Platelets and Complement Activation in Coronary Artery Bypass Graft Surgery (CABG)
Conditions: Cardiopulmonary Bypass, Inflammatory Response, Platelet Dysfunction
Sponsor: Medical University Innsbruck

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STUDY PROTOCOL 
 
 
Title: Platelets and complement activation in coronary artery bypass graft 
surgery (CABG) 
 
 
Protocol Code: 
 
 
PAC 
 
 
Version, Date: 
 
 
Version 2.1, 27.08.2018 
 
 
Sponsor: 
 
  
 
Medical University Innsbruck 
 
Clinic of Anaesthesiology and Intensive Care 
Medicine  
 
Anichstraße 35 
 
6020 Innsbruck 
 
 
Principal Investigator:   
Univ.-Prof. Dr. Judith Martini1 
Sub-Investigators:  
 
Univ.-Prof. Dr. Dietmar Fries2,  
Univ.-Doz. Dr. Petra Innerhofer1 
Univ.-Prof. Dr. Werner Streif3 
Dr. Nicole Innerhofer1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 Clinic of Anaesthesiology and Intensive Care Medicine, Medical University Innsbruck 
2 Clinic of General and Surgical Intensive Care Medicine, Medical University Innsbruck 
3 Clinic of Pediatrics I, Medical University Innsbruck 

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CONFIDENTIALITY 
The present study, including archiving of study documents, will be conducted according to this protocol, 
and in compliance with Good Clinical Practices, the Declaration of Helsinki in its latest version, the local 
laws and regulations and the applicable regulatory requirements. 
 
CONFIDENTIAL 
The information provided in this document is strictly confidential and is available for review to 
Investigators, potential Investigators, health authorities and appropriate ethics committees. No 
disclosure should take place without written authorization from the Sponsor, except to the extent 
necessary to obtain informed consent from potential subjects. Once signed, the terms of this protocol 
are binding for all parties. 
 
DECLARATION OF THE SPONSOR 
 
Study Title:  
Platelets and complement activation in  
coronary 
artery bypass graft surgery  
(CABG) factor assays  
Protocol Code Number:  
 PAC 
 
 
Sponsor: 
Principal Investigator:  
Medical University Innsbruck [MUI] 
Department of Anesthesiology  
Intensive Care Medicine 
Head: O. Univ.-Prof. Dr. Karl Lindner 
 
Anichstrasse 35, 6020 Innsbruck 
Phone: +43 512 504 22456 
E-Mail: Karl.Lindner@i-med.ac.at 
Univ.-Prof. Dr. Judith Martini 
Medical University Innsbruck 
Department of Anesthesiology  
Intensive Care Medicine 
 
Anichstrasse 35, 6020 Innsbruck 
Phone: +43 512 504 80478 
E-Mail: judith.martini@tirol-kliniken.at 
 
 
The present study protocol was subject to critical review. Its content is consistent with the current 
risk/benefit evaluation as well as with the moral, ethical and scientific principles of good clinical practice, 
the latest version of the Declaration of Helsinki, the local laws and the regulations and the applicable 
regulatory requirements. 
 
Univ.-Prof. Dr. Karl Lindner 
Department of Anesthesiology and 
Intensive Care Medicine 
Medical University Innsbruck 
Place, Date, Signature 
 
The signatories above confirm that they have read this study protocol and agree that it contains all 
information required for study performance. They also agree to conduct the study as set out in this 
protocol. It has been understood that all documentation previously not published will be kept in strictest 
confidence. 
 

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DECLARATION OF THE PRINCIPAL INVESTIGATOR AND THE SUB 
INVESTIGATOR, STUDY AUTHORS AND THE STATISTICIAN 
 
Study Title:  
Platelets and complement activation in CABG  
I have read the study protocol and agree that it contains all information required for study performance. I agree 
to conduct the study as set out in the protocol. In particular, I agree to adhere to the moral, ethical and scientific 
principles of good clinical practice, the latest version of the declaration of Helsinki, the local laws and regulations 
and the applicable regulatory requirements. 
In case of any changes which may concern the responsibility of each of the signatories, I will notify them. 
 
Prof. Dr. Judith Martini 
Principal Investigator, Study Author 
Department of Anaesthesia and Intensive Care 
Medicine 
Medical University Innsbruck  
Anichstraße 35, 6020 Innsbruck 
Austria 
  _____________________________________________  
Place, Date, Signature 
 
Ao.Prof. Dr. Dietmar Fries 
Sub-Investigator, Study-Author 
Department of Surgical and General Intensive 
Care Medicine 
Medical University Innsbruck  
Anichstraße 35, 6020 Innsbruck 
Austria 
  _____________________________________________  
Place, Date, Signature 
 
Univ.Doz. Dr. Petra Innerhofer 
Sub-Investigator, Study-Author 
Department of Anaesthesia and Intensive Care 
Medicine 
Medical University Innsbruck  
Anichstraße 35, 6020 Innsbruck 
 
  _____________________________________________  
Place, Date, Signature 
 
Dr. Nicole Innerhofer 
Sub-Investigator, Study Author 
Department of Anaesthesia and Intensive Care 
Medicine 
Medical University Innsbruck  
Anichstraße 35, 6020 Innsbruck Austria 
  _____________________________________________  
Place, Date, Signature 
 
Prof. Dr. Werner Streif 
Sub-Investigator, Study Author 
Department of Pediatrics 
Medical University Innsbruck  
Anichstraße 35, 6020 Innsbruck Austria 
  _____________________________________________  
Place, Date, Signature 
 

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Mirjam Bachler, PhD 
Study Author 
ISAG – UMIT Hall Medical Sciences 
Eduard-Wallnöfer-Zentrum 1, 6060 Hall in 
Tirol 
 
Tobias Hell, PhD 
Statistician 
Institute for Mathematics 
University Innsbruck Austria 
  _____________________________________________  
Place, Date, Signature 
 
 
 
 

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DECLARATION OF THE PRINCIPAL INVESTIGATOR 
Study Title: Platelets and complement activation in CABG  (“PAC”) 
 
I have read this study protocol and agree that it contains all the information required for study performance. I 
agree to conduct the study as set out in the protocol. In particular, I agree to adhere to the moral, ethical and 
scientific principles of good clinical practice, the latest version of the declaration of Helsinki, the local laws and 
regulations and the applicable regulatory requirements. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name 
 
 
 
 
 
 
 
Place, Date 
 
 
 
 
 
 
 
Signature 
 
 
 

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TABLE OF CONTENT 
 
STUDY PROTOCOL ......................................................................................................... 1 
CONFIDENTIALITY ......................................................................................................... 2 
DECLARATION OF THE SPONSOR .................................................................................... 2 
Declaration of the Principal Investigator and the SUB Investigator, Study Authors and the 
Statistician ..................................................................................................................... 3 
Declaration of the Principal Investigator ........................................................................... 5 
Synopsis ........................................................................................................................ 8 
Study Flow Chart .......................................................................................................... 11 
Abbreviations ............................................................................................................... 12 
Platelets after complement activation in cardiac surgery - PAC ......................................... 13 
1. Introduction ............................................................................................................. 13 
1.1. Background ................................................................................................................................. 13 
1.2. Need for the Study ..................................................................................................................... 13 
1.3. Risk Benefit analysis ................................................................................................................... 14 
2. Study Objectives ...................................................................................................... 14 
2.1. Primary Study Objective ............................................................................................................. 14 
2.2. Further Study Objectives ............................................................................................................ 14 
3. Investigational Plan .................................................................................................. 15 
3.1. General Design of the Study ....................................................................................................... 15 
3.2. Timetable .................................................................................................................................... 15 
3.3. Blood sampling ........................................................................................................................... 15 
3.3.1 Study related blood samples (T0-T8).................................................................................... 15 
3.3.2. Collection of routinely measured laboratory parameters, and clinical data (T0-T9) .......... 16 
3.4. Primary Study Endpoint .............................................................................................................. 16 
3.5. Secondary Study Endpoints ........................................................................................................ 16 
3.6. Number of patients .................................................................................................................... 17 
3.7. Timetable .................................................................................................................................... 17 
4. Study Population ...................................................................................................... 18 
4.1. Inclusion criteria: ........................................................................................................................ 18 
4.2. Exclusion criteria: ....................................................................................................................... 18 
5. Further Study Procedures .......................................................................................... 19 
5.1. Patient Withdrawal (Drop-out) .................................................................................................. 19 
5.2. Discontinuation of the Study ...................................................................................................... 19 

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5.3. Closure of the Study ................................................................................................................... 19 
6. Documentation and Data Management ....................................................................... 20 
6.1. Data Entry Form (CRF) ................................................................................................................ 20 
6.2. Data Management ...................................................................................................................... 20 
7. Statistics .................................................................................................................. 20 
Primary analysis ................................................................................................................................. 20 
Sample size estimation ...................................................................................................................... 21 
Secondary analysis ............................................................................................................................ 21 
8. Ethical, Legal and Administrative Issues ..................................................................... 21 
8.1. Good Clinical Practice and Declaration of Helsinki .................................................................... 21 
8.2. Approval of the Study Protocol .................................................................................................. 22 
8.3. Obtaining Informed Consent ...................................................................................................... 22 
8.4. Confidentiality ............................................................................................................................ 22 
8.5. Archiving of Study Records ......................................................................................................... 22 
9. Amendments ............................................................................................................ 22 
10. Scores ................................................................................................................... 23 
SOFA .................................................................................................................................................. 23 
EUROSCORE II .................................................................................................................................... 23 
11. References ............................................................................................................. 24 
 
 
 
 

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SYNOPSIS 
 
 
DESIGN OF THE CLINICAL STUDY 
Indication 
elective coronary artery bypass graft surgery (CABG) 
Study Design 
Prospective, observational, single center study 
Objective 
Primary:  
-To assess the impact of complement activation on platelet count. 
Secondary: 
- To assess the specific pathway of complement activation, mtDNA levels and their 
effects on platelet count and platelet function using mean platelet volume to platelet 
count ratio (MPV/PTC) and specific platelet function by fluorescence-activated cell 
sorting (FACS).  
- To explore whether these variables correlate with transfusion requirements, 
postoperative morbidity (need for revision surgery, cardiovascular events including 
thromboembolic events, sepsis, single or multiple organ failure according to SOFA 
Score) and in hospital mortality. 
Endpoints and 
Evaluation Parameters 
Primary endpoint:  
- Correlation of the change of C5b-9 and change of platelet count from baseline (T0) to 
after 15 minutes of administration of protamine (T2). 
 
Secondary endpoints: 
- correlation between levels of complement factors [c5b-9, C1q (C1r/C1s), C3a, C5a, 
MBL, Factor B, Factor D], mtDNA level [human NADH dehydrogenase 1 gene] and 
platelet function [MPV/PTC ratio and FACS for platelet activation factor 4 (PAF-4)]  
 
- correlation between levels of complement [see above] and concentration of coagulation 
factors [F I – F XIII, FXa, FXIIa, Kallikrein, Bradykinin, endogenous thrombin potential 
(ETP)] and transfusion requirements 
- correlation of levels of complement, mtDNA and platelet function to postoperative 
morbidity (need for revision surgery, cardiovascular events including thromboembolic 
events, sepsis, single or multiple organ failure according to SOFA Score) and in 
hospital mortality 
 
Number of Patients 
190 
Sponsor 
Medical University Innsbruck 
Center 
University Hospital Innsbruck 
Title 
Platelets and complement activation in CABG 
Protocol Code 
PAC 

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Duration of Patient 
Participation 
- day of surgery to postoperative day three (POD 3) 
- Follow-Up Period “Medical Monitoring”: until discharge or day 30 
Dates of Beginning 
and End of Study 
Date of beginning of study: 1.July 2018 
Date of end of study: 1.July 2020 
Inclusion Criteria 
I.1. Male and female subjects  18 years and < 85 years  
I.2. elective coronary artery bypass graft surgery on cardiopulmonary bypass (CPB) 
with or without valve surgery (max. 2 valves) and elective valve surgery (max. 2 
valves) 
I.3. ASA I - IV 
I.4. written informed consent 
Exclusion Criteria 
E.1.     emergency CABG with cardiac valve surgery and emergency valve surgery and 
emergency aortic dissection  
E.2.  
preexisting complement deficiency syndromes  
E.3.  
preexisting thrombocytopathy or thrombocytopenia (platelet count below 100 
G/L) 
E.4.    Known history of congenital coagulopathy 
E.5.   Patients that are known to be pregnant 
E.6. 
  Known participation in another interventional clinical trial 
Study-related 
Measurements and 
Data collection 
Study related measurements: complement factors, platelet function (FACS), mtDNA, 
concentrations of coagulation factors and thrombin generation will be obtained at 
baseline (T0) and at several time points (T1-T8) during and after CABG until POD3. 
Data collection: Details on patient’s condition before surgery, as well as all clinical data 
until discharge or day 30 
 
Study Procedure 
Except study related blood sampling patient’s care is not influenced by the study and 
follows clinical routine. The Visit time points are: 
T0  
baseline: after insertion of arterial cannula beginning surgery 
T1 
after re-opening aortic clamp on CPB 
T2 
15 minutes after protamine infusion 
T3 
end of surgery = admission lab at the postoperative intensive care unit 
T4 
4 hours after end of surgery 
T5 
12 hours after end of surgery 
T6 
24 hours after surgery begin 
T7 
48 hours after surgery begin 
T8 
72 hours after surgery begin 
T9 
30 days after surgery or hospital admission 
 
Study related blood sampling (T0-T8): 
▪ 
Levels of complement factors: c5-b9, C1q (C1r/C1s), C3a, C5a, MBL, Factor B, 
Factor D 

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▪ 
mtDNA (human NADH dehydrogenase 1 gene)  
▪ 
Platelet count, mean platelet volume, fluorescence-activated cell sorting 
(FACS) 
▪ 
Coagulation factors: FI – FXIII, FXa, FXIIa, Kallikrein, Bradykinin, endogenous 
thrombin potential (ETP) 
Routine laboratory blood sampling (T0-T9): 
activated clotting time (ACT), Rotem parameters, Multiplate test analysis, whole blood 
cell count, standard plasmatic coagulation tests, 
blood gas analysis, CRP, Procalcitonin (PCT), renal and liver function parameters, 
cardiac enzymes (CK, CK-MB, Myoglobin, Trop T, NTpro BNP) 
 
Clinical Data: 
The following parameters will be collected at baseline(T0): 
age, gender, body weight(kg) and size (cm), medical history (chronic renal 
impairment, Diabetes, Hypertension, previous myocardial infarction, previous stroke, 
previous percutaneous coronary intervention, previous CABG), pre-medication, 
Euroscore II. 
 
During surgery and until POD 3 following data will be collected if available: 
Details on CPB, Cell Saver blood volume, blood pressure (BP), hear rate (HR), central 
venous pressure (CVP), mean pulmonary artery pressure (MPAP),cardiac index (C.I.), 
cardiac output (C.O.), Oxygen Saturation (SpO2), Horowitz, Fraction of inspired 
Oxygen (FiO2), Diuresis per hour, Concomitant Medication, transfusion and coagulation 
products, need for haemofiltration , need for extracorporeal membrane oxygenation 
(ECMO) and/or need for inhalative NO. 
 
 
Risk-Benefit Analysis 
There is no risk for pain and infection since the blood is drawn from a routinely done 
cannula.  The total amount of blood sampling is 150 ml within 3 days and will not 
harm the patients. The patients benefit from the intensified diagnostics.  
Statistics 
Primary endpoint: Correlation of the change of C5b-9 and change of platelet count 
from baseline (T0) to after 15 minutes of administration of protamine (T2). 
 
 
 
 

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STUDY FLOW CHART 
 
 
 
T0 
Baseline 
Day of 
surgery 
Day 0 
T1 
CPB 
Opening 
Aortic 
crossclamp 
Day0 
T2 
CPB 
15min after 
Protamin 
Day0 
T3 
End of 
surgery 
Admission 
post Op 
ICU 
Day0 
T4 
PACU 
4h post 
surgery 
Day 0 
T5 
PACU 
12h post 
surgery 
Day 0 
T6 
PACU 
08:00 
Day1 
T7 
ICU 
08:00 
Day2 
T8 
ICU 
08:00 
Day3 
T9 
Ward/ICU 
Day 30 
Special 
laboratory1 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Routine 
laboratory2 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Clinical data3 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Concomitant 
Medication 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Transfusions/  
Coagulation 
products 
 
x 
x 
x 
x 
x 
x 
x 
x 
 
Scores 
Euroscore 
II 
 
 
 
SOFA* 
SOFA* 
SOFA* 
SOFA* 
SOFA* 
 
Final survey 
 
 
 
 
 
 
 
 
 
x 
 *calculation of SOFA if patient stays at ICU 
1 c5-b9, C1q (C1r/C1s), C3a, C5a, MBL, Factor B, Factor D, mtDNA, Platelet count, mean platelet volume, fluorescence-activated cell sorting (FACS), FI – FXIII, FXa, FXIIa, 
Kallikrein, Bradykinin, endogenous thrombin potential (ETP) 
2  activated clotting time (ACT), Rotem parameters, Multiplate test analysis, whole blood cell count, standard plasmatic coagulation tests, blood gas analysis, CRP, 
Procalcitonin (PCT), renal and liver function parameters, cardiac enzymes (CK, CK-MB, Myoglobin, Trop T, NTpro BNP)  
3 age, gender, body weight(kg) and size (cm), medical history (chronic renal impairment, Diabetes, Hypertension, previous myocardial infarction, previous stroke, 
previous percutaneous coronary intervention, previous CABG), pre-medication, Euroscore II, Details on CPB, Cell Saver blood volume, blood pressure (BP), hear rate (HR), 
central venous pressure (CVP), mean pulmonary artery pressure (MPAP),cardiac index (C.I.), cardiac output (C.O.), Oxygen Saturation (SpO2), Horowitz, Fraction of 
inspired Oxygen (FiO2), Diuresis per hour, Concomitant Medication, transfusion and coagulation products, , need for extracorporeal membrane oxygenation (ECMO) 
and/or need for inhalative NO. 
 
 

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ABBREVIATIONS 
 
ASA 
American Society of Anesthesiologists (ASA) Score is a global score 
that assesses the physical status of patients before surgery. 
BGA 
Blood gas analysis 
CA  
Competent Authority 
CABG 
Coronary Artery Bypass Graft 
CPB 
Cardiopulmonary Bypass 
CRF  
Case Report Form 
ECMO 
extracorporeal membrane oxygenation 
ETP 
endogenous thrombin potential 
FI - FXIII 
coagulation factor I - XIII 
FACS 
fluorescence-activated cell sorting 
GCP  
Good Clinical Practice 
ICH  
International Conference on Harmonization 
ICU  
Intensive Care Unit 
mtDNA 
mitochondrial DNA 
MPV 
mean platelet volume 
MPV ratio 
Mean platelet volume/mean platelet count 
NA  
not applicable 
ND  
not done 
POD 
postoperative day 
RIFLE Score 
Risk Injury Failure Loss Endstage kidney injury score 
SOFA Score 
sequential organ failure assessment score 
 

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PLATELETS AFTER COMPLEMENT ACTIVATION IN CARDIAC SURGERY 
- PAC 
 
 
1. INTRODUCTION 
1.1. Background 
Patients undergoing coronary artery bypass graft surgery (CABG) frequently exhibit 
postoperative bleeding complications which are still a major cause for morbidity and 
mortality (1). One major contributing factor is the loss of platelets and impaired platelet 
function. During cardiopulmonary bypass (CPB) blood comes in close contact with 
foreign surfaces which induces a series of reactions; especially the complement system 
as part of the innate immunity is highly activated (2). Due to the strong crosslink 
between complement system, platelet function and the plasmatic coagulation it is likely 
that complement activation during CPB has an impact on the overall process of clot 
formation. (3). Besides the activation of the complement system there is growing 
evidence that the occurrence of mitochondrial DNA (mtDNA) during CPB might be 
related to further platelet activation (4). Activated platelets may enhance micro-
thrombosis leading to organ failure and thereby contributing to postoperative 
morbidity. 
Several studies investigated the effect of complement activation in cardiac surgery 
patients (2;5;6-7) however, it is still unclear whether the activation of the complement 
system further increases platelet loss.  
 
1.2. Need for the Study 
One major complication during and after CABG surgery is bleeding requiring transfusion 
and even reoperation in about 2%- 8% of patients. 
A recent study shows that impaired hemostasis was observed in 9,5 - 21,5% of these 
cases (1). As bleeding complications increase patient morbidity and mortality, this study 
is designed to investigate, the possible mechanisms of platelet loss during CABG. 
We hypothesize that increased complement activation during CPB leads to platelet 
activation and loss of platelets. We further hypothesize that the degree of complement 
activation and levels of mtDNA correlate with postoperative bleeding, transfusion 
requirements and clinical outcome. 
 
 
 
 

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1.3. Risk Benefit analysis 
For blood withdrawal no additional arterial/venous puncture is needed.  
Blood samples will all be withdrawn from preexisting arterial cannulas. Only if there is 
no arterial line available, blood will be withdrawn of an also preexisting central venous 
catheter. 
The total amount of blood collected over the entire study period will be 150 ml, which 
seems to be a negligible risk compared to the average blood loss in CABG surgery.  
The findings of this study will help to understand further details of the mechanism of 
platelet drop and complement activation during and after CABG surgery. Furthermore, 
the results could help to develop new therapeutic strategies to avoid activation of the 
complement and hemostatic systems during CPB which may decrease bleeding 
complications in CABG patients.  
 
2. STUDY OBJECTIVES 
2.1. Primary Study Objective 
The aim of the study is to examine the correlation between complement activation (c5b-
9) and platelet decline during elective coronary artery bypass graft (CABG) with or 
without valve surgery (max. 2 valves) and elective valve surgery (max. 2 valves) on 
cardiopulmonary bypass (CPB). 
 
2.2. Further Study Objectives 
A secondary aim of the study is to investigate the specific pathway of complement 
activation, the mtDNA levels and the interaction with platelet function. 
Therefore we search for correlations between levels of complement factors [c5b-9, C1q 
(C1r/C1s), C3a, C5a, MBL, Factor B, Factor D], mtDNA level [human NADH 
dehydrogenase 1 gene] and platelet function [MPV/PTC ratio and FACS for platelet 
activation factor 4 (PFA)].  
 
Further it will be explored whether the level of complement activation and levels of 
mtDNA and the plasmatic coagulation system correlate with transfusion requirements, 
postoperative morbidity (need for revision surgery, cardiovascular events including 
thromboembolic events, sepsis, single or multiple organ failure according to SOFA 
Score), and in hospital mortality. 
We will correlate levels of complement [see above] and concentration of coagulation 
factors [F I – F XIII, FXa, FXIIa, Kallikrein, Bradykinin, endogenous thrombin potential 
(ETP)] and transfusion requirements and correlate levels of complement, mtDNA and 
platelet function to postoperative morbidity (need for revision surgery, cardiovascular 
events including thromboembolic events, sepsis, single or multiple organ failure 
according to SOFA Score) and in hospital mortality. 
 

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3. INVESTIGATIONAL PLAN 
3.1. General Design of the Study 
This is a prospective observational single center study in elective cardiac surgery 
patients undergoing coronary artery bypass grafts with or without valve surgery (max. 2 
valves) and elective valve surgery (max. 2 valves).  
Patient care is not influenced by the study and follows clinical routine. Patient follow up 
will be until hospital discharge or day 30 after surgery. 
 
3.2. Timetable 
T0  
baseline: after insertion of arterial cannula beginning surgery 
T1 
after re-opening aortic clamp on CPB 
T2 
15 minutes after protamine infusion 
T3 
end of surgery = admission lab at the postoperative intensive care unit 
T4 
4 hours after surgery/admission 
T5 
12 hours after surgery/admission  
T6 
24 hours after surgery begin 
T7 
48 hours after surgery begin 
T8 
72 hours after surgery begin 
T9 
30 days after surgery or hospital admission 
 
3.3. Blood sampling  
3.3.1 Study related blood samples (T0-T8) 
▪ Levels of complement factors: c5-b9, C1q (C1r/C1s), C3a, C5a, MBL, Factor B, 
Factor D 
▪ mtDNA (human NADH dehydrogenase 1 gene)  
▪ Platelet count, mean platelet volume, fluorescence-activated cell sorting (FACS) 
▪ Coagulation factors: FI – FXIII, FXa, FXIIa, Kallikrein, Bradykinin, endogenous 
thrombin potential (ETP) 
▪ Residual plasma and serum from blood samples will be stored at -80°C for further 
differentiated complement and mtDNA assays if useful 
 
 
 

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3.3.2. Collection of routinely measured laboratory parameters, and clinical 
data (T0-T9) 
The following routine laboratory parameters will be collected if available: 
activated clotting time (ACT), Rotem parameters, Multiplate test analysis, whole blood 
cell count, standard plasmatic coagulation tests, 
blood gas analysis, CRP, Procalcitonin (PCT), renal and liver function parameters, 
cardiac enzymes (CK, CK-MB, Myoglobin, Trop T, NTpro BNP)  
Study related blood sampling will overlap with routine blood sampling. 
Clinical Data 
The following parameters will be collected at baseline(T0): 
age, gender, body weight(kg) and size (cm), medical history (chronic renal impairment, 
Diabetes, Hypertension, previous myocardial infarction, previous stroke, previous 
percutaneous coronary intervention, previous CABG), pre-medication, Euroscore II. 
 
During surgery following data will be collected if available: 
Details on CPB, Cell Saver blood volume, blood pressure (BP), hear rate (HR), central 
venous pressure (CVP), mean pulmonary artery pressure (MPAP),cardiac index (C.I.), 
cardiac output (C.O.), Oxygen Saturation (SpO2), Horowitz, Fraction of inspired Oxygen 
(FiO2), Diuresis per hour, Concomitant Medication, transfusion and coagulation products, 
, need for extracorporeal membrane oxygenation (ECMO), need for inhalative NO  
As appropriate, the above mentioned clinical data will be collected until POD3 (T1-T9). 
3.4. Primary Study Endpoint 
The primary endpoint is the correlation of the change of C5b-9 and change of platelet 
count from baseline (T0) to after 15 minutes of administration of protamine (T2). 
 
3.5. Secondary Study Endpoints 
▪ levels of complement activation: c5b-9, C1q (C1r/C1s), C3a, C5a, MBL, Factor B, 
Factor D 
▪ Levels of mtDNA  
▪ platelet function as measured by FACS 
▪ Levels of coagulation factors FI – FXIII, FXa, FXIIa, Kallikrein, Bradykinin, 
endogenous thrombin potential (ETP) 
▪ transfusion requirements (red blood cell concentrates, fresh frozen plasma, 
platelet concentrate) and dosages of coagulation products administered during 
surgery, at ICU and until POD 3 

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▪ Need for revision surgery because of re-bleeding or hematoma formation within 
the first 24hours at ICU 
▪ hours/days on mechanical ventilation 
▪ Oxygenation index (Horowitz quotient) during surgery, at ICU until POD 3 if 
applicable 
▪ length of ICU stay 
▪ length of hospital stay 
▪ postoperative complications: 
o single and multiple organ failure according to SOFA score 
o acute kidney injury (RIFLE score) 
o diagnosed infection and sepsis  
▪ incidence and type of cardiovascular thromboembolic events  
▪ in hospital mortality  
 
3.6. Number of patients 
190 patients will be included after written informed consent.  
3.7. Timetable 
The duration of the planned recruitment phase is 2 years. For the single subject the 
duration of the participation 72 hours in the active phase and further 27 days (until 30 
days after inclusion) in the Follow-Up phase.  
ISSUE 
Time schedule 
Submission to the EC:  
 
Study Conduction:  
 
Laboratory Analysis:  
 
Final Report / Publication:  
 
 
 
 

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4. STUDY POPULATION 
All adult patients undergoing elective coronary artery bypass graft surgery with written 
informed consent meeting the following criteria: 
 
4.1. Inclusion criteria: 
I.1.  
 
Male and female subjects > 18 years and < 86 years  
I.2.  
 elective coronary artery bypass graft surgery with or without valve surgery 
(max. 2 valves) and elective valve surgery (max. 2 valves) on 
cardiopulmonary bypass (CPB) 
I.3.  
 
ASA I - IV 
I.4.  
 
written informed consent 
4.2. Exclusion criteria: 
E.1.  
 emergency CABG with or without cardiac valve surgery and emergency 
valve surgery and emergency aortic dissection 
E.2. 
 
preexisting complement deficiency syndromes  
E.3. 
 
preexisting thrombocytopathy or thrombocytopenia (platelet count below  
100 G/L) 
E.4. 
 
Known history of congenital coagulopathy 
E.5. 
  
Patients that are known to be pregnant 
E.6 
  
Known participation in another interventional clinical trial 
 

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5. FURTHER STUDY PROCEDURES  
5.1. Patient Withdrawal (Drop-out) 
Patients can drop-out of the study by their own request or the request of the patient’s 
authorized person or the patient’s legal guardian (whenever applicable) without 
mentioning any reasons and without any consequences for the subsequent treatment. 
Moreover, patients can be withdrawn from the study by the Investigator for health risk 
reasons. 
If the investigator decides to withdraw the patient from the study the reason  
will be documented in the CRF. It includes, but is not limited 
to: 
▪ Withdrawal of consent/ not giving consent 
▪ Lost to Follow-Up, etc 
 
If the patient decides to be withdrawn from the study the patient will be asked to agree 
to use the data collected in course of the study The patient’s decision will be recorded in 
the patient’s medical file. 
 
 
5.2. Discontinuation of the Study 
The Sponsor is authorized to discontinue the study because of relevant 
medical/administrative causes. The reasons for the discontinuation of the study have to 
be documented in detail. Patients who are still enrolled at the time of discontinuation, 
have to be examined for a final investigation, which will be documented in the CRF. If the 
Investigator has any ethical qualms concerning the continuation of the study, this has to 
be immediately reported to the Sponsor. 
 
The Sponsor is authorised to discontinue the study, if: 
▪ The recruitment rate of patients is not sufficient 
▪ Serious, non-resolvable problems of quality of the collected data evolve 
▪ New scientific findings during the study’s run-time do not allow a continuation of 
the study 
 
5.3. Closure of the Study 
The study must be closed after completion and the ethics committees will be informed 
when the study is closed. 
 
 
 
 
 

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6. DOCUMENTATION AND DATA MANAGEMENT 
The accomplishments of the study in agreement with the GCP-guidelines as well as the 
trueness of all data documented in the CRF are the responsibility of the Investigator. All 
collected data of this study have to be recorded in the CRF by appropriate authorized 
persons.  
The Investigator records the participation in a special identification list of patients. 
Additionally, the participation of the patient in this clinical study has to be recorded in 
the patient chart. 
 
6.1. Data Entry Form (CRF) 
Data of patients and investigation results will be recorded into Case Report Forms 
(CRF), which are especially developed for this study. 
Only the use of black ball pens is allowed. Corrections have to be made in such a way, 
that previous entries remain readable. Corrections have to be signed and dated by the 
authorized person, who made the corrections. Data which are not available or were not 
collected, have to be clearly identifiable as such (NA and ND). The reasons should be 
documented, if necessary. 
 
6.2. Data Management 
Data items from the CRF are entered centrally into the study database by authorized 
data management staff using appropriate entry techniques and probability check. 
Entered data are systematically checked by data management staff, using error 
messages printed from validation programs and database listings. Obvious errors or 
omissions are corrected by the data management personnel. After recording of all 
entries and clarification of all queries, the database will be closed at the completion of 
the study. This closure has to be documented. 
 
7. STATISTICS 
Primary analysis  
The primary endpoint of this study is the correlation of the change in C5b-9 and the 
change in platelet count from baseline (T0) to 15 minutes after administration of 
protamine (T2). The analyzed study population will consist of all patients with available 
measurements necessary to assess the primary endpoint. 
The two changes (C5b-9 and platelet count) are continuous variables and their 
correlation (Spearman's rank-order correlation denoted by ρ) will be assessed. 
Therefore, the hypotheses   
 
𝐻0: ρ = 0  
vs. 
𝐻1: ρ ≠0 

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will be tested using the Spearman rank-order correlation test, i.e. the null hypothesis 
that the Spearman correlation is zero against the alternative that it is not equal to zero. 
As C5b-9 levels and platelet count are assumed to be predictive for the clinical course of 
the patients, the association with secondary endpoints will be assessed. Moreover, as 
C5b-9 and platelet count will be documented at the study time points T0 to T8, their 
evolution over time will be analyzed using a linear mixed-effects model. 
 
Sample size estimation 
To the best of our knowledge, no study is available reporting the association of 
complement activity and change of platelets in cardiac patients. However, Qin et al. (4) 
reported a strong correlation (Pearson’s correlation coefficient) of r=0.683 between 
platelets (MPV/PTC ratio) and mtDNA levels in 68 patients who underwent CABG. As 
mtDNA levels are strongly associated with complement activation (9), we expect to 
observe a comparable correlation between the change of platelets and complement 
activation. Power analysis was performed to determine the sample size for Spearman's 
correlation using G*Power (version 3.1.9.2). Assuming an effect size corresponding to 
r=0.683, a sample size of 162 has a power of 95% to assess a significant difference from 
a large effect of r=0.5 with a type I error at a global 5% level (two-tailed). To account for 
drop-outs, a total of 190 patients will be included. 
 
Secondary analysis 
All secondary parameters will be summarized using descriptive statistics, i.e. no./total 
no. (%) for categorical variables and median (25th to 75th percentile) or mean (SD), 
however appropriate.  
Explorative data analyses will be performed as applicable to investigate the relationship 
between the comprehensive measurements. These might involve standard explorative 
techniques such as factor analysis (FA), principal component analysis (PCA), multiple 
factor analysis (MFA), homogeneity analysis and different clustering approaches as well 
as more recently developed techniques arising in the context of machine learning. 
 
8. ETHICAL, LEGAL AND ADMINISTRATIVE ISSUES 
8.1. Good Clinical Practice and Declaration of Helsinki 
The procedures set out in this study protocol are designed to ensure that the Sponsor 
and the Investigator abide the principles of the ICH guidelines on GCP (E6) 
recommended for adaptation on 1st of May 1996 by the ICH Steering Committee and the 
Declaration of Helsinki concerning the conduct, evaluation and documentation of the 
study.  
 

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8.2. Approval of the Study Protocol 
Prior to study start, the study protocol and/or other appropriate documents will be 
approved by the appropriate ethics committee and competent authorities. 
 
8.3. Obtaining Informed Consent 
Latest at the day before surgery the patient will be informed about the aim of the study 
and the need of study related blood sampling and collection of routine laboratory 
measurements and clinical data. 
The patient will be asked to agree on processing the data collected during their 
participation in the observational clinical study. The patient’s decision will be recorded 
in the patient’s medical file. 
 
8.4. Confidentiality 
All local legal requirements regarding data protection will be adhered to. All study 
findings and documents will be regarded as confidential. The Investigator and members 
of the research team must not disclose any information without prior written approval 
from the Sponsor.  
The anonymity of patients participating must be maintained. Throughout 
documentation and evaluation, the patients will be recognized on CRFs and other 
documents by identification number. Documents that identify the patient personally 
(e.g., the signed informed consent) will be maintained confidential by the Investigator. 
The patients will be told that all study findings will be handled in strictest confidence. 
 
8.5. Archiving of Study Records  
Essential documents will be retained for a minimum of 10 years after completion or 
discontinuation of the study.  
 
9. AMENDMENTS 
After the protocol has been submitted to an ethics committee (EC), any substantial 
change will require a formal amendment. Once the study has started, amendments will 
be made only in exceptional cases. The ethics committees must be informed of all 
amendments. 

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10. SCORES 
SOFA 
The SOFA is a six-organ dysfunction/failure score which is daily assessed. Each organ is graded 
from 0 (normal) to 4 (the most abnormal), providing a daily score of 0 to 24 points. Single organ 
failure is defined as SOFA score >2 for one organ and multiple organ daily arises with at least 2 
organ systems with >2 points. 
 
EUROSCORE II 
The abbreviation stands for European System for Cardiac Operative Risk Evaluation and 
calculate the estimated pre-operative mortality by using the following factors which can be seen 
in the graphic below. 
 
 
 
 
 

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11. REFERENCES 
 
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Blackstone, E. H., & Sabik, J. F. (2011). Morbidity of bleeding after cardiac surgery: 
Is it blood transfusion, reoperation for bleeding, or both? Annals of Thoracic 
Surgery, 91(6), 1780–1790. https://doi.org/10.1016/j.athoracsur.2011.03.105 
2. Oliver, W. C., Nuttall, G. A., Barash, P. G., Warren, O. J., Eng, M., Smith, A. J., … 
Athanasiou, T. (2009). The Inflammatory Response to Cardiopulmonary Bypass : 
Part 1 — Mechanisms of Pathogenesis. YJCAN, 23(2), 223–231. 
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3. Oikonomopoulou, K., Ricklin, D., Ward, P. A., & Lambris, J. D. (2012). Interactions 
between coagulation and complement - Their role in inflammation. Seminars in 
Immunopathology. https://doi.org/10.1007/s00281-011-0280-x 
4. Qin, C., Gu, J., Hu, J., Qian, H., Fei, X., Li, Y., … Meng, W. (2016). Platelets activation 
is associated with elevated plasma mitochondrial DNA during cardiopulmonary 
bypass. Journal of Cardiothoracic Surgery, 11(1), 1–9. 
https://doi.org/10.1186/S13019-016-0481-4 
5. Bruins, P., Velthuis, H. te, Yazdanbakhsh, A. P., Jansen, P. G. M., van Hardevelt, F. 
W. J., de Beaumont, E. M. F. H., … HackHome, C. E. (1997). Activation of the 
Complement System During and After Cardiopulmonary Bypass Surgery. 
Circulation, 96(10), 1–14. 
https://doi.org/http://dx.doi.org/10.1161/01.CIR.96.10.3542 
6. Pągowska-Klimek, I., Świerzko, A. S., Michalski, M., Głowacka, E., Szala-Poździej, 
A., Sokołowska, A., … Cedzyński, M. (2016). Activation of the lectin pathway of 
complement by cardiopulmonary bypass contributes to the development of 
systemic inflammatory response syndrome after paediatric cardiac surgery. 
Clinical and Experimental Immunology, 184(2), 257–263. 
https://doi.org/10.1111/cei.12763 
7. Hoedemaekers, C., van Deuren, M., Sprong, T., Pickkers, P., Mollnes, T. E., Klasen, 
I., & van der Hoeven, J. (2010). The Complement System Is Activated in a Biphasic 
Pattern After Coronary Artery Bypass Grafting. Annals of Thoracic Surgery, 89(3), 
710–716. https://doi.org/10.1016/j.athoracsur.2009.11.049 
8. Ricklin, D., Hajishengallis, G., Yang, K., & Lambris, J. D. (2010). Complement: A key 
system for immune surveillance and homeostasis. Nature Immunology. Nature 
Publishing Group. https://doi.org/10.1038/ni.1923 
9. Sandler, N., Kaczmarek, E., Itagaki, K., Zheng, Y., Otterbein, L., Khabbaz, K., … 
Hauser, C. J. (2018). Mitochondrial DAMPs Are Released During Cardiopulmonary 
Bypass Surgery and Are Associated With Postoperative Atrial Fibrillation. Heart 
Lung and Circulation, 27(1), 122–129. https://doi.org/10.1016/j.hlc.2017.02.014