early changes in immune, coagulation, and organ function

Institute of Clinical and Experimental Trauma Immunology,

Ulm University Hospital

Prof. Dr. med. Markus Huber-Lang

Early changes in immune, coagulation, and organ function after

clinical and experimental polytrauma

Dissertation submitted in partial fulfillment of the requirements for the degree

of ”Doctor rerum naturalium” (Dr. rer. nat.) of the International Graduate

School in Molecular Medicine Ulm

Rebecca Halbgebauer

Born in Ludwigsburg

2018

Current Dean of the Medical Faculty: Univ.-Prof. Dr. Thomas Wirth

Current Chairman of the Graduate School: Univ.-Prof. Dr. Michael Kühl

Thesis Advisory Committee:

- First supervisor: Univ.-Prof. Dr. Markus Huber-Lang

- Second supervisor: Univ.-Prof. Dr. Anita Ignatius

- Third supervisor: Univ.-Prof. Dr. Martijn van Griensven

External reviewer: Univ.-Prof. Dr. Michael Kirschfink

Day doctorate awarded: November 30, 2018

Results gained in my thesis have previously been published in the following publication:

Halbgebauer R, Braun CK, Denk S, Mayer B, Cinelli P, Radermacher P, Wanner GA,

Simmen HP, Gebhard F, Rittirsch D, Huber-Lang M: Hemorrhagic shock drives glycocalyx,

barrier and organ dysfunction early after polytrauma. J Crit Care 44: 229-237 (2017)

Permission for reuse in this dissertation, including modification of figures, has been obtained

from the publisher.

I

Table of contents

1 Introduction .................................................................................................................. 1

1.1 Acute posttraumatic inflammatory response ........................................................... 1

1.2 Mechanisms of acute trauma-induced coagulopathy .............................................. 3

1.3 Systemic response to severe blood loss ................................................................... 4

1.4 Development of trauma-induced organ dysfunction and failure ............................. 5

1.5 Lack of reliable immune and organ monitoring in trauma care .............................. 6

1.6 Aims of the study .................................................................................................... 7

2 Materials and methods ................................................................................................. 8

2.1 Consumables ........................................................................................................... 8

2.2 Buffers and media ................................................................................................... 8

2.3 Reagents and chemicals........................................................................................... 9

2.4 Primers................................................................................................................... 10

2.5 Antibodies and isotype controls ............................................................................ 10

2.6 Immunoassays ....................................................................................................... 11

2.7 Devices .................................................................................................................. 13

2.8 Software................................................................................................................. 13

2.9 PT study design at Ulm University Hospital ......................................................... 14

2.10 Plasma and serum collection ................................................................................. 14

2.11 Flow cytometric analysis of whole blood.............................................................. 14

2.12 TruCulture® assay ................................................................................................. 15

2.13 PAI-1 plasma detection ......................................................................................... 16

2.14 Neutrophil isolation ............................................................................................... 16

2.15 PAI-1 surface detection on stimulated neutrophils ............................................... 16

2.16 Gene expression analysis in neutrophils ............................................................... 16

2.17 Detection of surface PAI-1 and microvesicles after whole blood stimulation ...... 17

2.18 PT study design at Zurich University Hospital ..................................................... 18

II

2.19 Rotational thromboelastometry analysis ............................................................... 19

2.20 Testing of endothelial glycocalyx and intestinal mucus components in rotational

thromboelastometry ............................................................................................... 20

2.21 Murine model of PT and HS ................................................................................. 20

2.22 Statistical analyses ................................................................................................. 23

3 Results ........................................................................................................................ 24

3.1 Alterations in leukocyte surface molecules after trauma and correlation to shock

parameters ............................................................................................................. 24

3.2 PAI-1 in PT plasma and ex vivo on the granulocyte surface ................................. 34

3.3 Gene expression of PAI-1 and urokinase receptor in stimulated neutrophils ....... 38

3.4 Thromboelastic alterations in blood after PT ........................................................ 39

3.5 Cytokine response to bacterial stimulus in an ex-vivo whole blood model........... 43

3.6 Impact of HS on barrier disturbance and organ dysfunction after PT ................... 49

4 Discussion .................................................................................................................. 65

4.1 Leukocyte surface profiling in polytraumatized patients ...................................... 66

4.2 Coagulopathy in polytraumatized patients ............................................................ 68

4.3 Functional immune monitoring in trauma ............................................................. 69

4.4 HS as a major driver of barrier and organ dysfunction after PT ........................... 70

4.5 Conclusion ............................................................................................................. 73

5 Summary .................................................................................................................... 74

6 List of references ........................................................................................................ 76

III

List of abbreviations

AIS Abbreviated Injury Score

AKI Acute kidney injury

aPTT Abbreviated partial thromboplastin time

ANOVA Analysis of variance

BSA Bovine serum albumin

C3a Complement activation product 3a

C5a Complement activation product 5a

C5aR Complement activation product 5a receptor

CC16 Clara cell secretory protein

CD Cluster of differentiation

cDNA Complementary DNA

CRP C-reactive protein

Ctrl Control

DAMP Damage-associated molecular pattern

DNase Deoxyribonuclease

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent assay

FGF basic Basic fibroblast growth factor

FITC Fluorescein isothiocyanate

G-CSF Granulocyte colony-stimulating factor

GCS Glasgow Coma Scale

GM-CSF Granulocyte-macrophage colony-stimulating factor

Healthy Healthy volunteers

HR Heart rate

HMGB1 High mobility group box 1

HS Hemorrhagic shock

IV

I-FABP Intestinal fatty acid-binding protein

ICU Intensive care unit

IL Interleukin

IL1-RA Interleukin-1 receptor antagonist

IL-12 (p70) Interleukin -12 heterodimer

IP-10 Interferon gamma-induced protein 10

ISS Injury Severity Score

L-FABP Liver-type fatty acid-binding protein

LPS Lipopolysaccharide

MAP Mean arterial pressure

MCP-1 Monocyte chemoattractant protein 1

MDSC Myeloid-derived suppressor cell

MFI Mean fluorescence intensity

MIP-1 Macrophage inflammatory protein-1

MMP Matrix metalloproteinase

MODS Multiple organ dysfunction syndrome

MOF Multiple organ failure

MP Microparticle

mRNA Messenger RNA

MV Microvesicle

NET Neutrophil extracellular trap

NGAL Neutrophil gelatinase-associated lipocalin

PAI-1 Plasminogen-activator inhibitor-1

PAMP Pathogen-associated molecular pattern

PBS Phosphate-buffered saline

PCT Procalcitonin

PDGF-bb Platelet-derived growth factor subunit B

V

PICS Persistent low-grade inflammation, immunosuppression, and

high protein catabolism syndrome

PMA Phorbol 12-myristate 13-acetate

PRR Pattern recognition receptor

PT Polytrauma

PTC Polytrauma cocktail

qPCR (Real-time) quantitative polymerase chain reaction

R Pearson correlation coefficient

RAGE Receptor for advanced glycation end products

RBC Red blood cell

RISC Revised Injury Severity Classification

ROTEM® Rotational thromboelastometry

RPMI Roswell Park Memorial Institute medium

ROS Reactive oxygen species

SDHA Succinate dehydrogenase complex, subunit A, flavoprotein

variant

SIRS Systemic inflammatory response syndrome

SOFA score Sepsis-related or Sequential Organ Failure Assessment score

Sphingosine-1-P Sphingosine-1-phosphate

TASH score Trauma Associated Severe Hemorrhage score

TBI Traumatic brain injury

TCC Terminal complement complex

TIC Trauma-induced coagulopathy

TLR Toll-like receptor

TM Thrombomodulin

TNF Tumor necrosis factor

TREM Triggering receptor expressed on myeloid cells

TXT Thoracic trauma

VI

uPA Urokinase (plasminogen activator)

uPAR Urokinase receptor

VE-cadherin Vascular endothelial cadherin

VEGF Vascular endothelial growth factor

Introduction

1

1 Introduction

Physical injuries cause more than 5 million deaths worldwide every year, representing the

major cause of death in the population below the age of 44 years and accounting for 10% of

global mortality [89,123]. Despite improved public safety measures, rescue chains, as well

as emergency and intensive care, trauma remains a major burden of disease in all societies

[58]. In 2016, 33,000 people sustained injuries that required primary care in the emergency

room and a subsequent stay at the intensive care unit (ICU) in Germany alone. Patients were

predominantly male, mostly suffered from blunt trauma and stayed in hospital an average of

16 d. Furthermore, 20% developed multiple organ failure and 11.3% died in the hospital

[56]. Polytrauma (PT), defined by a complex injury pattern affecting several physical regions

or organ systems and including the severity of the injuries so that at least one injury or the

combination of several injuries are life-threatening (Injury Severity Score (ISS) ≥ 16)

[3,130,131,169], was present in 55% of patients [56].

1.1 Acute posttraumatic inflammatory response

After severe injury, an immediate systemic response occurs to the traumatic insult.

Breakdown of cellular structures and barriers results in the concomitant release of damage-,

but also pathogen-associated molecular patterns (DAMPs and PAMPs, respectively). These

molecules trigger the activation of different humoral serine protease systems, including the

coagulation and the complement system, with the aim of sealing off injured areas to stop

bleeding and recognizing invading microbes to stop their growth by transmitting the danger

signal to phagocytic leukocytes [27,35,53,85]. Leukocytes can also directly recognize

DAMPs and PAMPs via pattern recognition receptors [5,24,120]; complement and

leukocyte activation induces the secretion of pro-inflammatory cytokines and initiates a

systemic inflammatory response which can culminate in development of the systemic

inflammatory response syndrome (SIRS). Besides clinical parameters [8], SIRS is

characterized by local and systemic production and release of acute-phase proteins,

components of the contact phase and coagulation systems, complement factors, pro- and

anti-inflammatory cytokines, as well as an accumulation of immune cells at the site of tissue

damage [54,69,81]. An excessive immune reaction can reinforce dysfunction of cellular

barriers [86], allowing the entry and generation of more PAMPs and DAMPs, thereby

amplifying a vicious cycle of tissue injury and harmful immune processes [5,54,81] (Fig. 1).

However, in parallel to the pro-inflammatory response, there is also a marked induction of

suppressive processes immediately after injury which may render patients susceptible to

Introduction

2

infection [62,118,166]. It is therefore considered essential that the initial immune reaction

contains both pro- and anti-inflammatory aspects to establish an immune balance facilitating

clearance of the damaged tissues and induction of an effective regeneration [69] (Fig. 1).

Fig. 1: Posttraumatic immune response. Injury induces the release of molecular danger signals, so-called

pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs, respectively) after cell

damage and destruction of local barriers. These are sensed by activation of the complement and the coagulation

systems which may be reinforced by local hypoxia and acidosis. The danger signal is transmitted to leukocytes

via pattern recognition receptors (PRRs), inducing chemotaxis towards the inflammatory stimulus, release of

cytokines, reactive oxygen species (ROS), and microparticles (MPs), neutrophil extracellular trap (NET)

generation, and phagocytosis of opsonized particles. Effective immune control can aid to establish a balanced

reaction, allowing timely and effective clearance of damaged tissue, repair and regeneration. Adapted and

personalized trauma care may support the avoidance of an escalation of the danger response due to hemorrhage,

extended surgical interventions or nosocomial infection. Influenced by individual risk factors, the innate

immune response can become dysbalanced, with patients developing coagulopathy and barrier dysfunction,

resulting in edema formation, invasion of microorganisms, development of sepsis and multiple organ

dysfunction syndrome (MODS). From Huber-Lang et al. [69].

Apart from pre-existing physical conditions, exogenous factors such as the severity of the

injury itself (“first hit”) and surgical interventions (“second hit”) play an important role in

SIRS development and posttraumatic outcome [149]. Therefore, in the clinical setting,

additional tissue injury should be minimized during life-saving procedures and primary

Introduction

3

surgery immediately after trauma; secondary reconstructive surgery is recommended to be

delayed until after a stabilization phase ranging from a few days up to three weeks [4,54].

1.2 Mechanisms of acute trauma-induced coagulopathy

Tissue damage is almost always associated with rupture of blood vessels, and several studies

have reported that approximately 25% of PT patients arrive at the emergency department

with hemodynamic instability and acute traumatic coagulopathy, more recently termed

trauma-induced coagulopathy (TIC) [16,97-99]. About 40% of deaths after injury are caused

by bleeding [80]. Disintegration of macro- (e.g. the skin) and microbarriers such as

endothelial cell membranes immediately activates multiple hemostatic pathways of the

coagulation system, including fibrinolysis, with rapid consumption of coagulation factors

and thrombocytes [21]. This hypocoagulable state is mainly caused by tissue hypoperfusion

and predominantly involves protein C activation [13]. Moreover, platelets have been shown

to become hyporesponsive to pro-coagulatory stimuli with impaired clot assembly and

stability as early as 30 min after injury [90,180]. The risk of bleeding is reinforced by

hypoxia, acidosis, and hypothermia, also termed the “lethal triad” [106]. Furthermore,

increased secretion of catecholamines contributes to the activation of endothelial cells and

results in downregulation of thrombomodulin and secretion of plasminogen-activator

inhibitor-1 (PAI-1) [40,70,105,183]. Increased adrenaline levels and hypoperfusion also lead

to shedding of endothelial glycocalyx components and thereby amplify endothelial

dysfunction and coagulopathy [76]. Additionally, hyperfibrinolysis was demonstrated to be

a central risk factor for posttraumatic mortality [73,79,153]. Conversely, other studies

indicated that shutdown of fibrinolysis may be even more prominent than hyperfibrinolysis,

especially in severely injured patients, in regard to incidence and mortality [113,114]. As a

further driver of coagulation disturbances, fluid resuscitation especially with crystalloids

seems to increase the severity of TIC since the replaced volume was demonstrated to be

proportional to the degree of coagulopathy [98]. In other studies, however, TIC was also

present in patients who had received little or no fluids [13,14,34,51], suggesting that the

injury pattern and severity may also play a significant role. Currently, apart from addressing

hypoxia, acidosis, and hypothermia, treatment strategies such as balanced resuscitation,

antifibrinolytics, patient-tailored recombinant coagulation factors as well as fibrinogen

concentrates are employed. Guided by conventional coagulation assays or viscoelastic

functional testing, all of these can significantly improve the outcome [27].

Introduction

4

1.3 Systemic response to severe blood loss

Deaths due to bleeding occur for different reasons, ranging from delayed admission to

surgery to detrimental effects of prolonged shock periods despite successful control of

bleeding [15]. Upon arrival at the emergency room, 31% of patients with severe trauma as

defined by Paffrath et al. [130] and Pape et al. [131] present with shock (systolic blood

pressure ≤ 90 mmHg) and 24% with acidosis (base excess ≤ –6 mmol/l) as characteristics of

significant hemorrhage. Of note, in the presence of shock, mortality was increased to 37%

[56].

Severe blood loss and hypovolemia due to blunt or penetrating injury initiate a compensatory

response via sympathetic reflexes, inducing peripheral vasoconstriction in order to maintain

blood pressure. Blood flow in internal organs such as the liver, pancreas, kidney, and

gastrointestinal tract can be reduced (centralized) to such an extent that patients remain

normotensive despite severe shock [115]. Only after the loss of more than 30% of the blood

volume, decreases in cardiac output, blood pressure, and organ perfusion may become

evident [179]. The reduction in oxygen supply and transport of metabolites quickly results

in tissue hypoxia and an increase in lactate levels. In the following, sustained mitochondrial

anaerobic metabolization of glucose can lead to cellular destruction and metabolic acidosis

[19]. If rapid and effective restoration of normotension and removal of the source of bleeding

is not achieved, the accumulation of cell damage leads to disseminated intravascular

coagulopathy, microvascular damage, and SIRS with a mostly unfavorable outcome [115].

Recent studies have furthermore suggested hemorrhagic shock (HS) as a major driver of

systemic inflammation and disintegration of the blood-organ barrier. In rat models of

hemorrhage, a loss in intestinal tight-junction proteins and increased amounts of

mitochondrial DAMPs in the mesenteric lymph were detected, suggesting cellular

breakdown and a deterioration in barrier function [165,187]. These processes seem to be

mainly dependent on complement activation [49]. Shock has also been demonstrated to be a

potent inducer of mitochondrial DAMP release which in turn increases secretion of pro-

inflammatory mediators in neutrophils and other tissues [192]. Furthermore, posttraumatic

bleeding is also considered to play a central role in development of coagulopathy and

complementopathy [18,50,51,74] as well as organ failure [78]. These are presumed to be

especially driven by pathological activation of the endothelium, termed endotheliopathy. In

this regard, disturbances in the endothelial glycocalyx layer after trauma may depend on

shock severity: in severely injured patients, higher catecholamine levels were shown to lead

Introduction

5

to an increase in glycocalyx shedding from the endothelial surface [75,125,126] which is

likely to advance barrier failure and organ damage.

1.4 Development of trauma-induced organ dysfunction and failure

In all patients admitted to emergency departments in Germany, early mortality during the

first 24 h accounted for 5.1% of deaths while another 5.3% of patients died later during the

first 30 d after injury. On the ICU, 34% developed organ failure which culminated in

multiple organ failure (MOF) in 20% of patients [56]. After injury, the aforementioned

activation of serine protease cascades (such as the complement system) and release of

cytokines and other pro-inflammatory mediators from immune cells and tissues, especially

from the gut, prime and mobilize neutrophil granulocytes and other leukocytes

[100,112,122,155]. Neutrophil priming can be augmented by ischemia/reperfusion

conditions. Further iatrogenic contributors are represented by blood products which are

deemed to be immunoactive and contain considerable amounts of pro-inflammatory

coagulation and complement factors as well as cytokines and lipids [41,63]. The risk of MOF

is additionally increased in patients after severe hemorrhage due to tissue hypoperfusion

during the initial shock phase [167,175,178]. Early neutrophilia often turns into neutropenia,

suggesting organ sequestration based on increased adhesion molecules on neutrophils and

endothelium and transmigration into damaged tissues [12,26]. This is accompanied by

increased endothelial and epithelial permeability facilitating electrolyte and protein shifts

and subsequent edema formation. Degranulation of neutrophils in inflamed tissues releases

further pro-inflammatory cytokines, but also proteases and reactive oxygen species which

can damage host tissues and promote bystander injury [163,170]. These processes are

reinforced by ischemia and reperfusion; when substantial amounts of oxygen reenter the

tissue, accumulation of superoxide anions, hydrogen peroxide, and hydroxyl radicals causes

the peroxidation of cell membranes, inducing apoptosis and necrosis and stimulating the

local generation of inflammatory mediators [144,151,172]. In the context of the proceeding

compensatory anti-inflammatory response in parallel to SIRS development, Gentile et al.

established the concept of the persistent low-grade inflammation, immunosuppression, and

high protein catabolism syndrome (PICS). PICS is characterized by defects in innate and

adaptive immunity with decreased activity of dendritic cells, macrophages, and T effector

cells mediated by myeloid-derived suppressor cells (MDSCs), culminating in an increased

risk of late MOF [55].

Introduction

6

1.5 Lack of reliable immune and organ monitoring in trauma care

Despite considerable advances in trauma care in the last decades, some issues remain which

continue to complicate treatment and may impede further improvements in patient

management. There are several well-established techniques in order to monitor organ

function on the ICU, including clinical parameters and organ performance scores such as the

Sepsis-related or Sequential Organ Failure Assessment (SOFA) score [177]. Considering the

deleterious effects of extensive surgical interventions with substantial DAMP and PAMP

generation when the patient is currently at high risk of overwhelming inflammation or

immunosuppression, there is an obvious need for parallel monitoring of the immune function

during the time course after injury. Nevertheless, to date, no tests exist to provide the

clinician with reliable data on the remaining functional capacity of the immune system and

support decision-making in order to optimize timing of necessary interventions or definitive

surgical care. In this context, there is also a lack of information on the alterations in

peripheral blood leukocytes regarding their responsiveness to inflammatory or pathogenic

stimuli in order to implement an immune reaction after injury. Furthermore, not much is

known on how leukocytes may be involved in disturbed coagulation after trauma and

hemorrhage.

Although bleeding is a frequent concomitant condition in trauma patients, there is a lack of

studies assessing the influence of HS on the posttraumatic inflammatory response and

especially its impact on organ damage. For this purpose, reliable and specific organ damage

markers might provide better insight into pathophysiological processes particularly before

the onset of organ dysfunction. Given the central role that a loss in integrity of the endothelial

glycocalyx layer seems to play in posttraumatic coagulopathy and mortality, assessing the

extent of damage inflicted on the glycocalyx after traumatic hemorrhage could also support

an early recognition of barrier breakdown preceding loss of function with improved and

personalized treatment options for the severely injured. However, immune and organ

monitoring during the early response after severe trauma is not yet established neither

scientifically nor clinically.

Introduction

7

1.6 Aims of the study

The following hypotheses were tested in the course of this thesis:

Leukocyte surface molecules interacting with complement, DAMPs, and coagulation are

altered during the time course after injury.

Using a clinically applicable approach, the cellular immune response to an inflammatory

stimulus can be monitored.

The posttraumatic development of coagulopathy can be observed reliably using

functional thromboelastometry.

The organ injury after severe trauma is aggravated by an additional HS, and this damage

can be assessed by specific serum markers.

HS plays a significant role in the loss in endothelial glycocalyx and barrier function after

PT.

Materials and methods

8

2 Materials and methods

2.1 Consumables

BD FACS Clean BD Biosciences, Heidelberg, Germany

BD FACS Flow BD Biosciences, Heidelberg, Germany

BD FACS Shutdown Solution BD Biosciences, Heidelberg, Germany

Cannula, 25 G B. Braun Melsungen AG, Melsungen,

Germany

Centrifugation tube, 15 ml and 50 ml BD Biosciences, Heidelberg, Germany

Cup & Pin mini tem, Munich, Germany

Filter (0.2 µm pore size) GE Healthycare, Solingen, Germany

Nunc™ MicroWell™ 96-Well plate NUNC A/S, Roskilde, Denmark

Nunc™ MaxiSorp™ 96-Well ELISA plate NUNC A/S, Roskilde, Denmark

Mylar® polyester film, 50 µm Du Pont de Nemur, Bad Homburg,

Germany

Polyethylene catheter (0.62 mm) Föhr Medical Instruments GmbH,

Seeheim/Ober-Beerbach, Germany

Polystyrene tube, round bottom, 5 ml BD Biosciences, Heidelberg, Germany

Reaction tube, 0.5 ml, 1.5 ml and 2 ml Eppendorf, Hamburg, Germany

TruCulture® tube (Null, LPS) HOT Screen GmbH, Germany

S-Monovette® 7.5 ml, Clotting Activator/

Serum

Sarstedt, Nümbrecht, Germany

S-Monovette® 9 ml K3E (EDTA) Sarstedt, Nümbrecht, Germany

S-Monovette® 10 ml 9NC, Citrate 3.2%

(1:10)

Sarstedt, Nümbrecht, Germany

Safety Multifly cannula, 18 G Sarstedt, Nümbrecht, Germany

Silkam® 5/0 suture B. Braun Melsungen AG, Melsungen,

Germany

Syringe, 1 ml BD Biosciences, Heidelberg, Germany

2.2 Buffers and media

Aqua dest. Fresenius Kabi, Bad Homburg, Germany


9

DPBS-/- (Dulbecco’s Phosphate-Buffered

Saline without Ca2+/Mg2+)

Gibco BRL, Grand Island, NY, USA

HBSS+/+ (Hank´s Balanced Salt Solution

with Ca2+/Mg2+)


Jonosteril Fresenius Kabi, Bad Homburg, Germany

Sodium chloride solution (0.9%) Fresenius Kabi, Bad Homburg, Germany

RPMI 1640 (Roswell Park Memorial

Institute-Medium)


2.3 Reagents and chemicals

AffinityScript QPCR cDNA Synthesis Kit Agilent, Santa Clara, CA, USA

Annexin V-Alexa Fluor 647 Invitrogen, Carlsbad, CA, USA

Annexin V binding buffer (10x) BD Biosciences, Heidelberg, Germany

Bovine serum albumin Sigma-Aldrich, Steinheim, Germany

Brilliant III Ultra-Fast SYBR® Green QPCR

Master Mix

Agilent, Santa Clara, CA, USA

Buprenorphine (Temgesic®) Boehringer, Mannheim, Germany

C5a from human serum Complement Technology, Inc., Tyler, TX,

USA

CellFIX (10x, concentrate) BD Biosciences, Heidelberg, Germany

Chloroform (≥ 99%) Sigma-Aldrich, Steinheim, Germany

CountBright™ Absolute Counting Beads Invitrogen, Carlsbad, CA, USA

DEPC-treated water (RNase-free) Invitrogen, Carlsbad, CA, USA

DNase I Invitrogen, Carlsbad, CA, USA

Ethanol (pure) Sigma-Aldrich, Steinheim, Germany

Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich, Steinheim, Germany

Ex-tem Tem, Munich, Germany

FACS Lysing Solution BD Biosciences, Heidelberg, Germany

Fib-tem Tem, Munich, Germany

Ficoll-Paque GE Healthcare, Freiburg, Germany


10

Heparan sulfate Amsbio, Abingdon, UK

Human recombinant C3a Calbiochem Inc., San Diego, USA

Human recombinant interleukin-1 PeproTech, Rocky Hill, NJ, USA

Human recombinant interleukin-6 Biomol, Hamburg, Germany

Human recombinant interleukin-8 Biomol, Hamburg, Germany

Human recombinant syndecan-1 R&D, Wiesbaden-Nordenstadt, Germany

In-tem Tem, Munich, Germany

Isopropanol Sigma-Aldrich, Steinheim, Germany

Latex beads, amine-modified polystyrene,

fluorescent red (1.0 μm)

Sigma-Aldrich, Steinheim, Germany

Latex beads, carboxylate-modified

polystyrene, fluorescent red, (0.5 μm)


Latex beads, polystyrene (0.3 µm) Sigma-Aldrich, Steinheim, Germany

Lipopolysaccharides from Escherichia coli

O55:B5


Mucin from porcine stomach Type II Sigma-Aldrich, Steinheim, Germany

Norepinephrine Sanofi, Frankfurt am Main, Germany

Phorbol 12-myristate 13-acetate Sigma-Aldrich, Steinheim, Germany

Sevofluran (SevoraneTM) Abbott, Wiesbaden, Germany

TRIzol™ Reagent Thermo Fisher Scientific, Waltham, MA,

USA

2.4 Primers

Human PLAUR QuantiTect® Primer Assay QIAGEN, Hilden, Germany

Human SDHA QuantiTect® Primer Assay QIAGEN, Hilden, Germany

Human SERPINE1 QuantiTect® Primer

Assay

QIAGEN, Hilden, Germany

Human TBP QuantiTect® Primer Assay QIAGEN, Hilden, Germany

2.5 Antibodies and isotype controls

Mouse α-human C3aR, PE BioRad, Kidlington, UK


11

Mouse α-human TREM-1, PE BioRad, Kidlington, UK

Mouse IgG1 Negative Control, PE BioRad, Kidlington, UK

Mouse α-human C5L2, PE BioLegend, San Diego, CA, USA

Mouse IgG2a, κ Isotype Control, PE BioLegend, San Diego, CA, USA

Mouse α-human CD66b, FITC BD Biosciences, Heidelberg, Germany

Mouse IgM, κ Isotype Control, FITC BD Biosciences, Heidelberg, Germany

Mouse α-human CD88, FITC BioRad, Kidlington, UK

Mouse IgG2a Negative Control, FITC BioRad, Kidlington, UK

Mouse α-human CD142/Tissue factor, FITC BioRad, Kidlington, UK

Mouse IgG1 Negative Control, FITC BioRad, Kidlington, UK

Mouse α-human CD282 (TLR2), PE BioRad, Kidlington, UK

Mouse α-human CD284 (TLR4), PE BioRad, Kidlington, UK

Mouse IgG2a Negative Control, PE BioRad, Kidlington, UK

Mouse α-human PAR-2, PE Santa Cruz Biotechnology, Heidelberg,

Germany

Mouse IgG2a κ Isotype Control, PE Santa Cruz Biotechnology, Heidelberg,

Germany

Mouse α-human TCC, FITC Hycultec, Beutelsbach, Germany

Mouse IgG2a Isotype control, FITC Hycultec, Beutelsbach, Germany

Mouse α-human Thrombomodulin, FITC Abcam, Cambridge, UK

Mouse IgG1 Isotype Control, FITC Abcam, Cambridge, UK

Rabbit α-human PAI-1, FITC Bioss, Woburn, MA, USA

Rabbit α-human RAGE/AGER, FITC Bioss, Woburn, MA, USA

Rabbit IgG Isotype Control, FITC Bioss, Woburn, MA, USA

2.6 Immunoassays

2.6.1 Multiplex immunoassay

Bio-Plex Pro™ Human Cytokine 27-plex

Assay

BioRad, Hercules, CA, USA


12

2.6.2 ELISA kits for human proteins

ADM / Adrenomedullin ELISA Kit LifeSpan BioSciences, Seattle, WA, USA

Angiopoietin-2 DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

C-Reactive Protein/CRP Quantikine ELISA

Kit

R&D, Wiesbaden-Nordenstadt, Germany

CC16 (Clara Cell 16kD protein) ELISA Kit Elabscience, Bethesda, MD, USA

Claudin-5(CLDN5) ELISA kit Cusabio, College Park, MD, USA

FABP2/I-FABP DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

HS (Heparan Sulfate) ELISA Kit Elabscience Biotechnology Co., Houston,

TX, USA

IL-6 ELISA Set BD Biosciences, Heidelberg, Germany

L-FABP ELISA kit Hycultec, Beutelsbach, Germany

Lipocalin-2/NGAL DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

MUC2 ELISA Kit (Human) Aviva Systems Biology, San Diego, CA,

USA

Serpin E1/PAI-1 DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

Serum Albumin DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

Sphingosine 1 Phosphate ELISA Kit MyBiosource, San Diego, CA, USA

Syndecan-1 DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

Total MMP-9 DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

Total MMP-13 DuoSet ELISA R&D, Wiesbaden-Nordenstadt, Germany

2.6.3 ELISA kits for murine proteins

ALB/Serum Albumin ELISA Kit LifeSpan BioSciences, Seattle, WA, USA

Claudin-5 (CLDN5) ELISA kit Cusabio, College Park, MD, USA

Hspg2 (Basement membrane-specific

heparan sulfate proteoglycan core protein)

ELISA Kit

Amsbio, Abingdon, UK

MMP9/Gelatinase B ELISA Kit LifeSpan BioSciences, Seattle, WA, USA

MMP13 ELISA Kit LifeSpan BioSciences, Seattle, WA, USA


13

SDC1/Syndecan 1/CD138 ELISA Kit LifeSpan BioSciences, Seattle, WA, USA

THBD/CD141/Thrombomodulin ELISA Kit LifeSpan BioSciences, Seattle, WA, USA

2.7 Devices

Anesthesia device Vapor Drägerwerk AG, Lübeck,

Germany

Bio-Plex® 200 platform BioRad, Hercules, CA, USA

Blood pressure analyzer Data Sciences International, New

Brighton, MN, USA

FACSCanto II BD Biosciences, Heidelberg, Germany

Mx3000P QPCR System Agilent, Santa Clara, CA, USA

Vial Program 1 syringe pump BD Medical, Heidelberg, Germany

Qubit 2.0 Thermo Fisher Scientific, Waltham, MA,

USA

ROTEM® delta Tem, Munich, Germany

Tecan Sunrise Reader Tecan, Crailsheim, Germany

Temperature plate Föhr Medical Instruments, Seeheim/Ober-

Beerbach, Germany

Test tube rotator Snijders, Tilburg, Netherlands

ThermoMixer C Eppendorf, Hamburg, Germany

2.8 Software

BD FACSDivaTM Software (Version 6.1.2) Becton Dickinson, Heidelberg, Gemany

FlowJo (Version 10.2) FlowJo, LLC, Ashland, OR, USA

Ponemah (Version 5.0) Data Sciences International, New

Brighton, MN, USA

SAS (Version 9.3) SAS, Cary, NC, USA

SigmaPlot (Version 11.0) Systat Software, Erkrath, Germany

SigmaStat (Version 3.5) Systat Software, Erkrath, Germany

XFluor4 (Version 4.51) Tecan, Crailsheim, Germany


14

2.9 PT study design at Ulm University Hospital

A prospective clinical study was conducted in 12 patients after severe PT (ISS ≥ 32) that

were admitted to the University Hospital Ulm between December 2013 and May 2015. The

study protocol was approved by the Independent Local Ethics Committee of the University

of Ulm (approval numbers 244/11 and 94/14). The study was registered on

ClinicalTrials.gov, identifiers NCT00710411 and NCT02682550, and was performed in

accordance with the Declaration of Helsinki and its recent modifications. Exclusion criteria

were age < 18 years, pregnancy, infection with the human immunodeficiency virus,

cardiogenic shock as the primary underlying disease, underlying hematologic disease,

cytotoxic therapy given within the previous 6 months, and the presence of rapidly

progressing underlying disease anticipating death within the next 24 h. Whole venous blood

was drawn at the time of admission (0 h) and 4 h, 12 h, 24 h, 48 h, 5 d and 10 d after trauma

with a maximum divergence from time points of ± 10%. Seven healthy volunteers served as

a control group. Before inclusion, written informed consent was obtained from all patients

and volunteers; if the patient was incapable of making decisions because of intubation,

sedation or altered mental status, informed consent was obtained directly after recovery or

from the next of kin.

2.10 Plasma and serum collection

At all seven time points after injury, blood was drawn into serum, ethylenediaminetetraacetic

acid (EDTA) and sodium citrate tubes and transported on ice. Tubes with EDTA-chelated

and citrated blood were centrifuged immediately at 800 x g and 4°C for 10 min; supernatants

were centrifuged again at 16,000 x g at 4°C for 2 min to remove platelets. For serum

collection, blood was allowed to coagulate at 4°C for 30–120 min; tubes were centrifuged at

1,560 x g at 4°C for 10 min. All supernatants were aliquoted on ice and stored at –80°C until

further analysis.

2.11 Flow cytometric analysis of whole blood

To assess changes in surface molecules related to complement activation, modulation of

coagulatory processes and sensing of DAMPs and PAMPs on leukocytes, leukocytes in

whole blood were analyzed by flow cytometry during the course after trauma and compared

to healthy volunteers. 100 µl of EDTA-anticoagulated blood were stained for the cellular

receptors for the complement component 5a (C5a, C5aR1 and C5aR2), the terminal

complement complex (TCC), plasminogen activator inhibitor-1 (PAI-1), thrombomodulin,

receptor for advanced glycation end products (RAGE), toll-like receptors 2 and 4


15

(TLR-2/-4), and triggering receptor expressed on myeloid cells (TREM) using fluorescence-

labeled antibodies. Respective IgG isotype controls were used to correct for unspecific

binding. After staining at room temperature in the dark for 20 min, erythrocyte lysis was

performed for 12 min using FACS Lysing Solution. Cells were pelleted at 340 x g for 5 min,

washed with phosphate-buffered saline (PBS), resuspended in 100 µl CellFix and stored at

4°C for no more than 8 h until the analysis by flow cytometry. Light scatter characteristics

were used to distinguish granulocytes, lymphocytes, and monocytes, and at least 2,000 cells

of each population were recorded. The mean fluorescence intensity (MFI) for the cell

populations was analyzed using FlowJo (version 10.2, FlowJo, LLC, Ashland, OR, USA).

2.12 TruCulture® assay

Whole blood ex vivo stimulation was performed using TruCulture® tubes with blood taken

4 h, 24 h and 5 d after injury. Tubes were prefilled by the manufacturer under standardized

conditions with 2 ml culture medium, and contained unfractionated heparin as an

anticoagulant at a final concentration of 50 IU/ml. To assess the immune reaction to a

bacterial stimulus, tubes with or without 100 ng/ml lipopolysaccharide (LPS, from

Escherichia coli, O55:B5, or Null) were used. The TruCulture® system minimizes the risk

of contamination, but also reduces intra-individual variation by direct blood withdrawal

without additional pipetting steps [117]. Tubes were stored at –20°C and thawed in a water

bath at 37°C immediately before use. 1 ml of blood was drawn into blood collection tubes

from healthy volunteers and PT patients at indicated time points. After incubation at 37°C

for 24 h, sedimented cells were separated from the supernatant using a valve, and

supernatants were stored at –80°C until analysis. In order to increase clinical applicability,

a second set of tubes ± LPS was taken at the second time point (24 h after trauma), and

incubation at 37°C was reduced to 4 h before supernatant collection. Samples were analyzed

using a multiplexed sandwich immunoassay on a multiplex platform according to the

manufacturer’s recommendations. Since many samples were out of range for the pro-

inflammatory marker interleukin (IL)-6, they were re-assessed using the IL-6 Quantikine kit

(BD Biosciences, Germany). Concentrations of cytokines after LPS stimulus in supernatants

from PT patients were compared with those from healthy volunteers. To evaluate the shorter

incubation period, the differences in released mediators in stimulated and control samples

after 4 h incubation were compared to those after 24 h incubation.


16

2.13 PAI-1 plasma detection

PAI-1 concentrations in EDTA-plasma of PT patients and healthy volunteers were measured

using a commercially available enzyme-linked immunosorbent assay (ELISA) kit strictly in

accordance to the manufacturer’s protocol (R&D Systems and BD Biosciences,

respectively).

2.14 Neutrophil isolation

Venous blood from healthy volunteers was drawn into tubes containing 3.2% sodium citrate

from the antecubital vein. After mixing the blood with an equal volume of isotonic saline

(0.9% NaCl), 20 ml were layered carefully over 10 ml of Ficoll in a 50 ml centrifugation

tube and cells were separated at 340 x g for 30 min with slow brakes. The supernatant

containing plasma, mononuclear cells and Ficoll was removed, and the remaining pellet was

mixed with dextran in 0.9% NaCl at a final concentration of 1%. After sedimentation of

erythrocytes for 30 min, the supernatant was collected and neutrophils were pelleted by

centrifugation at 340 x g for 5 min. The remaining erythrocytes were lysed with distilled

water, followed by addition of 2.7% NaCl to restore isotonic conditions. Neutrophils were

pelleted and resuspended in medium at 5–10*106/ml.

2.15 PAI-1 surface detection on stimulated neutrophils

For surface staining, freshly isolated neutrophils were resuspended in RPMI 1640 + 0.1%

bovine serum albumin (BSA) at 5*106/ml. After addition of LPS (5 µg/ml) or the so-called

polytrauma cocktail (PTC) [65], containing complement component 3a (C3a, 500 ng/ml),

C5a (10 ng/ml), IL-1 (200 pg/ml), IL-6 (500 pg/ml), and IL-8 (150 pg/ml), cells were

incubated at 37°C for 1 h while rotating at 80 rpm to simulate blood circulation. After

centrifugation, 5*105 cells were stained with anti-PAI-1 antibody or the isotype control at

4°C for 20 min, followed by washing and fixation. Measurement of PAI-1 surface binding

on at least 10,000 cells was performed by flow cytometry and analyzed using FlowJo.

2.16 Gene expression analysis in neutrophils

For messenger RNA (mRNA) isolation, neutrophils isolated from citrated blood were

adjusted to 107/ml in Roswell Park Memorial Institute (RPMI) medium + 0.1% BSA and

stimulated as described in 2.15. After pelleting at 800 x g for 5 min at 4°C, 2*107/ml cells

per sample were lysed using Trizol, and mRNA was isolated following the manufacturer’s

protocol. In brief, the cell lysate was mixed with chloroform and centrifuged to separate the

aqueous phase containing RNA from the interphase and the phenol-chlorophorm phase

containing DNA and cellular proteins. RNA was precipitated with isopropanol, pelleted,


17

washed with 75% ethanol, dried and solubilized in ribonuclease-free water by incubation at

60°C for 10 min. Until further analysis, RNA was stored at –80°C.

RNA concentrations were determined using the Qubit fluorometer and the respective

quantitation assay, employing a dye that only produces a fluorescent signal when bound to

RNA. Genomic DNA was removed by deoxyribonuclease (DNase) I digestion of 1 µg RNA

per sample. RNA was mixed with 10x DNase I Reaction Buffer, DNase and diethyl

pyrocarbonate water and incubated for 15 min at room temperature. To inactivate the DNase,

EDTA was added to a concentration of 2.5 mM before heating the sample at 65°C for 10 min.

For reverse transcription of 550 ng RNA per sample, the AffinityScript QPCR cDNA

Synthesis Kit was used. After mixing RNA, first strand master mix, oligo(DT) primers and

AffinityScript RT/RNase Block enzyme mixture, samples were incubated at 25°C for 5 min

to allow primer annealing. Complementary DNA (cDNA) synthesis was performed at 42°C

for 15 min and the reaction was terminated at 95°C for 5 min. cDNA was diluted 1:5 with

water and stored at –20°C until further analysis.

Gene expression was measured using the Brilliant III Ultra-Fast SYBR® Green QPCR

Master Mix and commercially available primers in reaction volumes of 20 µl in duplicates

on the Mx3000P real-time quantitative PCR (qPCR) system. Expression was compared to

the neutrophil house-keeping genes succinate dehydrogenase complex, subunit A,

flavoprotein variant (SDHA) and TATA-binding protein [91] employing the 2–ΔΔCT method

[95]. For final analyses, SDHA was used as a reference.

2.17 Detection of surface PAI-1 and microvesicles after whole blood stimulation

In order to analyze membrane-bound molecules on leukocytes in a more physiological

setting, EDTA-anticoagulated blood was drawn from healthy volunteers. Blood was

stimulated with PTC, 5 µg/ml LPS or 20 ng/ml of the protein kinase C activator phorbol 12-

myristate 13-acetate (PMA) [23] at 37°C and rotating at 80 rpm to simulate blood circulation.

Whole blood was stained for PAI-1 for 20 min at room temperature. Following erythrocyte

lysis, cells were washed and fixed and fluorescence was detected by flow cytometry.

To assess microvesicles released by neutrophils during stimulation, plasma was obtained by

centrifugation at 2,200 x g for 15 min, and stored at –20°C. Plasma was mixed with Alexa

Fluor 647-labeled Annexin V, fluorescein isothiocyanate (FITC)-labeled anti-cluster of

differentiation (CD)66b antibody and CountBright™ Absolute Counting Beads in

Annexin V binding buffer (filtered with 0.2 µm pore size). 20 mM EDTA was added as a

control to inhibit Annexin V binding. After incubation for 15 min in the dark, samples were


18

washed with binding buffer and measured using the FACSCanto II flow cytometer. For

compensation, single stain controls were used. Gates were set using latex beads with defined

sizes (0.3 µm, 0.5 µm and 1 µm) and Annexin V-positive microvesicles with a diameter of

up to 1 µm were analyzed. The gating strategy is shown in Fig. 2. For volume control,

measurement was stopped after detecting 1000 counting beads corresponding to 2 µl of

plasma.

Fig. 2: Representative gating strategy for detection of microvesicles in plasma after whole-blood

stimulation. After gating for size according to light scatter characteristics (side scatter area, SSC-A, and

forward scatter area, FSC-A), annexin-V-positive vesicles were selected and mean fluorescence intensity as

well as percentage of fluorescein isothiocyanate (FITC)-positive microvesicles were analyzed. Counting beads

were used for volume control. Here, samples after control (Ctrl) or phorbol 12-myristate 13-acetate (PMA)

stimulus are shown.

2.18 PT study design at Zurich University Hospital

Since the low number of patients in the study Ulm PT group did not allow stratification for

the presence or absence of HS, a randomly selected subcohort of 30 patients from a mono-

centered, observational, prospective study at the University Hospital Zurich (Trauma Level

I Center) including a total of 104 patients (ClinicalTrials.gov identifier: NCT02508272,


19

[146,147]) was used. The study was approved by the Cantonal Ethic Commission Zurich

(StV 26–2007) and performed in accordance with local and international guidelines. Patients

with an ISS ≥ 18, age ≥ 18 y, and time passed since the injury < 6 h were included under

informed consent. Blood was drawn upon arrival at the emergency room (day 0) and daily

during the next 21 d.

The subcohort of 30 patients was stratified for signs of manifest HS [88,108,190] on day 0;

the presence of HS was assumed when at least one of the following criteria was fulfilled: a

Trauma Associated Severe Hemorrhage (TASH) score ≥ 10, base excess < −6 mmol/l, lactate

≥ 2.5 mmol/l, and/or red blood cell (RBC) transfusion during the first 24 h > 2 U. Venous

whole blood was drawn into serum or sodium citrate collection tubes and centrifuged at 2000

x g at 4°C for 10 min. Serum concentrations of IL-6, the organ damage markers clara cell

secretory protein (CC16), neutrophil gelatinase-associated lipocalin (NGAL), intestinal fatty

acid-binding protein (I-FABP), and liver-type fatty acid-binding protein (L-FABP), albumin,

sphingosine-1-phosphate, thrombomodulin, angiopoietin-2, matrix metalloproteinases

(MMP)-9 and -13, claudin-5, vascular endothelial (VE-) cadherin, syndecan-1, heparan

sulfate and mucin-2 were determined serially using commercially available sandwich ELISA

kits. After determining optimal dilution conditions, samples were incubated on ELISA plates

over night at 4°C; afterwards, samples were transferred to the next plates and ELISAs were

performed according to the manufacturers’ recommendations, allowing measurement of up

to 4 parameters in one sample. According to a previous publication, serial ELISA

measurements produce valid and reliable data [127]. Concentrations of fibrinogen, D-dimer,

soluble TCC and C5a in citrated plasma were determined by respective commercial

sandwich ELISA kits. To correct for dilution effects after mass transfusion, absolute serum

protein was determined using a bicinchonic acid assay (Pierce). Measurements in patients

with signs of HS were compared to those without shock; six healthy volunteers served as

controls.

2.19 Rotational thromboelastometry analysis

Due to the low sample volume available from PT patients and a required volume of 310 µl

for common rotational thromboelastometry (ROTEM®) analyses, small-volume ROTEM®

was established in collaboration with Prof. van Griensven, Munich; employing smaller cups

and pins allowed a reduction of the sample volume to 105 µl per analysis. To avoid variations

in measurements due to long time periods between patient inclusions, citrate plasma from

patients included in the Ulm PT study was stored at –80°C until analysis. For analysis,


20

plasma was pipetted into the cups, recalcified and mixed with thromboplastin as inducer of

coagulation via the extrinsic pathway. A pin was inserted into the cup and rotated while

detecting resistance by clot formation (Fig. 3). Reagents for extrinsic activation of

coagulation without (extem) and with (fibtem) inhibition of thrombocyte activation by

cytochalasin D were used. Measurements were performed on a ROTEM® delta device.

Results were compared to ROTEM® measurements of plasma from healthy volunteers.

Fig. 3: Exemplary depiction of a rotational thromboelastometry (ROTEM®) measurement with central

readout parameters. By courtesy of Instrumentation Laboratory, Bedford.

2.20 Testing of endothelial glycocalyx and intestinal mucus components in

rotational thromboelastometry

To assess whether dilution by volume replacement therapy after HS increases the

anticoagulatory potency of glycocalyx and mucus components, blood was drawn from

healthy volunteers and the respective hematocrit was determined by centrifugation at 340 x g

for 5 min. The hematocrit was adjusted to 33% or 25% with Jonosteril, and blood samples

with different hematocrit were incubated with heparan sulfate, human recombinant

syndecan-1 and mucin-2 for 30 min at room temperature. The activity of the intrinsic

pathway (in-tem test) was analyzed using a ROTEM® delta device.

2.21 Murine model of PT and HS

The murine PT model with and without HS published by Denk et al. [36] was performed in

adherence to national and international guidelines for the use of laboratory animals. Animals

were housed in groups of five in individually ventilated cages Type II long, with unrestricted

access to food and water. The study protocol was approved by the Federal Authorities for

Animal Research (Tübingen, Germany) under approval number 1194. 32 male C57BL/6


21

mice at the age of 8–9 weeks (Charles River WIGA GmbH, Sulzfeld, Germany) with a mean

body weight of 25 g (± 2.5 g) were randomly assigned to sham treatment, PT, HS, or

combined PT with HS. Anesthesia was induced by inhalation of 2.5% sevoflurane in oxygen

in a tube for narcosis induction and maintained during the experiment after transferring

animals to a face mask. Mice received 0.03 mg/kg body weight buprenorphine

subcutaneously for analgesia. Animals were placed on a feed-back loop temperature control

device with connected rectal probe to maintain a core temperature of 37°C. Chest and upper

abdomen, legs and head were shaved. Sham animals were catheterized and monitored, but

did not undergo trauma or HS. An overview of the study protocol is shown in Fig. 4.

Fig. 4: Overview of the study protocol. Animals in deep anesthesia underwent polytrauma including thoracic

trauma (TXT), traumatic brain injury (TBI), a closed fracture and soft tissue injury. After instrumentation, HS

was applied to achieve a mean arterial pressure (MAP) of 30 mmHg. MAP, heart rate (HR) and temperature

were monitored until termination of the experiment 240 min after trauma. From Denk et al. [36].

2.21.1 Trauma application

PT was induced by combining blunt bilateral chest trauma, traumatic brain injury and femur

fracture/soft tissue trauma. Animals were fixated in a supine position, and blunt chest trauma

was induced using a blast wave generator as established by Knöferl et al. [84]. Mice were

positioned 1.6 cm below the generator’s nozzle with its lower edge in alignment with the

costal margin. The upper section of the nozzle served as pressure reservoir which was

connected to a storage tank of compressed air and separated from the lower opening by a 50-

µm polyester film. The film would burst when the pressure in the upper part, created by

opening the storage tank, exceeded 17 bar, and generate a reproducible single blast wave

with a duration of 1−2 ms. The described protocol induces local and systemic inflammation,

but does not cause injuries of the bony thorax or of abdominal organs [83,84,94,133,134].


22

For induction of traumatic brain injury, animals were positioned on the abdomen and fixated.

The left galea aponeurotica frontoparietal was incised with a scalpel and the scalp was

exposed. A weight of 333 g was dropped on the cranial vault of the left hemisphere from a

height of 2.0 cm, inducing a contusion of the underlying brain tissue without injuring the

bony lamina interna [157].

A closed fracture was applied on the center of the right femur using a weight-drop device

modified from Bonnarens and Einhorn [10]. After positioning of the limb on the device, a

weight of 50 g was dropped from 120 cm. The resulting dynamic impulse induced a dynamic

three-point bending, leading to a transverse diaphyseal fracture and injury of the surrounding

soft tissue.

2.21.2 Induction of pressure-controlled HS

After trauma application, mice were instrumented aseptically by insertion of an arterial

polyethylene catheter (diameter 0.62 mm) in the distal left femoral artery, using a minimally

invasive dissection technique. The arterial catheter was connected to a device for blood

pressure monitoring. The same technique was employed to insert a catheter into the left

jugular vein for volume replacement and continuous infusion of norepinephrine regulated

by a perfusor. Hemorrhage was induced by controlled bleeding via the arterial catheter until

a mean arterial pressure of 30 mmHg ± 5 mmHg was reached; this was maintained over a

period of 60 min. Afterwards, animals were reperfused over the venous catheter with a bolus

of 400 µl Jonosteril over 5 min and per slower infusion of additional Jonosteril until the

fourfold volume of the blood drawn during hemorrhage had been given. Animals in the sham

or PT groups only received the bolus of 400 µl. After HS, mice were monitored for 120 min

after a standardized protocol, maintaining a mean arterial pressure of 50 mmHg by reducing

the depth of anesthesia or by norepinephrine support (0.01−0.12 µg/kg per min) via the

venous catheter. 240 min after PT or sham treatment, animals were exsanguinated by

thoracotomy and cardiac puncture. Blood was collected in EDTA tubes; plasma was

obtained by centrifugation at 800 x g and 4°C for 5 min; supernatants were centrifuged at

13,000 x g and 4°C for 2 min to remove platelets and stored at −80°C for further analyses.

2.21.3 Plasma analysis

Mouse plasma was analyzed using commercially available sandwich ELISA kits for serum

albumin, claudin-5, heparan sulfate proteoglycan core protein, MMP-9 and -13, syndecan-1

and thrombomodulin following the manufacturers’ recommendations.


23

2.22 Statistical analyses

Experimental results were compared using paired t-test for two groups or one-way analysis

of variance (ANOVA) followed by Student-Newman-Keuls post-hoc test for three or more

groups. Correlation analyses were performed using Pearson Product Moment Correlation.

Differences in leukocyte cytokine secretion after endotoxin stimulus were compared by

Student’s t-test. Clinical parameters of patients with and without HS were compared using

the Chi-square test in case of categorical variables and Student’s t-test in case of continuous

parameters. These statistical analyses were performed using SigmaPlot. To evaluate whether

the presence or absence of HS significantly changed overall plasma values of barrier and

organ molecules during the time course after trauma, repeated-measures ANOVA was used

employing SAS. For all ANOVA testing, no formal statistical test on normality was applied

due to its limited validity regarding the available sample size [140]. Results are presented as

mean ± standard error of the mean. A p-value < 0.05 was considered statistically significant.

Results

24

3 Results

3.1 Alterations in leukocyte surface molecules after trauma and correlation to

shock parameters

In order to analyze whether a severe trauma was reflected by changes in the surface

expression patterns of central receptors and molecules interacting with DAMPs/PAMPs,

complement, and coagulation, whole blood was stained during the course after injury and

the surface expression was measured by flow cytometry. Expression levels were then

correlated to clinical parameters with focus on hemorrhagic shock.

3.1.1 Complement receptors and terminal complement complex

C5aR1 expression was significantly lower on neutrophils and monocytes of injured patients

especially during the late phase after trauma as detected by flow cytometry (Fig. 5).

Fig. 5: Complement component 5a receptor 1 (C5aR1) on leukocytes after trauma. C5aR1 mean

fluorescence intensity (MFI) on A, neutrophils, B, lymphocytes, and C, monocytes in whole blood from healthy

volunteers (Healthy) or patients after polytrauma (PT). *, p < 0.05; #, p < 0.05 compared to Healthy; &, p < 0.05

compared to 0 h after PT; n = 7-12.

A

Time after trauma [h]

0 48 96 144 192 240

C5

aR

1 [

MF

I, a

rbit

rary

un

its

]

0

200

400

600

800

1000

1200PT

Healthy

##

# &


0 48 96 144 192 240

C5

aR

1 [

MF

I, a

rbit

rary

un

its

]

0

20

40

60

80

100PT

Healthy


0 48 96 144 192 240

C5

aR

1 [

MF

I, a

rbit

rary

un

its

]

0

100

200

300

400

500

600

PT

Healthy

**

#

A B

C

Neutrophils Lymphocytes

Monocytes

Results

25

Similarly, C5aR2 was reduced on neutrophils 10 d after injury (Fig. 6 A), and demonstrated

a significant decrease already after 4 h on monocytes when compared to controls (Fig. 6 B).

Of note, expression of both C5a receptors was considerably lower on lymphocytes compared

to granulocytes and monocytes (Fig. 6 C).

Fig. 6: Complement component 5a receptor 2 (C5aR2) on leukocytes after trauma. C5aR2 mean

fluorescence intensity (MFI) on A, neutrophils, B, lymphocytes, and C, monocytes in whole blood from healthy

volunteers (Healthy) or patients after polytrauma (PT). #, p < 0.05 compared to Healthy; &, p < 0.05 compared

to 0 h after PT; n = 4-7.


0 48 96 144 192 240

C5a

R2 [

MF

I, a

rbit

rary

un

its]

0

500

1000

1500

2000PT

Healthy

&


0 48 96 144 192 240

C5a

R2 [

MF

I, a

rbit

rary

un

its]

0

100

200

300

400PT

Healthy


0 48 96 144 192 240

C5a

R2 [

MF

I, a

rbit

rary

un

its]

0

500

1000

1500

2000

PT

Healthy

#

A B

C


Monocytes

Results

26

On neutrophils and lymphocytes, TCC could not be detected on the cell surface neither in

healthy volunteers nor in trauma patients. In contrast, on monocytes, considerable membrane

formation of the TCC was measured, which, after an early slight reduction, increased

significantly in PT patients 24 h and 48 h after injury, and returned to baseline levels on days

5 and 10 (Fig. 7).

Fig. 7: Terminal complement complex (TCC) on monocytes. Mean fluorescence intensity (MFI) on

monocytes in whole blood from healthy volunteers (Healthy) or patients after polytrauma (PT). *, p < 0.05; #,

p < 0.05 compared to Healthy; &, p < 0.05 compared to 0 h after PT; $, p < 0.05 compared to 240 h after PT; n

= 7-12.

The TCC formation at 4 h after injury was significantly associated with the number of

applied erythrocyte concentrates during the first day (Fig. 8).

Fig. 8: Pearson correlation between transfused blood products and terminal complement complex (TCC)

expression on monocytes. Number of applied erythrocyte concentrates during the first day in correlation with

TCC on monocytes as determined by mean fluorescence intensity (MFI). R, Pearson correlation coefficient.


0 48 96 144 192 240

TC

C [

MF

I, a

rbit

rary

un

its]

0

200

400

600

800

1000

1200

1400PT

Healthy

& &$ $

**

Erythrocyte concentrates [units]

0 2 4 6 8 10

TC

C [

MF

I, a

rbit

rary

un

its]

0

100

200

300

400

500

600

700

r = 0.87p = 0.005

Results

27

3.1.2 DAMP and PAMP receptors

While RAGE expression was detectable, but not significantly increased on neutrophils and

monocytes in patients during the later course after PT, there were no alterations in its surface

levels on lymphocytes (Fig. 9).

Fig. 9: Receptor for advanced glycation end products (RAGE) on peripheral leukocytes after trauma.

RAGE mean fluorescence intensity (MFI) on A, neutrophils, B, lymphocytes, and C, monocytes in whole

blood from healthy volunteers (Healthy) or patients after polytrauma (PT). N = 7-11.


0 48 96 144 192 240

RA

GE

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400

PT

Healthy


0 48 96 144 192 240R

AG

E [

MF

I, a

rbit

rary

un

its]

0

50

100

150

200

250

300PT

Healthy


0 48 96 144 192 240

RA

GE

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400

500PT

Healthy

A B

C


Monocytes

Results

28

However, RAGE expression on lymphocytes measured at the early time points after injury

correlated significantly with the base excess and the number of transfused erythrocyte

concentrates (Fig. 10 A-C). Furthermore, expression on monocytes 48 h and 5 d post trauma

correlated negatively with blood lactate at admission and positively with erythrocyte

concentrates given during the first day (Fig. 10 D, E).

Fig. 10: Pearson correlation between receptor for advanced glycation end products (RAGE) expression

on leukocytes and clinical parameters. Shown are correlations between initial base excess, the number of

applied erythrocyte concentrates during the first day, and blood lactate concentrations upon admission and

RAGE expression on lymphocytes 0 h (A), 4 h (B), and 12 h (C) after trauma and on monocytes 48 h (D) and

5 d (E) after injury as determined by mean fluorescence intensity (MFI). R, Pearson correlation coefficient.

Base excess [mmol/l]

-6 -4 -2 0 2 4RA

GE

MF

I [a

rbit

rary

un

its]

0

50

100

150

200

250

300

r = 0.64p = 0.04


-6 -4 -2 0 2 4RA

GE

MF

I [a

rbit

rary

un

its]

0

200

400

600

800

r = 0.92p = 0.001


0 2 4 6 8 10RA

GE

MF

I [a

rbit

rary

un

its]

0

100

200

300

400

r = 0.82p = 0.007


0 2 4 6 8 10RA

GE

MF

I [a

rbit

rary

un

its]

0

200

400

600

800

1000r = 0.72p = 0.04

Lactate [mmol/l]

0 1 2 3 4RA

GE

MF

I [a

rbit

rary

un

its]

0

100

200

300

400

500

600

r = 0.74

p = 0.03

A B

C D

E

Results

29

The expression of TLR-2 and TLR-4 was notably decreased on all leukocyte types to around

50% especially during the early time course after trauma, but alterations were not significant

compared to healthy volunteers (Fig. 11).

Fig. 11: Toll-like receptor (TLR) expression on leukocytes after trauma. Mean fluorescence intensity

(MFI) of TLR-2 (A, C, E) and TLR-4 (B, D, F) on granulocytes (A, B), lymphocytes (C, D) and monocytes

(E, F) of healthy volunteers (Healthy) and polytrauma patients (PT). N = 4-12.


0 48 96 144 192 240

TL

R-4

[M

FI, a

rbit

rary

un

its]

0

20

40

60

80

100

120

140

160PT

Healthy


0 48 96 144 192 240

TL

R-4

[M

FI, a

rbit

rary

un

its]

0

20

40

60

80

100

120

140

160PT

Healthy


0 48 96 144 192 240

TL

R-4

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400PT

Healthy


0 48 96 144 192 240

TL

R-2

[M

FI, a

rbit

rary

un

its]

0

20

40

60

80

100PT

Healthy


0 48 96 144 192 240

TL

R-2

[M

FI, a

rbit

rary

un

its]

0

10

20

30

40

50

60PT

Healthy


0 48 96 144 192 240

TL

R-2

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400

500PT

Healthy

A B

C D

E F

Neutrophils

Lymphocytes

Monocytes

Neutrophils

Lymphocytes

Monocytes

Results

30

Nevertheless, TLR-2 on monocytes 4 h and 12 h after injury revealed a significant positive

correlation with the patients’ TASH score upon admission (Fig. 12 A, B). Similarly, TLR-4

on monocytes 12 h after trauma correlated positively with the TASH score (Fig. 12 C).

Several negative correlations were found for TLR-4 expression: at 24 h on granulocytes and

monocytes with base excess, and on lymphocytes with erythrocyte concentrates (Fig. 12

D-F).

Fig. 12: Toll-like receptor (TLR) expression in correlation to clinical parameters. Pearson correlation

between Trauma Associated Severe Hemorrhage (TASH) score, base excess, and the number of applied

erythrocyte concentrates during the first day with TLR-2 (A, B) and TLR-4 (C-F) on monocytes 4 h (A) and

12 h (B, C) after trauma, and on granulocytes (D), monocytes (E), and lymphocytes (F) 24 h after injury as

determined by mean fluorescence intensity (MFI). R, Pearson correlation coefficient.

TASH score [points]

0 2 4 6 8 10 12 14TL

R-2

[M

FI,

arb

itra

ry u

nit

s]

0

100

200

300

400

500

600r = 0.71p = 0.03

TASH score [points]

0 2 4 6 8 10 12 14TL

R-2

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400

500 r = 0.66p = 0.04


-6 -4 -2 0 2 4TL

R-4

[M

FI,

arb

itra

ry u

nit

s]

0

50

100

150

200r = -0.68p = 0.03


-6 -4 -2 0 2 4TL

R-4

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400

500r = -0.7p = 0.03


0 2 4 6 8 10TL

R-4

[M

FI, a

rbit

rary

un

its]

0

20

40

60

80r = -0.72p = 0.02

TASH score [points]

0 2 4 6 8 10 12 14TL

R-4

[M

FI, a

rbit

rary

un

its]

0

100

200

300

400r = 0.68p = 0.03

A B

C D

E F

Results

31

While TREM-1 expression on granulocytes and lymphocytes was unaltered over the time

course after PT (Fig. 13 A, B), monocytes demonstrated some increase in surface TREM-1

already at the time of admission which was significant after 12 h compared to healthy

controls. After 10 d, TREM-1 values had returned to baseline levels (Fig. 13 C).

Fig. 13: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on peripheral leukocytes

during the time course after trauma. TREM-1 detected as mean fluorescence intensity (MFI) on A,

neutrophils, B, lymphocytes, and C, monocytes in whole blood from healthy volunteers (Healthy) or patients

after polytrauma (PT). *, p < 0.05; #, p < 0.05 compared to Healthy; &, p < 0.05 compared to 0 h after PT; n =

4-12.

A B

C


Monocytes


0 48 96 144 192 240

TR

EM

-1 [

MF

I, a

rbit

rary

un

its]

0

1000

2000

3000

4000PT

Healthy


0 48 96 144 192 240

TR

EM

-1[M

FI, a

rbit

rary

un

its

]

0

20

40

60

80PT

Healthy


0 48 96 144 192 240

TR

EM

-1 [

MF

I, a

rbit

rary

un

its

]

0

500

1000

1500

2000

2500

3000

PT

Healthy

#&

**

Results

32

In line with these results, monocyte TREM-1 expression at 4 h post injury correlated

significantly with lactate concentration and the number of transfused erythrocyte

concentrates at admission (Fig. 14).

Fig. 14: Triggering receptor expressed on myeloid cells-1 (TREM-1) correlated to clinical parameters.

Pearson correlation between initial lactate concentration (A) and the number of applied erythrocyte

concentrates during the first day (B) with TREM-1 expression as detected by mean fluorescence intensity

(MFI) on monocytes 4 h after trauma. R, Pearson correlation coefficient.

3.1.3 Molecules interacting with the coagulation system

Thrombomodulin was detectable only on monocytes, and surface expression was

significantly increased 24 h and 48 h after trauma compared to healthy volunteers and values

upon admission (Fig. 15). There were no significant correlations between thrombomodulin

and clinical parameters of shock.


0 48 96 144 192 240

TM

[M

FI, a

rbit

rary

un

its]

0

50

100

150

200

250PT

Healthy#&#&

Fig. 15: Thrombomodulin (TM) expression on monocytes after trauma. TM mean fluorescence intensity

(MFI) on monocytes in whole blood from healthy volunteers (Healthy) or patients after polytrauma (PT). #,

p < 0.05 compared to Healthy; &, p < 0.05 compared to 0 h after PT; n = 7-12.

Lactate [mmol/l]

0 1 2 3 4TR

EM

[M

FI,

arb

itra

ry u

nit

s]

0

500

1000

1500

2000

2500

3000

r = 0.73p = 0.04


0 2 4 6 8 10TR

EM

[M

FI,

arb

itra

ry u

nit

s]

0

500

1000

1500

2000

2500

3000

r = 0.73

p = 0.04

A B

Results

33

On the surface of all leukocytes, PAI-1 was detected in increased amounts after trauma

compared to healthy volunteers. While neutrophils and monocytes demonstrated a

significant increase only 5 d after trauma (Fig. 16 A, C), binding on lymphocytes was

significantly increased 24 h and 48 h compared to emergency room values, and days 5 and

10 only showed some increase compared to controls (Fig. 16 B).

Fig. 16: Surface plasminogen activator inhibitor-1 (PAI-1) on leukocytes during the time course after

trauma. PAI-1 mean fluorescence intensity (MFI) on A, neutrophils, B, lymphocytes, and C, monocytes in

whole blood from healthy volunteers (Healthy) or patients after polytrauma (PT). *, p < 0.05; #, p < 0.05

compared to Healthy; &, p < 0.05 compared to 0 h after PT; n = 7-12.


0 48 96 144 192 240

PA

I-1

[M

FI,

arb

itra

ry u

nit

s]

0

100

200

300

400

500

PT

Healthy

#

***

&


0 48 96 144 192 240P

AI-

1 [

MF

I, a

rbit

rary

un

its

]

0

10

20

30

40

50

60PT

Healthy

#

#

*

*


0 48 96 144 192 240

PA

I-1

[M

FI,

arb

itra

ry u

nit

s]

0

100

200

300

400PT

Healthy&

A B

C


Monocytes

Results

34

PAI-1 on granulocytes (Fig. 17 A) and on lymphocytes (Fig. 17 B, C) correlated significantly

with the initial number of needed erythrocyte concentrates.

Fig. 17: Plasminogen activator inhibitor-1 (PAI-1) in correlation to clinical parameters. Pearson

correlation between the number of applied erythrocyte concentrates during the first day with PAI-1 expression

as detected by mean fluorescence intensity (MFI) on granulocytes 4 h (A) and lymphocytes 4 h (B) and 12 h

(C) after injury. R, Pearson correlation coefficient.

3.2 PAI-1 in PT plasma and ex vivo on the granulocyte surface

Since PAI-1 was detected in increased amounts on all leukocytes especially on day 5 after

injury, soluble PAI-1 was measured in plasma samples to check whether higher plasma

concentrations could induce an increased surface binding. However, plasma PAI-1 was only

increased during the initial days post trauma (Fig. 18), therefore presumably not accounting

for increased surface binding on leukocytes.


0 2 4 6 8 10PA

I-1 [

MF

I, a

rbit

rary

un

its]

0

10

20

30

40

50

60

70r = 0.72p = 0.04


0 2 4 6 8 10PA

I-1 [

MF

I, a

rbit

rary

un

its]

0

50

100

150

200

250

300

350

r = 0.87p = 0.01


0 2 4 6 8 10PA

I-1 [

MF

I, a

rbit

rary

un

its]

0

10

20

30

40

50

60

r = 0.8p = 0.03

A B

C

Results

35


0 48 96 144 192 240P

AI-

1 [

ng

/ml]

0

100

200

300

400PT

Healthy

Fig. 18: Soluble plasminogen activator inhibitor-1 (PAI-1) increase early after trauma. PAI-1 in plasma

from healthy volunteers (Healthy) and from patients after polytrauma (PT). N = 7-12.

To analyze whether in vitro incubation with DAMPs/PAMPs stimuli related to trauma and/or

sepsis could alter the PAI-1 surface pattern similar to the clinical setting, freshly isolated

neutrophils from healthy volunteers were stimulated with PTC or LPS for 1 h and PAI-1 was

determined using flow cytometry. The amount of protein present on the cellular surface was

significantly increased after LPS and only slightly higher after PTC stimulus compared to

the control (Fig. 19 A). The percentage of PAI-1-positive cells was significantly increased

in both stimulated groups (Fig. 19 B).

Fig. 19: Surface plasminogen activator inhibitor-1 (PAI-1) on neutrophils ex vivo. PAI-1 on isolated

neutrophils after stimulation with polytrauma cocktail (PTC), lipopolysaccharide (LPS) or control treatment

(Ctrl) for 1 h. A, mean fluorescence intensity (MFI), and B, percentage of cells positive for PAI-1 as detected

by flow cytometry. *, p < 0.05; n=5-8.

Ctrl PTC LPS

PA

I-1

[M

FI,

arb

itra

ry u

nit

s]

0

100

200

300

400

500*

Ctrl PTC LPS

PA

I-p

os

itiv

e c

ell

s [

%]

0

2

4

6

8

10 **

A B

Results

36

Since isolated neutrophils demonstrate a considerable apoptosis rate after longer incubation

periods (own unpublished data), whole blood anticoagulated with EDTA was used for a

longer incubation protocol. As a positive control for cell stimulation, the protein kinase C

activator PMA was used. In contrast to the results for isolated neutrophils, stimulation of

whole blood by LPS or PTC resulted in no increase in surface PAI-1 expression. Only PMA

was able to induce a significant increase, and this was true for both 1 h (Fig. 20 A, B) and

the prolonged 24 h incubation time (Fig. 20 C, D).

Fig. 20: Surface plasminogen activator inhibitor-1 (PAI-1) on neutrophils after whole-blood stimulation.

Blood was incubated with polytrauma cocktail (PTC), lipopolysaccharide (LPS), phorbol 12-myristate 13-

acetate (PMA) or without stimulant (Ctrl) for 1 h (A, B) and 24 h (C, D). A, C, mean fluorescence intensity

(MFI) on neutrophils and B, D, percentage of neutrophils positive for PAI-1 as assessed by flow cytometry.

***, p < 0.001; n = 3.

To analyze whether this increase in surface PAI-1 on neutrophils stimulated in whole blood

was due to a reduced shedding of microvesicles from the cell membrane, microvesicle

number and origin in plasma were determined using flow cytometry. After 1 h, there were

no differences in the total microvesicle number (Fig. 21 A). Similarly, nor the absolute count

nor the percentage of CD66b-positive microvesicles was altered after either stimulus (Fig.

21 C, E). After 24 h, there was some increase in total microvesicle numbers (Fig. 21 B) and

Ctrl PTC LPS PMA

PA

I-1 [

MF

I, a

rbit

rary

un

its]

0

50

100

150

200

250 **

**

A B

C D

Ctrl PTC LPS PMA

PA

I-1

[M

FI, a

rbit

rary

un

its

]

0

200

400

600

800

1000

1200

1400

1600 ******

***

Ctrl PTC LPS PMA

PA

I-p

osit

ive c

ells [

%]

0

20

40

60

80

100*

***

Ctrl PTC LPS PMA

PA

I-p

osit

ive c

ells [

%]

0

20

40

60

80

100

*****

***

Results

37

a significant elevation in total and relative numbers of CD66b-positive microvesicles after

PMA incubation, but again, there were no changes in samples with PTC or LPS (Fig. 21 D,

F).

Fig. 21: Microvesicles (MV) in supernatant after whole-blood stimulation. Blood was incubated with

polytrauma cocktail (PTC), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) or without

stimulant (Ctrl) for 1 h (A, C, E) and 24 h (B, D, F). A, B, total microvesicles per µl plasma, C, D, number of

CD66b+ MV per µl plasma, and E, F, percentage of CD66b+ MV as assessed by flow cytometry. ***, p < 0.001;

n = 3.

Ctrl PTC LPS PMA

MV

[n

/µl p

las

ma

]

0

2000

4000

6000

8000

10000

Ctrl PTC LPS PMA

MV

[n

/µl p

las

ma

]

0

20000

40000

60000

80000

Ctrl PTC LPS PMA

CD

66

b+

MV

[n

/µl

pla

sm

a]

0

100

200

300

400

Ctrl PTC LPS PMA

CD

66

b+

MV

[n

/µl

pla

sm

a]

0

2000

4000

6000

8000***

******

Ctrl PTC LPS PMA

CD

66

b+

MV

[%

]

0

2

4

6

8

10

12 ******

***

Ctrl PTC LPS PMA

CD

66

b+

MV

[%

]

0

1

2

3

4

5

6

A B

C D

E F

1 h 24 h

Results

38

3.3 Gene expression of PAI-1 and urokinase receptor in stimulated neutrophils

To test the hypothesis that the increased PAI-1 detected on isolated neutrophils after

inflammatory stimuli was due to an increase in gene transcription, mRNA was isolated from

stimulated isolated neutrophils and expression of SERPINE1 (coding for PAI-1) and

PLAUR (coding for the urokinase receptor, uPAR) was analyzed using qPCR. There were

no changes in PAI-1 expression in neutrophils after PTC stimulation for 1 h and 4 h; in

contrast to the surface protein findings, incubation with LPS for 4 h lead to a significant

downregulation of PAI-1 expression (Fig. 22 A). When analyzing the cellular anchor for

PAI-1, uPAR, which is a receptor for urokinase (uPA) and thus facilitates PAI-1 binding, a

slight, but nonsignificant increase in uPAR expression was detected after 1 h in PTC and

LPS samples compared to unstimulated controls. However, the expression was

downregulated in cells after 4 h incubation (Fig. 22 B).

Fig. 22: Expression of plasminogen-activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) in

isolated neutrophils. A, SERPINE1 (coding for Pai-1) and B, PLAUR (coding for uPAR) in neutrophils 1 h

and 4 h after polytrauma cocktail (PTC) or lipopolysaccharide (LPS) stimulation as fold-expression compared

to control treatment (Ctrl) and normalized to the housekeeping gene. **, p = 0.001; ***, p < 0.001; n=2-8.

Results

39

3.4 Thromboelastic alterations in blood after PT

Citrated plasma was used to detect alterations in coagulation in PT patients compared to

healthy volunteers by performing ROTEM® analysis. When comparing measurements from

activation via the extrinsic pathway with (fibtem) and without (extem) inhibition of

thrombocyte activation, results were highly similar (Fig. 23), presumably due to the removal

of thrombocytes during centrifugation steps. For this reason, only results from extem

measurements are shown in the following.

Fig. 23: Analysis of platelet function in plasma samples. Comparison between representative

thrombelastograms from extem (without platelet inhibition, left panel) and fibtem (with platelet inhibition,

right panel) measurements of corresponding plasma samples from a patient 0 h, 4 h, 24 h, and 48 h after trauma.

Results

40

3.4.1 Coagulation activation and clot polymerization

During the first 10 d after PT, clotting time in patients was significantly higher than in

healthy controls, reaching a peak 48 h post injury (Fig. 24 A). Clot formation time was higher

in tendency, but comparison of results was difficult with a partly low n-size due to several

samples not reaching a 20 mm amplitude (Fig. 24 B). The -angle as the angle between the

baseline and the tangent to the clotting curve through the 2-mm point was significantly

reduced in trauma patients during the first 48 h, but then normalized on days 5 and 10 (Fig.

24 C). In line, the clot formation rate as the angle between the baseline and the tangent at

maximum slope was significantly lower at the early time point after trauma and returned to

normal values after 5 d (Fig. 24 D).

Fig. 24: Coagulation activation and clot polymerization after severe injury. Clotting time (A), clot

formation time (B), -angle (C), and clot formation rate (D) in citrated plasma from healthy volunteers (white

bars) and polytrauma patients (grey bars) at indicated time points after trauma. *, p < 0.05; #, p < 0.05 compared

to Healthy; §, p < 0.05 compared to 5 d; $, p < 0.05 compared to 10 d; n = 6-11 (A, C, D), n = 1-8 (B).

A B

C D

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

t fo

rmati

on

rate

[°]

0

20

40

60

80

100

#§$#§#§

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

-An

gle

[°]

0

20

40

60

80

100

#§$ # §$#§$

#§$

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

t fo

rmati

on

tim

e [

s]

0

200

400

600

800

1000

1200

1400

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

ttin

g t

ime [

s]

0

20

40

60

80

100

120

140

160 *

#

#

## # #

Results

41

3.4.2 Clot firmness

Besides analyzing the onset of coagulation, ROTEM® also allows to determine the clot

firmness during the course of measurement. The maximal clot firmness during the 90-min

assay was significantly reduced in patients upon admission to the emergency room compared

to healthy volunteers. The reduction remained present until 48 h after injury, and disappeared

on day 5 (Fig. 25 A). This pattern was already apparent 10 min and 30 min after starting the

assay (Fig. 25 B, C). Time to maximal clot firmness, however, was only slightly increased

in patients 48 h after injury (Fig. 25 D).

Fig. 25: Parameters of clot firmness after polytrauma. Maximal clot firmness (A), clot firmness after 10

min (B) and 30 min (C), and time to maximal clot firmness (D) in citrated plasma from healthy volunteers

(white bars) and polytrauma patients (grey bars) at indicated time points after trauma. *, p < 0.05; #, p < 0.05

compared to Healthy; §, p < 0.05 compared to 5 d; $, p < 0.05 compared to 10 d; n = 6-11.

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

t fi

rmn

ess [

mm

]

0

5

10

15

20

25

30

§$ §$

**

§$

#

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

t fi

rmn

ess [

mm

]

0

5

10

15

20

25

30

§ $

§$

*

§

#

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Clo

t fi

rmn

ess [

mm

]

0

5

10

15

20

25

30

§$§$

**

§$

#

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Tim

e t

o m

axim

al

clo

t fi

rmn

ess [

s]

0

500

1000

1500

2000

A B

C D

Results

42

3.4.3 Clot characteristics

As could be concluded from the lysis index at 30 min, there was no detectable clot lysis in

any of the samples (Fig. 26 A). Maximum velocity as the calculated maximum of the curve’s

first derivative was reduced in patient samples at all time points compared to healthy

volunteers (Fig. 26 B). In line with the results for clot firmness, the shear elastic modulus

strength was significantly reduced as early as upon hospital admission and remained low

until 24 h after trauma, returning to baseline levels after 48 h and even surpassing healthy

values on days 5 and 10 (Fig. 26 C).

Fig. 26: Clot characteristics in polytrauma patients. Lysis index after 30 min (A), maximum velocity (B),

and shear elastic modulus strength (C) in citrated plasma from healthy volunteers (white bars) and polytrauma

patients (grey bars) at indicated time points after trauma. *, p < 0.05; #, p < 0.05 compared to Healthy; §,

p < 0.05 compared to 5 d; $, p < 0.05 compared to 10 d; n = 6-11.

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Maxim

um

velo

cit

y [

mm

/min

]

0

20

40

60

80

# ## #

#

#

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Lysis

in

dex [

%]

0

20

40

60

80

100

120

Healthy 0 h 4 h 24 h 48 h 5 d 10 d

Sh

ear

ela

sti

c m

od

ulu

s s

tren

gth

0

500

1000

1500

2000

2500

*

§$§$

§

A B

C

Results

43

3.5 Cytokine response to bacterial stimulus in an ex-vivo whole blood model

Whole blood from healthy volunteers and from patients at different time points after PT was

incubated with LPS for 24 h and cytokines in the supernatant were analyzed. For all

cytokines, only results in stimulated samples were compared; unstimulated values are shown

as reference.

IL-6 generation was lower by tendency, but not significantly, in PT patients compared to

healthy volunteers (Fig. 27 A). TNF release was significantly reduced in patients 4 h and 5

d after injury (Fig. 27 B). IL-1 production and the IL-1/IL-RA ratio were significantly

decreased in stimulated patient groups at all time points (Fig. 27 C, D).

Fig. 27: Release of pro-inflammatory cytokines in whole blood after lipopolysaccharide (LPS) stimulus.

Interleukin (IL)-6 (A), tumor necrosis factor (TNF, B), and IL-1 (C) concentrations, and ratio between IL-1

and IL-1 receptor antagonist (IL1-RA) concentrations (D) in supernatants after whole blood stimulation with

(+) and without (-) LPS for 24 h. Healthy, healthy volunteers; PT, blood samples taken from polytrauma

patients at indicated time points after trauma. *, p < 0.05 compared to Healthy for analysis of stimulated

samples; unstimulated values are shown as reference. N = 5-7.

Results

44

Fig. 28: Cytokine release in whole blood after lipopolysaccharide (LPS) stimulus. Interferon- (IFN-, A),

interleukin (IL)-10 (B), IL-12 (C), IL-17 (D), eotaxin (E), basic fibroblast growth factor (FGF basic, F),

granulocyte colony-stimulating factor (G-CSF, G), and granulocyte-macrophage colony-stimulating factor

(GM-CSF, H) in supernatants after whole blood stimulation with (+) and without (-) LPS for 24 h. Healthy,

healthy volunteers; PT, blood samples taken from polytrauma patients at indicated time points after trauma.

Analysis was performed for stimulated samples; unstimulated values are shown as reference. N = 5-7.

Results

45

Although there were some alterations in the unstimulated samples after trauma, there were

no significant differences in the production of interferon-IFN- Fig. 28 A), IL-10 (Fig. 28

B), IL-12 (Fig. 28 C) or IL-17 (Fig. 28 D). Similarly, trauma also did not induce any

significant changes in the secretion of eotaxin (Fig. 28 E), basic fibroblast growth factor

(Fig. 28 F), granulocyte-colony stimulating factor (Fig. 28 G), or granulocyte-macrophage

colony-stimulating factor (Fig. 28 H).

In contrast, 24 h after trauma, there was a significantly increased release of IL-2 (Fig. 29 A),

IL-4 (Fig. 29 B), and IL-5 (Fig. 29 C). IL-7 production after LPS stimulus was also

significantly higher in patients 24 h and 5 d after trauma compared to healthy volunteers

(Fig. 29 D). IL-9 was slightly increased in patient samples 4 h and 5 d after trauma, and

significantly higher 24 h after injury (Fig. 29 E). IL-13 was elevated in trauma patients only

by tendency (Fig. 29 F). IL-15 secretion was significantly increased in patients at all time

points compared to healthy volunteers (Fig. 29 G). Monocyte chemoattractant protein 1

release after LPS stimulus was significantly higher in blood samples from patients 4 h and

24 h after injury (Fig. 29 H).

Results

46

Fig. 29: Release of inflammatory mediators in whole blood after lipopolysaccharide (LPS) stimulus.

Interleukin (IL)-2 (A), IL-4 (B), IL-5 (C), IL-7 (D), IL-9 (E), IL-13 (F), IL-15 (G), and monocyte

chemoattractant protein 1 (MCP-1, H) in supernatants after whole blood stimulation with (+) and without (-)

LPS for 24 h. Healthy, healthy volunteers; PT, blood samples taken from polytrauma patients at indicated time

points after trauma. *, p < 0.05 compared to Healthy for analysis of stimulated samples; unstimulated values

are shown as reference. N = 5-7.

Results

47

When comparing a shortened incubation time of 4 h in samples taken 24 h after trauma, there

was a highly similar pattern of cytokine release (Fig. 30).

Fig. 30: Comparison of differences in cytokine concentrations in stimulated and unstimulated samples

after 4 h and 24 h incubation. Data are shown as mean in pg/ml. FGF-basic, basic fibroblast growth factor;

G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor;

IFN- interferon-; IL, interleukin; IL1-RA, IL-1 receptor antagonist; IL-12 (p70), IL-12 heterodimer; IP-10,

interferon gamma-induced protein 10; MCP-1, monocyte chemoattractant protein 1; MIP-1, macrophage

inflammatory protein-1; PDGF-bb, platelet-derived growth factor subunit B; TNF, tumor necrosis factor;

VEGF, vascular endothelial growth factor.

IL-1βIL-1RA

IL-2

IL-4

IL-5

IL-6

IL-7

IL-9

IL-10

IL-12 (p70)

IL-13

IL-15IL-17

Eotaxin

FGF-basic

G-CSF

GM-CSF

IFN-γ

IP-10

MCP-1

MIP-1α

PDGF-bb

TNF-α

VEGF4 h

24 h

Results

48

A comparison of p-values for the difference between stimulated and unstimulated samples

for the two incubation periods is shown in Table 1.

Table 1: Comparison between sample incubation for 4 h or 24 h. Shown are p-values for differences

between stimulated and unstimulated samples.

Parameter p-value LPS vs. Ctrl

4 h incubation 24 h incubation

IL-1 0.018 0.009

IL1-RA 0.002 0.003

IL-2 < 0.001 0.006

IL-4 0.001 0.056

IL-5 0.005 0.010

IL-6 0.006 0.003

IL-7 0.532 0.021

IL-9 0.001 0.011

IL-10 0.002 0.009

IL-12 < 0.001 0.078

IL-13 0.015 0.030

IL-15 0.001 0.004

IL-17 0.003 0.014

Eotaxin 0.001 0.011

FGF basic 0.002 0.040

G-CSF 0.003 0.002

GM-CSF 0.021 0.019

IFN- < 0.001 0.009

IP-10 0.001 0.010

MCP-1 0.069 0.002

MIP-1 0.083 0.012

MIP-1 0.006 0.033

PDGF-bb 0.615 0.145

TNF 0.009 0.038

VEGF 0.003 0.031

Abbreviations: FGF-basic, basic fibroblast growth factor; G-CSF, granulocyte-colony stimulating factor; GM-

CSF, granulocyte-macrophage colony-stimulating factor; IFN- interferon-; IL, interleukin; IL1-RA, IL-1

receptor antagonist; IP-10, interferon gamma-induced protein 10; MCP-1, monocyte chemoattractant protein

1; MIP-1, macrophage inflammatory protein-1; PDGF-bb, platelet-derived growth factor subunit B;

TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

Results

49

3.6 Impact of HS on barrier disturbance and organ dysfunction after PT

Since there were several significant correlations between surface molecules on leukocytes

in our PT cohort and clinical parameters related to the presence or absence of HS (see 3.1),

the influence of an additional HS after severe trauma, especially focusing on inflammation,

vascular barrier and organ function, was analyzed. For this purpose, a PT patient cohort and

a murine model of PT and/or HS were studied.

3.6.1 Parameters of inflammation, barrier and organ dysfunction, and coagulation in

a human PT study and correlation with clinical data

A patient cohort from Zurich University Hospital was stratified for the presence or absence

of HS according to established parameters. The strategy identified 10 patients without and

20 patients with HS. The cohorts had similar injury patterns except for the prevalence of

abdominal trauma. Despite similar ISS values, patients with HS displayed a higher maximal

SOFA score during the course of observation and required a three-fold higher ICU and two-

fold increased in-hospital length of stay. Stratification criteria such as the TASH score,

numbers of packed RBC transfusion, and initial lactate levels were also significantly

different in the two groups. Further clinical parameters and a statistical comparison are

shown in Table 2.

Several inflammatory mediators and parameters related to barrier and organ (dys-)function

were analyzed over the course of the first 5 days after injury employing sandwich ELISAs.

P-values < 0.05 represent a significant difference between patients with compared to those

without HS over the 5-day period.

Results

50

Table 2: Descriptive statistics and comparison of demographic parameters in stratified groups. Statistical

comparison of stratified groups is shown as p-values. Modified from Halbgebauer et al. [60].

Polytrauma without HS

(n=10)

Polytrauma with HS

(n=20)

P-value

Stratification of

hemorrhagic shock

All criteria fulfilled:

base excess ≥ −6 mmol/l

lactate < 2.5 mmol/l

pRBC ≤ 2 units on day 0

TASH score < 10 points

One or more criteria

fulfilled:

base excess < −6 mmol/l

lactate ≥ 2.5 mmol/l

pRBC > 2 units on day 0

TASH score ≥ 10 points

Mean ± SEM Mean ± SEM

Demographics

Age [y] 44.4 ± 6.3 37.6 ± 3.8 0.45

Sex [male/total] 4/10 (40%) 17/20 (85%) 0.011

GCS 11.5 ± 1.5 12 ± 0.9 0.93

Traumatic brain injury (GCS ≤ 12) 4/10 (40%) 7/20 (35%) 0.789

AIS max. 4.6 ± 0.9 4.5 ± 0.8 0.7

AIS head/neck/cervical spine 2.9 ± 0.9 2.25 ± 0.4 0.45

AIS face 0.3 ± 0.3 0.8 ± 0.2 0.2

AIS thorax/thoracic spine 3.3 ± 0.6 3.7 ± 0.3 0.49

AIS abdomen/lumbar spine 1.1 ± 0.4 2.8 ± 0.3 0.01

AIS upper/lower extremity 2.3 ± 0.4 2.8 ± 0.2 0.23

ISS 36.2 ± 6.3 38.7 ± 2.8 0.54

SOFA score initial 4.6 ± 1.2 5.7 ± 0.7 0.4

SOFA score max. 6 ± 1.1 10.5 ± 1 0.009

Outcomes

RISC [% survival] 79.1 ± 10.2 80 ± 5.7 0.52

Survival 3/10 (30%) 4/20 (20%) 0.542

Hospital length of stay [d] 17.2 ± 3.2 34 ± 4.1 0.01

Intensive care unit length of stay [d] 5.9 ± 1.3 19.2 ± 2.8 0.001

Allogenic blood transfusions

TASH score [points] 4.6 ± 0.9 10.5 ± 1.2 0.004

Initial (d0) RBC transfusion [units] 1.1 ± 0.4 7.9 ± 1.5 0.002

Total RBC transfusion [units] 3.8 ± 0.5 18.2 ± 3.5 < 0.001

Massive transfusion rate 0/10 6/20 (30%) 0.053

Infectious complications

Nosocomial infections 4/10 (40%) 15/20 (75%) 0.872

Sepsis 0/10 6/20 (20%) 0.053

Hemostasis and blood gas analysis

Initial base excess [mmol/l] −2.2 ± 0.4 −3.6 ± 0.7 0.38

Initial lactate [mmol/l] 1.5 ± 0.2 2.7 ± 0.3 0.007

Initial Quick [%] 73.5 ± 5.7 53.4 ± 4.1 0.008

Initial aPTT [s] 29.7 ± 1.5 46.1 ± 6.4 0.04

Initial hematocrit [%] 33.5 ± 1.5 26.6 ± 1.7 0.013

Initial temperature [C] 35.1 ± 0.2 35.4 ± 0.3 0.58

P-values < 0.05 are bold.

Abbreviations: AIS, Abbreviated Injury Score; aPTT, abbreviated partial thromboplastin time; GCS, Glasgow

Coma Scale; HS, hemorrhagic shock; ISS, Injury Severity Score; RBC, red blood cells; PT, polytrauma; RISC,

Revised Injury Severity Classification; SOFA score, Sequential Organ Failure Assessment score; TASH score,

trauma associated severe hemorrhage score.

Results

51

IL-6 was higher in PT patients with HS as soon as they arrived at the hospital and further

increased until day 1; while PT only patients almost returned to baseline concentrations after

5 d, IL-6 after PT+HS remained elevated over the entire course of observation (Fig. 31 A).

In contrast, C-reactive protein (CRP) was initially low in both groups, then increased on days

1 and 2. PT+HS patients showed a further elevation of serum CRP while those without HS

had slightly decreased concentrations on day 5 (Fig. 31 B). Complement activation as

reflected by systemic C5a and TCC concentrations was initially low in both cohorts and

gradually increased over the days following trauma. Both C5a and TCC were slightly higher

in trauma patients with HS compared to those without (Fig. 31 C, D).

Fig. 31: Inflammatory markers during the early time course after severe trauma. Concentrations of A,

interleukin-6 (IL-6), B, C-reactive protein (CRP), C, complement activation product 5a (C5a), and D, terminal

complement complex (TCC) in plasma of healthy volunteers (Healthy, n = 6) and polytrauma patients without

(PT, n = 9-10) and with hemorrhagic shock (PT+HS, n = 17-20). Where p < 0.05, plasma values in the HS

group were significantly altered overall compared to PT alone. Modified from Halbgebauer et al. [60].

Time after trauma [d]

0 1 2 3 4 5

IL-6

[p

g/m

l]

0

500

1000

1500

2000PT+HS

PT

Healthy

p = 0.001


0 1 2 3 4 5

CR

P [

µg

/ml]

0

20

40

60

80

100

120

140

160

180PT+HS

PT

Healthy

p = 0.55


0 1 2 3 4 5

C5a

[p

g/m

l]

0

5000

10000

15000

20000PT+HS

PT

p = 0.155


0 1 2 3 4 5

TC

C [

ng

/ml]

0

1000

2000

3000

4000

5000

6000PT+HS

PT

Healthy

p = 0.17

A B

C D

Results

52

To assess the extent of organ dysfunction, markers specific for damage of lungs (CC16),

kidneys (NGAL), intestine (I-FABP) and liver (L-FABP) were analyzed. All showed

significantly higher concentrations in PT+HS patients compared to those with PT only, but

with a very distinct temporal pattern. CC16 was increased starting on day 1 after trauma and

gradually returned to healthy values until day 5 while PT patients demonstrated slightly

elevated concentrations only on day 0 (Fig. 32 A). In contrast, NGAL was initially low in

both groups, but then increased strongly in the PT+HS cohort over the course of observation

and remained high until day 5 (Fig. 32 B). I-FABP was initially high in PT+HS patients and

only slightly elevated in PT only; during the course of observation, the values approached

baseline which was reached on day 3 (Fig. 32 C). L-FABP was initially increased in both

groups; while after PT alone, concentrations returned to levels found in healthy volunteers

already on day 2, they remained elevated until day 5 in PT+HS (Fig. 32 D).

Fig. 32: Organ-damage markers during the early time course after severe trauma. Concentrations of A,

clara cell secretory protein (CC16), B, neutrophil gelatinase-associated lipocalin (NGAL), C, intestinal fatty

acid-binding protein (I-FABP), and D, liver-type fatty acid-binding protein (L-FABP) in plasma of healthy

volunteers (Healthy, n = 6) and polytrauma patients without (PT, n = 9-10) and with hemorrhagic shock

(PT+HS, n = 17-20). When p < 0.05, overall plasma values in the HS group were significantly altered

compared to PT alone. Modified from Halbgebauer et al. [60].


0 1 2 3 4 5

CC

16 [

pg

/ml]

0

200

400

600

800

1000

1200

1400PT+HS

PT

Healthy

p = 0.049


0 1 2 3 4 5

NG

AL

[p

g/m

l]

0

105

2x105

3x105

4x105

PT+HS

PT

Healthy

p = 0.0003


0 1 2 3 4 5

I-F

AB

P [

pg

/ml]

0

2000

4000

6000

8000

10000

12000

14000PT+HS

PT

Healthy

p = 0.02


0 1 2 3 4 5

L-F

AB

P [

pg

/ml]

0

2000

4000

6000

8000

10000

12000

14000 PT+HS

PT

Healthy

p = 0.004

A B

C D

Results

53

In order to determine whether certain clinical parameters revealed an influence on organ

damage, correlation analyses were performed between damage markers and clinical data.

CC16 on day 0 significantly correlated with the total number of transfused erythrocyte

concentrates during the first 21 d (Fig. 33 A), and on day 1, higher plasma concentrations

were associated with a higher fluid balance on day 2 (Fig. 33 B). NGAL on day 1 correlated

significantly with the AIS of the abdomen (Fig. 33 C), and on day 3 with concurrent

procalcitonin concentrations (Fig. 33 D). Upon arrival, I-FABP serum concentrations

correlated with lactate (Fig. 33 E) and with the fluid balance (Fig. 33 F). L-FABP on day 1

correlated negatively with Quick values (Fig. 33 G), and positively with procalcitonin two

days later (Fig. 33 H).

Results

54

Fig. 33: Pearson Product Moment Correlation of plasma concentrations of organ damage markers to

clinical parameters. Correlation analyses were performed for polytrauma patients without (PT) and with

hemorrhagic shock (PT+HS) on indicated days after injury. Significant correlations were found for clara cell

secretory protein (CC16) with A, total applied red blood cell (RBC) concentrates and B, the fluid balance,

neutrophil gelatinase-associated lipocalin (NGAL) with C, the abbreviated injury score (AIS) of the abdomen

and D, procalcitonin (PCT). Intestinal fatty acid-binding protein (I-FABP) concentrations correlated with E,

CC16 d0 [pg/ml]

0 103 2x103 3x103 4x103 5x103

RB

C t

ota

l [u

nit

s]

0

10

20

30

40

50

60

70PT+HS

PT

r = 0.62p = 0.001

NGAL d1 [pg/ml]

105 2x105 3x105 4x105 5x105

AIS

ab

do

men

0

1

2

3

4

5

6

PT+HS

PT

r = 0.66p < 0.001

NGAL d3 [pg/ml]

105 2x105 3x105 4x105 5x105

PC

T d

3 [

µg

/l]

0

2

4

6

8

10PT+HS

PT

r = 0.85p < 0.001

I-FABP d0 [pg/ml]

0 104 2x104 3x104 4x104

Lacta

te d

0 [

mm

ol/l]

0

2

4

6

8

PT+HS

PT

r = 0.71p < 0.001

I-FABP d0 [pg/ml]

0 104 2x104 3x104 4x104

Flu

id b

ala

nce d

0 [

ml]

-2000

0

2000

4000

6000

8000

10000

12000

PT+HS

PT

r = 0.51p = 0.008

L-FABP d1 [pg/ml]

5x103 104 2x104 2x104

Qu

ick d

1 [

%]

0

20

40

60

80

100

120PT+HS

PT

r = -0.45p = 0.01

L-FABP d1 [pg/ml]

5x103 104 2x104 2x104

PC

T d

3 [

µg

/l]

0

5

10

15

20PT+HS

PT

r = 0.80p < 0.001

CC16 d1 [pg/ml]

0 1000 2000 3000 4000

Flu

id b

ala

nce d

2 [

ml]

-2000

0

2000

4000

6000

8000

PT+HS

PT

r = 0.65p < 0.001

A B

C D

E F

G H

Results

55

blood lactate and F, the fluid balance; liver-type fatty acid-binding protein (L-FABP) was significantly

correlated with G, Quick values and H, PCT concentrations. R, Pearson correlation coefficient. Modified from

Halbgebauer et al. [60].

As could be expected from hemodilution conditions by resuscitation protocols, albumin

concentrations were lower in all patients compared to healthy volunteers, but there was no

significant difference between the patient cohorts (Fig. 34 A). Thrombomodulin as

endothelial damage marker was initially lower in patients, but then increased until day 5

especially in PT+HS patients (Fig. 34 B). The lipid mediator sphingosine-1-phosphate was

decreased in all patients compared to controls, but there was no significant difference

between the cohorts (Fig. 34 C). After lower concentrations in patients upon hospital

admission compared to healthy individuals, the vascular growth factor angiopoietin-2

normalized in both groups on day 1. Starting on day 2, there was an increase in serum

concentrations in PT+HS patients, and concentrations remained elevated until day 5 (Fig. 34

D). The vasodilator adrenomedullin was lower in PT only patients compared to controls

during the course of observation. In contrast, PT+HS patients revealed slightly increased

serum concentrations on day 0 which further increased until highest levels were reached on

day 2, then slowly decreasing until day 5 (Fig. 34 E).

Results

56

Fig. 34: Mediators of vascular permeability during the early time course after severe trauma.

Concentrations of A, albumin, B, thrombomodulin, C, sphingosine-1-phosphate, D, angiopoietin-2, and E,

adrenomedullin in plasma of healthy volunteers (Healthy, n = 6) and polytrauma patients without (PT, n = 9-

10) and with hemorrhagic shock (PT+HS, n = 17-20). Where p < 0.05, overall plasma values in the HS group

were significantly altered compared to PT alone. Modified from Halbgebauer et al. [60].


0 1 2 3 4 5

Alb

um

in [

mg

/ml]

0

10

20

30

40

50

60PT+HS

PT

Healthy

p = 0.3


0 1 2 3 4 5

Th

rom

bo

mo

du

lin

[p

g/m

l]

0

1000

2000

3000

4000

5000

6000PT+HS

PT

Healthy


0 1 2 3 4 5

An

gio

po

ieti

n-2

[p

g/m

l]

0

1000

2000

3000

4000PT+HS

PT

Healthy

p = 0.004


0 1 2 3 4 5Sp

hin

go

sin

e-1

-ph

osp

hate

[p

g/m

l]

0

2000

4000

6000

8000

10000

12000PT+HS

PT

Healthy

p = 0.03


0 1 2 3 4 5

Ad

ren

om

ed

ullin

[p

g/m

l]

0

100

200

300

400PT+HS

PT

Healthy

p = 0.047

p = 0.0005

A B

C D

E

Results

57

In both patient cohorts, MMP-9 was strongly increased upon arrival at the emergency room;

concentrations dropped already on day 1, but remained elevated until day 5 compared to

healthy volunteers (Fig. 35 A). MMP-13, however, showed a very different pattern; PT

patients had concentrations comparable to those in controls which only increased slightly on

day 5; levels in PT+HS patients were strongly increased on day 0 and slowly decreased after

that (Fig. 35 B). Claudin-5 as an important tight-junction molecule was initially increased in

both patient cohorts, although more pronounced in PT+HS patients; while concentrations

returned to healthy values in PT patients on day 5, they remained high in PT+HS patients

(Fig. 35 C). VE-cadherin was decreased in both patient cohorts over the observation period

compared to controls, but there was no difference between the cohorts (Fig. 35 D).

Fig. 35: Mediators and components of endothelial intercellular junctions during the early time course

after trauma. Concentrations of A, matrix metalloproteinase (MMP)-9, B, MMP-13, C, claudin-5, and D,

vascular endothelial (VE-) cadherin in plasma of healthy volunteers (Healthy, n = 6) and polytrauma patients

without (PT, n = 9-10) and with hemorrhagic shock (PT+HS, n = 17-20). Where p < 0.05, overall plasma

values in the HS group were significantly altered compared to PT alone. Modified from Halbgebauer et al.

[60].

p = 0.72


0 1 2 3 4 5

VE

-Ca

dh

eri

n [

ng

/ml]

0

50

100

150

200

250

PT+HS

PT

Healthy


0 1 2 3 4 5

MM

P-1

3 [

pg

/ml]

0

500

1000

1500

2000PT+HS

PT

Healthy

p = 0.001


0 1 2 3 4 5

Cla

ud

in-5

[p

g/m

l]

0

200

400

600

800PT+HS

PT

Healthy

p = 0.049


0 1 2 3 4 5

MM

P-9

[n

g/m

l]

0

20

40

60

80PT+HS

PT

Healthy

A B

C D

p = 0.58

p = 0.72

Results

58

Angiopoietin on day 3 correlated significantly with the number of transfused packed

erythrocyte units on day 0 (Fig. 36 A); similarly, higher claudin-5 concentrations on day 2

were associated with a higher number of erythrocyte concentrates (Fig. 36 B). Serum

claudin-5 over the entire observation period significantly correlated with blood lactate levels

(Fig. 36 C).

Fig. 36: Correlation between markers of barrier dysfunction with clinical parameters of shock. Pearson

Product Moment Correlation of angiopoietin and claudin-5 with clinical parameters in polytrauma patients

without (PT) and with hemorrhagic shock (PT+HS) on indicated days after injury. Significant correlations were

found for A, angiopoietin with applied red blood cell (RBC) concentrates, and between claudin-5 plasma

concentrations with B, applied RBC concentrates and C, blood lactate. R, Pearson correlation coefficient.

Modified from Halbgebauer et al. [60].

Syndecan-1 as a key marker for endotheliopathy was significantly higher in PT+HS patients

compared to the PT only cohort beginning on the day of admission, and concentrations

remained strongly elevated. In contrast, PT alone did only lead to increased syndecan-1 on

day 5 compared to healthy volunteers (Fig. 37 A). This result was confirmed when

normalizing to serum protein in order to correct for dilution effects after fluid resuscitation

(Fig. 37 B). The glycocalyx component heparan sulfate was slightly lower, but not

significantly altered in any patient cohort (Fig. 37 C). However, when normalized to serum

protein, PT+HS patients had significantly higher values compared to PT alone which only

Claudin-5 [pg/ml]

0 200 400 600 800 1000

La

cta

te [

mm

ol/l]

0

2

4

6

8d0

d1

d2

d3

d5

r = 0.62p = 0.001

Claudin-5 d2 [ng/ml]

0 500 1000 1500 2000

RB

C d

0 [

un

its]

0

5

10

15

20

25r = 0.62p < 0.001

PT+HS

PT

Angiopoietin d3 [ng/ml]

0 20 40 60 80 100

RB

C d

0 [

un

its

]

0

5

10

15

20

25r = 0.87p < 0.001

PT+HS

PT

A B

C

Results

59

slowly normalized until day 5 (Fig. 37 D). Mucin-2 as major component of the intestinal

mucosa was slightly, but not significantly lower in the PT cohort compared to PT+HS (Fig.

37 E); in contrast, normalization to total protein revealed significantly higher mucin-2

concentrations in PT+HS although levels were similar on day 5 (Fig. 37 F).

Fig. 37: Components of the endothelial glycocalyx and intestinal mucus after trauma. Plasma

concentrations of A, syndecan-1, C, heparan sulfate, and E, mucin-2 in plasma of healthy volunteers (Healthy,

n = 6) and polytrauma patients without (PT, n = 9-10) and with hemorrhagic shock (PT+HS, n = 17-20). B,

syndecan-1, D, heparan sulfate, and F, mucin-2 concentrations after normalization to total plasma protein. For

p < 0.05, overall plasma values in the HS group were significantly altered compared to PT alone. Modified

from Halbgebauer et al. [60].


0 1 2 3 4 5

Syn

de

ca

n-1

[p

g/m

l]

0

1000

2000

3000

4000

PT+HS

PT

Healthy

p < 0.0001


0 1 2 3 4 5

Syn

decan

-1 [

pg

/mg

]

0

20

40

60

80

100

120

140

PT+HS

PT

Healthy

p < 0.0001


0 1 2 3 4 5

He

pa

ran

su

lfa

te [

ng

/ml]

0

200

400

600

800

1000

1200PT+HS

PT

Healthy

p = 0.1


0 1 2 3 4 5

He

pa

ran

su

lfa

te [

ng

/mg

]

0

5

10

15

20

25

30PT+HS

PT

Healthy

p = 0.003


0 1 2 3 4 5

Mu

cin

-2 [

pg

/ml]

0

1000

2000

3000

4000

PT+HS

PT

Healthy

p = 0.07


0 1 2 3 4 5

Mu

cin

-2 [

pg

/mg

]

0

20

40

60

80

100

120

PT+HS

PT

Healthy

p = 0.0005

A B

C D

E F

Results

60

On day 1, higher MMP-13 concentrations correlated significantly with serum heparan sulfate

(Fig. 38 A), and syndecan-1 was significantly associated with the measured maximal SOFA

score (Fig. 38 B). Furthermore, over the entire observation period, syndecan-1 serum

concentrations correlated significantly with L-FABP (Fig. 38 C), NGAL concentrations

(Fig. 38 D), and the abbreviated prothrombin time (Fig. 38 E).

Fig. 38: Correlation analysis of markers of endothelial activation and clinical parameters. Pearson

Product Moment Correlation in polytrauma patients without (PT) and with hemorrhagic shock (PT+HS) on

indicated days after injury. A, correlation of matrix metalloproteinase 13 (MMP-13) with heparan sulfate;

syndecan plasma concentrations correlated to B, the maximal sequential organ failure score (SOFA max.)

during the course of observation, C, liver-type fatty acid-binding protein (L-FAPB), D, neutrophil gelatinase-

associated lipocalin (NGAL), and E, the abbreviated prothrombin time (aPTT). R, Pearson correlation

coefficient. Modified from Halbgebauer et al. [60].

MMP-13 d1 [pg/ml]

0 500 1000 1500 2000 2500

He

pa

ran

su

lfa

te d

1 [

ng

/ml]

0

200

400

600

800

1000

1200

1400

PT+HS

PT

r = 0.53p = 0.003

Syndecan [pg/ml]

0 2x103 4x103 6x103 8x103 104

aP

TT

[s]

0

20

40

60

80

100

120

140d0

d1

d2

d3

d5

r = 0.62p < 0.001

Syndecan d1 [pg/ml]

0 2x103 4x103 6x103 8x103

SO

FA

ma

x.

0

2

4

6

8

10

12

14

16

18

20

PT+HS

PT

r = 0.56p = 0.001

Syndecan [pg/ml]

0 2x103 4x103 6x103 8x103 104

L-F

AB

P [

pg

/ml]

0

104

2x104

3x104

4x104

d0

d1

d2

d3

d5

r = 0.39p < 0.001

Syndecan [pg/ml]

0 2000 4000 6000 8000

NG

AL

[p

g/m

l]

0

2x105

4x105

6x105

8x105

106

1x106

r = 0.4p < 0.001

d0

d1

d2

d3

d5

A B

C D

E

Results

61

Fibrinogen was significantly lower in PT+HS patients compared to the PT cohort, but

concentrations increased in both groups during the observation period (Fig. 39 A). The fibrin

degradation product D-dimer was lower in all patients compared to healthy controls, and

concentrations were significantly lower after HS compared to PT alone (Fig. 39 B). In an

experiment simulating hemodilution in patients after extensive fluid resuscitation,

adjustment of the hematocrit in the blood from healthy volunteers lead to a reduction in D-

dimer concentrations (Fig. 39 C).

Fig. 39: Fibrinogen and fibrin degradation after severe trauma and hemodilution. Plasma concentrations

of A, fibrinogen and B, D-dimer in plasma of healthy volunteers (Healthy, n = 6) and polytrauma patients

without (PT, n = 9-10) and with hemorrhagic shock (PT+HS, n = 13-16). P < 0.05 indicates a significant

alteration of overall plasma values in the HS group compared to polytrauma alone. C, D-dimer concentrations

in plasma from healthy volunteers after hematocrit adjustment.


0 1 2 3 4 5

0.0

0.5

1.0

1.5

2.0

2.5

PT+HS

PT

HealthyD

-dim

er

[µg

/ml]


0 1 2 3 4 5

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og

en

[µ

g/m

l]

0

2000

4000

6000

8000

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PT

Healthy

Adjusted hematocrit [%]

45 33 25

D-d

ime

r [t

urb

ido

me

tric

un

its

]

0.0

0.2

0.4

0.6

0.8

A B

C

p = 0.0004 p = 0.02

Results

62

In a similar experiment, dilution of whole blood from healthy volunteers induced a

significant increase in clotting time (Fig. 40 A) and clot formation time (Fig. 40 B).

Incubation of blood with normal hematocrit with heparan sulfate concentrations comparable

to those detected in patients lead to a significant delay in coagulation. This was also visible,

but not significant, in samples with lower hematocrit (Fig. 40 A). Addition of mucin-2 did

not alter clot formation time in blood with a normal hematocrit, but had an even stronger

effect on clot formation time in samples with the lowest hematocrit (Fig. 40 B).

Fig. 40: Influence of glycocalyx components on coagulation. Clotting time (A) and clot formation time (B)

in rotational thromboelastometry measurements of citrated blood from healthy volunteers with normal (45%,

n = 3-8) or adjusted (33% and 25%, n = 3-5) hematocrit after incubation with heparan sulfate and mucin-2 at

the indicated concentrations; *, p < 0.05 vs. hematocrit 45% without heparan sulfate/mucin-2; #, p=0.005 vs.

hematocrit 25% without mucin-2. Modified from Halbgebauer et al. [60].

Heparan sulfate [ng/ml]

0 100 500 1000

Clo

ttin

g t

ime

[s

]

180

200

220

240

260

280Hematocrit 45%

Hematocrit 33%

Hematocrit 25%

*

$

Mucin-2 [pg/ml]

0 100 1000 5000

Clo

t fo

rmati

on

tim

e [

s]

0

50

100

150

200

250Hematocrit 45%

Hematocrit 33%

Hematocrit 25%

#

$

A B

*

*

Results

63

3.6.2 Barrier disturbances in a murine model of traumatic HS

In order to investigate the impact of HS in trauma in a highly standardized and clinically

relevant model, mice underwent PT or HS or the combination of both; healthy animals

served as controls. Plasma was analyzed 4 h after trauma.

Plasma albumin was increased only in HS animals, but was significantly lower after

additional PT (Fig. 41 A). Thrombomodulin was significantly decreased in both groups

which were exposed to HS (Fig. 41 B).

Fig. 41: Modulators of endothelial permeability in a murine trauma model. Plasma albumin (A) and

thrombomodulin (B) in mice after polytrauma (PT), hemorrhagic shock (HS), combined PT+HS, or in control

(Ctrl) animals. *, p < 0.05; n = 5-8.

MMP-9 concentrations in plasma were significantly higher in PT+HS animals compared to

controls, but also increased to some extent after HS or PT alone (Fig. 42 A). MMP-13

showed the same pattern, but results were not significantly different (Fig. 42 B). Claudin-5

concentrations were similar to healthy values in HS animals and higher by tendency in both

PT groups (Fig. 42 C). Heparan sulfate was slightly, but not significantly increased in all

trauma groups compared to controls (Fig. 42 D). Syndecan-1 was elevated after HS or PT

alone, but only the combination lead to a significant increase compared to healthy animals

(Fig. 42 E).

Ctrl HS PT PT+HS

Alb

um

in [

mg

/ml]

0

10

20

30

40

50

60

*

Ctrl HS PT PT+HS

Th

rom

bo

mo

du

lin

[p

g/m

l]

0

5000

10000

15000

20000

25000

30000

* **

*

A B

Results

64

Fig. 42: Mediators and components of endothelial intercellular junctions in a murine model of severe

trauma. Plasma matrix metalloproteinase (MMP)-9 (A), MMP-13 (B), claudin-5 (C), heparan sulfate (D), and

syndecan (E) in mice after polytrauma (PT), hemorrhagic shock (HS), combined PT+HS, or in control (Ctrl)

animals. *, p < 0.05; n = 5-8.

Ctrl HS PT PT+HS

Cla

ud

in-5

[p

g/m

l]

0

50

100

150

200

250

300

Ctrl HS PT PT+HS

MM

P-9

[n

g/m

l]

0

10

20

30

40*

Ctrl HS PT PT+HS

MM

P-1

3 [

ng

/ml]

0

500

1000

1500

2000

2500

3000

Ctrl HS PT PT+HS

Syn

de

ca

n [

ng

/ml]

0

10

20

30

40*

A B

C D

E

Ctrl HS PT PT+HS

Hep

ara

n s

ulf

ate

[n

g/m

l]

0.0

0.4

0.8

1.2

1.6

Discussion

65

4 Discussion

In the present thesis, the influence of a severe injury on the molecular and cellular “first line”

danger response in peripheral blood and development of coagulopathy in PT patients was

evaluated. Furthermore, the role of an additional HS on endothelial and organ barrier

dysfunction after PT was investigated in a larger patient cohort and in a standardized murine

model of multiple injury. Main results found during the course of this thesis are summarized

in Fig. 43.

Fig. 43: Summary of main findings. Severe trauma leads to early changes in the surface expression of

molecules interacting with coagulation, receptors for complement activation products as well as endogenous

and exogenous danger signals on peripheral leukocytes, resulting in alterations in the intracellular response to

danger signals. Increased inflammation, augmented by an additional hemorrhagic shock, induces the

breakdown of tight junctions and of the endothelial glycocalyx, water efflux into extravascular tissues and

causes organ damage with a distinct spatial and temporal onset pattern which can be monitored using plasma

markers. Heparin-like structures released during cleavage of glycocalyx components can hinder coagulation,

resulting in trauma-induced coagulopathy. C5a, complement component 5 a; C5aR, C5a receptor; CC16, clara

cell secretory protein; IL-6, interleukin-6; I-FABP, intestinal fatty acid-binding protein; L-FABP, liver-type

fatty acid-binding protein; MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin;

PAI-1, plasminogen activator inhibitor-1; sphingosine-1-P, sphingosine-1-phosphate, TCC, terminal

complement complex; TLR, toll-like receptor; TM, thrombomodulin; TREM, triggering receptor expressed on

myeloid cells; uPA, urokinase plasminogen activator; uPAR, uPA receptor. Modified from Halbgebauer et al.

[60].

Discussion

66

4.1 Leukocyte surface profiling in polytraumatized patients

In the first part of this study, the role of leukocytes in the molecular danger response after

severe trauma was investigated. Confirming a previous study in polytraumatized patients

[1], there were significant reductions in the expression of complement receptors on

leukocytes from PT patients, possibly due to microvesicle shedding [171], cleavage from the

cell surface by neutrophil proteases [174], or internalization and subsequent degradation or

recycling processes [160]. Furthermore, TLR-2 and TLR-4 as the main receptors for

bacterial and fungal PAMPs [135,161], but also for endogenous DAMPs such as

extracellular histones and high mobility group box 1 (HMGB-1) [46,186] were rather

decreased on most leukocytes post trauma, possibly causing a reduced responsiveness to

microbial or DAMP stimulus. TREM-1, an amplifier of TLR activation by microbial

components [28], but also involved in sterile inflammation with potential ligands such as

HMGB-1 and heat-shock protein-70 [43,162] was only significantly higher on monocytes,

but mostly unaltered on granulocytes and lymphocytes in trauma patients. Strikingly, there

were several significant correlations of expression levels of RAGE, a multi-ligand receptor

for several endogenous molecules including advanced glycation end products [52,92], TLR-

2, TLR-4, and TREM-1 with parameters related to the presence of a HS, but not with injury

severity (data not shown), prompting the assumption that presence or absence of HS might

alter receptor expression in peripheral leukocytes. However, the relatively small number of

patients prevented us from a more detailed analysis in this regard.

All blood leukocytes were demonstrated to express complement regulatory proteins,

including CD59 as an inhibitor of TCC formation on healthy cells, and to increase their

surface levels in response to PT [1]. This may be the reason for a lack of detectable TCC on

neutrophils and lymphocytes. However, despite considerable amounts of CD59, TCC was

detectable on monocytes and there was a significant increase in patients compared to early

time points. Furthermore, early TCC surface levels correlated strongly with the number of

erythrocyte concentrates transfused during the first day after admittance, suggesting

increased complement activation after severe blood loss and extensive reperfusion therapy.

Sublytic amounts of TCC have been demonstrated to activate the inflammasome in

endothelial cells [168]. Further in vitro experiments will clarify whether this mechanism is

similar in monocytes and may induce a pro-inflammatory cellular phenotype.

Thrombomodulin is mainly expressed by endothelial cells; binding of thrombin strongly

reduces cleavage of fibrinogen and thereby clot formation, inhibits activation of endothelial

Discussion

67

thrombin receptors which may decrease vascular leakage, and enhances activation of

protein C with several anti-inflammatory and anti-coagulatory features [45,72,173].

Detection of thrombomodulin on monocytes and especially the strongly increased surface

expression in traumatized patients indicate a modulatory function of monocytes during

excessive inflammation and coagulation.

PAI-1 as the most potent inhibitor of fibrinolysis was bound to the surface of all leukocytes

and levels increased significantly especially during the later course of injury, suggesting that

peripheral leukocytes might be involved in altered coagulation and an increased risk of

thrombosis in severely injured patients [21]. In line, higher PAI-1 surface levels on

granulocytes and lymphocytes correlated with the numbers of given erythrocyte

concentrates, suggesting that patients with a higher blood loss showed higher binding of

PAI-1 on the leukocyte surface. In order to evaluate several possible causes for the

aforementioned increase in membrane-bound PAI-1 around day 5 after trauma,

concentrations in plasma from patients and healthy volunteers were measured. There was a

strong increase in circulating PAI-1 likely due to an increased expression in the liver [136]

during the early phase, but not in the later course after trauma, therefore presumably not

accounting for the increased surface detection on leukocytes. In a clinical study, a decrease

in active PAI-1 upon admission was detected in the 10% of patients after trauma with signs

of severe hyperfibrinolysis [22], which did not seem to be the case in our cohort. A more

recent study suggests that excessive tissue plasminogen activator release and complexation

of PAI-1 may be responsible for initial hyperfibrinolysis and trauma-induced coagulopathy

[25]. Of note, these studies only analyzed clotting disturbances in the very early phase and

did not monitor coagulation during the later time course after injury. As a next step, isolated

neutrophils were stimulated with LPS or a combination of inflammatory mediators (C3a,

C5a, IL-1, IL-6, IL-8) in trauma-relevant concentrations [65] in order to simulate the pro-

inflammatory conditions in severely injured patients, resulting in a fast increase in PAI-1

detectable on the cell surface. However, after stimulating cells in a more complex setting

using whole blood, this result could only be confirmed after stimulation with a potent

activator of the protein kinase C [23], but not with LPS or trauma-relevant mediators.

Similarly, alterations in microvesicles shed from the neutrophil surface were only detectable

after protein kinase C activation. As an additional approach, gene expression levels of PAI-

1 and its cellular anchor uPAR [6] were analyzed by qPCR. However, PAI-1 expression

levels were not increased after short-term exposure to inflammatory stimuli in contrast to

previous studies [145]. UPAR gene expression was by tendency higher after a short

Discussion

68

incubation with inflammatory cytokines or LPS, suggesting that a modest increase in uPAR

surface expression may be partly causative for elevated detection of PAI-1 on the cell

surface. Considering the observed changes especially in PAI-1, it is tempting to speculate

that blood leukocytes are involved in regulation of coagulation and may partially contribute

to thrombotic events, including venous thromboembolism, in patients after trauma [21] since

several leukocytes have been demonstrated to switch to pro-coagulatory phenotypes in

inflammatory (micro)environments [103,145,182]. Furthermore, several neutrophil

components, such as their cellular surface, shed microvesicles, granule proteins, or NETs,

have been established to be significantly involved in the modulation of coagulatory

processes [33,71,150] and can be presumed to play a role in coagulopathy after trauma.

Therefore, future experiments need to characterize in detail which factors in patient blood

might be responsible for changes in the expression of coagulation factors on peripheral

leukocytes. In this regard, a promising approach would also be the stimulation with

endogenous DAMPs such as HMGB-1, histones, or mitochondrial DNA which are able to

substantially activate pro-inflammatory processes in leukocytes.

4.2 Coagulopathy in polytraumatized patients

Disturbances in processes of coagulation represent a central risk factor for mortality in

trauma patients [16,97] and optimized transfusion strategies seem to be essential in order to

improve outcome [32,42,67]. To assess whether there was apparent coagulopathy in our PT

cohort, thromboelastometric analyses were performed in stored plasma samples. Patients

revealed impaired clotting as evident in delayed clot formation and a reduced clot stability

especially during the early time course after trauma. As summarized recently [15,176],

abnormal clot amplitude or MCF are indicators of coagulopathy and can be valuable tools

in the prediction of needed blood transfusion and mortality. The data in the present study

suggest substantial disturbances predominantly in initial activation of coagulation, although,

surprisingly, no hyperfibrinolysis as typically present after trauma [79,153] was detected in

this cohort. This may be due to a difficulty in diagnosing moderate fibrinolysis employing

thromboelastometric approaches by which the percentage of patients with fibrinolytic events

may be underestimated [139]. Since a differential analysis of the contribution of the cellular

blood components, including, but not limited to thrombocytes, was not possible due to

analysis of plasma samples, future coagulation analyses in whole blood from patients during

the time course after injury and corresponding correlations with flow cytometric data could

also shed more light on the earlier discussed role of leukocyte surface molecules in the

regulation of coagulatory and fibrinolytic processes.

Discussion

69

4.3 Functional immune monitoring in trauma

In a translational pilot study using a highly standardized ex vivo whole blood model, blood

from healthy volunteers and from polytraumatized patients was stimulated with LPS in order

to develop a tool for the diagnosis of remaining functional capacity of a patient’s immune

system. This experiment was performed with two aims: defining the changes in the

immediate immune reaction to a pathogenic stimulus after severe injury, and shortening the

incubation time in order to render this application of functional immune monitoring more

suitable for clinical use. Several studies have been performed focusing on the effects of

trauma, shock, or sepsis on peripheral blood cells and the alterations in their response to

bacterial stimuli [17,39,44,61,62,64,87,102,142,156,159]. However, those studies analyzed

isolated cells, not including effects of soluble plasma mediators or intercellular actions, or

used protocols with low standardization potential which are not suitable for testing in clinical

routine.

In the present study, when whole blood was stimulated with LPS, the secretion of

predominantly monocyte- and macrophage-, but also lymphocyte-derived cytokines was

significantly reduced during the time course after trauma in line with the literature

[11,68,81,129,156,158], although this effect was less distinct 24 h after injury. In contrast,

T cell function as reflected by the secretion of mediators mainly involved in T cell activation

after endotoxin incubation was not compromised, indicating that the observed alterations in

PAMP/DAMP surface receptors did not influence the lymphocytic capability to react to a

defined PAMP stimulus. As a major limitation, the small sample size, also due to the high

cost/sample ratio, and large interindividual variation [117] prevented a broad and definitive

assessment of immune functionality in this approach. Validation in a larger patient cohort

might also facilitate the detection of more subtle changes in the immune response as e.g.

mediated by MDSCs [31,119]. Furthermore, for some of the assessed cytokines (e.g. IL-9

and IL-12), there were no alterations in the secreted amounts upon stimulus since

concentrations in unstimulated samples were already higher. Nevertheless, this approach

provided a rather comprehensive analysis of the complex immune response in peripheral

blood. In addition, highly significant differences in stimulated and unstimulated samples

despite the markedly shortened incubation time demonstrated that this methodology could

provide a highly useful diagnostic tool for the immune surveillance of trauma patients and

may aid to optimize timing of surgical procedures during the posttraumatic immune response

[55,185]. Moreover, shortening stimulation time even further and analyzing only a few

Discussion

70

selected parameters relevant for the current functional immune status in clinical laboratories

might enable a valid and reliable immune monitoring and maximize clinical usefulness.

4.4 HS as a major driver of barrier and organ dysfunction after PT

In the past years, there has been accumulating evidence that severe blood loss may be one

of the central causes for posttraumatic barrier damage and organ dysfunction and failure

[49,78,154,188]. In order to assess whether the presence or absence of a HS was predictive

for organ damage during the early time course after injury, a larger PT patient cohort from a

clinical study at the University Hospital Zurich was stratified into those patients with and

those without HS according to established clinical parameters [88,108,190]. Despite equal

severity of injury in the two cohorts as reflected by the ISS and similar values in most

assessed inflammatory mediators except for IL-6, HS induced a spatially and temporally

specific injury of the lung, kidney, the intestine, and the liver as reflected by distinct serum

organ damage markers [7,143,184].

The present data did not confirm that CC16 correlates with the contused lung tissue volume

after PT [184] since there was no association with the AIS of the thorax; however, the

correlation of serum CC16 concentrations with transfused volume and positive fluid balance

suggest an increase in the permeability of the air-blood barrier [66], possibly leading to fluid

accumulation in the lung. A similar result was recently published in our murine trauma

model where only a combination of PT and HS induced a significant increase in plasma

CC16 [36].

The kidneys represent central organs in the pathophysiology after PT and HS as evidenced

by a report from a cohort of more than 2000 injured patients [181]; of those patients with

early acute kidney injury (AKI), 78% developed multiple organ failure and 27% died; both

rates are higher than those in patients with early lung, heart, or liver injury. NGAL has been

described to be produced by a number of tissues and cells, including the kidneys, neutrophils,

lungs, and liver [30,82,107,110]. HS is a strong inducer of renal hypoperfusion and hypoxia

[189] and release of NGAL from renal tubular cells was shown in response to ischemia-

reperfusion injury [116], concurrent with an early increase in serum NGAL in the HS patient

cohort in this study. This is indicative of an early remote kidney injury induced by prerenal

shock pathophysiology. Interestingly, NGAL values were associated with the initial severity

of abdominal injury although the kidneys are rarely affected by the trauma hit, suggesting

that HS significantly increases the risk of trauma-caused AKI and multiple organ failure.

This was also shown in our recent study in murine PT and HS [36]. Furthermore, correlation

Discussion

71

of serum NGAL with procalcitonin may be due to infectious complications since septic AKI

is often induced by high amounts of trauma-induced release of DAMPs and PAMPs. It is

noteworthy that NGAL was established as a marker for kidney damage with promising

results [7,109], but its specificity has ever since been discussed controversially [59,104].

Nevertheless, its validity in diagnosing AKI especially in combination with other markers

or in the urine remains high [2].

Abdominal injury was assessed using L-FABP, expressed in liver, intestine, kidney and

pancreas [38,93,96] as a reliable marker of abdominal and especially liver damage

[20,111,143], and I-FABP which is specifically expressed in the small intestine [132]. L-

FABP was higher in HS patients and correlated with coagulation parameters as well as

procalcitonin, likely due to the high blood loss in patients with abdominal injury, also

reflected by the association of HS with higher AIS of the abdomen, and a well-established

connection between abdominal trauma and procalcitonin [101,152]. A disruption of the

intestinal barrier was indicated by an immediate increase in I-FABP serum concentrations

in HS patients, and levels correlated with lactate and the fluid balance, again suggesting a

connection with the shock-induced loss in intraluminal blood volume. A recent study in burn

patients with multiple organ dysfunction demonstrated a highly similar serum I-FABP

profile, most likely due to injury-induced intestinal ischemia and barrier breakdown [128].

It can therefore be presumed that HS is detrimental in the course of posttraumatic abdominal

ischemia and organ damage.

A breakdown in endothelial barrier function is likely to play a significant role in the

development of posttraumatic organ pathology. Of note, the different organ damage markers

showed very distinct time patterns, possible due to differential alterations in the respective

blood-organ barriers. Barrier permeability can be regulated by a large number of molecules.

The lipid mediator sphingosine-1-phosphate, besides its involvement in activation of the

immune system, seems to play a significant role in this regulation especially when stabilized

by binding to plasma albumin [124,191]. Therapeutically, a functional sphingosine-1-

phosphate analogue has already been applied in the context of trauma/HS [9]. Lower serum

albumin upon admission has recently been linked to endotheliopathy in severely injured

patients [148]. Concentrations of both sphingosine-1-phosphate and albumin clearly

decreased early in patients after trauma, although HS only seemed to play a minor role here.

Interestingly, HS alone in the murine trauma setting rather induced an increase in serum

albumin, while a reduction was only visible after combined PT+HS. Furthermore, a decrease

Discussion

72

in plasma thrombomodulin, detected immediately after injury in the PT+HS cohort and in

both mouse groups which had undergone HS, can indicate the activation of endothelial cells

[70]. These data are suggestive of an initiation of endotheliopathy with disturbances in

anticoagulatory surface properties and barrier function as soon as patients arrive at the

emergency room. Angiopoietin-2 as a mediator involved in pro-inflammatory processes and

vascular leakage [57], but also adrenomedullin with rather opposite effects [164] were

increased only in the HS patient cohort and rather at the later time points after trauma. These

findings and the correlation of angiopoietin with the number of initially transfused

erythrocyte concentrates point to a strong activation of pathways regulating endothelial

permeability exclusively in patients after HS. This is also supported by initial increases in

MMP-9 (albeit in both patient cohorts) and MMP-13, matrix proteases which have been

shown to be involved in cleaving components of the apical glycocalyx layer off endothelial

cells [138,191]. In line, increased levels of syndecan-1 and heparan sulfate were detected

especially in the HS cohort; similar results were obtained in the murine PT setting where an

isolated HS already induced a clear enhancement of MMP-9, -13, and syndecan-1 plasma

concentrations compared to controls, but animals with combined PT+HS had highest

corresponding concentrations. Shedding of glycocalyx components is likely to be

responsible for increased vascular permeability after trauma and HS [137]. In this context,

syndecan-1 and its fragment heparan sulfate have been increasingly used as markers of

endothelial glycocalyx degradation especially by the group around P.I. Johansson

[75,76,125,148] and may induce a so-called “autoheparinization” effect due to their

anticoagulatory features [126,141]. This was assessed in an ex vivo experiment in blood

samples with artificially adjusted hematocrits similar to those in healthy volunteers (45%),

PT (33%), and PT+HS (25%) patients where heparan sulfate significantly delayed clotting.

Furthermore, correlations of syndecan-1 with serum markers of organ dysfunction indicate

a causal relationship between endothelial damage and organ failure. Mechanistically, release

of (nor-)epinephrine induced by sympathoadrenal activation may be a central driver of

endotheliopathy [77,125]. As a limitation of the present study, catecholamine levels were

not measured in this patient cohort. However, in addition to endogenous production, PT

patients especially after HS are likely to have received catecholamine support during the

initial phase after trauma as part of the standard emergency care protocols. Plasma fibrinogen

was lower in HS patients throughout the entire observation period; D-dimers were initially

decreased in both cohorts, but an additional effect of HS was not detectable, presumably due

to plasma dilution after extensive fluid replacement with crystalloids.

Discussion

73

Increased endothelial permeability was furthermore assessed by measurement of circulating

intercellular tight-junction and adherens-junction molecules. As recently published, we

found an early increase in plasma JAM-1, a central component of tight junctions, in humans

and mice after PT [37]. Claudin-5 as another molecule involved in regulation of tight-

junction permeability, especially of the blood-brain barrier, but also in other organs and

tissues [29,47,121], was increased in PT+HS patients and correlated with markers of HS,

suggesting that barrier disturbances after HS not only include the apical glycocalyx, but also

intercellular junctions. Unexpectedly, we also detected increased amounts of mucin-2, a

central component of intestinal mucus, in the circulation, indicating a disturbed blood-gut

barrier possible due to a disruption of intestinal mucus layers by pancreatic enzymes post

trauma [48]. Similarly to heparan sulfate, mucin-2 in concentrations measured in our patient

cohorts (up to 6000 pg/ml) prolonged coagulation in blood samples with low hematocrit,

suggesting that components of the intestine released by a direct or indirect gut barrier damage

may interfere with coagulation and could be partly responsible for coagulopathy in severely

injured patients.

4.5 Conclusion

Severe trauma induces several alterations in leukocyte surface molecules and is associated

with cell type-specific changes in the immune response to bacterial stimuli. This study

demonstrated that coagulation is significantly impaired predominantly during the early phase

after trauma, and leukocyte surface molecules may play a detrimental role. Furthermore, a

clinically relevant testing system was established to allow functional bed-side monitoring of

the patient’s remaining immune capacity which revealed a deficit especially in monocyte

function. Lastly, the differential analysis of a larger patient cohort revealed HS as a major

driver of posttraumatic organ damage. In addition, several molecules associated with barrier

(dys-)function were significantly altered by posttraumatic HS with very distinct time

patterns, underlining the importance of effective management of bleeding and reliable

monitoring of barrier function in order to treat latent and evident organ damage before onset

of leakage syndrome and organ failure.

Summary

74

5 Summary

Despite the considerable progress made in public safety measures, emergency and intensive

care in the last decades, trauma remains a major medical issue and is responsible for a large

number of deaths worldwide particularly in young people. In regard to the posttraumatic

immune response, development of disturbances in coagulation, and detection and monitoring

of end organ damage, several issues remain unresolved. Therefore, this thesis focused on the

alterations in peripheral blood leukocytes as “first line of defense” after severe injury

employing static and functional analyses. Furthermore, disturbances in coagulation were

observed over a 10-day time course after trauma. In a larger patient cohort, the impact of an

additional hemorrhagic shock on inflammation, organ damage, and breakdown of the

endothelial glycocalyx was assessed. These findings were re-translationally evaluated in a

murine model of isolated and combined trauma and hemorrhage.

In patients after severe trauma, peripheral leukocytes rapidly changed their surface

expression profile of several receptors for complement activation products as well as

damage- and pathogen-associated molecular patterns, involved in regulating the immune

response, but also modulators of coagulation. In a pilot study, a highly standardized ex vivo

testing system revealed defects in production of early pro-inflammatory cytokines such as

tumor-necrosis factor and interleukin-1 upon incubation with microbial endotoxin in

monocytes, but not in the lymphocyte function after severe injury. A shortened standardized

exposure time (4 h) resulted in a similar immune response compared to the established

incubation protocol. After further validation and optimization, this method might offer a

useful tool to monitor the immune system’s remaining capacity to react to inflammatory or

pathogenic stimuli. In a similar approach, functional monitoring of coagulation over the time

course after injury detected striking defects in coagulation immediately after trauma which

partly lasted until 10 days later, but no hyperfibrinolysis was found.

In a larger patient cohort, hemorrhagic shock was demonstrated to significantly contribute

to posttraumatic inflammation and organ damage. Despite comparable injury severity,

patients after blood loss had increased serum concentrations of specific damage markers of

the lung (Clara cell secretory protein), liver (liver-type fatty acid-binding protein), kidneys

(neutrophil gelatinase-associated lipocalin) and the intestine (intestinal fatty acid-binding

protein) in distinct spatial and temporal patterns. Furthermore, presumably by increased

generation of matrix metalloproteinases-9 and -13, there was an elevation of circulating

components of the glycocalyx such as syndecan-1 and heparan sulfate, suggesting

Summary

75

endothelial damage and increased permeability. Interestingly, increased concentrations of

elements of the intestinal mucus (mucin-2) were detected after trauma/hemorrhage, implying

a deterioration in intestinal barrier function. In vitro experiments revealed that the detected

amounts of glycocalyx as well as intestinal mucus components were able to interfere with

coagulation, indicating that the posttraumatic barrier breakdown might also impair clotting

and increase the risk of bleeding. These findings were mostly confirmed in a murine model

of PT and hemorrhage, where shock alone already demonstrated marked impact on barrier

dysfunction and glycocalyx breakdown. The data is indicative of an early development of

barrier and organ damage, presumably hours to days before manifestation of barrier

breakdown and organ dysfunction. Monitoring the posttraumatic course using novel organ

damage markers in addition to traditional functional scores may aid to detect subpar

tendencies before onset of (multiple) organ dysfunction.

In conclusion, severe trauma leads to significant alterations in leukocyte and immune

function, coagulation, and considerable barrier and organ dysfunction which is worsened in

the presence of a hemorrhagic shock. Reliable monitoring of the clinical course using valid

functional markers and assays as well as rapid implementation into clinical routine and

advanced treatment strategies may be decisive for improved outcome.

List of references

76

6 List of references

1. Amara U, Kalbitz M, Perl M, Flierl MA, Rittirsch D, Weiss M, Schneider M, Gebhard

F, Huber-Lang M: Early expression changes of complement regulatory proteins and

C5A receptor (CD88) on leukocytes after multiple injury in humans. Shock 33: 568-

575 (2010)

2. Asada T, Isshiki R, Hayase N, Sumida M, Inokuchi R, Noiri E, Nangaku M, Yahagi

N, Doi K: Impact of clinical context on acute kidney injury biomarker performances:

differences between neutrophil gelatinase-associated lipocalin and L-type fatty acid-

binding protein. Sci Rep 6: 33077 (2016)

3. Baker SP, O'Neill B, Haddon W, Jr., Long WB: The injury severity score: a method

for describing patients with multiple injuries and evaluating emergency care. J Trauma

14: 187-196 (1974)

4. Bhandari M, Tornetta P, III, Sprague S, Najibi S, Petrisor B, Griffith L, Guyatt GH:

Predictors of reoperation following operative management of fractures of the tibial

shaft. J Orthop Trauma 17: 353-361 (2003)

5. Billiar TR, Vodovotz Y: Time for trauma immunology. PLoS Med 14: e1002342

(2017)

6. Binder BR, Mihaly J, Prager GW: uPAR-uPA-PAI-1 interactions and signaling: a

vascular biologist's view. Thromb Haemost 97: 336-342 (2007)

7. Bolignano D, Donato V, Coppolino G, Campo S, Buemi A, Lacquaniti A, Buemi M:

Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. Am

J Kidney Dis 52: 595-605 (2008)

8. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM,

Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of

innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee.

American College of Chest Physicians/Society of Critical Care Medicine. Chest 101:

1644-1655 (1992)

9. Bonitz JA, Son JY, Chandler B, Tomaio JN, Qin Y, Prescott LM, Feketeova E, Deitch

EA: A sphingosine-1 phosphate agonist (FTY720) limits trauma/hemorrhagic shock-

induced multiple organ dysfunction syndrome. Shock 42: 448-455 (2014)

List of references

77

10. Bonnarens F, Einhorn TA: Production of a standard closed fracture in laboratory

animal bone. J Orthop Res 2: 97-101 (1984)

11. Boomer JS, To K, Chang KC, Takasu O, Osborne DF, Walton AH, Bricker TL, Jarman

SD, Kreisel D, Krupnick AS, Srivastava A, Swanson PE, Green JM, Hotchkiss RS:

Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA

306: 2594-2605 (2011)

12. Botha AJ, Moore FA, Moore EE, Sauaia A, Banerjee A, Peterson VM: Early

neutrophil sequestration after injury: a pathogenic mechanism for multiple organ

failure. J Trauma 39: 411-417 (1995)

13. Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie RC, Pittet JF: Acute

traumatic coagulopathy: initiated by hypoperfusion: modulated through the protein C

pathway? Ann Surg 245: 812-818 (2007)

14. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, Pittet JF: Acute

coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and

hyperfibrinolysis. J Trauma 64: 1211-1217 (2008)

15. Brohi K, Eaglestone S: Traumatic coagulopathy and massive transfusion: improving

outcomes and saving blood. Programme Grants for Applied Research 5: (2017)

16. Brohi K, Singh J, Heron M, Coats T: Acute traumatic coagulopathy. J Trauma 54:

1127-1130 (2003)

17. Bulger EM, Cuschieri J, Warner K, Maier RV: Hypertonic resuscitation modulates the

inflammatory response in patients with traumatic hemorrhagic shock. Ann Surg 245:

635-641 (2007)

18. Burk AM, Martin M, Flierl MA, Rittirsch D, Helm M, Lampl L, Bruckner U, Stahl

GL, Blom AM, Perl M, Gebhard F, Huber-Lang M: Early complementopathy after

multiple injuries in humans. Shock 37: 348-354 (2012)

19. Cairns CB: Rude unhinging of the machinery of life: metabolic approaches to

hemorrhagic shock. Curr Opin Crit Care 7: 437-443 (2001)

20. Cakir OO, Toker A, Ataseven H, Demir A, Polat H: The Importance of Liver-Fatty

Acid Binding Protein in Diagnosis of Liver Damage in Patients with Acute Hepatitis.

J Clin Diagn Res 11: OC17-OC21 (2017)

List of references

78

21. Cap A, Hunt B: Acute traumatic coagulopathy. Curr Opin Crit Care 20: 638-645

(2014)

22. Cardenas JC, Matijevic N, Baer LA, Holcomb JB, Cotton BA, Wade CE: Elevated

tissue plasminogen activator and reduced plasminogen activator inhibitor promote

hyperfibrinolysis in trauma patients. Shock 41: 514-521 (2014)

23. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y: Direct activation

of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting

phorbol esters. J Biol Chem 257: 7847-7851 (1982)

24. Cekic C, Linden J: Purinergic regulation of the immune system. Nat Rev Immunol 16:

177-192 (2016)

25. Chapman MP, Moore EE, Moore HB, Gonzalez E, Gamboni F, Chandler JG, Mitra S,

Ghasabyan A, Chin TL, Sauaia A, Banerjee A, Silliman CC: Overwhelming tPA

release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured

trauma patients. J Trauma Acute Care Surg 80: 16-23 (2016)

26. Ciesla DJ, Moore EE, Johnson JL, Burch JM, Cothren CC, Sauaia A: A 12-year

prospective study of postinjury multiple organ failure: has anything changed? Arch

Surg 140: 432-438 (2005)

27. Cohen MJ, Christie SA: Coagulopathy of Trauma. Crit Care Clin 33: 101-118 (2017)

28. Colonna M, Facchetti F: TREM-1 (triggering receptor expressed on myeloid cells): a

new player in acute inflammatory responses. J Infect Dis 187 Suppl 2: S397-S401

(2003)

29. Cooper I, Cohen-Kashi-Malina K, Teichberg VI: Claudin-5 expression in in vitro

models of the blood-brain barrier. Methods Mol Biol 762: 347-354 (2011)

30. Cowland JB, Borregaard N: Molecular characterization and pattern of tissue

expression of the gene for neutrophil gelatinase-associated lipocalin from humans.

Genomics 45: 17-23 (1997)

31. Cuenca AG, Delano MJ, Kelly-Scumpia KM, Moreno C, Scumpia PO, Laface DM,

Heyworth PG, Efron PA, Moldawer LL: A paradoxical role for myeloid-derived

suppressor cells in sepsis and trauma. Mol Med 17: 281-292 (2011)

32. Curry NS, Davenport RA, Hunt BJ, Stanworth SJ: Transfusion strategies for traumatic

coagulopathy. Blood Rev 26: 223-232 (2012)

List of references

79

33. Darbousset R, Thomas GM, Mezouar S, Frere C, Bonier R, Mackman N, Renne T,

Dignat-George F, Dubois C, Panicot-Dubois L: Tissue factor-positive neutrophils bind

to injured endothelial wall and initiate thrombus formation. Blood 120: 2133-2143

(2012)

34. Davenport R, Manson J, De'Ath H, Platton S, Coates A, Allard S, Hart D, Pearse R,

Pasi KJ, MacCallum P, Stanworth S, Brohi K: Functional definition and

characterization of acute traumatic coagulopathy. Crit Care Med 39: 2652-2658 (2011)

35. Degn SE, Thiel S: Humoral pattern recognition and the complement system. Scand J

Immunol 78: 181-193 (2013)

36. Denk S, Weckbach S, Eisele P, Braun CK, Wiegner R, Ohmann JJ, Wrba L, Hoenes

FM, Kellermann P, Radermacher P, Wachter U, Hafner S, McCook O, Schultze A,

Palmer A, Braumuller S, Gebhard F, Huber-Lang M: Role of Hemorrhagic Shock in

Experimental Polytrauma. Shock 49: 154-163 (2018)

37. Denk S, Wiegner R, Hones FM, Messerer DA, Radermacher P, Weiss M, Kalbitz M,

Ehrnthaller C, Braumuller S, McCook O, Gebhard F, Weckbach S, Huber-Lang M:

Early Detection of Junctional Adhesion Molecule-1 (JAM-1) in the Circulation after

Experimental and Clinical Polytrauma. Mediators Inflamm 2015: 463950 (2015)

38. Derikx JP, Poeze M, van Bijnen AA, Buurman WA, Heineman E: Evidence for

intestinal and liver epithelial cell injury in the early phase of sepsis. Shock 28: 544-

548 (2007)

39. Dimofte G, Alexander A, Carlson G, Little R, Irving M: TNF alpha and IL-6

involvement in surgical trauma. II. In vitro cytokine production. Rev Med Chir Soc

Med Nat Iasi 105: 493-498 (2001)

40. Dirkmann D, Radu-Berlemann J, Gorlinger K, Peters J: Recombinant tissue-type

plasminogen activator-evoked hyperfibrinolysis is enhanced by acidosis and inhibited

by hypothermia but still can be blocked by tranexamic acid. J Trauma Acute Care Surg

74: 482-488 (2013)

41. Dunne JR, Malone DL, Tracy JK, Napolitano LM: Allogenic blood transfusion in the

first 24 hours after trauma is associated with increased systemic inflammatory

response syndrome (SIRS) and death. Surg Infect (Larchmt ) 5: 395-404 (2004)

List of references

80

42. Eastridge BJ, Mabry RL, Seguin P, Cantrell J, Tops T, Uribe P, Mallett O, Zubko T,

Oetjen-Gerdes L, Rasmussen TE, Butler FK, Kotwal RS, Holcomb JB, Wade C,

Champion H, Lawnick M, Moores L, Blackbourne LH: Death on the battlefield (2001-

2011): implications for the future of combat casualty care. J Trauma Acute Care Surg

73: S431-S437 (2012)

43. El MR, El GM, Seeds MC, McCall CE, Dreskin SC, Nicolls MR: Endogenous signals

released from necrotic cells augment inflammatory responses to bacterial endotoxin.

Immunol Lett 111: 36-44 (2007)

44. Ertel W, Kremer JP, Kenney J, Steckholzer U, Jarrar D, Trentz O, Schildberg FW:

Downregulation of proinflammatory cytokine release in whole blood from septic

patients. Blood 85: 1341-1347 (1995)

45. Esmon CT: Protein C anticoagulant system--anti-inflammatory effects. Semin

Immunopathol 34: 127-132 (2012)

46. Fan J, Li Y, Levy RM, Fan JJ, Hackam DJ, Vodovotz Y, Yang H, Tracey KJ, Billiar

TR, Wilson MA: Hemorrhagic shock induces NAD(P)H oxidase activation in

neutrophils: role of HMGB1-TLR4 signaling. J Immunol 178: 6573-6580 (2007)

47. Fernandez AL, Koval M, Fan X, Guidot DM: Chronic alcohol ingestion alters claudin

expression in the alveolar epithelium of rats. Alcohol 41: 371-379 (2007)

48. Fishman JE, Sheth SU, Levy G, Alli V, Lu Q, Xu D, Qin Y, Qin X, Deitch EA:

Intraluminal nonbacterial intestinal components control gut and lung injury after

trauma hemorrhagic shock. Ann Surg 260: 1112-1120 (2014)

49. Fleming SD, Phillips LM, Lambris JD, Tsokos GC: Complement component C5a

mediates hemorrhage-induced intestinal damage. J Surg Res 150: 196-203 (2008)

50. Frith D, Davenport R, Brohi K: Acute traumatic coagulopathy. Curr Opin Anaesthesiol

25: 229-234 (2012)

51. Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson PI,

Stanworth S, Thiemermann C, Brohi K: Definition and drivers of acute traumatic

coagulopathy: clinical and experimental investigations. J Thromb Haemost 8: 1919-

1925 (2010)

52. Fritz G: RAGE: a single receptor fits multiple ligands. Trends Biochem Sci 36: 625-

632 (2011)

List of references

81

53. Ganter MT, Brohi K, Cohen MJ, Shaffer LA, Walsh MC, Stahl GL, Pittet JF: Role of

the alternative pathway in the early complement activation following major trauma.

Shock 28: 29-34 (2007)

54. Gebhard F, Huber-Lang M: Polytrauma--pathophysiology and management

principles. Langenbecks Arch Surg 393: 825-831 (2008)

55. Gentile LF, Cuenca AG, Efron PA, Ang D, Bihorac A, McKinley BA, Moldawer LL,

Moore FA: Persistent inflammation and immunosuppression: a common syndrome

and new horizon for surgical intensive care. J Trauma Acute Care Surg 72: 1491-1501

(2012)

56. German Trauma Society (DGU), Committee on Emergency Medicine ICaTMSN,

AUC - Academy for Trauma Surgery: TraumaRegister DGU® - Annual Report 2017.

<[11] Journal> <[12] Volume>: (9-15-2017)

57. Giamarellos-Bourboulis EJ, Kanellakopoulou K, Pelekanou A, Tsaganos T,

Kotzampassi K: Kinetics of angiopoietin-2 in serum of multi-trauma patients:

correlation with patient severity. Cytokine 44: 310-313 (2008)

58. Haagsma JA, Graetz N, Bolliger I, Naghavi M, Higashi H, Mullany EC, Abera SF,

Abraham JP, Adofo K, Alsharif U, Ameh EA, Ammar W, Antonio CA, Barrero LH,

Bekele T, Bose D, Brazinova A, Catala-Lopez F, Dandona L, Dandona R, Dargan PI,

De LD, Degenhardt L, Derrett S, Dharmaratne SD, Driscoll TR, Duan L, Petrovich

ES, Farzadfar F, Feigin VL, Franklin RC, Gabbe B, Gosselin RA, Hafezi-Nejad N,

Hamadeh RR, Hijar M, Hu G, Jayaraman SP, Jiang G, Khader YS, Khan EA,

Krishnaswami S, Kulkarni C, Lecky FE, Leung R, Lunevicius R, Lyons RA, Majdan

M, Mason-Jones AJ, Matzopoulos R, Meaney PA, Mekonnen W, Miller TR, Mock

CN, Norman RE, Orozco R, Polinder S, Pourmalek F, Rahimi-Movaghar V, Refaat A,

Rojas-Rueda D, Roy N, Schwebel DC, Shaheen A, Shahraz S, Skirbekk V, Soreide K,

Soshnikov S, Stein DJ, Sykes BL, Tabb KM, Temesgen AM, Tenkorang EY, Theadom

AM, Tran BX, Vasankari TJ, Vavilala MS, Vlassov VV, Woldeyohannes SM, Yip P,

Yonemoto N, Younis MZ, Yu C, Murray CJ, Vos T: The global burden of injury:

incidence, mortality, disability-adjusted life years and time trends from the Global

Burden of Disease study 2013. Inj Prev 22: 3-18 (2016)

59. Haase M, Bellomo R, Devarajan P, Schlattmann P, Haase-Fielitz A: Accuracy of

neutrophil gelatinase-associated lipocalin (NGAL) in diagnosis and prognosis in acute

List of references

82

kidney injury: a systematic review and meta-analysis. Am J Kidney Dis 54: 1012-1024

(2009)

60. Halbgebauer R, Braun CK, Denk S, Mayer B, Cinelli P, Radermacher P, Wanner GA,

Simmen HP, Gebhard F, Rittirsch D, Huber-Lang M: Hemorrhagic shock drives

glycocalyx, barrier and organ dysfunction early after polytrauma. J Crit Care 44: 229-

237 (2017)

61. Hassett S, Moynagh P, Reen D: TNF-alpha is a mediator of the anti-inflammatory

response in a human neonatal model of the non-septic shock syndrome. Pediatr Surg

Int 22: 24-30 (2006)

62. Hazeldine J, Naumann DN, Toman E, Davies D, Bishop JRB, Su Z, Hampson P,

Dinsdale RJ, Crombie N, Duggal NA, Harrison P, Belli A, Lord JM: Prehospital

immune responses and development of multiple organ dysfunction syndrome

following traumatic injury: A prospective cohort study. PLoS Med 14: e1002338

(2017)

63. Hebert PC, Wells G, Blajchman MA, Marshall J, Martin C, Pagliarello G, Tweeddale

M, Schweitzer I, Yetisir E: A multicenter, randomized, controlled clinical trial of

transfusion requirements in critical care. Transfusion Requirements in Critical Care

Investigators, Canadian Critical Care Trials Group. N Engl J Med 340: 409-417 (1999)

64. Heftrig D, Sturm R, Oppermann E, Kontradowitz K, Jurida K, Schimunek L, Woschek

M, Marzi I, Relja B: Impaired Surface Expression of HLA-DR, TLR2, TLR4, and

TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma

Patients. Mediators Inflamm 2017: 2608349 (2017)

65. Hengartner NE, Fiedler J, Schrezenmeier H, Huber-Lang M, Brenner RE: Crucial role

of IL1beta and C3a in the in vitro-response of multipotent mesenchymal stromal cells

to inflammatory mediators of polytrauma. PLoS One 10: e0116772 (2015)

66. Hermans C, Knoops B, Wiedig M, Arsalane K, Toubeau G, Falmagne P, Bernard A:

Clara cell protein as a marker of Clara cell damage and bronchoalveolar blood barrier

permeability. Eur Respir J 13: 1014-1021 (1999)

67. Holcomb JB, McMullin NR, Pearse L, Caruso J, Wade CE, Oetjen-Gerdes L,

Champion HR, Lawnick M, Farr W, Rodriguez S, Butler FK: Causes of death in U.S.

Special Operations Forces in the global war on terrorism: 2001-2004. Ann Surg 245:

986-991 (2007)

List of references

83

68. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med

348: 138-150 (2003)

69. Huber-Lang M, Lambris JD, Ward PA: Innate immune responses to trauma. Nat

Immunol (2018)

70. Hunt BJ, Jurd KM: Endothelial cell activation. A central pathophysiological process.

BMJ 316: 1328-1329 (1998)

71. Iba T, Miki T, Hashiguchi N, Tabe Y, Nagaoka I: Is the neutrophil a 'prima donna' in

the procoagulant process during sepsis? Crit Care 18: 230 (2014)

72. Ito T, Kakihana Y, Maruyama I: Thrombomodulin as an intravascular safeguard

against inflammatory and thrombotic diseases. Expert Opin Ther Targets 20: 151-158

(2016)

73. Ives C, Inaba K, Branco BC, Okoye O, Schochl H, Talving P, Lam L, Shulman I,

Nelson J, Demetriades D: Hyperfibrinolysis elicited via thromboelastography predicts

mortality in trauma. J Am Coll Surg 215: 496-502 (2012)

74. Jenkins DH, Rappold JF, Badloe JF, Berseus O, Blackbourne L, Brohi KH, Butler FK,

Cap AP, Cohen MJ, Davenport R, DePasquale M, Doughty H, Glassberg E, Hervig T,

Hooper TJ, Kozar R, Maegele M, Moore EE, Murdock A, Ness PM, Pati S, Rasmussen

T, Sailliol A, Schreiber MA, Sunde GA, van de Watering LM, Ward KR, Weiskopf

RB, White NJ, Strandenes G, Spinella PC: Trauma hemostasis and oxygenation

research position paper on remote damage control resuscitation: definitions, current

practice, and knowledge gaps. Shock 41 Suppl 1: 3-12 (2014)

75. Johansson PI, Henriksen HH, Stensballe J, Gybel-Brask M, Cardenas JC, Baer LA,

Cotton BA, Holcomb JB, Wade CE, Ostrowski SR: Traumatic Endotheliopathy: A

Prospective Observational Study of 424 Severely Injured Patients. Ann Surg 265: 597-

603 (2017)

76. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR: A high admission syndecan-

1 level, a marker of endothelial glycocalyx degradation, is associated with

inflammation, protein C depletion, fibrinolysis, and increased mortality in trauma

patients. Ann Surg 254: 194-200 (2011)

List of references

84

77. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR: High circulating adrenaline

levels at admission predict increased mortality after trauma. J Trauma Acute Care Surg

72: 428-436 (2012)

78. Johnson JL, Moore EE, Kashuk JL, Banerjee A, Cothren CC, Biffl WL, Sauaia A:

Effect of blood products transfusion on the development of postinjury multiple organ

failure. Arch Surg 145: 973-977 (2010)

79. Kashuk JL, Moore EE, Sawyer M, Wohlauer M, Pezold M, Barnett C, Biffl WL,

Burlew CC, Johnson JL, Sauaia A: Primary fibrinolysis is integral in the pathogenesis

of the acute coagulopathy of trauma. Ann Surg 252: 434-442 (2010)

80. Kauvar DS, Wade CE: The epidemiology and modern management of traumatic

hemorrhage: US and international perspectives. Crit Care 9 Suppl 5: S1-S9 (2005)

81. Keel M, Trentz O: Pathophysiology of polytrauma. Injury 36: 691-709 (2005)

82. Kjeldsen L, Johnsen AH, Sengelov H, Borregaard N: Isolation and primary structure

of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem

268: 10425-10432 (1993)

83. Knoferl MW, Liener UC, Perl M, Bruckner UB, Kinzl L, Gebhard F: Blunt chest

trauma induces delayed splenic immunosuppression. Shock 22: 51-56 (2004)

84. Knoferl MW, Liener UC, Seitz DH, Perl M, Bruckner UB, Kinzl L, Gebhard F:

Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a

novel mouse model of blunt chest trauma. Shock 19: 519-525 (2003)

85. Kohl J: The role of complement in danger sensing and transmission. Immunol Res 34:

157-176 (2006)

86. Kottke MA, Walters TJ: Where's the Leak in Vascular Barriers? A Review. Shock 46:

20-36 (2016)

87. Kremer JP, Jarrar D, Steckholzer U, Ertel W: Interleukin-1, -6 and tumor necrosis

factor-alpha release is down-regulated in whole blood from septic patients. Acta

Haematol 95: 268-273 (1996)

88. Kruse O, Grunnet N, Barfod C: Blood lactate as a predictor for in-hospital mortality

in patients admitted acutely to hospital: a systematic review. Scand J Trauma Resusc

Emerg Med 19: 74 (2011)

List of references

85

89. Kung HC, Hoyert DL, Xu J, Murphy SL: Deaths: final data for 2005. Natl Vital Stat

Rep 56: 1-120 (2008)

90. Kutcher ME, Redick BJ, McCreery RC, Crane IM, Greenberg MD, Cachola LM,

Nelson MF, Cohen MJ: Characterization of platelet dysfunction after trauma. J Trauma

Acute Care Surg 73: 13-19 (2012)

91. Ledderose C, Heyn J, Limbeck E, Kreth S: Selection of reliable reference genes for

quantitative real-time PCR in human T cells and neutrophils. BMC Res Notes 4: 427

(2011)

92. Lee EJ, Park JH: Receptor for Advanced Glycation Endproducts (RAGE), Its Ligands,

and Soluble RAGE: Potential Biomarkers for Diagnosis and Therapeutic Targets for

Human Renal Diseases. Genomics Inform 11: 224-229 (2013)

93. Lieberman JM, Sacchettini J, Marks C, Marks WH: Human intestinal fatty acid

binding protein: report of an assay with studies in normal volunteers and intestinal

ischemia. Surgery 121: 335-342 (1997)

94. Liener UC, Bruckner UB, Knoferl MW, Steinbach G, Kinzl L, Gebhard F: Chemokine

activation within 24 hours after blunt accident trauma. Shock 17: 169-172 (2002)

95. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408 (2001)

96. Lowe JB, Boguski MS, Sweetser DA, Elshourbagy NA, Taylor JM, Gordon JI: Human

liver fatty acid binding protein. Isolation of a full length cDNA and comparative

sequence analyses of orthologous and paralogous proteins. J Biol Chem 260: 3413-

3417 (1985)

97. MacLeod JB, Lynn M, McKenney MG, Cohn SM, Murtha M: Early coagulopathy

predicts mortality in trauma. J Trauma 55: 39-44 (2003)

98. Maegele M, Lefering R, Yucel N, Tjardes T, Rixen D, Paffrath T, Simanski C,

Neugebauer E, Bouillon B: Early coagulopathy in multiple injury: an analysis from

the German Trauma Registry on 8724 patients. Injury 38: 298-304 (2007)

99. Maegele M, Schochl H, Cohen MJ: An update on the coagulopathy of trauma. Shock

41 Suppl 1: 21-25 (2014)

100. Maier B, Lefering R, Lehnert M, Laurer HL, Steudel WI, Neugebauer EA, Marzi I:

Early versus late onset of multiple organ failure is associated with differing patterns of

List of references

86

plasma cytokine biomarker expression and outcome after severe trauma. Shock 28:

668-674 (2007)

101. Maier M, Wutzler S, Lehnert M, Szermutzky M, Wyen H, Bingold T, Henrich D,

Walcher F, Marzi I: Serum procalcitonin levels in patients with multiple injuries

including visceral trauma. J Trauma 66: 243-249 (2009)

102. Majetschak M, Krehmeier U, Ostroverkh L, Blomeke B, Schafer M: Alterations in

leukocyte function following surgical trauma: differentiation of distinct reaction types

and association with tumor necrosis factor gene polymorphisms. Clin Diagn Lab

Immunol 12: 296-303 (2005)

103. Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD: Complement and

coagulation: strangers or partners in crime? Trends Immunol 28: 184-192 (2007)

104. Martensson J, Bellomo R: The rise and fall of NGAL in acute kidney injury. Blood

Purif 37: 304-310 (2014)

105. Martini WZ, Pusateri AE, Uscilowicz JM, Delgado AV, Holcomb JB: Independent

contributions of hypothermia and acidosis to coagulopathy in swine. J Trauma 58:

1002-1009 (2005)

106. Mikhail J: The trauma triad of death: hypothermia, acidosis, and coagulopathy. AACN

Clin Issues 10: 85-94 (1999)

107. Minami K, Saito T, Narahara M, Tomita H, Kato H, Sugiyama H, Katoh M, Nakajima

M, Yokoi T: Relationship between hepatic gene expression profiles and hepatotoxicity

in five typical hepatotoxicant-administered rats. Toxicol Sci 87: 296-305 (2005)

108. Minei JP, Cuschieri J, Sperry J, Moore EE, West MA, Harbrecht BG, O'Keefe GE,

Cohen MJ, Moldawer LL, Tompkins RG, Maier RV: The changing pattern and

implications of multiple organ failure after blunt injury with hemorrhagic shock. Crit

Care Med 40: 1129-1135 (2012)

109. Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, Zahedi K,

Shao M, Bean J, Mori K, Barasch J, Devarajan P: Neutrophil gelatinase-associated

lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet

365: 1231-1238 (2005)

List of references

87

110. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P:

Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary

biomarker for ischemic renal injury. J Am Soc Nephrol 14: 2534-2543 (2003)

111. Monbaliu D, de VB, Crabbe T, van HE, Verwaest C, Roskams T, Fevery J, Pirenne J,

Buurman WA: Liver fatty acid-binding protein: an early and sensitive plasma marker

of hepatocellular damage and a reliable predictor of graft viability after liver

transplantation from non-heart-beating donors. Transplant Proc 37: 413-416 (2005)

112. Moore EE, Johnson JL, Cheng AM, Masuno T, Banerjee A: Insights from studies of

blood substitutes in trauma. Shock 24: 197-205 (2005)

113. Moore HB, Moore EE, Gonzalez E, Chapman MP, Chin TL, Silliman CC, Banerjee

A, Sauaia A: Hyperfibrinolysis, physiologic fibrinolysis, and fibrinolysis shutdown:

the spectrum of postinjury fibrinolysis and relevance to antifibrinolytic therapy. J

Trauma Acute Care Surg 77: 811-817 (2014)

114. Moore HB, Moore EE, Liras IN, Gonzalez E, Harvin JA, Holcomb JB, Sauaia A,

Cotton BA: Acute Fibrinolysis Shutdown after Injury Occurs Frequently and Increases

Mortality: A Multicenter Evaluation of 2,540 Severely Injured Patients. J Am Coll

Surg 222: 347-355 (2016)

115. Moore K: The physiological response to hemorrhagic shock. J Emerg Nurs 40: 629-

631 (2014)

116. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, Schmidt-Ott KM, Chen

X, Li JY, Weiss S, Mishra J, Cheema FH, Markowitz G, Suganami T, Sawai K,

Mukoyama M, Kunis C, D'Agati V, Devarajan P, Barasch J: Endocytic delivery of

lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion

injury. J Clin Invest 115: 610-621 (2005)

117. Mueller SC, Marz R, Schmolz M, Drewelow B: Intraindividual long term stability and

response corridors of cytokines in healthy volunteers detected by a standardized

whole-blood culture system for bed-side application. BMC Med Res Methodol 12: 112

(2012)

118. Munford RS, Pugin J: Normal responses to injury prevent systemic inflammation and

can be immunosuppressive. Am J Respir Crit Care Med 163: 316-321 (2001)

List of references

88

119. Nacionales DC, Szpila B, Ungaro R, Lopez MC, Zhang J, Gentile LF, Cuenca AL,

Vanzant E, Mathias B, Jyot J, Westerveld D, Bihorac A, Joseph A, Mohr A,

Duckworth LV, Moore FA, Baker HV, Leeuwenburgh C, Moldawer LL, Brakenridge

S, Efron PA: A Detailed Characterization of the Dysfunctional Immunity and

Abnormal Myelopoiesis Induced by Severe Shock and Trauma in the Aged. J Immunol

195: 2396-2407 (2015)

120. Netea MG, Balkwill F, Chonchol M, Cominelli F, Donath MY, Giamarellos-

Bourboulis EJ, Golenbock D, Gresnigt MS, Heneka MT, Hoffman HM, Hotchkiss R,

Joosten LAB, Kastner DL, Korte M, Latz E, Libby P, Mandrup-Poulsen T, Mantovani

A, Mills KHG, Nowak KL, O'Neill LA, Pickkers P, van der Poll T, Ridker PM,

Schalkwijk J, Schwartz DA, Siegmund B, Steer CJ, Tilg H, van der Meer JWM, van

de Veerdonk FL, Dinarello CA: A guiding map for inflammation. Nat Immunol 18:

826-831 (2017)

121. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S: Size-

selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol

161: 653-660 (2003)

122. Niu CY, Li JC, Zhao ZG, Zhang J, Shao XH: Effect of intestinal lymphatic circulation

blockage in two-hit rats. World J Gastroenterol 12: 5805-5812 (2006)

123. Norton R, Kobusingye O: Injuries. N Engl J Med 368: 1723-1730 (2013)

124. Obinata H, Hla T: Sphingosine 1-phosphate in coagulation and inflammation. Semin

Immunopathol 34: 73-91 (2012)

125. Ostrowski SR, Henriksen HH, Stensballe J, Gybel-Brask M, Cardenas JC, Baer LA,

Cotton BA, Holcomb JB, Wade CE, Johansson PI: Sympathoadrenal activation and

endotheliopathy are drivers of hypocoagulability and hyperfibrinolysis in trauma: A

prospective observational study of 404 severely injured patients. J Trauma Acute Care

Surg 82: 293-301 (2017)

126. Ostrowski SR, Johansson PI: Endothelial glycocalyx degradation induces endogenous

heparinization in patients with severe injury and early traumatic coagulopathy. J


127. Osuchowski MF, Remick DG: The repetitive use of samples to measure multiple

cytokines: the sequential ELISA. Methods 38: 304-311 (2006)

List of references

89

128. Osuka A, Kusuki H, Yoneda K, Matsuura H, Matsumoto H, Ogura H, Ueyama M:

Glycocalyx Shedding is Enhanced by Age and Correlates with Increased Fluid

Requirement in Patients with Major Burns. Shock (2017)

129. Osuka A, Ogura H, Ueyama M, Shimazu T, Lederer JA: Immune response to traumatic

injury: harmony and discordance of immune system homeostasis. Acute Medicine &

Surgery 1: 63-69 (2014)

130. Paffrath T, Lefering R, Flohe S: How to define severely injured patients? -- an Injury

Severity Score (ISS) based approach alone is not sufficient. Injury 45 Suppl 3: S64-

S69 (2014)

131. Pape HC, Lefering R, Butcher N, Peitzman A, Leenen L, Marzi I, Lichte P, Josten C,

Bouillon B, Schmucker U, Stahel P, Giannoudis P, Balogh Z: The definition of

polytrauma revisited: An international consensus process and proposal of the new

'Berlin definition'. J Trauma Acute Care Surg 77: 780-786 (2014)

132. Pelsers MM, Hermens WT, Glatz JF: Fatty acid-binding proteins as plasma markers

of tissue injury. Clin Chim Acta 352: 15-35 (2005)

133. Perl M, Gebhard F, Braumuller S, Tauchmann B, Bruckner UB, Kinzl L, Knoferl MW:

The pulmonary and hepatic immune microenvironment and its contribution to the early

systemic inflammation following blunt chest trauma. Crit Care Med 34: 1152-1159

(2006)

134. Perl M, Gebhard F, Bruckner UB, Ayala A, Braumuller S, Buttner C, Kinzl L, Knoferl

MW: Pulmonary contusion causes impairment of macrophage and lymphocyte

immune functions and increases mortality associated with a subsequent septic

challenge. Crit Care Med 33: 1351-1358 (2005)

135. Poltorak A, He X, Smirnova I, Liu MY, Van HC, Du X, Birdwell D, Alejos E, Silva

M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B: Defective

LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science

282: 2085-2088 (1998)

136. Raeven P, Salibasic A, Drechsler S, Weixelbaumer KM, Jafarmadar M, van GM,

Bahrami S, Osuchowski MF: A non-lethal traumatic/hemorrhagic insult strongly

modulates the compartment-specific PAI-1 response in the subsequent polymicrobial

sepsis. PLoS One 8: e55467 (2013)

List of references

90

137. Rahbar E, Cardenas JC, Baimukanova G, Usadi B, Bruhn R, Pati S, Ostrowski SR,

Johansson PI, Holcomb JB, Wade CE: Endothelial glycocalyx shedding and vascular

permeability in severely injured trauma patients. J Transl Med 13: 117 (2015)

138. Ramnath R, Foster RR, Qiu Y, Cope G, Butler MJ, Salmon AH, Mathieson PW,

Coward RJ, Welsh GI, Satchell SC: Matrix metalloproteinase 9-mediated shedding of

syndecan 4 in response to tumor necrosis factor alpha: a contributor to endothelial cell

glycocalyx dysfunction. FASEB J 28: 4686-4699 (2014)

139. Raza I, Davenport R, Rourke C, Platton S, Manson J, Spoors C, Khan S, De'Ath HD,

Allard S, Hart DP, Pasi KJ, Hunt BJ, Stanworth S, MacCallum PK, Brohi K: The

incidence and magnitude of fibrinolytic activation in trauma patients. J Thromb

Haemost 11: 307-314 (2013)

140. Razali NM, Wah YB: Power comparisons of shapiro-wilk, kolmogorov-smirnov,

lilliefors and anderson-darling tests. Journal of statistical modeling and analytics 2:

21-33 (2011)

141. Rehm M, Bruegger D, Christ F, Conzen P, Thiel M, Jacob M, Chappell D,

Stoeckelhuber M, Welsch U, Reichart B, Peter K, Becker BF: Shedding of the

endothelial glycocalyx in patients undergoing major vascular surgery with global and

regional ischemia. Circulation 116: 1896-1906 (2007)

142. Reikeras O, Sun J, Wang JE, Foster SJ, Aasen AO: Differences in LPS and PepG

induced release of inflammatory cytokines in orthopedic trauma. J Invest Surg 21: 255-

260 (2008)

143. Relja B, Szermutzky M, Henrich D, Maier M, de Haan JJ, Lubbers T, Buurman WA,

Marzi I: Intestinal-FABP and liver-FABP: Novel markers for severe abdominal injury.

Acad Emerg Med 17: 729-735 (2010)

144. Remick DG, Villarete L: Regulation of cytokine gene expression by reactive oxygen

and reactive nitrogen intermediates. J Leukoc Biol 59: 471-475 (1996)

145. Ritis K, Doumas M, Mastellos D, Micheli A, Giaglis S, Magotti P, Rafail S, Kartalis

G, Sideras P, Lambris JD: A novel C5a receptor-tissue factor cross-talk in neutrophils

links innate immunity to coagulation pathways. J Immunol 177: 4794-4802 (2006)

146. Rittirsch D, Schoenborn V, Lindig S, Wanner E, Sprengel K, Gunkel S, Blaess M,

Schaarschmidt B, Sailer P, Marsmann S, Simmen HP, Cinelli P, Bauer M, Claus RA,

List of references

91

Wanner GA: An Integrated Clinico-transcriptomic Approach Identifies a Central Role

of the Heme Degradation Pathway for Septic Complications after Trauma. Ann Surg

264: 1125-1134 (2016)

147. Rittirsch D, Schoenborn V, Lindig S, Wanner E, Sprengel K, Gunkel S, Schaarschmidt

B, Marsmann S, Simmen HP, Cinelli P, Bauer M, Claus RA, Wanner GA:

Improvement of prognostic performance in severely injured patients by integrated

clinico-transcriptomics: a translational approach. Crit Care 19: 414 (2015)

148. Rodriguez EG, Cardenas JC, Lopez E, Cotton BA, Tomasek JS, Ostrowski SR, Baer

LA, Stensballe J, Holcomb JB, Johansson PI, Wade CE: Early Identification of the

Patient with Endotheliopathy of Trauma by Arrival Serum Albumin. Shock (2017)

149. Rotstein OD: Modeling the two-hit hypothesis for evaluating strategies to prevent

organ injury after shock/resuscitation. J Trauma 54: S203-S206 (2003)

150. Ruf W, Ruggeri ZM: Neutrophils release brakes of coagulation. Nat Med 16: 851-852

(2010)

151. Sasaki M, Joh T: Oxidative stress and ischemia-reperfusion injury in gastrointestinal

tract and antioxidant, protective agents. J Clin Biochem Nutr 40: 1-12 (2007)

152. Sauerland S, Hensler T, Bouillon B, Rixen D, Raum MR, Andermahr J, Neugebauer

EA: Plasma levels of procalcitonin and neopterin in multiple trauma patients with or

without brain injury. J Neurotrauma 20: 953-960 (2003)

153. Schochl H, Frietsch T, Pavelka M, Jambor C: Hyperfibrinolysis after major trauma:

differential diagnosis of lysis patterns and prognostic value of thrombelastometry. J

Trauma 67: 125-131 (2009)

154. Seitz DH, Perl M, Liener UC, Tauchmann B, Braumuller ST, Bruckner UB, Gebhard

F, Knoferl MW: Inflammatory alterations in a novel combination model of blunt chest

trauma and hemorrhagic shock. J Trauma 70: 189-196 (2011)

155. Senthil M, Brown M, Xu DZ, Lu Q, Feketeova E, Deitch EA: Gut-lymph hypothesis

of systemic inflammatory response syndrome/multiple-organ dysfunction syndrome:

validating studies in a porcine model. J Trauma 60: 958-965 (2006)

156. Seshadri A, Brat GA, Yorkgitis BK, Keegan J, Dolan J, Salim A, Askari R, Lederer

JA: Phenotyping the Immune Response to Trauma: A Multiparametric Systems

Immunology Approach. Crit Care Med (2017)

List of references

92

157. Stahel PF, Shohami E, Younis FM, Kariya K, Otto VI, Lenzlinger PM, Grosjean MB,

Eugster HP, Trentz O, Kossmann T, Morganti-Kossmann MC: Experimental closed

head injury: analysis of neurological outcome, blood-brain barrier dysfunction,

intracranial neutrophil infiltration, and neuronal cell death in mice deficient in genes

for pro-inflammatory cytokines. J Cereb Blood Flow Metab 20: 369-380 (2000)

158. Stoecklein VM, Osuka A, Lederer JA: Trauma equals danger--damage control by the

immune system. J Leukoc Biol 92: 539-551 (2012)

159. Sturm R, Heftrig D, Mors K, Wagner N, Kontradowitz K, Jurida K, Marzi I, Relja B:

Phagocytizing activity of PMN from severe trauma patients in different post-traumatic

phases during the 10-days post-injury course. Immunobiology 222: 301-307 (2017)

160. Suvorova ES, Gripentrog JM, Miettinen HM: Different endocytosis pathways of the

C5a receptor and the N-formyl peptide receptor. Traffic 6: 100-115 (2005)

161. Takeuchi O, Akira S: Pattern recognition receptors and inflammation. Cell 140: 805-

820 (2010)

162. Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC: TREM-1 and

its potential ligands in non-infectious diseases: from biology to clinical perspectives.

Pharmacol Ther 177: 81-95 (2017)

163. Tanaka H, Sugimoto H, Yoshioka T, Sugimoto T: Role of granulocyte elastase in

tissue injury in patients with septic shock complicated by multiple-organ failure. Ann

Surg 213: 81-85 (1991)

164. Temmesfeld-Wollbruck B, Hocke AC, Suttorp N, Hippenstiel S: Adrenomedullin and

endothelial barrier function. Thromb Haemost 98: 944-951 (2007)

165. Thuijls G, de Haan JJ, Derikx JP, Daissormont I, Hadfoune M, Heineman E, Buurman

WA: Intestinal cytoskeleton degradation precedes tight junction loss following

hemorrhagic shock. Shock 31: 164-169 (2009)

166. Timmermans K, Kox M, Vaneker M, van den Berg M, John A, van LA, van der

Hoeven H, Scheffer GJ, Pickkers P: Plasma levels of danger-associated molecular

patterns are associated with immune suppression in trauma patients. Intensive Care

Med 42: 551-561 (2016)

List of references

93

167. Tran DD, Cuesta MA, van Leeuwen PA, Nauta JJ, Wesdorp RI: Risk factors for

multiple organ system failure and death in critically injured patients. Surgery 114: 21-

30 (1993)

168. Triantafilou K, Hughes TR, Triantafilou M, Morgan BP: The complement membrane

attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome

activation. J Cell Sci 126: 2903-2913 (2013)

169. Tscherne H: [The treatment of the seriously injured at an emergency station]. Chirurg

37: 249-252 (1966)

170. Tsukamoto T, Chanthaphavong RS, Pape HC: Current theories on the pathophysiology

of multiple organ failure after trauma. Injury 41: 21-26 (2010)

171. Unnewehr H, Rittirsch D, Sarma JV, Zetoune F, Flierl MA, Perl M, Denk S, Weiss M,

Schneider ME, Monk PN, Neff T, Mihlan M, Barth H, Gebhard F, Ward PA, Huber-

Lang M: Changes and regulation of the C5a receptor on neutrophils during septic

shock in humans. J Immunol 190: 4215-4225 (2013)

172. Vajdovich P: Free radicals and antioxidants in inflammatory processes and ischemia-

reperfusion injury. Vet Clin North Am Small Anim Pract 38: 31-123, v (2008)

173. Van de Wouwer M, Collen D, Conway EM: Thrombomodulin-protein C-EPCR

system: integrated to regulate coagulation and inflammation. Arterioscler Thromb

Vasc Biol 24: 1374-1383 (2004)

174. van den Berg CW, Tambourgi DV, Clark HW, Hoong SJ, Spiller OB, McGreal EP:

Mechanism of neutrophil dysfunction: neutrophil serine proteases cleave and

inactivate the C5a receptor. J Immunol 192: 1787-1795 (2014)

175. van Wessem KJP, Leenen LPH: Reduction in Mortality Rates of Postinjury Multiple

Organ Dysfunction Syndrome: A Shifting Paradigm? A Prospective Population-Based

Cohort Study. Shock 49: 33-38 (2018)

176. Veigas PV, Callum J, Rizoli S, Nascimento B, da Luz LT: A systematic review on the

rotational thrombelastometry (ROTEM(R)) values for the diagnosis of coagulopathy,

prediction and guidance of blood transfusion and prediction of mortality in trauma

patients. Scand J Trauma Resusc Emerg Med 24: 114 (2016)

177. Vincent JL, Moreno R, Takala J, Willatts S, De MA, Bruining H, Reinhart CK, Suter

PM, Thijs LG: The SOFA (Sepsis-related Organ Failure Assessment) score to describe

List of references

94

organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related

Problems of the European Society of Intensive Care Medicine. Intensive Care Med 22:

707-710 (1996)

178. Vogel JA, Liao MM, Hopkins E, Seleno N, Byyny RL, Moore EE, Gravitz C, Haukoos

JS: Prediction of postinjury multiple-organ failure in the emergency department:

development of the Denver Emergency Department Trauma Organ Failure score. J


179. Waechter J, Kumar A, Lapinsky SE, Marshall J, Dodek P, Arabi Y, Parrillo JE,

Dellinger RP, Garland A: Interaction between fluids and vasoactive agents on

mortality in septic shock: a multicenter, observational study. Crit Care Med 42: 2158-

2168 (2014)

180. Wohlauer MV, Moore EE, Thomas S, Sauaia A, Evans E, Harr J, Silliman CC, Ploplis

V, Castellino FJ, Walsh M: Early platelet dysfunction: an unrecognized role in the

acute coagulopathy of trauma. J Am Coll Surg 214: 739-746 (2012)

181. Wohlauer MV, Sauaia A, Moore EE, Burlew CC, Banerjee A, Johnson J: Acute kidney

injury and posttrauma multiple organ failure: the canary in the coal mine. J Trauma

Acute Care Surg 72: 373-378 (2012)

182. Wojta J, Kaun C, Zorn G, Ghannadan M, Hauswirth AW, Sperr WR, Fritsch G, Printz

D, Binder BR, Schatzl G, Zwirner J, Maurer G, Huber K, Valent P: C5a stimulates

production of plasminogen activator inhibitor-1 in human mast cells and basophils.

Blood 100: 517-523 (2002)

183. Wolberg AS, Meng ZH, Monroe DM, III, Hoffman M: A systematic evaluation of the

effect of temperature on coagulation enzyme activity and platelet function. J Trauma

56: 1221-1228 (2004)

184. Wutzler S, Lehnert T, Laurer H, Lehnert M, Becker M, Henrich D, Vogl T, Marzi I:

Circulating levels of Clara cell protein 16 but not surfactant protein D identify and

quantify lung damage in patients with multiple injuries. J Trauma 71: E31-E36 (2011)

185. Xiao W, Mindrinos MN, Seok J, Cuschieri J, Cuenca AG, Gao H, Hayden DL,

Hennessy L, Moore EE, Minei JP, Bankey PE, Johnson JL, Sperry J, Nathens AB,

Billiar TR, West MA, Brownstein BH, Mason PH, Baker HV, Finnerty CC, Jeschke

MG, Lopez MC, Klein MB, Gamelli RL, Gibran NS, Arnoldo B, Xu W, Zhang Y,

Calvano SE, McDonald-Smith GP, Schoenfeld DA, Storey JD, Cobb JP, Warren HS,

List of references

95

Moldawer LL, Herndon DN, Lowry SF, Maier RV, Davis RW, Tompkins RG: A

genomic storm in critically injured humans. J Exp Med 208: 2581-2590 (2011)

186. Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT: Extracellular histones are

mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol

187: 2626-2631 (2011)

187. Yi J, Slaughter A, Kotter CV, Moore EE, Hauser CJ, Itagaki K, Wohlauer M, Frank

DN, Silliman C, Banerjee A, Peltz E: A "Clean Case" of Systemic Injury: Mesenteric

Lymph After Hemorrhagic Shock Elicits A Sterile Inflammatory Response. Shock 44:

336-340 (2015)

188. Younger JG, Sasaki N, Waite MD, Murray HN, Saleh EF, Ravage ZB, Hirschl RB,

Ward PA, Till GO: Detrimental effects of complement activation in hemorrhagic

shock. J Appl Physiol (1985 ) 90: 441-446 (2001)

189. Yu L, Seguro AC, Rocha AS: Acute renal failure following hemorrhagic shock:

protective and aggravating factors. Ren Fail 14: 49-55 (1992)

190. Yucel N, Lefering R, Maegele M, Vorweg M, Tjardes T, Ruchholtz S, Neugebauer

EA, Wappler F, Bouillon B, Rixen D: Trauma Associated Severe Hemorrhage

(TASH)-Score: probability of mass transfusion as surrogate for life threatening

hemorrhage after multiple trauma. J Trauma 60: 1228-1236 (2006)

191. Zeng Y, Adamson RH, Curry FR, Tarbell JM: Sphingosine-1-phosphate protects

endothelial glycocalyx by inhibiting syndecan-1 shedding. Am J Physiol Heart Circ

Physiol 306: H363-H372 (2014)

192. Zhang Q, Itagaki K, Hauser CJ: Mitochondrial DNA is released by shock and activates

neutrophils via p38 map kinase. Shock 34: 55-59 (2010)

VII

Declaration

I hereby declare that I wrote the present dissertation with the topic

Early changes in immune, coagulation, and organ function after clinical and

experimental polytrauma

independently and used no other aids that those cited. In each individual case, I have clearly

identified the source of the passages that are taken word for word or paraphrased from other

works.

I also hereby declare that I have carried out my scientific work according to the principles

of good scientific practice in accordance with the current „Satzung der Universität Ulm zur

Sicherung guter wissenschaftlicher Praxis“ [Rules of the University of Ulm for Assuring

Good Scientific Practice].

Ulm, 28.03.2018

VIII

Acknowledgements

The acknowledgements were removed for data privacy protection reasons.

IX

Curriculum vitae

Personal information

Name Rebecca Halbgebauer, née Wiegner

Year of birth 1989

Place of birth Ludwigsburg, Germany

Education

Since 04/2014 PhD student in the International Graduate School in Molecular

Medicine Ulm (IGradU)

Since 10/2013 Doctoral thesis in the Institute of Clinical and Experimental

Trauma Immunology, Ulm University Hospital

Head: Prof. Dr. med. Markus Huber-Lang

Topic: Early immune reaction after severe trauma

10/2011-07/2013 Master of Science in Molecular Medicine, University of Ulm

Master thesis at the Institute of Orthopaedic Research and

Biomechanics, University of Ulm

Head: Prof. Dr. med. vet. Anita Ignatius

Title: Role of neutrophils in fracture healing

10/2008-09/2011 Bachelor of Science in Molecular Medicine, University of

Ulm

09/1999 - 07/2008 Abitur at Gymnasien im Ellental, Bietigheim-Bissingen,

Germany

X

Oral presentations

10/2017 Halbgebauer R, Denk S, Taylor R, Weiss M, Huber-Lang M

Komplement-induzierte Veränderung der Neutrophilenmorphologie während

der Entzündung

German Congress of Orthopaedics and Trauma Surgery (DKOU), Berlin,

Germany

05/2017 Wiegner R, Denk S, Gebhard F, Huber-Lang M

Early Barrier Dysfunction in Polytrauma

Retreat of the Collaborative Research Centre 1149, Oberstdorf, Germany

02/2017 Wiegner R, Denk S, Eisele P, Weckbach S, Braun CK, Cinelli P,

Radermacher P, Wachter U, Hafner S, Palmer A, Braumüller S, Wanner GA,

Simmen HP, Gebhard F, Rittirsch D, Huber-Lang M

Hämorrhagischer Schock im Polytrauma

Gathering of the Trauma Research Network (Netzwerk Traumaforschung,

NTF), Frankfurt, Germany

10/2016 Wiegner R, Denk S, Kalbitz M, Weiß M, Gebhard F, Huber-Lang M

Leukozyten als Interaktionsplattform zwischen Komplement- und

Gerinnungssystem bei polytraumatisierten Patienten

German Congress of Orthopaedics and Trauma Surgery (DKOU), Berlin,

Germany

06/2016 Wiegner R, Denk S, Perl M, Huber-Lang M

Immune monitoring in trauma

IgradU Student Retreat 2016, Kleinwalsertal, Austria

04/2016 Wiegner R, Brenner RE, Weiss M, Lampl L, Huber-Lang M

Role of stem cells after polytrauma

Student Symposium on Molecular Medicine, Ulm, Germany

02/2016 Wiegner R, Lampl L, Weiss M, Brenner R, Huber-Lang M

Rolle von VSELs im Polytrauma



XI

09/2015 Wiegner R, Hönes F, Kalbitz M, Weiss M, Gebhard F, Huber-Lang M,

Denk S

Leukocytes mediate the crosstalk between the complement and coagulation

system in polytrauma patients

XVI. Congress of the European Shock Society, Cologne, Germany

04/2015 Wiegner R, Denk S, Weiss M, Gebhard F, Huber-Lang M

Coagulation-complement crosstalk on leukocytes in polytrauma patients



03/2014 Wiegner R, Kovtun A, Ignatius A

Role of neutrophils in fracture healing

German Congress of Surgery (DGCH), Berlin, Germany

Poster presentations

09/2017 Wiegner R, Braun CK, Denk S, Cinelly P, Radermacher P, Gebhard F,

Rittirsch D, Huber-Lang M

Hemorrhagic shock drives organ dysfunction in polytrauma patients

XVI. Congress of the European Shock Society, Paris, France

06/2015 Wiegner R, Hönes F, Kalbitz M, Weiss M, Braumüller S, Huber-Lang M,

Denk S

Cellular crosstalk between the complement and coagulation system in

polytrauma patients

15th European Meeting on Complement in Human Disease, Uppsala, Sweden

Awarded with a Travel Award

05/2015 Wiegner R, Denk S, Hönes F, Kalbitz M, Gebhard F, Huber-Lang M

Immunophenotyping of severely injured patients

IgradU Student Retreat 2015, Como, Italy

06/2014 Wiegner R, Perl M, Denk S, Braumüller S, Huber-Lang M

Important role of Hsp70 in complement component C5a-induced delayed

apoptosis of neutrophil granulocytes

37th Annual Conference on Shock, Charlotte, NC, USA

XII

Original articles

van Griensven M, Ricklin D, Denk S, Halbgebauer R, Braun CK, Schultze A, Hones F,

Koutsogiannaki S, Primikyri A, Reis E, Messerer D, Hafner S, Radermacher P, Biglarnia

AR, Resuello RRG, Tuplano JV, Mayer B, Nilsson K, Nilsson B, Lambris JD, Huber-Lang

M: Protective Effects of the Complement Inhibitor Compstatin CP40 in Hemorrhagic Shock.

Shock (2018). Doi: 10.1097/SHK.0000000000001127

Denk S, Weckbach S, Eisele P, Braun CK, Wiegner R, Ohmann JJ, Wrba L, Hoenes FM,

Kellermann P, Radermacher P, Wachter U, Hafner S, McCook O, Schultze A, Palmer A,

Braumuller S, Gebhard F, Huber-Lang M: Role of Hemorrhagic Shock in Experimental

Polytrauma. Shock 49: 154-163 (2018)

Halbgebauer R, Braun CK, Denk S, Mayer B, Cinelli P, Radermacher P, Wanner GA,

Simmen HP, Gebhard F, Rittirsch D, Huber-Lang M: Hemorrhagic shock drives glycocalyx,

barrier and organ dysfunction early after polytrauma. J Crit Care 44: 229-237 (2017)

Braun CK, Kalbitz M, Halbgebauer R, Eisele P, Messerer DAC, Weckbach S, Schultze A,

Braumuller S, Gebhard F, Huber-Lang MS: Early structural changes of the heart after

experimental polytrauma and hemorrhagic shock. PLoS One 12: e0187327 (2017)

Wiegner R*, Rudhart NE*, Barth E, Gebhard F, Lampl L, Huber-Lang MS, Brenner RE:

Mesenchymal stem cells in peripheral blood of severely injured patients. Eur J Trauma

Emerg Surg (2017). doi: 10.1007/s00068-017-0849-8. *, equally contributing first authors

Denk S, Taylor RP, Wiegner R, Cook EM, Lindorfer MA, Pfeiffer K, Paschke S, Eiseler T,

Weiss M, Barth E, Lambris JD, Kalbitz M, Martin T, Barth H, Messerer DAC, Gebhard F,

Huber-Lang MS: Complement C5a-Induced Changes in Neutrophil Morphology During

Inflammation. Scand J Immunol 86: 143-155 (2017)

Denk S, Neher MD, Messerer DAC, Wiegner R, Nilsson B, Rittirsch D, Nilsson-Ekdahl K,

Weckbach S, Ignatius A, Kalbitz M, Gebhard F, Weiss ME, Vogt J, Radermacher P, Kohl J,

Lambris JD, Huber-Lang MS: Complement C5a Functions as a Master Switch for the pH

Balance in Neutrophils Exerting Fundamental Immunometabolic Effects. J Immunol 198:

4846-4854 (2017)

XIII

Kovtun A, Bergdolt S, Wiegner R, Radermacher P, Huber-Lang M, Ignatius A: The crucial

role of neutrophil granulocytes in bone fracture healing. Eur Cell Mater 32: 152-162 (2016)

Denk S, Wiegner R, Hones FM, Messerer DA, Radermacher P, Weiss M, Kalbitz M,

Ehrnthaller C, Braumuller S, McCook O, Gebhard F, Weckbach S, Huber-Lang M: Early

Detection of Junctional Adhesion Molecule-1 (JAM-1) in the Circulation after Experimental

and Clinical Polytrauma. Mediators Inflamm 2015: 463950 (2015)

Review articles

Halbgebauer R, Schmidt CQ, Karsten CM, Ignatius A, Huber-Lang M: Janus face of

complement-driven neutrophil activation during sepsis. Semin Immunol (2018). doi:

10.1016/j.smim.2018.02.004

Huber-Lang M, Ekdahl KN, Wiegner R, Fromell K, Nilsson B: Auxiliary activation of the

complement system and its importance for the pathophysiology of clinical conditions. Semin

Immunopathol 40: 87-102 (2018)

Wiegner R, Chakraborty S, Huber-Lang M: Complement-coagulation crosstalk on cellular

and artificial surfaces. Immunobiology 221: 1073-1079 (2016)

Huber-Lang M, Gebhard F, Schmidt CQ, Palmer A, Denk S, Wiegner R: Complement

therapeutic strategies in trauma, hemorrhagic shock and systemic inflammation - closing

Pandora's box? Semin Immunol 28: 278-284 (2016)

Huber-Lang M, Wiegner R, Lampl L, Brenner RE: Mesenchymal Stem Cells after

Polytrauma: Actor and Target. Stem Cells Int 2016: 6289825 (2016)

early changes in immune, coagulation, and organ function

Documents