Briefly, plasma fibrinogen fractional syn- gen breakdown rate from 1-13C-phenylalanine would have
thesisrate(FSR)wascalculatedusingtheformula: beenthesameasthatfromd -phenylalanineinthisstudyif
5
(cid:2) (cid:3) (cid:2) (cid:3) 1-13C-phenylalaninewasinfusedfor4hoursandstoppedat
FSR¼ EB (cid:2)EB = EF(cid:3)t ð1Þ
ðt2Þ ðt1Þ 5 hours. In our calculation, it was assumed that the plasma
where EB is the enrichment of fibrinogen-bound amino fibrinogen pool acted like a single pool for calculation of
(t)
acids and EF is the enrichment of plasma amino acids FBR.Previousstudieshaveuseda2-poolmodeltodescribe
(precursor enrichment). Enrichment is defined as tracer fibrinogen turnover and have concluded that the 2 pools
(labeledaminoacids)totracee(unlabeledaminoacids)ratio represent intravascular and extravascular spaces [18-21].
(TTR). However,thesestudieswereconductedoveraperiodof6to
Plasma fibrinogen fractional breakdown rate (FBR) was 15 days and the effect from the extravascular pool on the
calculated using the formula: intravascularpoolcouldonlybedetectedafterdays[18-21],
(cid:2) (cid:3) (cid:2) (cid:3) indicating that it takes more than 1 day for the single-pool
FBR¼ EB (cid:2)EB = EB (cid:3)t ð2Þ
pred act ðt1Þ
assumption to be invalid. Thus, considering the time frame
used in this study (7 hours), it is reasonable to assume that
EB ¼EB þFSR(cid:3)EF(cid:3)t ð3Þ
pred ðt1Þ the plasma fibrinogen pool acts like a single pool.
whereEB isthepredictedfibrinogen-boundd -phenylal-
pred 5 2.4. Thrombin generation kinetics
anine enrichment, EB is the actual measured fibrinogen-
act
bound d -phenylalanine enrichments, EB is the starting Thrombin generation was assessed by measuring throm-
5 (t1)
fibrinogen-bound phenylalanine enrichment when d -phe- bin–antithrombinIII(TAT)complexfromfreshwholeblood
5
nylalanine infusion is stopped, EF is the enrichment of samples, following the procedure described by Rand et al
plasma phenylalanine, and t is the amount of time that has [22].Briefly,thrombingenerationanalysiswasperformedin
elapsed from the starting fibrinogen-bound phenylalanine a closed temperature-controlled chamber. Chamber temper-
W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221 217
Table1 2.6. Analytical methods
Physiologicchangesafterhypothermia(328C)inpigs
Blood chemistry was determined by standard clinical
Meanarterial Heartrate Cardiac
methods (Dimension Clinical Chemistry System, Dade
pressure(mmHg) (bpm) output(L/min)
Behring,Newark,DE).Plateletcountsweremeasuredusing
Controlgroup
Baseline(0h) 87F4 107F6 3.6F0.4 an ABX Pentra 120 Hematology Analyzer (ABX Diagnos-
End(7h) 80F5 115F5 3.2F0.3 tics, Irvine, CA). Plasma fibrinogen concentration was
Hypothermiagroup determined based on clotting functional changes using the
Baseline(0h) 85F3 108F5 3.9F0.6
Blood Coagulation System (Dade Behring, Deerfield, IL).
End(7h) 53F34 66F144 1.8F0.24
For assessment of plasma free amino acid enrichments
DataareexpressedasmeansFSE. from the infusion of 1-13C-phenylalanine and d -phenylala-
4 P b.05comparedwithcorrespondingbaselinevalues. 5
nine, 0.2 mL plasma was acidified by 0.3 mL 15%
sulfosalicylicacidandtheacidified plasma supernatantwas
loaded onto a cation exchange column (AG 50W-X8 resin,
atures were set and maintained to match animal body
200-400 mesh, H+ form, Bio-Rad Laboratories, Richmond,
temperature at the time blood samples were withdrawn.
CA). Amino acids were separated after elution with 6 N
Thus, measured thrombin generation reflected that of the
ammoniumhydroxide.The extractsweredried under speed
animal. Fresh whole blood samples were aliquoted into
vacuumandderivatizedbyN-methyl-N-(tert-butyldimethyl-
12tubescontaining20mmol/LHEPES,150mmol/LNaCl,
and 5 mmol/L CaCl . A bquenchQ solution (50 mmol/L silyl)-trifluoroacetamide at 1008C for 1 hour. Plasma
2
fibrinogen was isolated by adding 0.5 mol/L CaCl and
EDTA and 10 mmol/L benzamidine in HEPES-buffered 2
thrombin to form fibrin clot, following the procedure
saline) was added to each of the aliquots at different time
described by Stein et al [27]. The purity of fibrinogen by
intervalstostopclotformation.Thequenchedsampleswere
usingthisprocedurehasbeenvalidatedpreviouslybyaffinity
centrifuged and supernatants were collected for TAT
chromatography[28]andbypolyacrylamidegelelectropho-
concentration measurement using commercially available
resis[29].Theclotwasthenwashedandhydrolyzedin6N
ELISA kits (Enzygnost TAT, Dade Behring, Deerfield, IL).
HCl at 1108C for 24 hours and dried under speed vacuum.
The TAT concentrations from the supernatant samples
The released amino acids after hydrolysis were isolated,
reflect thrombin content generated from fresh whole blood
dried,andderivatizedinthesamemannerasforplasmafree
samplesbeforequenchtimepoints.Thistechniquehasbeen
aminoacids.Theenrichmentsofphenylalaninefromplasma
used to assess thrombin generation kinetics in various
free amino acid pool and in fibrinogen protein were
studies [22-24].
determined by gas chromatography-mass spectrometry
2.5. Coagulation function (model5973,Hewlett-Packard,PaloAlto,CA)intheelectron
impactionizationmode.Aselectiveion-monitoringmethod
Thromboelastograph is an instrument designed to mon-
wasusedatnominalmass-to-chargeratio(m/z)of336(m+0),
itor and analyze the coagulation state of a blood sample
337(m+1),338(m+2),339(m+3),340(m+4),and341(m+5)
[25]. The TEG measures the changes in clot physical
forphenylalanineaspreviouslydescribed[17].
strength using a special stationary cylindrical cup, which is
oscillated with rotation cycles. When a blood sample is 2.7. Statistical analysis
added to the cup, force changes in rotation movements are
All results are expressed as means F SE. Comparisons
converted by a mechanical-electrical transducer to an
between the control and hypothermia groups in fibrinogen
electrical signal, which is recorded by a computer as a
synthesis and breakdown measurements were made with
clotting curve. The clotting curve provides a coagulation
Student t test. Comparisons over time in substrate concen-
profile from a measured blood sample. For example,
trations, thrombin generation, and coagulation parameters
reaction time (R time) represents the latency time for initial
detectable clot formation. Angle a measures the rapidity of
fibrin clot buildup and cross-linking. Maximum amplitude
(MA) represents maximum strength or stiffness of the clot,
andLY indicatesthepercentageofclotlysisat60minutes
60
after MA is achieved.
In this study, coagulation functionwas assessed by TEG
(TEG 5000 Hemostasis Analyzer, Hemoscope, Niles, IL)
using fresh whole blood samples and pig thromboplastin
(extracted from pig brain tissue following procedures
described previously from our laboratory, reference [26]) at
thecoretemperaturesoftheanimalsat0hourandat5hours
duringtheinfusion.Bloodsamples(3mLeach)weregently
Fig. 2. Effects of hypothermia (328C) on fibrinogen synthesis and
withdrawntominimizeshear-inducedplateletactivation. breakdowninpigs.*P b.05comparedwithcontrolvalues.
218 W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221
and1.4%F0.1%/hinthehypothermiagroup(P b.05).The
absolute synthesis rate, calculated by multiplying FSR with
plasmavolumeandfibrinogenconcentration,wasdecreased
fromthecontrolvalueof2.6F0.4to1.2F0.2mgkg(cid:2)1h(cid:2)1
byhypothermia(P b.05,Fig.2).
Fibrinogen FBRs were calculated based on the changes
offibrinogenphenylalanineenrichmentTTR(m+5)afterthe
cessationoftracerd -phenylalanineinfusion.Thecalculated
5
FBRs were 3.6% F 1.0%/h in the control group and 4.0 F
0.6%/h in the hypothermia group (P N .05). The ABRs,
calculated by multiplying FBR with plasma volume and
Fig. 3. Changes in thrombin generation kinetics in hypothermic (328C) fibrinogen concentration, were not significantly different
pigs. *P b .05 compared with corresponding quench time values from between the hypothermia and control groups (Fig. 2).
baselinesamplesinthesamegroup.
3.3. Thrombin generation kinetics
were made using Bonferroni multiple comparisons test
Thrombingenerationwasmeasuredatbaselineandatthe
relative to baseline. Statistical significance was set at
end of the study. There were no differences in thrombin
the .05 level.
generationkineticsatbaseline(0hour)betweenthe2groups.
In the control group, there were no changes in thrombin
3. Results generation from baseline to the end of the study. However,
there was a delay in the initiation phase of thrombin
3.1. Hemodynamics and substrate concentration
generation by hypothermia. At the 3-, 4-, and 5-minute
Hypothermiawassuccessfullyinducedinabout1hourin quenchtimepointsfromthesamplestakenattheendofthe
the hypothermia group. All 12 pigs survived the 6-hour study, thrombin generation in the hypothermia group
isotope infusion study. There were no differences in all decreased to 11% F 3%, 30% F 9%, and 65% F 14%
baseline measurements between the 2 groups. There were of the corresponding baseline values from the same group
also no significant changes in the hemodynamics in the (allP b.05,Fig.3).Beyondthe5-minutequenchtime,there
controlgroupovertheentireexperimentalperiod.Uponthe were no significant differences in thrombin generation
completionofhypothermiainduction,thereweresignificant between the samples taken at baseline (0 hour) and at the
decreases in mean arterial pressure, heart rate, and cardiac end of the study (Fig. 3). Thus, hypothermia primarily
output (Table 1). These measurements remained unchanged inhibitedtheinitiationphaseofthrombingeneration.
during the remainder of the study.
At baseline (0 hour), plasma fibrinogen concentration
wassimilarinthecontrol(173F9mg/dL)andhypothermia
(161 F 13 mg/dL) groups. Plasma total protein was also
similarinthecontrol (5.5F0.3g/dL) andthehypothermia
(5.6 F 0.2 g/dL) groups. Platelet counts at baseline were
311F34103/lLinthecontrolgroupand325F39103/lLin
the hypothermia group. During the 6-hour isotope infusion,
there were no significant changes in fibrinogen concentra-
tion, total protein content, or platelet count in either group
(Fig.1).Plasmavolume,measuredbyindocyaninegreendye
at5hoursduringtheinfusion,was50.0F0.5mL/kginthe
control group and 55.4 F 2.0 mL/kg in the hypothermia
group(P b.05).
3.2. Fibrinogen synthesis and breakdown
TheenrichmentofplasmafreephenylalanineTTR(m+1)
reached plateau values in both animal groups after 1-hour
infusion of 1-13C-phenylalanine (21.23% F 0.58% in the
control group and 25.68% F 1.03% in the hypothermia
group, P b .05). Plasma fibrinogen-bound phenylalanine
enrichmentTTR(m+1)increasedlinearlyduringtheinfusion
of 1-13C-phenylalanine. Fibrinogen FSRs, calculated from
the increased slope of fibrinogen-bound phenylalanine
Fig.4.Effectsofhypothermia(328C)oncoagulationfunctionbyTEGin
enrichment(m+1),was2.7%F0.2%/hinthecontrolgroup pigs.*P b.05comparedwithbaselinevaluesinthesamegroup.
W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221 219
3.4. Coagulation function amplified in clinical settings when the multiple factors of
hypothermia, hemorrhage, resuscitation, and tissue injury
Coagulation was measured at 0 hour and at 5 hours
are all involved. Additional efforts are needed to define the
during the infusion. There were no changes in TEG
individual and combined effects of these factors on
measurements in the control group during the study. In the
coagulation defects. Nonetheless, our kinetic results from
hypothermia group, the initial clotting time (R time) was
this study demonstrate that there is a potential deficit in
prolongedfromthebaselinevalueof3.01F0.13to4.30F
fibrinogen availability from hypothermia alone due to the
0.24 minutes (P b .05, Fig. 4). Clotting rapidity (angle a)
consequences of inhibited production without changes
was decreased from the baseline value of 72.30 F 0.90 to
in utilization.
65.34 F 1.07 (P b .05, Fig. 4). There were no significant
Thrombin, catalyzing fibrinogen to fibrin clot, plays an
changes in clot strength (MA) and fibrinolysis (LY ) by
60 important role in maintaining hemostasis. In this study,
hypothermia (data not shown).
hypothermia of 328C significantly delayed the initiation
phase in thrombin generation, but had minimal effects on
the propagation phase. Because the initial thrombin is
4. Discussion
generatedupontheformationoftissuefactorandfactorVIIa
Sufficientfibrinogenavailabilityisessentialforeffective complex, our data led us to speculate that the observed
clotting. Using our newly established stable isotope inhibition in thrombin generation by hypothermia is
technique [17], we investigated hypothermia effects on primarily in the formation (or activation) of tissue factor
fibrinogen metabolism and coagulation function. Our data and factor VIIa complex. Additional studies are needed to
showed, for the first time, that hypothermia of 328C confirm or deny this speculation because we did not
decreased fibrinogen synthesis by about 50% with no measure plasma levels of tissue factor or factor VIIa in this
changes in fibrinogen breakdown, indicating a potential study. Interestingly, we observed a reverse sequence in the
deficit in fibrinogen availability. This deficit may become effectsofacidosisonthrombingeneration[31].Inpigswith
more problematic when massive blood loss and resuscita- acidosisofpH7.1,wefoundthatthrombingenerationinthe
tion are present because fibrinogen levels may already be propagation phase was inhibited by acidosis by as much as
nearorbelowthethresholdfornormalcoagulationfunction 50%, with no changes in the initiation phase [31]. Thus,
before hypothermia fully develops. In addition, because hypothermia and acidosis inhibit thrombin generation
fibrinogen synthesis under normal circumstances is very kinetics via different mechanisms. Although the mecha-
slow relative to its pool size (about 3%/h) [17,30], it may nisms underlying these differential changes remain to be
take a long time to reflect synthesis changes in fibrinogen elucidated, it appears that different approaches should be
concentration. For example, when there is a 50% inhibition used to recover thrombin activities in hypothermic and
in synthesis and no change in breakdown, it would take acidotic coagulopathy.
more than 6 hours to result in detectable changes in Using cell-based in vitro clotting assays, Wolberg et al
fibrinogen concentration (about 10%). Thus, the severe [32]andMengetal[33]investigatedthesystemiceffectsof
inhibition imposed by hypothermia on fibrinogen synthesis hypothermiaonenzymeactivities,thrombingeneration,and
islikelytobemaskedduetothelackofimmediatechanges coagulation function. The authors reported that there were
infibrinogenconcentration.Therefore,ourmetabolicresults no significant changes in tissue factor activities on
maysupportthecurrentpracticeofimmediaterewarmingas phospholipids and monocytes when temperature dropped
well as the prescription of blood products to replenish from 378C to 338C, but significant inhibitions occurred
coagulation factors in hypothermic trauma patients. when temperature dropped to less than 338C [31,32].
Ourobservationofinhibitedfibrinogensynthesiswithout Thrombin generation decreased by 5% and 64% of the
significant changes in fibrinogen breakdown indicates that valueat378Cwhentemperaturedroppedfrom378Cto338C
fibrinogen synthesis and degradation are independently and 238C, respectively [32]. Similarly, we observed
regulated in response to cooling. The significant decreases inhibitions in thrombin generation by hypothermia in our
in fibrinogen synthesis shown in this study were expected study, using an in vivo model and a different thrombin
because protein synthesis requires a series of signals for generation assay. Furthermore, the inhibition appears to be
gene expression activation as well as translation activation, likely in the initiation phase (during the formation of factor
whereas protein breakdown only requires the presence of VII and tissue factor complex). Thus, findings from
proteases. Thus, temperature is likely to have more impact references [32] and [33] and the present study support the
on fibrinogen synthesis than on fibrinogen breakdown. notionthatrecombinantfactorVIIaisapotentialtherapyfor
Interestingly, we observed the opposite in pigs with reversing hypothermia coagulopathy.
hemorrhagic shock [17]; after a moderate hemorrhage, Our measurements of fibrinogen metabolism and throm-
fibrinogen degradation was accelerated but synthesis bin generation correlated well with coagulation functional
remained unchanged in pigs [17]. Apparently, complex assessmentbyTEG.Reactiontime(R)inTEGisthelatency
mechanisms may be involved in regulating fibrinogen timeforinitialdetectableclotformation.AprolongedRtime
synthesis and degradation. This complexity may be further represents a deficiency in enzyme activity or clotting factor
220 W.Z.Martini/MetabolismClinicalandExperimental56(2007)214–221
levels. Angle a measures the rapidity of fibrin buildup and of fibrinogen degradation in normal humans are not
cross-linking. It is affected by the availability of fibrinogen available at present. Thus, we are not able to compare
and platelets, but mostly by thrombin activity (amount). In degradation rates between pigs and humans. However, the
this study, we observed a delayed onset of thrombin values between synthesis and degradation in our control
generation, which was consistent with a prolonged R time pigsindicatethatpigsareareasonablemodelforfibrinogen
andadecreasedainTEGmeasurements.Becausefibrinogen metabolic studies. With respect to coagulation, the pig has
synthesisrateisverylowcomparedwithtotalfibrinogenpool becometheestablishedanimalmodelduetothesimilarities
size, the inhibition in fibrinogen synthesis by hypothermia in human and porcine coagulation processes [36,37].
wasnotreflectedinthechangeoffibrinogenlevelswithinthe Thromboelastography has been used to assess blood
shorttimeframeofthisstudy(6-hourhypothermia).Evenso, coagulation profiles for several decades. Its high sensitivity
we observed coagulation changes of delayed thrombin in detecting and monitoring coagulation changes has been
generation, prolonged clotting time, and decreased clotting recognized in clinical studies since the 1970s [38]
rapidity. Thus, if hypothermia is prolonged, depleted Comparable values of clotting formation time, MA, and
fibrinogen levels would be present and the observed clotting rapidity have been reported by Velik-Salchner et al
coagulationchangeswouldlikelybeamplified. [39] in blood samples from human and pigs. However,
In this study, fibrinogen synthesis rate is the amount of Landskroner et al [40] recently showed a difference in ex
fibrinogen (expressed as milligram per kilogram of body vivo lysis assays between human and pig plasma samples,
weight) that is synthesized and released to circulation in indicating possible differences in fibrinolysis between
1 hour. Fibrinogen breakdown rate is the total loss of human and pigs. Although the validity remains to be clari-
fibrinogen (expressed as milligram per kilogram of body fied, a swine model appears to be a reasonable choice for
weight) from plasma, which includes removal from plasma coagulation studies at this time.
by cells and loss to the extravascular space. Because our In summary, we investigated the effects of hypothermia
method does not allow the quantification of fibrinogen on the coagulation process in a swine model. Differential
breakdown from a specific pathway, we were not able to effects of hypothermia were observed on fibrinogen
differentiate the effects of hypothermia on fibrinogen synthesis and degradation, as well as on the initiation and
breakdown by cell removal, or loss to the extravascular propagation phases in thrombin generation. Hypothermia
space. Recently, Kamler et al [34] reported that edema inhibited fibrinogen production, delayed initial thrombin
formation by the peripheral vascular system of rats was generation, and consequently compromised the clotting
markedly increased during hypothermia, indicating that the process. Based on our results, rewarming is highly
lossofplasmaprotein(includingfibrinogen)totheextravas- recommended due to the potential deficit in fibrinogen
cular space might be increased by hypothermia. Additional availability and thrombin generation inhibition caused by
studies are needed to clarify the effects of hypothermia on hypothermia.Inaddition,recombinantfactorVIIamaybea
individualpathwaysinvolvedinfibrinogenbreakdown. beneficialadjunctinhypothermicpatientswithbleedingdue
Normal coagulation process requires a dynamic balance toitspotentialtoreleasetheinhibitionintheinitiationphase
of complex regulations in procoagulant, anticoagulant, and of thrombin generation.
fibrinolyticprocesses.Togetherwithprocoagulantenzymes,
platelets and fibrinolysis play important roles in blood
coagulation. In this study, we did not observe changes in Acknowledgments
TEG LY (fibrinolysis). Similar findings have been
60 This study was supported by the US Army Medical
reported by Watts et al [16] in hypothermic trauma patients
ResearchandMedicalCommand.Theopinionsorassertions
and by Kurrek [12] in hypothermic pigs. We also did not
contained hereinaretheprivateviews ofthe author andare
observe changes in platelet counts, which was consistent
not to be construed as official or as reflecting the views of
with unchanged MA in TEG measurements. Similarly,
theDepartmentoftheArmyortheDepartmentofDefense.
Staab et al [15] reported that temperature changes in mini-
TheauthorthanksSusanneChristensen,DouglasCortez,
pigs did not cause changes in platelet aggregations.
andMichaelSchererfortechnicalassistance;AmyNewland
However, Watts et al [16] showed significant alterations in
for editing the manuscript; and the Veterinary Service
plateletfunctionbyTEGinhypothermicpatients.Although
Support Branch and Laboratory Support Branch at the US
speciesdifferencesandtraumainjurymightcontributetothe
Army Institute of Surgical Research.
discrepancy, additional investigation is required to clarify
the effects of hypothermia on platelet function.
A swine model was used in this study to investigate the
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