Disseminated intravascular coagulation;
Development and standardization of a non-clinical
rabbit model
Ph.D. Thesis
Line Olrik Berthelsen, DVM
Department of Small Animal Clinical Sciences
Faculty of Life Sciences
Copenhagen University
&
Haemostasis Pharmacology
Novo Nordisk A/S
Copenhagen, Denmark
2010
TABLE OF CONTENTS:
PREFACE.……………………………………………………………………………………………………………………..3
ABBREVIATIONS…………………………………………………………………………………………………………….4
SUMMARY (ENGLISH)………………………………………………………………………………………………………5
SAMMENDRAG (DANSK)…………………………………………………………………………………………………...7
1
INTRODUCTION, HYPOTHESES AND OBJECTIVES .................................................................................. 9
2
BASIC MECHANISMS OF HAEMOSTASIS AND THROMBOSIS ............................................................... 11
2.1
PHYSIOLOGIC HAEMOSTASIS...................................................................................................... 11
2.2
REGULATION OF COAGULATION .................................................................................................. 13
2.2.1
Anticoagulation ................................................................................................................ 13
2.2.2
Fibrinolysis ....................................................................................................................... 14
2.2.2.1
2.2.2.2
Activation of plasmin .................................................................................................................. 14
Regulation of the fibrinolytic system .......................................................................................... 14
2.3
ACQUIRED PROCOAGULANT DISORDERS OF HAEMOSTASIS ......................................................... 15
2.4
DISSEMINATED INTRAVASCULAR COAGULATION .......................................................................... 15
2.4.1
Aetiology .......................................................................................................................... 15
2.4.2
Pathophysiology .............................................................................................................. 16
2.4.3
Interaction between inflammation and haemostasis........................................................ 16
2.4.4
Diagnosis of DIC .............................................................................................................. 17
2.4.4.1
2.4.4.2
2.4.4.3
2.4.4.4
3
Detection of Activation of Coagulation ....................................................................................... 17
Inhibitor consumption ................................................................................................................. 17
Fibrinolytic activity ...................................................................................................................... 18
ISTH classification of overt and non-overt human DIC .............................................................. 18
EXPERIMENTAL THROMBOSIS AND DIC IN ANIMAL MODELS .............................................................. 19
3.1
3.2
3.3
METHODS IN DIAGNOSIS OF EXPERIMENTAL MICROTHROMBOSIS ................................................... 19
STANDARDIZATION AND TRANSLATIONAL ASPECTS OF DIC IN ANIMAL MODELS .............................. 20
ANIMAL MODELS OF DIC AND THEIR RELEVANCE TO HUMAN DIC – A SYSTEMATIC REVIEW ................
(PAPER I) .................................................................................................................................. 20
4
DEVELOPMENT OF A RABBIT MODEL OF DIC AND IMPLEMENTATION OF NEW PARAMETERS FOR
EARLY DIAGNOSIS OF MICROTHROMBOSIS IN DIC ...................................................................................... 21
4.1
VALIDATION OF A PULMONARY FUNCTION TEST IN A RABBIT MODEL OF EMBOLISATION MIMICKED BY
MICROSPHERES .................................................................................................................................... 22
4.1.1
4.1.1.1
4.1.2
4.1.2.1
4.1.2.2
4.1.2.3
4.1.2.4
4.1.2.5
4.1.3
4.1.3.1
Background ...................................................................................................................... 22
Multiple Inert Gas Elimination Technique (MIGET) .................................................................... 22
Materials and methods .................................................................................................... 23
Animal procedures ..................................................................................................................... 23
Microspheres ............................................................................................................................. 23
Ventilation-perfusion relationships ............................................................................................. 24
Experimental protocol ................................................................................................................ 24
Preparation of tissue specimens ................................................................................................ 24
Results ............................................................................................................................. 25
Histological examination ............................................................................................................ 26
4.1.4
Discussion and conclusion .............................................................................................. 26
4.1.5
Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary
thrombosis (Paper II) ...................................................................................................................... 27
4.2
IMPLEMENTATION OF NEW PARAMETERS IN EARLY DIAGNOSIS OF DIC .......................................... 28
4.2.1
Background ...................................................................................................................... 28
4.2.2
Materials and methods .................................................................................................... 28
4.2.2.1
4.2.2.2
4.2.2.3
4.2.2.4
4.2.2.5
Animals ...................................................................................................................................... 28
Animal procedures ..................................................................................................................... 29
Cardiac troponin I (cTnI) measurements ................................................................................... 29
Thromboelastography (TEG) ..................................................................................................... 29
Experimental design .................................................................................................................. 30
4.2.3
Results ............................................................................................................................. 30
4.2.4
Discussion and conclusion .............................................................................................. 32
4.3
ESTABLISHMENT OF A RABBIT MODEL OF THROMBOPLASTIN INDUCED DIC .................................... 33
1
4.3.1
4.3.2
Background ...................................................................................................................... 33
Development of a model of thromboplastin induced DIC in rabbits ................................ 33
4.3.2.1 Background................................................................................................................................ 33
4.3.2.2 Materials and methods .............................................................................................................. 34
4.3.2.2.1 Experimental design ............................................................................................................. 34
4.3.2.3 Results ....................................................................................................................................... 34
4.3.2.4 Conclusion ................................................................................................................................. 35
5
IMPLEMENTATION OF THE ISTH CLASSIFICATION OF NON-OVERT DIC IN A THROMBOPLASTIN
INDUCED RABBIT MODEL (PAPER III) .............................................................................................................. 35
6
CHARACTERISATION AND PURIFICATION OF THROMBOPLASTIN...................................................... 36
6.1
6.2
6.3
6.4
BACKGROUND ........................................................................................................................... 36
MATERIALS AND METHODS ......................................................................................................... 37
RESULTS .................................................................................................................................. 37
CONCLUSION ............................................................................................................................ 37
7
PURIFIED THROMBOPLASTIN CAUSES HAEMOSTATIC ABNORMALITIES BUT NOT OVERT DIC IN
AN EXPERIMENTAL RABBIT MODEL (PAPER IV) ............................................................................................ 38
8
DISCUSSION AND CONCLUSION .............................................................................................................. 39
9
PERSPECTIVES ........................................................................................................................................... 42
10
REFERENCES .............................................................................................................................................. 44
11
PUBLICATIONS ............................................................................................................................................ 54
Paper I:
“Animal models of DIC and their relevance to human DIC – A systematic review” (Submitted)
Paper II:
“Cardiovascular and haemostatic changes following microsphere injection in a rabbit model of acute pulmonary
microvascular thromboembolism” (Submitted)
Paper III:
“Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model”.
(Thrombosis Research, 2009; Vol. 124, Issue 4, Pages 490-497)
Paper IV:
“Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model”
(Thrombosis Research, 2010; in Press; DOI: 10.1016/j.thromres.2010.06.022)
2
Preface
“Whether you believe you can do a thing or not - you’re right”
Henry Ford
This thesis is the culmination of years of hard work, ups and downs and believing.
Early on in life I discovered my passion for the detail. I take every chance I get to learn and
understand what is possible on a specific subject. However, the thought of doing a PhD did not settle
in my mind until almost the finishing of my master studies (probably because I was too focused on the
details of my master studies), but to be given the opportunity to take this detailed journey into PhD and
DIC land has been fantastic. It has also been hard work - hard in other ways than I imagined. I
expected it to be mostly scientifically challenging, but realized that working with my own response to
set backs, frustrations and worries has been the hardest - and most rewarding.
“There are two kinds of problems; those you can solve - so don’t worry about them, and those you
can’t solve - so don’t worry about them”
Author unknown
I want to thank my colleagues at Novo Nordisk. Everywhere I went I was met by open minds and an
eagerness to help. I specifically wish to thank my supervisors. Thanks to Henrik Duelund Pedersen
(Novo Nordisk A/S), my supervisor for the first 1½ years, for teaching me focus on the experiments
and the data and not worry so much about forms and conformities and for advancing me from student
to colleague. Thanks to Annemarie (KU Life) for many high-level, high-speed meetings with extremely
valuable professional inputs. Mikael (Novo Nordisk A/S) - in a rather chaotic switch from one
department to another you quickly identified my strengths and weaknesses and used this
constructively to create solutions where I could do my best. Thanks for always being there, being
ready, listening and taking action.
My family and friends have had to put up with my absence in periods - especially during this spring,
where the work has been most intense. Thank you for your patience.
And a special thank goes to my fiancé Martin and our son Viktor, who have felt the tough times as
much as I have. Martin thank you for pushing, lifting and nursing me through the hard times of this
PhD project - without you I wouldn’t have come this far (No, I wouldn’t !).
3
Abbreviations
ABP
APC
aPTT
AT
Arterial blood pressure
Activated protein C
activated partial thromboplastin time
Antithrombin
cTnI
Cardiac troponin I
DIC
Disseminated intravascular coagulation
ECG
Electrocardiogram
FDP
FV
FVa
FVII
FVIIa
FVIII
FVIIIa
FIX
FIXa
FX
FXa
FXI
FXIa
Fibrin degradation products
Factor V
Activated Factor V
Factor VII
Activated Factor VII
Factor VIII
Activated Factor VIII
Factor IX
Activated Factor IX
Factor X
Activated Factor X
Factor XI
Activated Factor XI
HE
Haematoxylin eosin
ISTH
International Society on Thrombosis and Haemostasis
LPS
Lipopolysaccharide
MIGET
Multiple inert gas elimination technique
PaCO2
PaO2
PAI-1
PC
PS
PT
PTAH
Arterial CO2 tension
Arterial O2 tension
Plasminogen activator inhibitor 1
Protein C
Protein S
Prothrombin time
Phosphotungstic acid haematoxylin
RVP
Right ventricular pressure
TAT
TEG
TF
TFPI
TM
tPA
Thrombin-antithrombin
Thromboelastography
Tissue factor
Tissue factor pathway inhibitor
Thrombomodulin
tissue plasminogen activator
uPA
urokinase type plasminogen activator
V/Q
Ventilation-perfusion ratio
vWF
von Willebrand factor
4
Summary (English)
Disseminated intravascular coagulation (DIC) is a syndrome occurring secondary to a wide range of
predisposing diseases. DIC is a pathological process with widespread thrombosis in the
microcirculation causing organ damage and having a poor prognosis. Comparison of animal models of
DIC to human DIC is crucial in order to translate findings in research models to treatment modalities
for DIC in humans.
The aims of the current thesis were to establish a rabbit in vivo model of thromboplastin induced DIC
and evaluate the implementation of a wide panel of markers for pulmonary, cardiovascular and
haemostatic function in the early identification of microthrombosis. It was hypothesized that a DIC
scoring system can be applied to a rabbit model of thromboplastin induced DIC to standardize and
score the rabbit model of DIC according to the ISTH scoring system of DIC in humans.
This thesis is composed of a general introduction to coagulation and its regulation and the syndrome
DIC including an overview of animal models of DIC presented in paper I.
The core work of the project is then presented including the methods applied and results obtained.
These include the use of microspheres to establish a mechanical model of pulmonary embolism which
is further described in paper II and a number of pilot studies developing a thromboplastin induced
rabbit model of DIC with determination of a wide panel of markers for cardiovascular and haemostatic
function including early markers of microthrombosis and finally characterising non-washed and purified
thromboplastin. The validation and applicability of these methods are described in the set up of a nonclinical rabbit model of DIC in paper III and IV.
Finally the conclusion and perspectives are presented based on four accompanying papers.
Four papers are included in the thesis:
Paper I “Animal models of DIC and their relevance to human DIC – A systematic review” (submitted
for publication) provides an extensive literature search on established animal models of DIC. An
overview of distribution of animal species, inducers, measurements and treatments are given for the
many identified studies. Generally a high variability was found and it was concluded that
standardization of animal models of DIC by for example application of a DIC score as has been
developed for the diagnosis of human DIC by ISTH is recommendable. Furthermore it was identified
that the majority of studies testing treatment of DIC only evaluated the prophylactic effect of such,
though prophylaxis is in general irrelevant for cases of human DIC. The large amount of data compiled
in this study is useful for the interpretation and comparison of responses in animal models of DIC and
implementation of the recommendations may significantly improve the clinical relevance of animal
models within this research area.
In paper II “Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary
thrombosis” (submitted for publication), microspheres were used to validate the response of
5
cardiovascular parameters (RVP and systemic blood pressure) to fixed size pulmonary thrombi in the
rabbit. Haemostatic parameters were evaluated in vivo and in vitro to test whether the microspheres
are truly inert. A dose dependent response on cardiovascular parameters was observed in vivo,
cumulating in a lethal effect of the highest in vivo dose of microspheres. An in vitro setup evaluating
thromboelastographic effects of microspheres in rabbit whole blood spiked with equivalent doses to
the in vivo study demonstrates that the highest dose resulted in a procoagulant effect. Haemostatic
parameters showed no significant procoagulant effect of any of the doses of microspheres in vivo and
it was concluded that non-lethal doses of microspheres result in inert pulmonary fixed size emboli.
Paper III “Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced
rabbit model” [1] describes the establishment of a rabbit model of thromboplastin induced non-overt
DIC. Bolus injections of 1.25 and 2.5 mg thromboplastin/kg non-washed thromboplastin in rabbits
resulted in non-overt DIC diagnosed by a modified score based on the ISTH score of non-overt DIC in
humans, injection of higher thromboplastin doses were lethal and induction of overt DIC was not
accomplished in this study.
In paper IV “Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an
experimental rabbit model” [2], it is reasoned that the purification of thromboplastin would decrease
variability and enable injection of higher non-lethal doses resulting in overt DIC. However injection of a
2.5 mg/kg bolus followed by a 1.25 mg/kg infusion of thromboplastin resulted in less severe
procoagulant activation than for the previous study, though a lethal effect was also observed within
this study indicating an even more narrow window between procoagulant and lethal effect than for the
injection of non-washed thromboplastin. These results underscores the difficulties in comparisons
between animal models using different inducers of DIC and emphasizes that induction of DIC,
experimentally as well as clinically, may call for different treatment approaches.
6
Sammendrag (Dansk)
Dissemineret intravaskulær koagulation (DIC) er et syndrom, der forekommer sekundært til en bred
vifte af predisponerende sygdomme. DIC er en patologisk proces med udbredt trombose i
microcirkulationen, hvilket forårsager organskade og giver en dårlig prognose. Sammenligning af
dyremodeller for DIC med human DIC er altafgørende for at kunne translatere fund i forsøgsmodeller
til behandlingsmodaliteter for human DIC.
Målsætningen for denne afhandling var at etablere en kanin in vivo model for thromboplastin induceret
DIC og evaluere implementeringen af et bredt panel af markører for pulmonær, kardiovaskulær og
hæmostatisk funktion i den tidlige identifikation af microtrombose. Hypotesen var, at et DIC
scoringssystem kan anvendes i en kaninmodel for thromboplastin-induceret DIC til at standardisere og
score kaninmodellen for DIC ifølge ISTH scoringssystemet for human DIC.
Denne afhandling omfatter en generel introduktion til koagulation og regulering af koagulationen og
syndromet DIC inkluderende et overblik over dyremodeller for DIC præsenteret i artikel I.
Kernearbejderne i dette projekt præsenteres herefter og inkluderer de anvendte metoder og opnåede
resultater. Disse inkluderer anvendelsen af microspherer til etablering af en mekanisk model for
pulmonær embolisme, som beskrives nærmere i artikel II, samt et antal pilot studier med udvikling af
en thromboplastin induceret kaninmodel for DIC med bestemmelse af et bredt panel af markører for
kardiovaskulære og hæmostatiske funktioner inkluderende tidlige markører for microtrombose og
endeligt karakterisering af ikke-vasket samt renset thromboplastin. Valideringen og anvendelsen af
disse metoder er beskrevet i artikel III og IV.
Endelig præsenteres konklusionen og perspektiveringen baseret på 4 vedlagte artikler.
4 artikler indgår i afhandlingen:
Artikel I ”Animal models of DIC and their relevance to human DIC – A systematic review” (indsendt)
omfatter et bredt litteraturstudie af etablerede DIC dyremodeller. Fordeling af dyrearter,
induktionsmetoder, målinger og behandlinger oplyses for de mange identificerede studier. Generelt
sås en høj variation i henhold til disse parametre og det blev konkluderet at standardisering af
dyremodeller for DIC ved for eksempel anvendelse af en DIC score som tilsvarende er udviklet af
ISTH til diagnosticering af human DIC anbefales. Ydermere bliver der vist, at hoveddelen af studierne,
der tester behandling af DIC, udelukkende evaluerede den profylaktiske effekt af disse, selvom
profylakse generelt er irrelevant for tilfælde af human DIC. Den store mængde data indsamlet til dette
studie er brugbar for fortolkning og sammenligning af respons i DIC dyremodeller og implementering
af disse anbefalinger kan signifikant forbedre den kliniske relevans af dyremodeller indenfor dette
forskningsområde.
I artikel II ”Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary
thrombosis” (indsendt), anvendes microspherer til at validere det kardiovaskulære respons (RVP og
7
systemisk blodtryk) til pulmonære tromber af fast størrelse i kaninen. Hæmostatiske parametre blev
evalueret in vivo og in vitro for at teste om microsphererne med sikkerhed er inerte. Et dosisafhængigt respons af kardiovaskulære parametre observeredes in vivo, kumulerende til en letal effekt
af den højeste in vivo dosis af microspherer. Et in vitro setup, evaluerende tromboelastografisk effekt
af den højeste in vivo dosis af microspherer i kaninfuldblod spiked med doser equivalent til in vivo
studiet, demonstrerede at den højeste dosis resulterede i en prokoagulant effekt. Hæmostatiske
parametre viste ingen signifikant prokoagulant effekt for nogen af microsphere-doserne in vivo og det
konkluderedes, at non-letale doser af microspherer resulterer i inerte pulmonære emboli af fast
størrelse.
Artikel III ”Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced
rabbit model” beskriver etableringen af en kaninmodel for tromboplastin-induceret non-overt DIC.
Bolusinjektioner af 1.25 og 2.5 mg tromboplastin/kg ikke-vasket tromboplastin i kaniner resulterede i
non-overt DIC diagnoseret af en modificeret score baseret på ISTH scoren for non-overt human DIC,
injektioner af højere tromboplastin-doser var letale og induktion af overt DIC blev ikke opnået i dette
studie.
I artikel IV ”Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an
experimental rabbit model” argumenteredes det, at rensning af tromboplastin ville mindske variationen
og muliggøre injektion af højere non-letale doser resulterende i overt DIC. Men injektion af 2.5 mg/kg
bolus fulgt a 1.25 mg/kg infusion af tromboplastin resulterede i svagere prokoagulant aktivering end i
det tidligere studie, selvom en letal effekt også observeredes i dette studie, hvilket indikerer et endnu
smallere vindue mellem prokoagulant og letal effekt end for injektionen af ikke-vasket tromboplastin.
Disse resultater understreger problemerne ved sammenligning af dyremodeller, der bruger forskellige
inducere af DIC og underbygger, at induktion af DIC, eksperimentelt som klinisk, kan kræve forskellige
behandlingsstrategier.
8
1 Introduction, hypotheses and objectives
Disseminated intravascular coagulation (DIC) is a complex and dynamic haemostatic syndrome
occurring secondary to a wide range of underlying diseases [3-5]. DIC is characterized by variable
imbalances of the components of the coagulation and fibrinolytic systems and the clinical signs vary
considerably ranging from no overt signs of disease (non-overt DIC) [6;7], accompanied by minor
changes in haemostatic parameters, to clinical symptoms of organ failure, associated with microvascular thrombosis in vital organs, to fulminant DIC with bleeding symptoms (overt DIC) [6].
Diagnosis of DIC early in the non-overt stage may increase the chances of survival in the DIC patient,
because early and aggressive intervention through supportive and antithrombotic therapy, besides
treatment of the underlying disease, may minimize or prevent thrombo-embolic complications and
progression to overt DIC [8].
Clinically relevant animal models of DIC are crucial in the research and development of novel
therapeutic interventions as well as in understanding the pathogenesis of DIC. Standardization of
animal models of DIC is necessary in order to compare findings between species and to increase the
translational aspect in non-clinical testing of therapeutics for DIC in humans.
Simple diagnostic scoring systems for diagnosis of DIC in humans, such as the algorithms for overt
and non-overt DIC developed by the International Society on Thrombosis and Haemostasis (ISTH)
[7;9] are valuable standardized approaches to the characterization of DIC in humans and have been
proven to have a high diagnostic accuracy [10-12]. As the approach taken by ISTH in scoring of DIC
has been successfully applied in dogs suffering from DIC [13], application of similar scoring systems in
experimental animal models of DIC may help to increase the clinical relevance and standardization of
these non-clinical models.
The overall hypothesis of the ph.d.-study was that a DIC scoring system can be applied to a rabbit
model of thromboplastin induced DIC. Since a similar scoring system for diagnosis of DIC in man has
been validated as an accurate diagnostic tool, such an approach could lead to a standardized and
clinical relevant animal model of non-overt and overt DIC.
9
Furthermore, it was hypothesized that determination of several cardiovascular and haemostatic
parameters would help to diagnose DIC in this model at an early stage and that the use of pulmonary
function tests would add in the early diagnosis of microthromboembolism, an important complication of
DIC.
The specific objectives of the proposed investigations were:

To develop and standardize a non-clinical rabbit model of DIC with determination of a wide
panel of markers for cardiovascular and haemostatic function including early markers of
microthrombosis

To investigate whether a rabbit model of DIC could be standardized through application and
scoring of the observed changes in laboratory markers according to the ISTH scoring system
of DIC in humans
10
2 Basic Mechanisms of Haemostasis and Thrombosis
Knowledge of basic mechanisms of haemostasis are important to understand the complexity of
thrombo-hemorrhagic disease processes, to interpret laboratory testing modalities and to understand
the mechanism of action of pharmacological agents being tested in non-clinical animal models.
2.1 Physiologic Haemostasis
Coagulation is a complex process resulting in formation of clots. It is an important part of haemostasis,
wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop
bleeding and begin repair of the damaged vessel wall [14;15].
Haemostasis starts almost instantly after damage to the endothelium as exposure of the blood to
proteins such as tissue factor (TF) initiates changes to platelets and fibrinogen [16]. Platelets
immediately form a plug at the site of injury [17], simultaneously coagulation proteins in the blood
plasma respond to form fibrin strands, which strengthen the platelet plug [18;19].
The revision of the cascade model of coagulation [20], which explains fibrin formation by two different
pathways; the extrinsic and intrinsic pathway, led to the suggestion that TF is the initiating factor [21]
and to the development of a cell-based model of haemostasis by Hoffman and Monroe [22], where
haemostasis is divided into three overlapping stages – initiation, amplification and propagation.
Initiation:
Coagulation is initiated by activation of FVII to FVIIa via TF from TF-bearing cells exposed to the blood
during vessel wall damage. FVII binds to TF and is activated to FVIIa by several of the coagulation
proteases when coagulation is ongoing [23]. Initially however, small trace amounts of FVIIa circulating
in the blood [24] functions as an autoactivator of FVII complexed with TF [25]. The TF/FVIIa complex
activates FIX to FIXa and FX to FXa. FXa can combine with FVa and produce small amounts of
thrombin [22;26] (figure 1).
Figure 1 Initiation of coagulation. TF-bearing cells exposed to the blood bind factor VIIa. The TF:VIIa complex activates factor X
to Xa, which together with factor Va produce small amounts of thrombin
Amplification:
Platelets adhere to collagen and von Willebrand Factor (vWF) at the site of vascular injury [27].
Thrombin generated during initiation amplifies the coagulation by activation of platelets via protease-
11
activated receptors (PAR) [28]. Thrombin also binds to non-PAR platelet receptors and activates FV
released from the alpha granules of the activated platelets [29]. Furthermore circulating FVIII bound to
vWF attaches to the platelets, where thrombin cleaves FVIII from vWF and activates it to FVIIIa (figure
2). Importantly, the procoagulant negatively charged phospholipid, phosphatidyl-serine becomes
increasingly available during the platelet activation process [30].
Figure 2 Amplification of coagulation. Platelets are activated by the thrombin generated during initiation. Thrombin furthermore
dissociates FVIII from vWF and activates FVIII, FV and FXI on the surface of the platelets
Propagation:
During the propagation stage, the generated FIXa move from the TF-bearing cell and bind to the
negatively charged surface of the activated platelet [31] as does FXI from plasma [32]. Thrombin
activates FXI to FXIa [33] that further activates FIX on the platelet surface. FIXa bind to FVIIIa in what
is called the ‘tenase’ complex and further activate FX. The ‘prothrombinase’ complex is formed
between FXa and FVa on the platelet surface; it cleaves prothrombin and leads to a burst of thrombin,
which then cleaves fibrinogen leading to the formation of a haemostatic fibrin clot [22] (figure 3).
Figure 3 Propagation of coagulation. The tenase complex (IXa:VIIIa) lead to the formation of the prothrombinase complex
(Xa:Va) via FX activation which facilitates the thrombin burst
12
2.2 Regulation of Coagulation
2.2.1 Anticoagulation
Anticoagulation and fibrinolysis keep platelet activation, coagulation and clot formation in balance and
restricted to the site of vascular injury to prevent inappropriate escalation, leading to systemic
coagulation and disseminated fibrin formation [34].
Three pathways: the antithrombin (AT) pathway, the tissue-factor pathway inhibitor (TFPI) pathway
and the protein C (PC) pathway are the main players in anticoagulation.
Antithrombin (AT):
The serine protease inhibitor AT is a major inhibitor of coagulation proteases such as thrombin [35],
FXa [36], FIXa, FXIa [37] and FVIIa in complex with TF [38]. The serine proteases bind irreversibly to
AT, and the complexes are subsequently removed from the circulation by clearance in the liver [36].
The inhibitory effect of AT is markedly enhanced in presence of heparin [39].
Tissue-factor pathway inhibitor (TFPI):
TFPI is a Kunitz-type plasma protease inhibitor that regulates FVIIa/TF activity by inhibition of FXa
[40]. TFPI is produced by endothelial cells and can be bound hereto; however TFPI mainly circulates
as a complex with plasma lipoproteins [41]. The inhibition of FXa and FVIIa/TF is a two step process.
TFPI directly inhibits FXa by binding near its active site. Secondly, this binding induces conformational
changes which allow the TFPI-FXa complex to bind to FVIIa/TF [42]. TFPI thereby rapidly down
regulates the direct activation of FXa by FVIIa/TF preventing thrombogenesis [43].
Protein C (PC)/protein S:
The antithrombotic serine protease PC is converted to activated PC (APC) by thrombin bound to
thrombomodulin (TM), an endothelial cell surface receptor [44]. This interaction blocks the clot
promoting capacity of thrombin and enhances the specificity of thrombin to protein C [45]. In
conjunction with protein S, the activated PC (APC) inhibits the tenase and prothrombinase complex
formed in the propagation stage by inactivation of FVa and FVIIIa [46].
13
Figure 4 Anticoagulation. Antithrombin (AT), protein C (PC) and tissue-factor pathway inhibitor (TFPI) keep the coagulation in
balance by inhibiting key factors of the coagulation. Dotted lines indicate inhibition, double lines indicate activation. AT inhibits
several serine proteases such as thrombin and factor VIIa, IXa, Xa and XIa. Thrombin activates PC to activated PC (APC),
which inhibits factor VIII and V activation. TFPI binds and inhibits factor X, which leads to the binding and inhibition of TF:FVIIa
2.2.2 Fibrinolysis
The formed clot is reorganised and removed from the injured tissue by a process referred to as
fibrinolysis. Fibrinolysis is initiated already while the fibrin clot is being formed and the central factor in
the fibrinolytic system is plasmin, which is responsible for the degradation of fibrin.
2.2.2.1 Activation of plasmin
The zymogen plasminogen is converted into the active enzyme plasmin by tissue-plasminogen
activator (tPA) and urokinase type plasminogen acticator (uPA) [47]. tPA is the physiological activator
of fibrinolysis and is found in most tissues, it is synthesized by endothelial cells and released slowly
upon tissue damage [48]. In the presence of fibrin the affinity of tPA to plasminogen is increased and
this localizes plasmin to the clot [49]. The other plasminogen activator uPA is synthesized in the liver
or by macrophages and circulates freely in plasma [50]. The functions of uPA overlaps those of tPA
[47], however uPA is important in activation of plasminogen in tissues and during wound healing
whereas tPA is of primary importance for the lysis of fibrin clots in the circulation [51].
When fibrin is degraded by plasmin, a number of soluble fibrin degradation products (FDPs) are
produced [48]. Thus, FDP measurements are used as an indicator that coagulation and fibrinolysis are
activated. FDP’s compete for thrombin, and thus slow down the conversion of fibrinogen to fibrin and
inhibit clot formation [48]. D-Dimers are a unique form of FDP, and products of cross-linked fibrinderived degradation [52], thus D-Dimer more specifically signify clot lysis.
2.2.2.2 Regulation of the fibrinolytic system
The fibrinolytic system is primarily regulated by 2-antiplasmin and plasminogen activator inhibitor 1
(PAI-1) [47]. The 2-antiplasmin is secreted by the liver and binds and neutralizes plasmin rapidly
when circulating in plasma, it is also cross-linked to fibrin in the clot [53], where however the inhibition
of plasmin is less effective [48]. PAI-1 is released from the endothelium upon vascular injury and
14
inhibits tPA and uPA [47]. The close proximity of plasminogen activator, plasminogen and fibrin in the
clot prevents inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1) [48]. Furthermore,
thrombin-activatable fibrinolytic inhibitor (TAFI) is activated by thrombomodulin-bound thrombin [54]
and can prevent plasmin to bind to fibrin strands by cleaving plasminogen binding sites and thereby
slow down the fibrinolysis rate [53;55].
In summary, normal haemostasis is dependent on localization of both pro- and anti-coagulant
activities, with perfect maintenance of the delicate balance between coagulation and fibrinolysis.
2.3 Acquired Procoagulant Disorders of Haemostasis
Acquired procoagulant disorders of haemostasis covers any insult that tips the balance between
coagulation and fibrinolysis towards thrombosis [56;57], i.e. surgery, trauma [58], cancer [59],
obstetrical complications [60;61], infections [62] etc. Thrombosis is the pathological and uncontrolled
development of blood clots occurring as a result of impairment of the systems normally restricting clot
formation to the local site of vascular injury [17]. In classical terms, thrombosis is caused by one of the
corners of Virchow’s triad: hypercoagulability, vascular wall injury or circulatory stasis [63;64].
Clinical effects of thrombosis can range from no symptoms to lethality, dependent on the occurrence
of complications, which are caused either by the effects of local obstruction of the vessel, embolization
of thrombotic material or consumption of haemostatic elements [65]. Thrombosis is classified as either
venous, arterial, cardiac or systemic [66]. Venous thrombi in humans usually occur in the lower limbs
and can cause late complications such as pulmonary emboli [67]. Arterial thrombi usually occur in
association with pre-existing vascular disease and induce tissue ischemia by obstructing flow or by
embolizing into the distal microcirculation [68]. However both venous and arterial thrombosis may
share a number of risk factors [69]. Intracardiac thrombi usually form on damaged valves, are usually
asymptomatic, but may produce serious complications if they embolize [70]. Systemic thrombosis of
the microcirculation is a complication of DIC in which microthrombi cause ischemic necrosis and/or
consumption of platelets and clotting factors resulting in bleeding tendencies [66].
2.4 Disseminated Intravascular Coagulation
DIC, also known as consumptive coagulopathy, is a complex syndrome characterised by considerable
activation of the coagulation and fibrinolytic system with increasing loss of localisation or compensated
control [6]. However, the degree to which these systems are activated depends upon the triggering
event, host response and contemporary conditions [71]. Consequently the clinical signs may range
from no obvious disease to overt signs of bleeding and organ failure [72]. DIC is difficult to diagnose
and treat, and is associated with a poor prognosis as it plays a significant role in organ failure and
related mortality [72-74].
2.4.1 Aetiology
DIC occurs as a response to a variety of disorders and is neither characterised as a disease nor a
symptom [75]. It is one of the most severe complications seen in patients suffering from sepsis [76],
15
cancer [77-79], acute leukaemia [80], abruption of the placenta [81;82] and trauma [83;84] etc.
Although a huge number of agents and disorders are associated with DIC [73] the basic aetiology is
activation of the coagulation system by production or exposure of tissue factor [75;85-87].
2.4.2 Pathophysiology
Though the disorders predisposing to DIC are numerous, several seem to share overall mechanisms
of initiation of DIC. Trauma, some malignancies and obstetric calamities may cause a sudden release
of large amounts of tissue factor or placental tissues [6]. Infections cause a proinflammatory cytokine
release and tissue factor expression from macrophages, monocytes and endothelial cells [88], thus
activation of the coagulation system is mediated. Lipopolysaccharides has also been shown to induce
tissue factor expression in experimental animal models [86;87;89;90]. More rare conditions such as
snake bits and heat stroke initiate DIC via procoagulant or profibrinolytic mediators [91;92].
Regardless of the type of underlying disease or mechanism, continuous TF expression from either
endogenous production or endothelial damage, massive enough to overwhelm the anticoagulant
devices, seem to be the most important activator of blood coagulation in DIC [93;94]. The enhanced
and widespread fibrin formation and deposition cause systemic microthrombosis, which trap platelets
and coagulation factors [94]. The status of the fibrinolytic system plays a significant role in the fate of
the formed fibrin and thereby the clinical picture of DIC [47]. If sufficient tPA is released from injured
endothelium or other tissues rich in tPA such as placenta [95] and brain tissue [96] clots may be
dissolved in the microcirculation by plasmin, leading to increase in circulating FDPs. On the other
hand, if more thrombin than plasmin is present, lodging of clots in the microvasculature leads to tissue
ischemia and organ failure [69]. The latter is typically seen in patients with septic DIC, where an
initially profibrinolytic response is immediately followed by a suppression of fibrinolytic activity due to a
sustained increase in plasma levels of PAI-1 [97] as confirmed in a chimpanzee model of endotoxemia
[98].
The complexity of DIC is therefore caused by the variations in dysregulation of both coagulation and
fibrinolysis, which challenges the diagnosis and treatment of this syndrome.
2.4.3 Interaction between inflammation and haemostasis
Once activated, the inflammatory and coagulation pathways interact with one another [99;100].
Cytokines and pro-inflammatory mediators stimulate pro-coagulants (increase fibrinogen levels and
induce TF expression) [88], inhibit anticoagulants (down regulate thrombomodulin) [101] and inhibit
fibrinolysis (by PAI-1 release) [97;98]. Thrombin as well as FXa and probably tissue factor/FVIIa
complex can interact with protease-activated receptors on cell surfaces to promote further activation
and additional inflammation. Thus thrombin stimulates cytokine production, has chemotactic effect on
monocytes and mitogenic effect on lymphocytes [102]. This vicious circle between inflammation and
coagulation further amplifies the response to initiation of DIC. The complexity of these interactions
may limit treatment effects in the clinic and affect the outcome of experimental trials if underestimated
[103].
16
2.4.4 Diagnosis of DIC
The overall progressive but dynamic complex patho-physiologic changes in DIC calls for a panel of
diagnostic markers as, until now, no single laboratory test has been able to establish or rule out the
diagnosis of DIC [104]. The classic DIC patient often presents with bleeding as the predominant
symptom and concurrent micro-thrombosis may be missed [6]. Thus, the whole clinical picture, such
as established diagnoses, clinical condition and laboratory results, must be taken into account.
2.4.4.1 Detection of Activation of Coagulation
Activation of coagulation can be detected by either decreases in the level of coagulation factors or
abnormalities in global coagulation tests such as prothrombin time (PT) and activated partial
thromboplastin time (aPTT).
PT and aPTT:
The PT and aPTT, measures of tissue factor induced clotting or phospholipid and calcium induced
clotting respectively, are often prolonged at some point during the course of DIC, mainly due to the
consumption of coagulation factors, but may also be a result of inflammation [105]. The PT and aPTT
can provide important evidence of the degree of coagulation factor consumption and activation,
though interestingly PT is only prolonged in 50-75% of cases and aPTT in 50-60% of cases, and
hence normal or even shortened values can not be used to rule out the diagnosis of DIC [6].
Platelet count:
Thrombocytopenia is found in up to 98% of DIC cases, the platelet count is even <50 x 109/l in
approximately 50% of these [106]. Serial measurements of platelet count increases the sensitivity of
this marker in the diagnosis of DIC, because a single measure of the platelet count within the normal
range can be difficult to interpret in the dynamic context of DIC. A continuous drop in the platelet
count, even within a normal range, indicates active thrombin generation, whereas a stable platelet
count suggests that thrombin formation has stopped. Although a decrease in platelet count has a high
sensitivity of DIC it is not very specific; a decrease in platelet count is associated with many of the
diseases predisposing to DIC, and often thrombocytopenia is present without the development of DIC
[107].
Fibrinogen:
Decreased levels of fibrinogen are often used as a measure of coagulation factor consumption [108].
However, fibrinogen acts as a positive acute-phase reactant [109;110], hence its plasma concentration
increases during inflammation and cytokine release, which can cause the plasma fibrinogen levels to
remain within the normal range despite considerable coagulation activation and fibrin formation. Thus,
hypo-fibrinogenemia is only common in severe cases of human DIC [108].
2.4.4.2 Inhibitor consumption
The inhibitors of coagulation; AT and PC previously described are the markers most commonly
evaluated for determination of inhibitor consumption [108].
17
AT/TAT:
AT forms irreversible complexes with the generated thrombin, as well as other serin proteases, this
causes the AT levels to rapidly decline during DIC, whereas thrombin-antithrombin (TAT) complex
levels increase [111]. The persistent decrease in AT levels has been shown to have a very high
prognostic value for prediction of death in human septic DIC [112]. TAT is an early marker of in vivo
thrombin generation, but not a specific marker of DIC; it can however be useful in the early diagnosis
of DIC when evaluated in combination with other markers of coagulation and fibrinolysis.
Decreased PC/PS:
PC is rapidly converted to APC in DIC and then consumed during its regulation of fibrinolysis. This is
reflected in a decrease in endogenous PC levels and transient increase in endogenous APC levels as
observed in both human DIC patients [113] and an animal model of DIC [114]. The reduction of PC
levels is consistent and can be used prognostically in human DIC patients [112].
2.4.4.3 Fibrinolytic activity
In addition to enhanced thrombin formation, fibrinolytic activity is often also increased in DIC.
However, in some cases inhibition of fibrinolysis may dominate the picture [115]. It is possible to test
for specific markers of fibrinolysis such as tPA, plasminogen, 2-antiplasmin, plasmin-antiplasmin
complexes, fibrin and fibrinogen degradation products (FDP) and PAI-1. Except for FDP and D-Dimer
assays these tests are not commonly used in the clinic.
FDP/D-Dimer:
Fibrin degradation can be evaluated by assays measuring FDP levels or D-Dimer levels [52;116]. As a
product of fibrin degradation, FDPs reveal that both thrombin and plasmin was present, whereas DDimers, from degradation of cross-linked fibrin, show that a fibrin clot was formed and degraded [117].
FDP levels are increased in 85 to 100% of DIC patients and D-Dimer assays show abnormal values in
93% of DIC patients [6]. These highly sensitive DIC parameters have a limited specificity though;
indications of increased thrombin and plasmin activity are not diagnostic of thrombo-embolism and
DIC, these assays therefore mainly represent a negative predictive value in the diagnosis of DIC.
Soluble fibrin monomers (SF) reflect the direct action of thrombin on fibrinogen and may present an
advantage in diagnosis of DIC. SF are elevated in 90-100% of DIC cases [52], its generation is limited
to the intravascular compartment and therefore not influenced by extravascular fibrin formation from
trauma or inflammation. However, as most other markers of DIC it has a very low specificity [52].
2.4.4.4 ISTH classification of overt and non-overt human DIC
Although the syndrome DIC has been known for decades, the complexity of the syndrome has brought
along a lack of consensus on the definition and diagnosis of DIC. Choice of assays for monitoring
imbalances of coagulation and fibrinolysis in DIC has often been random and dependent on local
availability and routines. In 2001, The International Society of Thrombosis and Haemostasis (ISTH)
sub-committee of the Scientific and Standardization Committee (SSC) on DIC recommended the use
18
of a scoring system for overt and non-overt DIC in humans. With the lack of a gold standard for
diagnosis of DIC, these recommendations are based on consensus statements from specialist sub
committees [7]. The ISTH criteria utilizes a combination of platelet count, measurement of prothrombin
time, measurement of fibrinogen and fibrin degradation products in a 5-step diagnostic algorithm to
calculate an overt DIC score, the scoring system for non-overt DIC furthermore incorporates a scoring
of abnormal trends by repeated DIC scoring and addition of specific criteria such as thrombinantithrombin and protein C levels. Parameters included in the ISTH scoring system are based on
simple laboratory tests available in most hospital laboratories [7]. Included in the recommendations
are that this scoring system should not be used if no underlying disorder known to be associated with
DIC is present.
Prospective evaluations from pilot studies using these scoring systems have shown a strong
correlation between increasing DIC score and mortality and a high sensitivity and specificity of DIC
[10-12].
3 Experimental thrombosis and DIC in animal models
DIC affects numerous components in the blood circulation; blood cells, coagulation factors, vessel
wall, blood flow and pH, and trigger compensatory capabilities of the organism (changes in respiration,
heart rate and blood pressure) [6]. The complexity of DIC makes ex vivo mimicking very difficult and
inconsistency in administration of treatment and supportive care causes high variability in the course
of the syndrome in the human DIC patients, hence relevant and predictable animal models of DIC are
extremely valuable for investigation of pathophysiologic mechanisms and treatment of the syndrome.
Though no single animal model can ever duplicate all aspects of DIC occurring in humans, animal
models still provide an opportunity to gain insight in pathogenesis and progression of the syndrome
within a living organism, with all the complexities this gives rise to.
Multiple inducers have been used in experimental animal models of DIC reflecting the many identified
predisposing conditions associated with DIC in humans [6]. Thus the characteristics of the different
models may vary considerably according to dynamics and end-points. Also selection of parameters for
diagnosis of DIC differs between animal models of DIC; however most studies use the occurrence of
micro-thrombosis as evidence of DIC, and until now this is the closest to a golden standard of DIC
diagnosis in animal DIC models.
3.1 Methods in diagnosis of experimental microthrombosis
The above mentioned tests of coagulation activation, fibrinolysis activation and inhibitor consumption
indicates the occurrence of thrombosis and are widely used in diagnosis of DIC in animal models. In
addition hereto, a great advantage of experimental animal DIC studies as compared to studies in
human DIC patients is the possibility to apply more invasive methods in order to increase the
diagnostic accuracy of DIC. In this context histopathology is a valuable tool for the visualization of
disseminated thrombus formation, one of the key parameters of DIC.
19
In animal models of DIC histopathology is often used to determine the presence and number of
microthrombi. Various staining methods can be used to get a morphological overview and identify
components of the fibrin clot [1;71;118-120]. Alternative ways to identify and visualize the thrombi is by
radiolabelling of components included in the thrombus formation, especially fibrin [121-123] or by
immunohisto/cytochemichal staining [118;122;124]. Besides identification of thrombi in different
tissues, several studies furthermore quantify the microthrombi either by manual or computerized
counting methods. As an example, the number of thrombi in the kidneys can be assessed by the
percentage glomerular fibrin deposition (%GFD), and this is used as a DIC parameter in some
experimental animal models [125;126]. Thrombosis scores based on light microscopy counts from
selected tissue sections [1;127;128] or by computerized counting [129] have also been used in animal
models of DIC.
3.2 Standardization and translational aspects of DIC in animal
models
Various attempts have been made to establish relevant animal models of disseminated intravascular
coagulation during the last 4-5 decades. A wide range of animal species, inducers of DIC and dosing
regimens have been applied in the process of imitating the various predisposing conditions and
complex dynamics of DIC. To phylogenetically mimic the human situation the chimpanzee (primates)
are the preferred species [130]). For physiological or cardiovascular similarities to humans the pig
[131] or the rabbit [132] may be chosen and finally species may be chosen because of size or
availability of genetically modified specimens (mouse [124;133;134]).
Non-clinical testing in animal models of DIC is aimed at development or safety testing of products for
use in human DIC patients. It is of outmost importance that non-clinical and clinical researchers strive
to increase predictability and translational aspects of animal models of DIC.
Standardization of DIC among several different species may be challenging due to lack of availability
or access to species specific assays for the different parameters. It is important to keep in mind that
cross-reactivity of immunoassays between man and animal species is generally poor [135], and
species specific assays should preferably be used for evaluation of coagulation parameters in the
respective animal models if no validation of human assays exist [136].
Though consensus on definition and diagnosis of DIC and non-clinical testing setup is currently
lacking in the work with experimental animal models of DIC, the methods for development of similar
scoring systems as the ISTH human DIC score are available, as are experiences from application of
such a scoring system in dogs suffering from spontaneous DIC [13].
3.3 Animal models of DIC and their relevance to human DIC – A
systematic review (Paper I)
In paper I, the established animal models of DIC were reviewed and an overview of species, inducers,
and dosing regimens was given. Furthermore diagnostic approaches were compared in the light of the
20
recent ISTH scoring system for human DIC as a means to standardize experimental animal models of
DIC. Finally treatments tested in animal models of DIC were reviewed.
The rat is by far the preferred species amongst animal models of DIC and lipopolysaccharides (LPS)
the preferred inducer of DIC; in addition, species such as rabbit, dog and mouse, and inducers such
as tissue factor, live bacteria and thrombin have been used most often. The induction of DIC in animal
models usually tend to imitate the pathogenesis of DIC developing from a specific predisposing
disease seen in humans or to mimic a more isolated part of the general DIC pathogenesis.
Confirmation of DIC in experimental animal models was mainly based on histological evidence of
thrombosis and changes in a series of haemostatic parameters; however there was no agreement on
which parameters should be evaluated for a definitive diagnosis of DIC in the reviewed experimental
animal models. An overview of the reporting of ISTH DIC scoring parameters revealed that only about
25% of the studies measure all of the four parameters necessary for the implementation of the scoring
system. About half of the studies scored 2 or 3 of the 4 parameters and could probably easily be
standardized to include the parameters in the ISTH score of human DIC, which could increase the
ability to compare DIC amongst animal models and also potentially improve the translational aspects
of treatments tested.
A large number of studies testing treatment modalities for DIC in different animal models were
identified and an overview and description of the most frequently tested compounds is provided, this
includes antithrombin (AT), platelet activating factor (PAF)-antagonist, thrombomodulin (TM), thrombin
inhibitors, heparin, FX-inhibitor, APC, TFPI and urokinase. For a number of the compounds various
effects have been reported, but all of the compounds have at some point shown a positive effect in the
treatment or prevention of DIC in experimental animal models. Unfortunately these positive results are
rarely replicated in human clinical trials and so far only APC has been approved for treatment of septic
DIC in humans. Explanations as to why compounds effective in non-clinical animal models fail in
clinical trials appear to be based in the fact that most treatment compounds tested in animal models of
DIC are administered prophylactically, which is not the case in human DIC patients, and in the fact
that the complexity of DIC has lead to the use of inappropriate animal models, which do not accurately
reflect the complex syndrome of DIC in humans.
Despite the challenges in extrapolation of data from non-clinical models to setup of human clinical
trials it is believed that animal models of DIC are valuable tools investigating pathogenesis as well as
treatment possibilities, however it was concluded that it is recommended that the animal models of
DIC are standardized so that comparison between species may increase the translational aspects
within this field.
4 Development of a rabbit model of DIC and
implementation of new parameters for early diagnosis of
microthrombosis in DIC
Based on the introduction and the review of DIC and animal models of DIC, experiments were
conducted to establish an animal model of DIC with application of a standardized diagnostic scoring
21
system and investigation of new markers valuable in the early diagnosis of DIC. In the following an
overview of the studies performed during the ph.d. is provided.
4.1 Validation of a pulmonary function test in a rabbit model of
embolisation mimicked by microspheres
4.1.1 Background
The developed microthrombi in the initial stage of DIC most often get caught in the lung vasculature,
which acts as a fine-meshed net for all thrombi developed with embolization from the venous side of
circulation, or in the renal glomeruli for thrombi formed at the arterial side of circulation [94]. Thrombi
blocking the small pulmonary vessels can cause impairment of the gas-exchange and result in
retention of carbon dioxide and insufficient oxygenation of arterial blood [137]. Pulmonary function
testing has been used as a diagnostic tool for both respiratory and pulmonary cardiovascular
dysfunction in both human patients [138] and in animal models of DIC to detect gas exchange
impairment caused by pulmonary thrombi [139]. It offers the ability to measure the diffusion of gas
across the alveolar capillary membrane and the perfusion of the lung by blood. Optimal oxygenation of
blood, vital for maintenance of nearly all tissue function, is extremely dependent on normal physiology
of both ventilation and perfusion.
4.1.1.1 Multiple Inert Gas Elimination Technique (MIGET)
The multiple inert gas elimination technique (MIGET) is a pulmonary function test. MIGET measures
the distribution of inert gasses in the blood entering and leaving the pulmonary circulation and in the
expiratory air [140]. A ventilation-perfusion ratio (V/Q) is calculated based on these measures [141].
Hospitals equipped to run MIGET use this technique to monitor gas exchange during several
pulmonary; vascular and cardiac diseases in human patients [138].
In order for a pulmonary embolus to affect the gas exchange it has to be large enough to block a
vessel that supplies a functional lung unit, a term used for distal alveoli grouped so that gas can
diffuse between them [142]. With microthrombosis as a key parameter of DIC success in early
detection of DIC by application of MIGET depends on the size of pulmonary thrombi. MIGET has been
applied to animal models of experimental pulmonary thromboembolism and proven effective in early
diagnosis of pulmonary emboli of a certain minimum size dependent on the individual species, e.g.
100-150 µm in the dog [143] and about 60 µm in the pig [142]. MIGET has also been applied to
rabbits, however not for detection of pulmonary emboli [144].
We hypothesized that MIGET can detect very early changes in pulmonary function caused by
microthrombi of rabbits.
Based on the information on MIGET and as part of an overall aim to identify markers for early
diagnosis of DIC, the effect of MIGET in early detection of microthrombi was validated in an
exploratory pilot study, where pulmonary embolization was mimicking by injection of different sizes of
microspheres to rabbits.
22
4.1.2 Materials and methods
4.1.2.1 Animal procedures
Six male New Zealand White rabbits (2.8-3.3 kg) were included in the study. The rabbits were sedated
with Hypnorm® i.m. (0.3 ml/kg) and anaesthetized with Stesolid® i.v. (0.2 ml/kg). Anaesthesia was
maintained with Stesolid®/Hypnorm® mixture (1:6). The rabbits were intubated and connected to a
ventilator, and the respiratory frequency maintained at 30 breaths/min. The left ear artery was
catheterized for blood sampling and measurement of arterial blood pressure (ABP). 0.5 ml Xylocain®
was injected intracutaneously in the neck, the right internal jugular vein was catheterised and the
catheter was advanced to the right ventricle to measure right ventricular pressure (RVP) and to obtain
venous blood samples. Catheters were placed in the ear vein of both ears – one for infusion of
dissolved inert gasses and one for the maintenance of anaesthesia. ECG electrodes were placed,
allowing the recording of 3 ECG limb leads (lead I, II and III). This technique was performed in
collaboration with the staff at Uppsala University Hospital, Sweden, Department of Medical Sciences,
Clinical Physiology.
All animal studies were conducted according to guidelines and approvals of the animal research
ethical committee of Uppsala University.
4.1.2.2 Microspheres
From previous pilot studies, performed at Novo Nordisk A/S, testing the effect of 45 µm microspheres
on blood pressure (BP) and right ventricular pressure (RVP) in rabbits, it was known that a dose of
250 µl microspheres was not lethal and caused a significant increase in RVP. Thus these data
determined the starting point of this exploratory pilot study which additionally tested larger
microspheres as the effect of MIGET was expected to depend on the blocking of a functional lung unit.
Copolymer microspheres in aqueous suspension (Duke Scientific Corporation, Palo Alto, CA, USA)
were used for embolization. Different sizes of microspheres were used (45, 98 and 222 µm) (See
Table 1) and each animal received only one size of microspheres. Care was taken to shake the
aqueous suspension of microspheres to prevent aggregation at the time of injection, as aggregated
microspheres might give misleading results.
Table 1
Microsphere dosing in six rabbits
Animal ID
1
2
3
4
5
6
Animal weight
(kg)
3.0
3.2
3.1
3.2
2.8
3.3
Microsphere
diameter (µm)
45
45
45
98
45
222
Microsphere
volume (µl)
250
250
250
250
1800
1500
23
4.1.2.3 Ventilation-perfusion relationships
To assess the ventilation-perfusion ratio, MIGET was used. A solution of six inert gases with different
solubility in blood was dissolved in saline and infused into an ear vein. The infusion was maintained for
at least 45 minutes before first measurement.
The MIGET technique was conducted as previously described [142], in brief; three millilitre samples of
arterial and mixed venous blood and 20 ml mixed expired gas were collected simultaneously. The
blood and gas samples were collected in tight glass syringes and the retention and excretion of each
gas were measured by gas chromatography [141] to calculate a V/Q ratio. In addition 2 ml blood was
collected for blood gas analysis and haematology.
In a normal situation ventilation and perfusion are matched to reach an optimal gas exchange, this is
characterised by a V/Q equal to 1. Conditions decreasing the ventilation (i.e. asthma or pneumonia)
cause decreases of the V/Q, whereas conditions decreasing the perfusion (i.e. thrombosis in DIC)
cause increases of the V/Q.
4.1.2.4 Experimental protocol
Continuous infusion of inert gases was started once rabbits were anaesthetized and instrumented.
MIGET, ABP, RVP, ECG, blood gas levels and blood cell counts were evaluated at two baseline time
points and 15 and 60 minutes after microsphere injection. The rabbits were used as their own controls
for evaluation of the effect of anaesthesia on the V/Q ratios by the two baseline evaluations. The
rabbits were euthanized by exanguination at the end of the study (Figure 5).
As the initial doses of 250 µl 45 µm microspheres failed to cause any increase in RVP two rabbits
were injected with microspheres until a rise in RVP was seen in order to compare this effect of
pulmonary embolism with MIGET and blood gas measures. This resulted in 3 boluses of microsphere
injection for one rabbit and it was chosen to perform only one baseline measurement followed by a
MIGET measurement 15 minutes following each of the bolus injections, as only 4 MIGET measures
could be performed on each rabbit.
Figure 5. Study time line
4.1.2.5 Preparation of tissue specimens
Heart, lung and kidneys were harvested and fixed with 10% buffered formalin for 4 weeks. Sections
were stained with hematoxylin and eosin (HE) for general overview and with Phosphotungsten acid
hematoxylin (PTAH) for demonstration of fibrin. Sections were studied under the light microscope to
24
determine microsphere location, extent of microsphere clustering, and detect possible fibrin deposition
around the beads.
4.1.3 Results
Baseline cardiovascular and blood gas levels were within reference values for rabbits [145] (data not
shown). Injection of 250 µl of 45 or 98 µm microspheres had no effect on any of the measured
parameters.
As a consequence the approach of administering microspheres until RVP effect was taken and this is
reflected in the increase in RVP seen for animal 5 and 6 (Fig. 6A).
RVP seemed to depend on the dose and not the size of microspheres, a tendency supported by
earlier pilot studies (unpublished by Olrik Berthelsen et al. 2009) and studies in the pig [142]. Other
cardiovascular parameters were not affected by the microsphere boluses.
Changes in V/Q ratios, with a dose dependent increase in occurrence of high V/Q regions, only
developed in rabbit 6 receiving the largest sized microspheres (222µm), whereas none of the other
animals showed any increase in ventilation of high V/Q ratios (Fig. 6B).
B
60
20
*
40
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Animal 6
20
High VA/Q areas
% of Total ventilation
10
*
5
0
3
af
te
rb
Time (min)
15
15
m
m
in
in
ol
us
60
*
15
0
-7
0
3
af
te
rb
ol
us
60
*
15
0
-1
0
-7
0
0
Time (min)
*For animal 6; 15 min after bolus 2
*For animal 6; 15 min after bolus 2
D
300
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Animal 6
200
100
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Animal 6
50
*
40
30
20
10
3
rb
ol
us
60
*
te
af
in
*For animal 6; 15 min after bolus 2
15
m
in
m
15
*For animal 6; 15 min after bolus 2
15
0
0
-1
0
Time (min)
af
Time (min)
-7
3
te
rb
ol
us
60
*
15
0
-1
-7
0
0
0
0
60
Arterial P CO2 (mmHg)
C
Arterial P O2 (mmHg)
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Animal 6
15
-1
0
Mean systolic RVP (mmHg)
A
Figure 6 Effect of microspheres on A) systolic right ventricular pressure (RVP), B) presence of high V/Q areas, C) arterial PO2
and D) arterial PCO2 according to time points (min). Bolus of microspheres is injected at time point 0. For animal 6 a bolus of
microspheres was given 15 minutes before each of the last three measurements. Each symbol represents one measurement in
one animal
25
There was a slight decrease in PaO2 and increase in PaCO2 for animal 5 and 6 following embolisation,
however not until 60 minutes after injection of 45 µm microspheres and 15 minutes after the third
bolus of 222 µm microsphere injection (Fig. 6C and 6D).
4.1.3.1 Histological examination
Microspheres were easily located in lung vessels, and 222µm microspheres were visible
macroscopically as well. Microspheres were found widely distributed in the lung tissue. No
microspheres were found in kidney or heart sections. In none of the sections studied microspheres
were found to aggregate two or more in width. No fibrin formation was observed in relation to
microspheres.
4.1.4 Discussion and conclusion
In the current pilot study it was possible to apply the MIGET technique to a rabbit model mimicking
pulmonary embolism. Sampling for MIGET measures, blood gas analysis and hematologic evaluation
in this study resulted in a removal of 8 ml of blood at each sampling. As we were able to apply MIGET
measures 4 times for each rabbit, samplings resulted in removal of 19% of the total blood volume (in a
3 kg rabbit) during the study (with a circulating blood volume of 56ml/kg [146]. This is a considerable
amount of blood removal and it can not be excluded that it might have affected the cardiovascular and
hematologic parameters.
No fibrin depositions were observed around the microspheres caught in the pulmonary vessels. Also
no microspheres were found to aggregate, which would have caused blockage of vessels twice (or
more) the intended size. This supports that the changes in the measured parameters relate to
pulmonary emboli of the stated microsphere diameters and not larger emboli as could have been
formed by either fibrin attachment or microsphere aggregation. The acute effect on gas exchanging
capabilities of the lung indicates that the microspheres cause a total blockage of the pulmonary
vessels, however we can not exclude that blood may be able to pass around the microsphere when
for example blood pressure increases, as indicated by others [147].
The blood gas measurements seem not to be as sensitive of the V/Q changes as the MIGET
measurements, and the RVP for that matter, this shows, that the organism to a large extent is able to
secure an optimal oxygenation of the blood despite obvious changes in the small gas exchanging
functional lung units, and that changes in blood gas levels reflect serious damage to larger gas
exchanging areas.
It should be emphasized, that this study is an exploratory pilot study and the results described are only
observed in very few animals. However, each animal was used as its own control and several
samplings from the individual animals all pointed to the same conclusions, thus we believe the results
are of high value.
26
In conclusion it was possible to apply the MIGET technique to rabbits for detection of pulmonary
emboli. It was shown that MIGET detects pulmonary emboli very early on and is more sensitive of
changes in the pulmonary gas exchanging capabilities than blood gas measurements. MIGET
detected instant and dose dependent changes in the V/Q-ratio following the injection of 222 µm
microspheres and showed that high doses of 45 µm microspheres did not affect the V/Q ratio.
We believe MIGET has high value as a clinical tool for early detection and monitoring of DIC, however
due to the amount of specially modified equipment and technical expertise needed, it will probably not
be used on a regular basis in pre-clinical and clinical settings.
Although it was decided to discontinue the validation of MIGET for early diagnosis of microthrombosis,
the use of microspheres to mimic pulmonary embolism proved very valuable in this setting and it was
decided to further develop this model in the attempt to find novel markers of early DIC.
4.1.5 Cardiovascular and haemostatic changes in a rabbit microsphere
model of pulmonary thrombosis (Paper II)
We hypothesized that a rabbit model of fixed size pulmonary thromboembolism would enable
investigations of the mechanical consequences of pulmonary microvascular thrombosis in DIC and
possibly the discovery of useful markers for early diagnosis hereof.
In paper II the aim was to establish a mechanical model of pulmonary thromboembolism in the rabbit
by venous injection of various doses of microspheres in order to test the response of cardiovascular
parameters such as RVP, systemic blood pressure and ECG. Furthermore in vitro and in vivo testing
of haemostatic parameters such as TEG, PT, aPTT, platelet count, fibrinogen levels, TAT and FDP
were included to investigate whether the microspheres are truly inert even at high doses. Histological
evaluation of kidneys, heart, brain and lung was performed to determine microsphere location and
evaluate fibrin formation in relation to microspheres caught in the vessels.
Microspheres of 45 µm in diameter were injected. Three groups of eight rabbits received one dose of
0.3, 6 or 16 µl microspheres/ml blood and one group of only three rabbits received one dose of 32 µl
microspheres/ml blood as a lethal effect of this dose was shown. No effect on any of the parameters
was shown for the small dose (0.3 ul microspheres/ml blood) of microspheres. Administration of 6 and
16 µl microspheres/ml blood resulted in minor and significant increases in cardiovascular parameters
with rapid and dose dependent effects on RVP and systemic blood pressure. No changes in
haemostatic parameters were shown with administration of 6 µl microspheres/ml blood whereas 16 µl
microspheres/ml blood caused minor initial haemostatic changes. TEG showed minimal haemostatic
in vitro changes in rabbit whole blood spiked with the highest dose of microspheres (32 µl
microspheres/ml blood). Microspheres were only located in the pulmonary vasculature except in
animals receiving a lethal dose of microspheres, where few microspheres were found in the
myocardium and renal cortex. No fibrin formation was observed around the microspheres.
27
It was concluded that administration of microspheres to the rabbit enabled early detection of changes
in RVP and systemic blood pressure caused by mimicked fixed sized mechanical pulmonary
thromboembolism.
The results from this study of mechanical pulmonary thromboembolism showing the response of the
cardiovascular parameters; systemic blood pressure and RVP, to various doses of fixed size
pulmonary emboli indicated that these parameters could be valuable in early diagnosis of pulmonary
microvascular thromboembolism in experimental animal models of DIC.
4.2 Implementation of new parameters in early diagnosis of DIC
4.2.1 Background
As widespread microthrombosis in DIC can cause myocardial damage due to microthrombi caught in
coronary vessels [148;149], early detection of myocardial ischemia might be valuable in an animal
model of DIC. The cardiac isoform of troponin I is a recognized biomarker for acute myocardial
damage with a high sensitivity and specificity [150] and the effect of cTnI measurements in detection
of myocardial ischemia was tested in a pilot study of non-invasive medically induced myocardial
infarcts in the rabbit by injection of Isoproterenol and Vasopressin as previously described by Pinelli A.
et al. [151].
Furthermore a separate purpose of this pilot study was to evaluate different settings of TEG and
determine the most optimal design for TEG measurements in a rabbit model of DIC.
TEG is a global haemostatic test that has the ability to evaluate all steps of coagulation and fibrinolysis
in whole blood containing all the intravascular coagulation factors, inhibitors and cells, thus only
missing the contribution of the vessel wall. Parameters describing the whole blood clotting stages are
attained by measuring the forces acting on a pin in the rotating TEG cup [152]. TEG has been shown
valuable in the diagnosis of DIC in dogs [13;153], however the use of TEG in human patients mostly
involves monitoring of haemostatic function during cardiovascular surgery [154] or management of
coagulopathies following injuries [155] and though able to show haemostatic abnormalities in septic
patients, the diagnostic specificity and sensitivity for DIC has not been analyzed [108].
4.2.2 Materials and methods
4.2.2.1 Animals
New Zealand White rabbits of 2-3 kg were obtained from Charles River, Sulzfeld, Germany. The
animals were kept in a barriered facility and acclimated for a period of 5 weeks which included
preventive treatment with coccidiostatic drugs during the second and fourth week (Esbetre® Vet,
Novartis, Denmark). The rabbits were housed in multiple pens of 8-10 animals each with free access
to drinking water and standard rodent pelleted diet (Altromin 2113, Altromin GmbH, Lage, Germany)
fed ad libitum. Rabbits were used non-fasted. The study was approved by the Danish Animal
Experiments Inspectorate, the Ministry of Justice.
28
4.2.2.2 Animal procedures
Three rabbits were pre-anaesthetized with Diazepam 5 mg/ml (Stesolid®, Alpharma, Oslo, Norway)
0.4 mg/kg and anaesthetized with pentobarbital sodium 5% in sterile water (Nomeco, Copenhagen,
Denmark) intravenously through an ear vein, pentobarbital sodium was supplemented as needed. The
other ear vein and an ear artery were catheterized for injection of test compound and blood sampling
respectively. 0.5 ml Xylocain® 10 mg/ml (Astra Zeneca, Albertslund, Denmark) was injected
intracutaneously in the neck. The right heart ventricle was catheterized via the right internal jugular
vein to measure right ventricular pressure (RVP) and the left carotid artery was catheterized for
measurement of systemic blood pressure and blood sampling. ECG electrodes were placed, allowing
the recording of 3 ECG limb leads (lead I, II and III).
4.2.2.3 Cardiac troponin I (cTnI) measurements
Serum levels of cTnI were tested by an ELISA assay validated for rabbit cTnI (Life Diagnostics, West
Chester, PA)
4.2.2.4 Thromboelastography (TEG)
Citrated whole blood was obtained from either the carotid artery or an ear artery and left to stabilize for
30 minutes. To determine the most optimal TEG setup for future studies 1). effects of blood sampling
site and 2). of kaolin as a trigger source on the coagulation profile were evaluated. TEG analyses were
performed in duplicates by adding calcium, different doses of kaolin (China Clay, Haemoscope®,
USA) and citrated whole blood to the computerized thromboelastograph (TEG 5000, software version
4.1.73, Haemoscope, USA). The reaction time (R) depicts the time from placement of blood in the
TEG cup until initial fibrin formation and is primarily related to plasma clotting factors and inhibitor
activity. The clotting time (K) measures the time to clot formation from the end of R until predetermined
amplitude of 20 mm and is related to clotting factors, fibrinogen and platelets. The angle () shows the
speed of fibrin cross-linking, dependent on platelets, fibrinogen and clotting factors and the maximum
amplitude (MA) represents the strength of the formed fibrin clot [156]. In this study data were recorded
continuously until a value for maximum strength of the clot (MA) was reached. TEG parameters
reported are reaction time (R), velocity of clot formation (angle, ) and maximum strength of the clot
(MA). The tracing of TEG are depicted as exemplified in figure 7.
29
Figure 7 TEG parameters. The parameters reaction time (R) (latency from placement of blood in the cup until TEG tracings
reach amplitude of 2 mm), clotting time (K) (measured from R until TEG tracing amplitude reaches 20 mm), alpha angle ()
(slope of TEG tracing from R to K) and maximum amplitude (MA) (greatest amplitude of the TEG tracing) is graphically depicted
4.2.2.5 Experimental design
Blood samples for thromboelastography were taken from the carotid artery before induction of
myocardial infarction, which was induced as previously described [151]. Briefly Isoproterenol
(Isoproterenol Hydrochloride, Sigma-aldrich, Schnelldorf, Germany) 3 mg/kg was injected i.p. at time
point 0 min and Vasopressin (Arg8-vasopressin acetate, Sigma-aldrich, Schnelldorf, Germany) 0.3
mg/kg/5 min was injected i.v. at time point 10-15 min. Samples were taken at baseline and 20, 30, 40
and 55 min. (fig. 8). All animal studies were conducted according to guidelines and approvals of the
Danish Animal Experiments Inspectorate, the Ministry of Justice.
Figure 8 Time line for induction of myocardial ischemia and sampling
Due to dramatic effect on blood pressure, heart rate and respiration, the experiment was stopped at
13 minutes for one rabbit and only half the vasopressin dose was injected to the next rabbit for which
the experiment then was completed as planned. In the last rabbit the whole vasopressin dose was
injected and the experiment was extended to last 5 hours with blood samples every hour.
4.2.3 Results
Injection of Isoproterenol caused a significant increase in heart rate and systolic blood pressure for all
3 rabbits. The following injection of vasopressin worsened the blood pressure either by increasing the
diastolic pressure or lowering both systolic and diastolic blood pressure significantly. The respiration
became rapid and superficial in response to the circulatory changes.
30
For the last rabbit running the full protocol, serum levels of cTnI significantly increased from 120 min
till the end of the experiment (fig. 9). For the two other rabbits no increase in serum cTnI levels were
measured at any time point (data not shown).
cTnI (ng/ml)
6
4
2
0
-5 20 55
120
180
240
300
Time (min)
Figure 9 Serum levels of cTnI for one rabbit receiving Isoproterenol (3 mg/kg) at time point 0 min and Vasopressin (0.3 mg/kg/5
min) at time point 10-15 min.
Evaluation of TEG with regard to effect of trigger addition and blood sampling site (figure 10) showed
that 1). Blood sampling from the ear artery resulted in much greater variations for the TEG parameters
than blood sampling from the carotid artery and 2). different doses of kaolin all resulted in a very short
reaction time leaving very little room for hypercoagulant detection, whereas a wider window was
achieved by pure recalcification of the citrated blood samples.
31
80
20
60
Angle(deg)
Reaction time (R) (min)
25
15
10
20
5
0
Maximum Amplitude (MA) (mm)
0
6
Clotting time (K) (min)
40
4
2
0
80
60
40
20
0
Carotid artery (pure recalc)
Carotid artery (kaolin 1%)
Ear artery (pure recalc)
Figure 10 TEG parameters R, angle, K and MA for citrated rabbit whole from either the carotid artery (pure recalcification or
triggered with kaolin 1%) or the ear artery (pure recalcification)
4.2.4 Discussion and conclusion
No macroscopic or histopathologic evaluation was made of the hearts from the rabbits. The correlation
between ischemic injury and cTnI would be highly relevant as a marker of microthrombosis in animal
models of DIC; however it can be speculated if ischemic myocardial injury due to early microthrombi
formation in DIC is too subtle to be detected by this assay. Anyway, in the current pilot study the rabbit
specific cTnI assay detected increasing levels of cTnI in response to the non-invasive myocardial
infarction.
In animal models it is possible to take blood by several more or less invasive methods. Blood sampling
from the ear artery is less invasive than from the carotid artery and could be valuable for sampling in
non-anaesthetized rabbits. However as a consequence of the great variations in TEG parameters of
blood sampled from the ear artery it was decided to use blood samples from the carotid artery for
determination of TEG parameters. Furthermore, because of the significant shortening of reaction time
(R) with kaolin as a trigger it was decided to run purely recalcified TEG samples in future experiments.
32
4.3 Establishment of a rabbit model of thromboplastin induced DIC
4.3.1 Background
Few animal models of TF induced DIC have been established and the pathophysiology of these
models has not been as thoroughly evaluated as for the LPS induced animal models of DIC. It has
however been shown, that intravenous injection of thromboplastin gives a more immediate and fast
response with a short-lasting systemic thrombin generation compared with LPS induced DIC [157].
Tissue factor has been suggested as the most important factor for the onset of DIC. It has been shown
to be expressed and released in cancer including certain leukemias and solid tumour cells [5].
Furthermore trauma and obstetrical diseases can also cause a sudden release of large amounts of
thromboplastin like material [6;158]. The mechanisms for induction of DIC by these underlying
diseases are different from DIC associated with infectious diseases such as sepsis. Thus we find it
important to establish an animal model of DIC with tissue factor as the inducer to enable further
investigations of this mechanism involved in the onset of DIC.
Thromboplastin, a mixed product of tissue factor and phospholipids, was chosen as the inducer in the
establishment of a new acute rabbit model of DIC. The rabbit is widely used in other areas of research
[159;160], and therefore well characterized with respect to hematologic and biochemical values and
normal physiology [145;161].
Pilot studies were performed to determine the optimal dosing regimens in the balance between no
response and lethal effect of procoagulant activation. Also the thromboplastin was purified of
endotoxin and both non-washed and purified thromboplastin was characterised with regard to tissue
factor activity, endotoxin level and procoagulant effect. Finally, in addition to the more classical assays
of coagulation, an ELISA assay for measurement of a marker of myocardial damage (cardiac troponin
I) and a global haemostatic profile test (thromboelastography) were implemented to explore the
applicability in the early diagnosis of DIC.
4.3.2 Development of a model of thromboplastin induced DIC in rabbits
4.3.2.1 Background
We hypothesized that intravenous bolus injection or infusion of thromboplastin would result in
widespread activation of coagulation with a concurrent decrease in platelet count.
Several pilot studies were performed to optimize the dosing regimen of thromboplastin in a rabbit
model of DIC. Based primarily on platelet counts, effect of different bolus doses and infusion regimens
were tested in several combinations. As platelet counts above 100x10 9/l are not scored according to
the ISTH score of DIC in humans it was attempted to cause the platelet count of the rabbits to
decrease below this limit.
33
4.3.2.2 Materials and methods
Choice of reagents and initial dosing level of thromboplastin in this pilot study was based on previous
pilot studies of thromboplastin administration to rabbits performed at Novo Nordisk A/S.
Vials of 4 mg thromboplastin (Thromboplastin rabbit brain, Fluka, Sigma-aldrich, Switzerland) were
obtained and dissolved in 2 ml of sterile physiological saline to reach a concentration of 2 mg/ml.
Platelet counts were determined in EDTA stabilized whole blood by an automatic counter, Medonic CA
620 (Boule Nordic, Kastrup, Denmark).
4.3.2.2.1 Experimental design
New Zealand White rabbits (Charles River, Sulzfeld, Germany) were anaesthetized with Diazepam
and Pentobarbital and the left carotid artery was catheterized to measure systemic blood pressure as
previously described. The many different dosing regimens tested are listed in table 2, dose and length
of a single bolus injection and effect of bolus injection followed by infusion were evaluated.
Measurement of platelet count was generally performed at baseline and at 5, 15, 30, 60 and 90
minutes. For a few of the rabbits receiving only a bolus injection of thromboplastin, measurements
were only performed at baseline and 5 min after bolus to test whether the attempt to decrease the
platelet count below 100x109/l succeeded.
4.3.2.3 Results
Effect on platelet count within the dosing groups varied. High bolus doses of thromboplastin injected
over a short time span were immediately lethal or caused so severe dyspnea that assisted ventilation
was necessary for the initial survival of the rabbit (dosing group A, C and D). The non-lethal bolus
doses and bolus and infusion combinations caused an overall dose dependent decrease in platelet
count; however platelet counts below 100x109/l were rarely observed (table 2 and fig 11).
Table 2 Overview of thromboplastin dosing testing and effects
Dosing
group
A
B
C
D
E
F
G
H
I
J
Bolus
dose
(mg/kg)
5
2.5
3.125
5
.
2.5
2.5
2.5
3
1.56
Bolus
time
(min)
0-2
0-2
0-2
0-10
.
0-2
0-2
0-2
0-2
0-0.15
Infusion
dose
(mg/kg)
.
.
.
.
15
5
2.5
1.25
1.25
.
Infusion
time (min)
.
.
.
.
0-30
10-40
10-40
10-40
10-40
.
N
1
2
2
4
1
3
1
2
1
5
Lethal
effect
1/1
.
1/2
1/4
1/1
2/3
1/1
.
.
.
Average
platelet count
at 5 min (x109/l)
171
116
165
179
197
103
255
114
85
219
34
Platelet count (109/l)
300
B (n=2)
C (n=1)
D (n=3)
F (n=3)
H (n=2)
I (n=1)
J (n=5)
200
100
-5
5
15
Time (min)
Figure 11 Immediate effects on platelet count of non-lethal boluses. Each combined pair of data points represents
measurements in one rabbit
A general tendency for the platelet count to slowly recover was observed with the single bolus
injections. Infusions following a bolus of thromboplastin did not result in further decrease in the platelet
count, but low infusion doses appeared to prevent the platelet count recovery and thereby stabilize the
platelet count at the level reached by the thromboplastin bolus (data not shown). High infusion doses
following a bolus injection were found to be lethal (dosing group E and F).
4.3.2.4 Conclusion
Great variations were seen within the different dosing groups and a clear decision of optimal dosing
regimen for future studies was difficult to make. As there seemed to be a very narrow window between
significant procoagulant effect and mortality the dosing regimen for group B and H (receiving a single
2.5 mg/kg bolus and a 2.5 mg/kg bolus followed by a 1.25 mg/kg infusion, respectively) were
determined as the most optimal in the establishment of a rabbit model of DIC.
5 Implementation of the ISTH classification of non-overt
DIC in a thromboplastin induced rabbit model (Paper III)
We hypothesized that a rabbit model of DIC could be established by intravenous injection of
thromboplastin and that it was possible to apply a non-overt DIC score based on the ISTH score of
non-overt DIC in humans. We further hypothesized that measurement of thromboelastography (TEG),
TAT levels, Troponin I and right ventricular pressure (RVP) would add to an early diagnosis of DIC in
this rabbit model.
In paper III the aim was to test cardiovascular and haemostatic effect of the determined optimal
thromboplastin doses supplemented with surrounding thromboplastin doses. The applicability of a
non-overt DIC score based on major and specific criteria used in the ISTH DIC score was also tested
35
and finally the responses in TEG, TAT, cTnI and RVP as early markers of the initial microthrombotic
stage of DIC were evaluated.
Twenty-four rabbits included in the study were administered saline and different doses of rabbit brain
thromboplastin. At baseline and fixed time points following thromboplastin or saline administration (5,
30, 60 and 90 minutes) evaluation of cardiovascular parameters (BP, RVP, ECG and cTnI) and
haemostatic parameters (complete blood counts, PT, aPTT, TAT and FDP) were performed on all
rabbits. Thromboelastographic tests on whole blood were applied to half of the rabbits in each test
group. For all rabbits PT, platelet count, fibrinogen level, FDP and TAT levels were scored at all time
points for diagnosis of non-overt DIC, the number of thrombi in key organs were scored by
histopathology and fibrin formation described.
The effect on cardiovascular and haemostatic parameters was shown to be dose dependent and
resulted in a lethal effect for the highest dose group. For the two middle doses of thromboplastin the
rabbits could be scored as having non-overt DIC according to the diagnostic non-overt DIC score,
which was found valuable in the evaluation of severity and progression of DIC during the study.
It was concluded that administration of 1.25 – 2.5 mg thromboplastin/kg to rabbits induces non-overt
DIC and that the severity of non-overt DIC can be scored according to the ISTH scoring system of DIC
in humans. Measurement of TAT levels was found important for the early diagnosis of non-overt DIC
in combination with the other parameters of the non-overt DIC score (Platelet count, PT, Fibrinogen
and FDP). TEG was considered valuable in the detection of early hypercoagulability in response to
thromboplastin injection, although further investigations were found necessary due to the few TEG
samples in the current study resulting in large variations.
The non-washed thromboplastin was used as inducer of DIC in this study. As rabbits are generally
very sensitive to endotoxin we speculated that purified thromboplastin might provide the opportunity to
administer larger non-lethal doses of thromboplastin, in order to reach an overt stage of DIC in the
rabbit and to further determine the contribution of the endotoxin to the current findings.
6 Characterisation and purification of thromboplastin
6.1 Background
Rabbit brain thromboplastin is per definition hard to produce without any contamination somewhere in
the process. Consequently a significant and variable endotoxin level of the thromboplastin exists and
when injected in a rabbit, most likely affects the coagulation response.
We hypothesized that any endotoxin contamination of the thromboplastin product could be removed
by purification of the thromboplastin and aimed to produce a purified pool of thromboplastin to be
compared with a non-purified product and to thoroughly characterise these with regard to tissue factor
activity, endotoxin level and procoagulant effect.
36
6.2 Materials and methods
Thromboplastin was obtained from Sigma-aldrich, Switzerland and contains 4 mg thromboplastin.
A purified thromboplastin pool was produced by centrifugation of the pooled thromboplastin vials
dissolved in saline followed by removal of the supernatant and finally resuspension with sterile saline,
all steps performed under aseptic conditions. The tissue factor activity was measured by a
chromogenic assay using a known level of FVIIa, a modification of the method previously described
[162] and endotoxin levels were determined by a lysate assay as described elsewhere [1].
Procoagulant
effect
of
non-washed
and
purified
thromboplastin
was
evaluated
by
thromboelastography. Citrated blood from female New Zealand White rabbits (Charles River, Sulzfeld,
Germany), was spiked with 0.045 mg non-washed or purified thromboplastin or buffer. The dose of
thromboplastin added was calculated to be equivalent to a final concentration of 2.5 mg
thromboplastin/kg injected as a bolus in a rabbit with a blood volume of 56 ml/kg [146]. TEG was run
as described previously, in brief, rabbit blood spiked with either buffer, non-washed or purified
thromboplastin was placed in the TEG cup containing CaCl2. Parameters reported are reaction time
(R), clotting time (K) and angle. All samples were tested in duplicates.
6.3 Results
Tissue factor activity of the non-washed and purified thromboplastin was determined to be 2.46 nM
and 2.59 nM respectively and endotoxin levels were decreased from 454 EU/ml for the non-washed
thromboplastin to <20 EU/ml for the purified thromboplastin.
A significant decrease in R and K and increase in angle was determined for both non-washed and
purified thromboplastin compared to control samples (fig. 12). Differences between R, K and angle
values for non-washed and purified thromboplastin were insignificant, however a tendency towards a
higher procoagulant effect of the non-washed thromboplastin was observed.
2
1
60
40
20
od
lo
lo
o
F/
m
lb
m
lb
F/
gT
45
m
0.
0
04
0.
0.
0
d
he
d
as
-w
on
N
he
as
N
on
-w
d
)
fe
r
5m
gT
on
C
gT
45
m
tr
ol
F/
m
(b
lb
uf
lo
lo
o
m
lb
F/
5m
gT
04
ed
ifi
d
he
as
-w
on
N
0.
0.
0
0.
ed
ifi
Pu
r
od
)
fe
r
uf
on
C
gT
45
m
tr
ol
F/
m
F/
5m
gT
04
(b
lb
m
lb
lo
lo
o
od
)
fe
r
uf
(b
tr
ol
on
C
0
d
0
d
0
ns
80
3
ed
5
***
ns
ifi
10
***
100
***
Angle(deg)
Clotting time (K) (min)
ns
Pu
r
Reaction time (R) (min)
***
4
***
Pu
r
***
15
Figure 12 In vitro TEG results of procoagulant effect of non-washed and purified thromboplastin
6.4 Conclusion
The purification procedure reduced the endotoxin level dramatically and preserved tissue factor
activity. Both the non-washed and the purified thromboplastin furthermore demonstrated a significant
37
procoagulant effect in spiked whole rabbit blood measured by thromboelastography. No significant
differences were determined between non-washed and purified thromboplastin.
Based on these experiments and the results obtained it was decided to pursue the establishment of a
thromboplastin induced rabbit model of overt DIC including evaluation of cardiovascular and
haemostatic parameters and scored according to the ISTH score of overt DIC in humans.
7 Purified thromboplastin causes haemostatic
abnormalities but not overt DIC in an experimental rabbit
model (Paper IV)
We hypothesized that an overt DIC model could be established in rabbit by using purified
thromboplastin as inducer and that it was possible to apply an overt DIC score based on the ISTH
score of overt DIC in humans. We further hypothesized that measurement of TEG, TAT levels,
Troponin I and RVP would increase the diagnostic sensitivity of early overt DIC.
In paper IV the objectives were to test effect on cardiovascular and haemostatic parameters of purified
thromboplastin bolus administration followed by purified thromboplastin infusion. As for the established
rabbit model of non-overt DIC diagnosed by a non-overt DIC score based on ISTH parameters of
human DIC an overt DIC score was accordingly implemented in this study as well as measurement of
TEG, TAT, cTnI and RVP responses.
Eleven rabbits included in the study were administered saline or thromboplastin as a bolus of 2.5 mg
thromboplastin/kg followed by a 15 minutes infusion of 1.25 mg thromboplastin/kg. Cardiovascular and
haemostatic responses and thromboelastographic profiles were evaluated at baseline and fixed time
points following thromboplastin or saline administration (5, 30, 60 and 90 minutes). For all rabbits PT,
platelet count, fibrinogen level and FDP were scored at all time points for diagnosis of overt DIC and
key organs were evaluated by histopathology and described with regard to occurrence of microthrombi
and fibrin formation.
Slight effects on cardiovascular and haemostatic parameters were observed for rabbits injected with
thromboplastin; however this effect was less severe than for the previous study of non-overt DIC in
rabbits and none of the rabbits in the current study reached a score to be diagnosed as having overt
DIC according to the ISTH scoring system.
It was concluded that administration of purified thromboplastin to rabbits under the current settings can
not cause overt DIC. The purified thromboplastin was described as having less procoagulant effect in
vivo than the non-washed thromboplastin as indicated by the in vitro comparison of the two
thromboplastin products. The implementation of TEG was found valuable as TEG parameters were
able to reflect the procoagulant changes early on.
38
8 Discussion and Conclusion
The overall aim and main hypothesis of this thesis that a DIC scoring system can be applied to a
rabbit model of thromboplastin induced DIC was confirmed with the establishment of a rabbit model of
non-overt DIC diagnosed by a scoring system based on the ISTH scoring system of DIC in humans.
The specific objectives of the current thesis were:
1. To develop and standardize a non-clinical rabbit model of DIC with determination of a wide panel of
markers for cardiovascular and haemostatic function including early markers of microthrombosis. The
application of a pulmonary function test revealed a high sensitivity in the early diagnosis of pulmonary
thromboembolism as hypothesized, however due to practical and technical obstacles, this test was not
used in the rabbit model of thromboplastin induced non-overt DIC. It was demonstrated that
intravenous bolus administration of thromboplastin rapidly induced non-overt DIC in the anaesthetized
rabbit; however an overt DIC stage could not be reached in the current acute setting despite
exploration of numerous dosing regimes. We evaluated a number of cardiovascular and haemostatic
parameters to diagnose DIC at an early stage including the classic parameters from the ISTH scoring
system (platelet counts, PT, fibrinogen and FDP) as well as alternative methods including Troponin I
and whole blood Thromboelastography. These parameters were applied in a model of fixed size
mechanical pulmonary embolization as well as in the thromboplastin induced rabbit model of non-overt
DIC.
2. To standardize and score a rabbit model of DIC according to the ISTH scoring system of DIC in
humans. Application of a DIC scoring system to the established thromboplastin induced rabbit model
of non-overt DIC provided a standardized and clinically relevant diagnosis and monitoring of DIC as
hypothesized.
The reader is kindly referred to the papers in the appendix for discussions of specific results. Here the
studies of the project are generally discussed and concluded upon.
Microspheres were used to establish a mechanical model of pulmonary embolism. Application of
MIGET, a pulmonary function test, revealed that this method rapidly detects pulmonary thrombi of a
certain size able to block a functional lung unit. MIGET was found more sensitive in detecting impaired
gas exchange capabilities than blood gas measurements. Though promising, implementation of this
technique in a rabbit model of DIC was not further pursued due to the challenges of access to the
specialized equipment and technical expertise. Furthermore groups of rabbits receiving sizes of
microspheres between 45 and 222µm as well as a higher number of rabbits in each group needs to be
added for final validation of the use of MIGET in early detection of pulmonary embolism in the rabbit.
Though MIGET is not a handy bed-side method, it may be a valuable parameter for monitoring DIC
patients in hospitals, especially in cases dominated by thromboembolic events.
The microspheres were further used to validate the response of cardiovascular parameters (RVP and
systemic blood pressure) to fixed size pulmonary thrombi in the rabbit. Haemostatic parameters were
39
evaluated in vivo and in vitro to test whether the microspheres were truly inert. A dose dependent
response on cardiovascular parameters was observed in vivo, cumulating in a lethal effect of the
highest in vivo dose of microspheres. An in vitro setup evaluating thromboelastographic effect of
microspheres in rabbit whole blood spiked with equivalent doses as the in vivo study showed that the
highest dose resulted in a procoagulant effect. Other haemostatic parameters showed no significant
procoagulant effect of any of the doses of microspheres in vivo and it was concluded that non-lethal
doses of microspheres result in inert pulmonary fixed size emboli. The finding of microspheres in the
myocardium and kidneys in rabbits receiving a lethal dose of microspheres intravenously were
puzzling and clarification of the mechanism enabling microspheres to transit the lung are interesting in
understanding the basic physiology of the response of lung vasculature to pulmonary embolism.
The use of microspheres for mimicking pulmonary emboli is debatable and the in vivo cardiovascular
effect in rabbits and in vitro thromboelastographic effect in rabbit whole blood of microspheres have to
my knowledge not previously been investigated. The microspheres are assumed to be inert, and as
histopathology confirms that the pulmonary emboli are caused purely by single microspheres we
argue that this model is relevant for validation of acute cardiovascular responses to fixed size
pulmonary emboli. However, though we did not observe any signs of coagulation activation in relation
to the microspheres caught in the pulmonary vasculature, the decrease in platelet count, fibrinogen
level and white blood cells indicate that coagulation was activated at some point, hence if this
approach was to be used for imitating chronic pulmonary embolism further investigations of a possible
in vivo haemostatic and inflammatory effect of high microsphere doses are necessary. One approach
could be to apply scientigraphy studies to the current setting to monitor the distribution of 111In
labelled platelets as previously described [163]. Furthermore, a plausible explanation to mechanisms
of cardiovascular arrest in the animals injected with the highest dose of microspheres would be fatal
pulmonary vascular obstruction and measurement of cardiac output might add further insight to this
effect.
Validation of cTnI measurements and optimization of thromboelastography as new parameters in early
diagnosis of DIC was demonstrated in a rabbit model of non-invasive myocardial infarction showing an
increase in cTnI within two hours of myocardial infarction. Levels of cTnI have previously been
correlated by histology to the degree of myocardial ischemia induced by this non-invasive approach
and cTnI levels were shown to increase within 45 minutes of vasopressin injection [151]. cTnI is
regarded as the most cardiac-specific biochemical marker of myocardial injury [164]. During both
myocardial infarction and thromboplastin induced DIC studies performed, no thrombi were observed in
coronary or myocardial vessels and no significant increase in cTnI levels were determined in rabbits
with thromboplastin induced DIC during a 90 minute time line. However in a more chronic
experimental setup with long term induction of DIC, coronary thrombi might develop and result in a
significant myocardial ischemia and increase in cTnI or the analysis might show insensitive for
detection of microinfarcts.
40
Determination of a relevant dosing regimen for thromboplastin induction of DIC was investigated in a
wide-ranging pilot study. A narrow window between lethality and a significant procoagulant effect of
thromboplastin injection in rabbits was found. Comparison of thromboplastin induction of DIC in animal
models is difficult due to the non-standardized reagents with large variations in endotoxin levels and
probably tissue factor activity and phospholipid content of the thromboplastin.
Characterisation of non-washed and purified thromboplastin revealed a high endotoxin level of the
non-washed thromboplastin. As many studies of tissue factor induced DIC in animal models use a
source of lung or brain thromboplastin without further characterisation it is most likely that these
animal models of DIC are based on a contaminated source of thromboplastin. Although these model
are still relevant as models of aspects of DIC in humans, where predisposing disorders may often
involve tissue factor and endotoxin exposure, thorough characterization of the inducer of DIC is
important for the standardization, reproducibility and comparison of animal models of DIC.
In the establishment of a rabbit model of thromboplastin induced non-overt DIC, bolus injections of
1.25 and 2.5 mg thromboplastin/kg non-washed thromboplastin in rabbits resulted in non-overt DIC
diagnosed by a modified score based on the ISTH score of non-overt DIC in humans, injection of
higher thromboplastin doses were lethal and induction of overt DIC was not accomplished in this
study.
It was hypothesized that the purification of thromboplastin would decrease variability and enable
injection of higher non-lethal doses resulting in overt DIC. However, injection of a 2.5 mg/kg bolus
followed by a 1.25 mg/kg infusion of thromboplastin resulted in less severe procoagulant activation
than for the previous study, though a lethal effect was also observed within this study indicating an
even more narrow window between procoagulant and lethal effect than for the injection of non-washed
thromboplastin. These results underscore the difficulties in comparisons between animal models using
different inducers of DIC and emphasize that the inducer of DIC, experimentally as well as clinically,
may call for different treatment approaches.
The narrow window between lethality and significant procoagulant effect observed in the pilot study
testing different thromboplastin dosing regimens was also evident in these in vivo studies with injection
of non-washed thromboplastin and even more so with injection of purified thromboplastin. These
gathered results indicate that induction of overt DIC with the current inducer and acute setup is not
possible without further pathology involved.
As a DIC scoring system has not previously been applied to an animal model of DIC the present data
can not be compared to previous findings.
In conclusion, a rabbit model of thromboplastin induced non-overt DIC with early detection of
microthromboembolism has been established and standardized according to the ISTH scoring of DIC
in humans. This model can be useful in efficacy and safety testing of drugs as well as for investigation
of pathophysiological events of DIC. No animal model of acute overt DIC was established and animal
studies of long term induction of DIC are needed to further explore the long term aspects of
thromboplastin induction of DIC.
41
9 Perspectives
Experiences and ideas evolved during the studies in this ph.d. are here placed in perspective and
speculations on future investigations of interest based on the data obtained are given.
Two overall perspectives arise from the studies performed during this project, which regards 1) the
optimization and expansion of the rabbit DIC model and future implications of such and 2) the future
implementation of the established rabbit model of non-overt DIC in safety or efficacy studies.
Ideally animal models of DIC should reflect the different stages of human DIC; the non-overt and overt
stage as defined by the ISTH scoring system. Because of the narrow window between no effect and
lethal effect with thromboplastin injections a rabbit model of overt DIC appears difficult to establish, at
least in the acute setting. The next step in the attempt to establish a rabbit model of overt DIC could
be to include extended studies in the evaluation of long term effect of low dose thromboplastin
injections in order to reach an overt stage of experimental DIC. An overt DIC stage might otherwise be
established in the rabbit by two consecutive endotoxin injections, known as the Generalized
Shwartzmann reaction (GSR) [165], which has been generally accepted as the counterpart of DIC in
humans [90] but, to our knowledge, never evaluated according to a standardized score of DIC in
humans.
Due to the complexity of DIC in humans, comparison between several similarly standardized animal
models of DIC would allow the thorough exploration of differences in pathogenesis and severity
relating to varying predisposing diseases and underlying conditions observed with DIC in humans. In
the work with experimental animal modelling of human DIC, the many different settings explored have
generally been conceived as minor modifications of similar animal models of DIC. The experiences
from the current studies, and the provided overview of animal models of DIC, indicate that significant
differences exist in pathogenesis and severity of DIC models, depending on the inducer and species
and hence, the different setups should be perceived as distinct animal models of DIC. Though some
authors have published studies on experimental DIC covering the use of different species and
inducers [166-168] the differences in pathophysiology between their settings have not been directly
addressed, with the exception of one scientific group comparing pathophysiology of endotoxin and
thromboplastin induced DIC in rats [71;157;169-172]. Further investigations into the differences in
pathogenesis and severity of thromboplastin and endotoxin induced DIC in different animal species
could be valuable in the understanding, standardization and comparison of animal models of DIC.
Standardized diagnosis and monitoring of DIC as described in the current thesis may be transferred to
other settings and species to help understand the impact of selected setup criteria on the
pathophysiology of DIC in distinct animal models.
A rabbit model of non-overt DIC was validated and standardized including the implementation of a
non-overt DIC score based on the ISTH scoring of DIC in humans. This model and the methods
applied could provide a valuable tool for safety and efficacy testing of haemostatic drugs in research
and development of new therapeutic interventions in the complex pathology of DIC.
42
Safety evaluations are important in development of treatments for DIC as the dynamic properties of
DIC often result in treatment schedules aimed at thrombosis and bleeding simultaneously [4], which
potentially could have adverse effects if not correctly balanced. Hence, it would be interesting to
implement this standardized rabbit model of DIC in non-clinical safety evaluations of new procoagulant drugs in order to increase the prediction of safe doses, toxicity values, identify potential
target organs of toxicity and determine reversibility of adverse effects for patients at risk of developing
DIC. A knowledge base on safety aspects of different pro-coagulant drugs in animal models of DIC
with different species and settings standardized as the established rabbit model of non-overt DIC
could potentially prove valuable.
Optimization of efficacy of drugs in the non-clinical stage of development is primary of ethical but also
economical importance as these drugs otherwise may fail in clinical trials or cause hazards to patients
enrolled in these trials. Efficacy testing of both haemostatic drugs in development and reference
compounds could be performed in the established rabbit model of non-overt DIC and in future
modified and expanded animal models of DIC applying a similar standardized diagnostic DIC scoring.
Regarding the translational aspects of drug effect evaluations in animal models of DIC, it is important
to mention that testing of prophylactic drug effects are generally clinically irrelevant, rather effect of a
compound on the fully developed DIC should be tested. Besides modification, with different species
and inducers, including prolonged induction and observation time, this rabbit model of non-overt DIC
could furthermore be adapted to include predisposing conditions of DIC and administration of
supportive care. Such interventions might increase the prediction of exposure and activity of relevant
drugs during various conditions in the clinic. A predisposing condition of DIC seen in humans is
atherosclerosis, where rupture of atherosclerotic plaques cause release of tissue factor [173], this
condition has been imitated in either a spontaneous [174] or diet-induced [175] rabbit model, it would
be interesting to combine atherosclerosis and thromboplastin induction of DIC in a rabbit model to
further test efficacy and safety of haemostatic drugs.
43
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11 Publications
Paper I:
“Animal models of DIC and their relevance to human DIC – A systematic review” (Submitted)
Paper II:
“Cardiovascular and haemostatic changes following microsphere injection in a rabbit model of acute
pulmonary microvascular thromboembolism” (Submitted)
Paper III:
“Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model”.
(Thrombosis Research, 2009; Vol. 124, Issue 4, Pages 490-497)
Paper IV:
“Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit
model” (Thrombosis Research, 2010; in Press; DOI: 10.1016/j.thromres.2010.06.022)
54