Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1299 Monocytes, Tissue Factor and Heparin-coated Surfaces Clinical and Experimental Studies BY MATILDA JOHNELL ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003 ! " # $% $& %&'%( ) ! ) ) *! ! +" ) , -. /! 0 10!. 2 ! ,. $&. , / " 3 4 1) . 5 6 1 . . %$77. 8( . . 91:# 7%4((4(;;74; 5 +5*:- 0 ! ) ) . 3 ) ! 5*: ! 0 ! ) ! ) . /! )) ) 0 ! ) ! 5 3 1) +531- 0 ! . /! 531 ) 0 ! 5*: ! ) ! . /! ) ) ) 0 ) ! ) 531. : ! ) ! 0 ! ! ! 4 ) ) ) . *! ) ! ) ! 531 ! ) ! ! ) < 0! 0 ! ! ) . ) = ! ! . > ! )) .. ) ) 531 0 4 . /! ) ! ! ) ) ) ! 531 ) ) . , ) +/"- ) ! ! ) ) . /" ) ) ?99 +"?99 - < ) ) ! . @ ! )) ) ! 0 ! ! ) ) /"A"?99 . /! /"A"?99 *B"4::4 ) 9C4D /#"4E . /! ! ) ) ) ) . ) ! 4 ) ) ! ! !" " #" $%&'()' F , 2 ! $& 911# $D$4;;8 91:# 7%4((4(;;74; ' ''' 4&; +! 'GG ..G H I ' ''' 4&;- ! " # $ % % $ &'((') * + , -'.&')/ 0'-,0' ! " # % % $ &'((') * * 1 % 02&2)/ 03-,034 ! % $ , & ) 5 ! % $ 6 + 65 7#6,88, ,9 :6,; & ) Contents Introduction.....................................................................................................1 Monocytes .............................................................................................1 Tissue Factor .........................................................................................1 TF gene regulation and protein structure ..........................................1 TF expression....................................................................................2 TF·FVIIa signalling...........................................................................3 Haemostasis...........................................................................................5 Blood coagulation process ................................................................5 Cell-based model of coagulation..................................................6 Contact activation pathway ..........................................................8 Inhibitors of the coagulation process ................................................8 Fibrinolysis .......................................................................................8 Molecular markers of coagulation and fibrinolysis ..........................9 Coagulation and inflammation – integrated processes ..........................9 Endothelial transmigration..............................................................10 The atherotic plaque and atherothrombosis ....................................11 Biomaterials.........................................................................................12 Biocompatibility .............................................................................12 Surface modifications .....................................................................13 Heparin............................................................................................13 Heparin-coated surfaces .............................................................14 Corline Heparin Surface.............................................................15 Heart surgery and cardiopulmonary bypass....................................15 Oxygenators ...............................................................................15 Shed blood and retransfusion .....................................................15 Reduced anticoagulation protocol..............................................16 AIMS ............................................................................................................17 PATIENTS, MATERIALS AND METHODS.............................................18 Patients (Papers I and II).................................................................18 Heparin surfaces (Papers I-III)........................................................18 Oxygenator (Papers I and II)...........................................................19 Cardiopulmonary bypass (Papers I and II) .....................................19 Anticoagulation and operative procedures (Papers I and II)...........19 Blood sampling and analysis (Papers I and II) ...............................20 The Chandler loop model and sampling (Paper III)........................20 Preparation of mononuclear cells, culture and stimulation (Paper IV)...................................................................................................20 RNA preparation and cDNA synthesis (Papers II-IV)....................21 Real-time quantification PCR TFmRNA (Papers II-IV) ...............21 Flow cytometry ...............................................................................22 Analysis of markers of coagulation, fibrinolysis and inflammation24 Chemotaxis assay (Paper IV)..........................................................25 Statistical analysis...........................................................................25 RESULTS AND DISCUSSION...................................................................27 TF-expression, coagulation and inflammation during CABG using devices coated with heparin according to a new principle (papers I and II) .........................................................................................................27 Adhesion ....................................................................................27 Cell activation and TF-expression..............................................27 Coagulation activation................................................................29 Shed mediastinal blood ..............................................................31 The influence of different heparin surface concentrations and antithrombin-binding capacity on inflammation and coagulation (paper III)........................................................................................................32 Cell activation ............................................................................32 Monocytic TF-expression and thrombin generation ..................35 The effects of active site-inhibited FVIIa (FFR-FVIIa).............35 Formation of the proteolytically active tissue factor·FVIIa complex leads to enhanced PDGF-BB-stimulated chemotaxis and production of IL-8 and TNF-α in monocytes (paper IV) ...........................................36 TF-expression and cytokine production.....................................36 Chemotaxis.................................................................................38 Concluding remarks......................................................................................40 SUMMARY..................................................................................................43 SAMMANFATTNING PÅ SVENSKA.......................................................45 ACKNOWLEDGEMENTS..........................................................................47 REFERENCES .............................................................................................49 Abbreviations ABP Actin binding protein ACT Activated clotting time AP-1 Activating protein-1 AT Antithrombin BFA BrefeldinA C1-INH Complement C1-esterase inhibitor CABG Coronary artery bypass grafting CBAS Carmeda BioActive Surface CD Cluster of differentiation CD40L CD40 ligand cDNA Complementary DNA CHS Corline Heparin Surface CPB Cardiopulmonary bypass CRP C-reactive protein EDTA Ethylene-diamine-tetraacetic acid Egr Early growth reponse ELISA Enzyme-linked immunosorbent assay ERK Extracellular signal-regulated kinase F1+2 Prothrombin fragment 1+2 FAK Focal adhesion kinase FAM 6-carboxyfluorescein FBS Fetal bovine serum FDA Fluorescein-di-acetate FDP Fibrin degradation products FITC Fluorescein-isothiocyanate fMLP Formyl-Met-Leu-Phe HMWK High molecular weight kininogen IFN Interferon IL Interleukin JAK Janus kinase/Just another kinase LBP LPS binding protein LDL Low density lipoprotein LMWH Low molecular weight heparin LPS Lipopolysaccharide; endotoxin LRE LPS response element MAPK mitogen-activated protein kinase MCP-1 Monocyte chemotactic protein-1 M-CSF Macrophage colony-stimulating factor MDCK Madin-Darby canine kidney MFI Mean channel fluorescence intensity MPO Myeloperoxidase mRNA Messenger RNA NF Nuclear factor PAI Plasminogen activator inhibitor PAP Plasmin-antiplasmin complex PBS Phosphate buffered saline PC Phosphorylcholine PCR Polymerase chain reaction PDGF Platelet derived growth factor PE Phycoerythrin PI3K Phosphatidyl-inositide-3-kinase PKB Protein kinase B PLC Phospholipase C PMA Phorbol-12-myristate-13-acetate PSGL P-selectin glycoprotein ligand PVC Polyvinylchloride RPMI Roswell Park Memorial Institute RT-PCR Reversed transcription-PCR SDS Sodium-dodecyl-sulphate sF Soluble fibrin sIL2R soluble IL-2 receptor SMA Surface modifying additive SMB Shed mediastinal blood SP-1 Stimulating protein-1 SRR Serum response region STAT Signal transducer and activator of transcription TAFI Thrombin-activatable fibrinolysis