1



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 Activation of plasmin 14

2.2.2.2 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 Detection of Activation of Coagulation 17

2.4.4.2 Inhibitor consumption 17

2.4.4.3 Fibrinolytic activity 18

2.4.4.4 ISTH classification of overt and non-overt human DIC 18

3 Experimental thrombosis and DIC in animal models 19

3.1 Methods in diagnosis of experimental microthrombosis 19

3.2 Standardization and translational aspects of DIC in animal models 20

3.3 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 Background 22

4.1.1.1 Multiple Inert Gas Elimination Technique (MIGET) 22

4.1.2 Materials and methods 23

4.1.2.1 Animal procedures 23

4.1.2.2 Microspheres 23

4.1.2.3 Ventilation-perfusion relationships 24

4.1.2.4 Experimental protocol 24

4.1.2.5 Preparation of tissue specimens 24

4.1.3 Results 25

4.1.3.1 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 Animals 28

4.2.2.2 Animal procedures 29

4.2.2.3 Cardiac troponin I (cTnI) measurements 29

4.2.2.4 Thromboelastography (TEG) 29

4.2.2.5 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

4.3.1 Background 33

4.3.2 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 Background 36

6.2 Materials and methods 37

6.3 Results 37

6.4 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)

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 !).

Abbreviations

ABP Arterial blood pressure

APC Activated protein C

aPTT activated partial thromboplastin time

AT Antithrombin

cTnI Cardiac troponin I

DIC Disseminated intravascular coagulation

ECG Electrocardiogram

FDP Fibrin degradation products

FV Factor V

FVa Activated Factor V

FVII Factor VII

FVIIa Activated Factor VII

FVIII Factor VIII

FVIIIa Activated Factor VIII

FIX Factor IX

FIXa Activated Factor IX

FX Factor X

FXa Activated Factor X

FXI Factor XI

FXIa Activated Factor XI

HE Haematoxylin eosin

ISTH International Society on Thrombosis and Haemostasis

LPS Lipopolysaccharide

MIGET Multiple inert gas elimination technique

PaCO2 Arterial CO2 tension

PaO2 Arterial O2 tension

PAI-1 Plasminogen activator inhibitor 1

PC Protein C

PS Protein S

PT Prothrombin time

PTAH Phosphotungstic acid haematoxylin

RVP Right ventricular pressure

TAT Thrombin-antithrombin

TEG Thromboelastography

TF Tissue factor

TFPI Tissue factor pathway inhibitor

TM Thrombomodulin

tPA tissue plasminogen activator

uPA urokinase type plasminogen activator

V/Q Ventilation-perfusion ratio

vWF von Willebrand factor

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 non-clinical 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 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.

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 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 dosis-afhængigt 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.

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 micro-vascular 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.

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

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.

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).

[pic]

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-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].

[pic]

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).

[pic]

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

2 Regulation of Coagulation

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].

[pic]

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 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.

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 fibrin-derived degradation [52], thus D-Dimer more specifically signify clot lysis.

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 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.

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].

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].

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], 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 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.

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].

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.

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 ................
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