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
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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 inhibitor TAMRA 6-carboxy-tetramethyl-rhodamine TAT Thrombin-antithrombin complex TCC Terminal complement complex TF Tissue factor TNF Tumour necrosis factor t-PA Tissue type plasminogen activator u-PA Urokinase type plasminogen activator
Introduction
Monocytes The monocyte is a mononuclear leukocyte derived from a multipotent stem cell in the bone marrow that gives rise to a bipotent stem cell, progenitor of the granulocyte and mononuclear phagocyte cell lines. In 1970 the promonocyte was described, a direct precursor of the monocyte1 and in 1975 the monoblast2. The mature monocyte migrates from the bone marrow to the blood from where it, after approximately one day, migrates further into the tissue and differentiates to a macrophage. The monocytes represent 5-10% of the total leukocytes in the blood. Both monocytes and macrophages are phagocytes that engulf and destroy dead cells and tissue, bacteria and other particles. They are involved in inflammation and coagulation; among other properties they produce a variety of cytokines and tissue factor.
Tissue Factor Tissue factor (TF), the main initiator of blood coagulation in vivo, is a 47 kDa, type I transmembrane glycoprotein3. TF is receptor for the zymogen factor (F) VII and the active serine protease FVIIa4. TF is constitutively expressed in cells surrounding blood vessels and large organs to form a haemostatic barrier but can also be induced in vascular cells in response to a variety of inflammatory stimuli5. Also, total lethality among homozygous TF knock-out mouse embryos gives convincing evidence for TF to be indispensable for life6. In addition to its role in coagulation, TF is shown to be a signalling receptor7,8 involved in various biological processes, such as development, inflammation, angiogenesis and tumour metastasis.
TF gene regulation and protein structure The TF gene is located on chromosome 1 and extends over 12,4 kbp9,10. The gene contains six exons separated by five introns. In the promoter region two regions are well defined, the LPS-response element (LRE) and the serum response region (SRR), regulating mainly the induced and constitutive expression, respectively. The LRE contains two activating protein (AP)-1
1 sites and one nuclear factor (NF)-κB site, activated by bacterial lipo- polysaccharide (LPS; endotoxin), cytokines and integrins to induce transcription of TF. AP-1 is binding site for the heterodimer c-Fos/c-Jun whereas the NF-κB binds c-Rel/p65 that is regulated through complex formation with a cytoplasmic inhibitor IκBα. Upon LPS stimulation the complex is phosphorylated and degraded which permits nuclear translocation of the transcription factors11. The effect on SRR is regulated by serum, phorbol-ester (PMA) and shear stress via three early growth response (Egr)-1 and five stimulating protein (Sp)-1 binding sites. The transcription factor Sp-1 is constantly expressed and thought to be responsible for basal TF expression whereas Egr-1 mediate induced expression11. The full length TF-protein is 263 amino acids where the N-terminal part is a 219 amino acids extracellular domain that share structural homology with type II cytokine receptors, most related to interferon (IFN)-α, β and γ and interleukin (IL)-10 receptors12. The extracellular domain binds coagulation FVIIa and the complex generates its proteolytic activity. The C- terminal part of TF consists of a single transmembrane domain of 23 amino acids and a short cytoplasmic tail of 21 amino residues with three serine residues that are potential phosphorylation sites3. TF is identified as a growth related immediate early gene as induction at the transcriptional level occurs quickly after activation. Increasing levels of TF mRNA in human monocytes are detected 15 minutes after LPS stimulation in vitro13. The de novo synthesised TF protein expression occurs 1h after stimulation and reaches maximum after 4-6 h13,14.
TF expression Under normal physiological conditions TF is expressed at extravascular sites. High expression is normally recorded in the brain and the placenta5 but also in many malignant tumours15. Moreover, TF is highly expressed in cells within the atherosclerotic plaque16. During the last years it has clearly been demonstrated that TF is expressed in the circulating blood in microparticles derived from activated or apoptotic monocytes and platelets17. These intravascular microparticles can tether to activated platelets and endothelial cells and be concentrated at sites of injury18. Cells in contact with blood do not normally express TF, however, monocytes and endothelial cells can be induced by a variety of stimuli. The most potent and well studied inducer of TF-expression in vivo and in vitro is lipopolysaccharide (LPS)19, the main component of the outer membrane of Gram-negative bacteria. LPS binds to LPS-binding protein (LBP) in plasma and the complex binds to the cell surface receptor CD14. The signalling is mediated through toll like receptor-4 and its accessory protein MD2, leading
2 to upregulation of TF and many inflammatory mediators11. In vivo initiation of coagulation by LPS can lead to disseminated intravascular coagulation, DIC, a severe complication of septicaemia20. Other proinflammatory mediators like immune complexes21, complement22, oxidised low density lipoprotein (oxLDL)23, tumour necreosis factor (TNF)-α, IL-1α24, monocyte chemoattractant protein-1 (MCP-1)14, platelet derived growth factor (PDGF)-BB14, C-reactive protein (CRP)25 and tethering to adhesion molecules26 have also been shown to induce TF-expression in monocytes in vitro. In a whole blood environment, however, only LPS, immune complexes and adhesion seems to be inducers in monocytes27,28. Monocytes bonded to platelets, lymphocytes and endothelial cells via P-selectin glycoprotein ligand-1 (PSGL-1), CD4013,29 and CD11b/CD1830, express TF. Also, upon adhesion of monocytes to artificial surfaces the expression of TF, is enhanced14. This has also been demonstrated in in vitro studies with whole blood circulating in extracorporeal tubings and oxygenator circuits26,28.
TF·FVIIa signalling The structural similarities with cytokine receptors indicate that TF may be a signalling receptor12. The first study confirming a role for the TF·FVII complex in signal transduction was presented in 199531. Binding of FVIIa induced transient cytosolic Ca2+-signals in cell lines with either constitutive or transfected expression of TF and in human endothelial cells induced to synthesize TF31. This response is shown to be dependent on the proteolytic activity of FVIIa but independent of the cytoplasmic domain32. Also, Ca2+- oscillations were found to be mediated via phosphatidyl inositol-specific phospholipase C (PLC) in monocyte-derived macrophages and MDCK- cells32,33. TF·FVIIa signal transduction via the mitogen-activated protein kinase (MAPK) pathway was first demonstrated by transient activation of p42/44MAPK phosphorylation34, known to be associated with cell growth and differentiation. The activation of MAPK is dependent on the activation of the upstream MAPK kinase, extracellular signal-regulated kinase (ERK)1/2 and functional FVIIa since both the MAPK kinase inhibitor PD98059 and inactivated FVIIa abolish this signalling34. Activation of this pathway was shown to be independent of the cytoplasmic tail of TF35. Thus, these results suggest binding of TF·FVIIa to another receptor mediating this signal transduction pathway. Phosphorlation of p38MAPK was also demonstrated to be involved in the TF·FVIIa signalling36. The physiological relevance of FVIIa/TF-induced p38MAPK signalling is, however, not known but may be a part of TF- associated inflammation. A third MAPK, c-Jun N-terminal kinase (JNK), activated during cell growth, inflammation and differentiation, is also demonstrated to be
3 activated upon FVIIa stimulation leading to upregulation of the early growth response gene-1 (egr-1)36. However, this observation could not be confirmed by others37, but the contradictory results may be explained by cell-specific differences. TF·FVIIa signal transduction is also shown to induce activation of the Src-like kinases, c-Src, Lyn and Yes, leading to phosphatidyl-inositide-3- kinase (PI-3K) activity37. Members of the Src family are proto-oncogenes and involved in inflammatory responses38. PI-3K activity is also essential for FVIIa-mediated activation of the anti-apoptotic protein kinase B (PKB; c- Akt) and activation of the small GTPases Raf, Rac and Cdc4237, important for cytoskeletal reorganization39,40. Also, Raf and Rac are required for activation of p42/p44MAPK and p38MAPK, respectively. The function of Cdc42 in this picture is, however, still unknown. Furthermore, other pro- inflammatory pathways may be involved in TF·FVIIa signalling41. Accumulating data demonstrate that activation of coagulation, signalling inducing Ca2+-oscillation, MAPK phosphorylation and gene induction are independent of the cytoplasmic domain, indicating binding to another receptor that subsequently initiates the intracellular signal8,32,35. The involvement of a secondary target for the proteolytic activity of the TF·FVIIa complex initiating the intracellular signalling is, however, a subject of debate. The protease activated receptor (PAR)-family recognised as seven-transmembrane domain G-protein linked receptors, are activated upon proteolytic cleavage of their extracelllular domain42. The PAR-2 has been suggested as the candidate for TF·FVIIa signalling32,43,44. However, others have demonstrated contradictory results45. An alternative pathway for TF·FVIIa signalling may be generated directly via the cytoplasmic tail of TF that has three serine residues, potential docking sites for important signalling proteins upon phosphorylation. The cytoplasmic tail seems essential for some Ca2+-oscillations33 and also for generation of VEGF46. Moreover, the cytoplasmic domain has been demonstrated to have a high affinity binding site for actin-binding protein (ABP)-28047 that leads to cytoskeletal rearrangements of importance for cell migration48. Furthermore, actin-filament organisation induced by TF- mediated adhesion leads to phosphorylation of focal adhesion kinase (FAK), a central nonreceptor tyrosine kinase involved in cytoskeleton-dependent signalling47 and migration of cells49. TF seems to be localised in actin- filament-rich membrane areas of cells50. These observations together indicate involvement in processes like cell-matrix adhesion, cell-cell contact and cellular motility mediated by the TF·FVIIa complex dependent on the cytoplasmic domain of TF. Very recently, we have demonstrated that the cytoplasmic part of TF is mandatory for migration. Porcine aorta endothelial (PAE) cells transfected with a construct with deletion of the intracellular
4 domain totally abolished the migration response to a growth factor51. In fact, the complexity of different pathways through which actin cytoskeleton is influenced, may be due to the importance of TF signal transduction in controlling cell morphology and motility. We have shown that the TF·FVIIa signalling increased the sensitivity to PDGF-BB-induced migration by a 100-fold in fibroblasts and vascular smooth muscle cells. This response is dependent on the proteolytic activity of TF·FVIIa inducing PLC-activity but independent of PI3K52. In cultured keratinocytes, TF·FVIIa alters the expression of a number of genes encoding growth factors, proinflammatory cytokines and proteins involved in cellular reorganisation and migration53,54. All these data clearly indicate that the TF·FVIIa signalling and cellular functions beyond coagulation are closely related to inflammatory events.
Haemostasis The main functions of the haemostatic system are to prevent loss of blood upon damage of a blood vessel and to keep the blood in a fluid phase. To obtain this a state of equilibrium between clot formation and dissolving of the clot must prevail. This is dependent of the interaction between blood vessels and platelets, the primary haemostasis, the coagulation phase and fibrinolytic system, the humoral haemostasis55. The primary haemostasis involves vasoconstriction and the formation of a platelet plug via activation and aggregation of platelets. The coagulation phase results in formation of fibrin strand that will bind and stabilise the weak platelet plug. Finally the fibrinolytic system dissolves the clot. A delicate balance between these systems is needed as an imbalance inevitably will result in either thrombosis or bleeding.
Blood coagulation process The coagulation factors are highly glycosylated inactive plasma proteins found in mostly low concentrations in the blood. The formation of a fibrin clot can be described as a cell-bound system with proteolytic cleavage of inactive zymogens forming active enzymes which in turn cleave other zymogens with amplification in each step56,57. Many coagulation factors are dependent of Ca2+ and a phospholipid surface as cofactors for their activity and also to localise the clot formation to the site of injury. The blood coagulation is most easily described in two parts, a cell-based model and the contact activation pathway although it is probably more correct to regard the coagulation system as an interactive network of amplifiers and inhibitors.
5 Cell-based model of coagulation This is today considered to be the main coagulation model in vivo and can be described in three overlapping phases: initiation, priming and propagation58. Upon injury, TF is exposed from subendothelial tissue, and binds FVII- FVIIa. The proteolytic complex TF·FVIIa activates FX and FIX. FXa activates plasma FV on the TF-expressing cell and in combination with cofactor FVa they cleave prothrombin (FII) to form small amounts of thrombin, the main enzyme in the coagulation process (Figure 1a).
INITIATION TFPI X II
Xa Va Xa
VIIa V TF IIa
VIII IX TF monocyte XI
VIIa
IXa A
Figure 1a. Initiation of the coagulation process
In the priming phase, thrombin activates platelets at the site of injury, to release FV from their α-granule. A positive feed-back loop is initiated, where thrombin activates the released FV and FVIII, bound to von Willebrand factor, and the activated factors bind to the activated platelets. Thrombin also activates FXI bound to the platelet surface. The coagulation activity of TF is inhibited by TF pathway inhibitor (TFPI), produced by endothelial cells. When a certain amount of FXa is formed, TFPI is bound, together forming a quarternary complex with TF and FVIIa. TFPI is a unique inhibitor with two active sites, for both FXa and FVIIa59 (Figure 1b). In the propagation phase, the phospholipid surface of the activated platelet will act as cofactor for the activation of the complexes FVa·FXa, “prothrombinase”, and FVIIIa·FIXa, “Xase” that will accelerate the formation of thrombin and FXa, respectively. In addition, FXIa on the platelet surface activates FIX to form more Xase. Thrombin is the last enzyme in the coagulation phase and cleaves fibrinogen to fibrin. The
6 soluble fibrin is finally stabilised by FXIIIa, also activated by thrombin, to form a fibrin network, a thrombus57,58 (Figure 1c).
TFPI PRIMING
VIIa Xa vW F
TF IIa VIII/vWF XI
V platelet VIIIa XIa VIIIa XIa Va Va
activated platelet
P-selectin gpIIb/IIIa CD40L B
Figure 1b. Priming of the coagulation process.
IXa PROPAGATION X fibrinogen
IX II soluble fibrin XIII IXa XIIIa
Xa VIIIa IIa XIa Va activated platelet fibrin network C
Figure 1c. Propagation of the coagulation process
7 Contact activation pathway The contact activation pathway involves FXII, XI, plasma prekallikrein and high-molecular-weight kininogen (HMWK) and is mainly involved in in vitro coagulation60. FXII is auto-activated by an internal proteolytic cleavage of the protein initiated by contact with a negatively charged surface. Prekallikrein in complex with HMVK is activated by FXIIa and the formed kallikrein in turn activates more FXII. FXIIa also axtivates FXI, activating FIX. The two pathways then converge when FIXa activates FX, an enzyme shared with the TF-pathway. The significance of the contact activation pathway in coagulation in vivo is unknown as deficiencies of FXII, prekallikrein or HMWK are not associated with increased bleeding tendency61,62.
Inhibitors of the coagulation process The coagulation is regulated by several inhibitors. As previously described the main inhibitor of the TF·FVIIa complex is TFPI. Thrombomodulin (TM) is a transmembrane receptor expressed mainly by endothelial, but also other cells. Thrombin undergoes conformational change upon binding to TM that inhibits its procoagulant functions63,64. The TM·thrombin complex generates another anticoagulant effect through activation of protein C that, together with its cofactor protein S, inactivates FVa and FVIIIa65. The activation of protein C by TM·thrombin is potentiated by protein C binding to endothelial protein C receptor (EPCR)66. The main inhibitor of thrombin is, however, antithrombin (AT) that inactivates mainly thrombin and FXa but also FXIa and FXIIa. FXIa and FXIIa can also be inhibited by complement C1-esterase inhibitor (C1 INH)67, an inactivator of C1 subcomponents that also binds kallikrein. The efficiency of antithrombin is dramatically increased in the presence of glycosaminoglycans such as heparan sulphate, found on the surface of endothelial cells68. The same effect is exerted by heparin69.
Fibrinolysis The fibrinolytic system degrades the fibrin clot and dissolves the thrombus. The main enzyme of this system is plasmin that degrades fibrin into fibrin degradation products, FDPs. The proenzyme plasminogen is activated through proteolytic cleavage by tissue-type plasminogen activator, secreted by endothelial cells. In the absence of fibrin, t-PA activates plasminogen at a low rate and is inactivated by plasminogen activator inhibitor type-1 (PAI- 1). When fibrin is present, both t-PA and plasminogen bind to fibrin and plasmin is formed70. Also another pathway can activate plasminogen, the urokinase type plasminogen activator, u-PA. The activation of u-PA is mediated by FXIIa, kallikrein and also by a feed-back loop of plasmin. FXIIa and kallikrein are, as mentioned, considered as part of the coagulation
8 system but may be more important in fibrinolysis. However, the importance of the u-PA dependent pathway of fibrinolysis is not fully understood. The fibrinolytic activity of plasmin is inhibited by α2-antiplasmin (AP), secreted by the liver, via formation of the inactive complex plasmin·antiplasmin, PAP. Also thrombin inhibits the fibrinolysis via thrombin activated fibrinolysis inhibitor, TAFI, strongly enhanced by the formation of TM·thrombin. TAFI acts by degrading the binding sites for plasminogen and t-PA in fibrin71.
Molecular markers of coagulation and fibrinolysis The activity of the coagulation cascade and fibrinolysis can be determined at several stages by the use of specific markers; mainly with ELISA techniques. During formation of thrombin with cleavage of prothrombin by FXa, the prothrombin fragment 1+2 (F1+2) is released. The activity of thrombin can be reflected by analysis of the formation of soluble fibrin (sF). The inactivation of thrombin by formation of thrombin·AT complex (TAT) and other inhibitory complexes formed with AT can be analysed, like FXIa·AT and FXIIa·AT. The inactivation of plasmin can be detected by determination of PAP complex.
Coagulation and inflammation – integrated processes Inflammation is a protective response of vascularized tissue that is characterised by a complex humoral and cellular interaction of several pathways. Complement activation, production and release of cytokines, expression of adhesion molecules and thrombin generation are some of the results of the activation of these pathways72. In cardiac surgery with CPB these processes contribute to postoperative complications as myocardial dysfunction, respiratory failure, neurological complications and bleeding disorders. The activation of coagulation and inflammation is integrated through a network of components73. Inflammation-induced thrombin generation can be mediated through cytokine-activated mononuclear cells leading to expression of TF. Thrombin is a central enzyme during haemostasis but can also induce cellular responses involved in inflammation74. Thrombin receptor activation on leukocytes and endothelium results in production and release of chemotactic and inflammatory cytokines such as IL-1, Il-6, IL-8 and MCP-1 and upregulation of adhesion molecules P-selectin, E-selectin and ICAM-175,76. Thrombin is also a potent chemottractant for monocytes and activator of platelets. As a consequence of activation platelets release their granule contents, express gpIIb/IIIa, P-selectin and CD40L on their surface. Leukocytes, particularly monocytes, adhere to the activated platelets
9 via bridging of fibrinogen to CD11b/CD18, PSGL-1 or CD40, respectively77 thus forming platelet-leukocyt complexes. Cross-talk between the cells in the complexes via P-selectin and CD40L leads to TF expression and cytokine release 78,79.
Endothelial transmigration During inflammation, leukocytes are recruited to the site of infection or inflammation via transmigration through the activated endothelium. Before transmigration, the leukocytes must be captured from the main blood stream and arrested at the endothelium. Tethering reflects the first contact of a leukocyte with the vessel wall (Figure 2). Members of the selectin family, mainly P-selectin, and also E- selectin on endothelial cells and L-selectins on leukocytes, are involved in this process. When leukocytes are captured, they transiently adhere to the endothelium via interaction between P-selectin and PSGL-1, and begin to roll. Bonds between the selectins and their ligands are formed at the leading edge and broken at the trailing edge of the rolling cell80.
Tethering Rolling Adhesion Transmigration platelet- leukocyte complex
P-selectin L-selectin ICAM/ VCAM PECAM E-selectin integrins Fig.2. Endothelial transmigration.
Rolling leukocytes adhere by engagement of upregulated integrins on the activated leukocytes and their counter receptors on the endothelium. The firm adhesion of monocytes mainly involves the β2-integrins or lymphocyte function-associated antigen-1 (LFA;CD11a/CD18) and Mac-1 (CD11b/CD18) with their common receptor intracellular adhesion molecule- 1 (ICAM)81. Also the β1-integrin very late antigen-4, (VLA;CD49d/CD29) and the receptor vascular adhesion molecule-1 (VCAM) play a role. The transmigration across the endothelium into the site of inflammation has been shown to be dependent of both Mac-1/ICAM-1 and platelet/endothelial cell adhesion molecule-1 (PECAM;CD31)82. Chemotaxis is a dose-dependent directed migration stimulated by chemoattractant. Many chemoattractants are known to be involved in the accumulation of
10 leukocytes during inflammation e.g. formyl-Met-Leu-Phe (fMLP), CRP, TNF-α and chemokines like MCP-1, IL-883 and direct the leukocytes to the site of inflammation.
The atherotic plaque and atherothrombosis The atherosclerotic process is a chronic inflammatory disease84 that often leads to cardiovascular disease after decades without symptoms. Arterial thrombosis is considered to arise, after a vessel is damaged, from the interaction of TF in the vascular wall with platelets and coagulation factors in the circulating blood. The atherosclerotic plaque is a thickening in the arterial wall. During the development of a plaque, monocytes and lymphocytes are recruited to the inflamed endothelium and transmigrate into the intima. Cytokines generated locally in the response to stimuli, like oxLDL, may induce upregulation of selectins and VCAM-1. The interactions with integrins on the leukocytes recruit the cells to endothelium. Proinflammatory cytokines like MCP-1 and IL-8 and the acute phase protein CRP are expressed within the plaque, providing a chemotactic stimulus and direct the migration into the atheroma85,86. The transmigration process is suggested to induce the monocytic differentiation to macrophages in the tissue82. Monocyte-derived macrophages upregulate scavenger receptors and phagocyte lipids in the plaque, and may transform into TF-expressing macrophage foam cells stimulated by macrophage colony-stimulating factor (M-CSF)87. This uptake of lipoproteins by macrophages in the plaque may also be CRP-mediated88,89. Accumulation of foam cells, fatty streaks, is an early, reversible phase in the formation of an atherosclerotic plaque. As the inflammatory process continues, accumulation of more leukocytes from the blood is accompanied by smooth muscle cells from the media, stimulated by PDGF-BB and transforming growth factor (TGF)-β from stimulated endothelial cells and T- lymphocytes90. Progression of the plaque to an advanced, complicated lesion is characterised by the formation of a fibrous cap towards the lumen of the vessel. Smooth muscle cells, stimulated to collagen biosynthesis by fibrogenic growth factors as TGF-β and PDGF-BB, take part in the formation of the cap91. The fibrous cap covers a mixture of leukocytes, lipids, and cell debris called the lipid core. This is highly thrombogenic due to both extracellular and cell-bound TF and production of pro-inflammatory cytokines from activated cells. The lipid core may transform into a necrotic core as a result of apoptosis, necrosis, increased proteolytic activity and lipid accumulation. The stability of the plaque is dependent on the thickness and content of the fibrous cap. If the content of collagen is high the plaque is stable,
11 however, leukocytes and smooth muscle cells within the plaque can produce matrix-degrading enzymes. Interaction between CD40 and CD40L stimulates to production of cytokines like IFN-γ that influence smooth muscle cell growth and matrix-degrading proteases92. If the balance between synthesis of collagen and degradation by matrix proteins is disturbed the plaque can rupture, exposing the thrombogenic lipid core. Rupture often occurs at the edge of the lesion where the fibrous cap is thinnest. Plaque rupture may be triggered by mechanical and haemodynamic forces i.e. increased blood pressure or pulse rate93. Upon rupture, the thrombogenic content of the plaque is exposed and a thrombus is immediately formed.
Biomaterials A variety of biomaterials are used in the human body for either treatment of various diseases, after trauma or for cosmetic reasons. Usual biomaterials are ceramics, composite, metals and polymers, where metals and polymers are the most common used for treatment, diagnosis or replacement of a tissue, organ or functions in or outside the body. Metals are most often used in surgical implants for hard tissue replacement but also for coronary artery stents however, often covered by polymers94,95 whereas polymers replace soft tissues. Polymers, especially polyvinyl chloride (PVC), are used in tubing for extracorporeal circulation and polypropylene is often used in the membranes of the oxygenator. The evolution of biomaterials has evolved from inert, to bioactive, with a further development toward biointeractive, able to generate functional tissue96.
Biocompatibility Biocompatibility, the characteristics of a biomaterial that are necessary for the material to work in a biologic tissue, depend on the purpose for the material. A hip implant needs to be able to integrate with the bone, whereas the material in a medical device for temporary contact with the blood needs to be as inert as possible. The main biocompatibility problem with the polymers used today is that they stimulate absorption, adhesion and activation processes. The contact with blood will inevitably lead to an instant absorption of proteins to the surface97 leading to activation of different cascade systems in the body activating coagulation and inflammation. Cells adhering to the surface interacts with this spontaneously adsorbed layer of proteins rather than with the material itself97,98. Thus, the interaction between the phagocyte integrin Mac-1 and the platelet integrin GpIIb/IIIa (CD41/CD61) and fibrinogen, partly explains the accumulation of cells on biomaterial. Chemical modifications of the artificial surface can be made in order to reduce adhesion and activation of cells.
12 Surface modifications Different coating techniques has been established to improve the biocompatibility of biomaterials99. Heparin-coating is the most developed, studied and used method but new surface coating techniques are evolving. To mimic biomembranes, coupling of derivatives of phosphorylcholine (PC), the major lipid component found in the outer surface of a cell membrane, to synthetic surfaces has been developed and shown to reduce platelet deposition, anastomotic neointimal hyperplasia, and neointimal cell proliferation in a dog model of arteriovenous grafts100,101. Another kind of surface modification is not a coating technique in the usual sense. It is a surface modifying additive (SMA), contributed to the synthetic material in the production phase. During the polymerization process, the copolymer distributes itself in the basal material and moves to the surface as it cools, dramatically changing the outermost surface molecular layers influencing the biocompatibility102. The SMA surface is found to reduce platelet deposition, coagulation activity and delay contact activation, but did not influence complement activation or leukocyte cells activation in both an ex vivo canine study102 and a patient study where the SMA surface was compared with an uncoated CPB device used during CABG103.
Heparin Heparin, produced by mast cells, is a heterogenous group of sulphated glycosaminoglycan (GAG) that consists of linear polymers of repeating disaccharide subunits of variable size ranging from 3-30 kDa, with an average of 15 kDa representing 40-50 saccharides in length69. One anticoagulant effect of heparin is mediated through activation of AT where heparin accelerates AT’s inhibition of thrombin and FXa by ~2000-fold104. The interaction between heparin and AT is dependent of a specific pentasaccharide sequence, the natural pentasaccharide105. Heparin also increases the effect and release of TFPI from vascular endothelium106,107 and accelerates the inhibitory effect of AT on the TF·FVIIa complex108. Moreover, heparin can inhibit binding of fibrinogen, factor X, iC3b and ICAM-1 to CD11b/CD18109. This may result in alteration of leukocyte functions involved in activation of coagulation and inflammation. The activity of heparin is dependent of the length of the molecule. Inhibition of thrombin requires a polysaccharide of at least 18 saccharides compared to the inhibition of FXa where the pentasccharide sequence result in better inhibition then a longer molecule110,111. Clinically, low molecular weight heparins (LMWH) is mostly used. The LMWHs are prepared from unfractionated heparin resulting in a mean molecular weight of 4-6,5 kDa with a total range of 2-10 kDa. They are
13 characterised by a high bioavailability, a longer half-life and lower affinity for plasma proteins and cells than unfractionated heparin, thus more easy to dosage. However, the available LMWHs differ in the distribution of molecular weights, bioavailability and anti-Xa activity112, consequently do their pharmacodynamic behavior, recommended dose and efficacy113. Heparin is used in large doses during coronary artery bypass surgery (CABG) with cardiopulmonary bypass (CPB). Even though unfractionated heparin is efficiently eliminated in the body, these large doses need to be antagonised. Protamine, administered at the end of surgery, forms a stable, inactive complex with heparin. Heparin is also used to modify surfaces of biomaterials.
Heparin-coated surfaces The aim of coating an artificial surface with heparin is to mimic the antithrombogenic effect of heparansulfate at the endothelium-intravascular surface to generate a biocompatible surface. The development of heparin- coated surfaces started in the early 1960s114 and today about ten different heparin surfaces are available on the market99,115. At the beginning of the 1980s the Carmeda Bioactive Surface (CBAS) was developed116 where heparin is covalently bound by the technique of end-point immobilization onto a modified artificial surface resulting in a stable heparin surface. The AT-binding sequence of heparin is not affected, thus generating a bioactive surface structure. The CBAS has been used in a number of clinical studies in extracorporeal devices, oxygenators and stents presenting various results. It can be concluded that in some studies the CBAS surface generate a reduction of the complement activation117, modification of leukocyte and platelet activation118,119, but no reduction of thrombin generation118,119. However, in other studies no differences were found compared to unmodified surfaces120,121. A few years later, the Duraflo II surface from Baxter was developed, with a heparin (-benzalkonium-chloride)-complex ionically bound to the artificial surface resulting in a relatively firm connection122. Also this surface has been used in numerous clinical studies with varying results. Reduction of complement activation123,124, reduced leukocyte response125 reduced thrombin generation126 has been reported but also studies where no advantages were detected127-129. The BioLine Coating, developed by Jostra, is prepared by an immobilized polypeptide, uniformly attached to the artificial surface onto which heparin is both covalently and ionically bonded that results in an active and stable heparin surface. The BioLine Coating was shown to preserve platelets and reduce inflammation130,131 but did not improve platelet activation, coagulation or fibrinolytic activity131.
14 Corline Heparin Surface The Corline Hepain Surface (CHS), studied in this thesis, is prepared by a macromolecular heparin conjugate, irreversibly attached onto a conditioning layer of polymeric amine. The specificity of the covalent linkages ensures that the AT-binding pentasaccharide sequence of heparin is left intact. The CHS is stable with no leakage of heparin into the blood (data submitted to the Swedish Medical Products Agency).
Heart surgery and cardiopulmonary bypass During open heart surgery with CPB the body is subject to an extensive trauma and the blood is exposed to a large artificial surface leading to activation of inflammation and coagulation. Many factors during CPB can induce complex inflammatory response involving complement and cellular activation along with the production of cytokines. It has also been suggested that the systemic inflammatory response in CABG patients is caused mainly by the surgical procedure per se132. Thrombin generation during cardiac surgery using CPB may be induced by the surface of the extracorporeal unit with the oxygenator, pump and connectors between the patient and the extracorporeal device133. The surgical trauma with exposure of TF, is also suggested to be of major importance for thrombin generation134-137. Previously, it was thought that the activation of coagulation during CPB occurred mainly through contact activation138. However, current evidence suggests that activation of the TF induced pathway of coagulation can be the most important mechanism of thrombin generation during this type of surgery128,139,140. To prevent clotting in the extracorporeal circuit during cardiac surgery, large doses of systemic heparin are administered, and heparin-coating has been reported to improve the biocompatibility of artificial surfaces in contact with circulating blood.
Oxygenators The oxygenator has a large area, i.e. approximately 2m2, to support gas- exchange during CPB. It represents half of the total area in the extracorporeal device and is thus, a potential surface for adherence of proteins and cells. Consequently, when adhesion is studied in simulated extracorporeal circulation of during CABG usually the oxygenator is used26,28.
Shed blood and retransfusion Retransfusion of shed mediastinal blood (SMB) was introduced by Schaff and colleges in 1978141 and has become a widely used method during and after cardiac surgery142,143. However, a number of studies have shown that the composition of the shed blood is far from normal144,145. SMB after
15 CABG, is characterised by extraordinary activated coagulation and fibrinolysis 121,146-148. Furthermore, high levels of proinflammatory cytokines are found in shed blood 146. However, the interpretations of the outcome of retransfusion of this blood differs; some studies have concluded that retransfusion of shed blood deteriorates haemostasis, fibrinolysis and inflammation while others suggest that it does not 144-149.
Reduced anticoagulation protocol The enhanced biocompatibility achieved by coating the surface of the extracorporeal device with heparin has initiated studies where the dose of systemic heparin has been reduced. However, these studies have presented conflicting results. Some have shown a reduction of granulocyte activation150 and lower incidence of homologous transfusions151 while others found it clinically safe but not in favour to standard heparin levels in combination with available heparin surfaces152,153. Yet others have concluded that a reduction of systemic heparin should not be made due to an increased formation of thrombin154. An important difference between various clinical studies is the use of different surfaces and heparin doses at the same time129,154. This design makes it hard to distinguish between the effects of heparin coating and those induced by reduced anticoagulation.
16 AIMS
The general aim of this project was to further explore the regulation of monocyte tissue factor expression at contact with biomaterial and heparin- coated surfaces. Elucidate the interactions between inflammation and coagulation induced in blood at contact with these surfaces. In the context of monocyte activation, study some cellular functions induced by TF·FVIIa signal transduction. More specific aims were;
• to investigate the biocompatibility of a newly developed heparin surface, the Corline Heparin Surface, used in cardio pulmonary bypass during coronary artery bypass grafting;
• to study the influence of different systemic heparin doses in combination with the heparin-coated surface on coagulation, fibrinolysis and cell activation in patients during coronary artery bypass grafting;
• to study the influence of Corline Heparin Surface in combination with different levels of systemic heparin on adhesion and activation of leukocytes, with focus on monocytic TF, and platelets in oxygenators used during coronary artery bypass grafting;
• to investigate, in an experimental ex vivo model, different surface concentrations of heparin in the Corline Heparin Surface with respect to cell activation, coagulation and inflammation;
• to investigate the influence of FVIIa and active-site inhibited FVIIa- binding to TF-expressing human monocytes on cytokine production and cell migration;
• to study the role of the cytoplasmic tail of TF in monocyte migration.
17 PATIENTS, MATERIALS AND METHODS
Patients (Papers I and II) The randomised studies presented in paper I and II were performed on the same group comprising 60 patients scheduled for elective coronary artery bypass grafting (CABG). Patients over 75 years of age or with inflammatory or renal disease or severely low ejection fraction were not considered for the study. The patients undergoing CABG with cardiopulmonary bypass (CPB) were randomly assigned to four equally large groups with either an uncoated circuit (Jostra Medizintechnic AG) or a completely Corline Heparin Surface- coated circuit (Corline Systems AB) with one of three different levels of systemic heparin; standard, low or high. No autotransfusion of shed mediastinal blood (SMB) was performed during this study. Informed consent was obtained from all patients and the study was approved by the Swedish Medical Product Agency and the ethical committee of the Medical Faculty at Uppsala University and the Swedish National Board for Health and Welfare.
Heparin surfaces (Papers I-III) In papers I and II the Corline Heparin Surface (CHS) was applied to a complete set of an extracorporeal circuit (tubing, cannula, oxygenator and reservoir). The CHS is prepared by a conditioning layer of polymeric amine onto which a macromolecular heparin conjugate is irreversibly attached by multiple ionic interactions. In the heparin conjugate, approximately seventy heparin molecules are covalently linked to a polymer carrier with a molecular weight of 50 kD. The specificity of the covalent linkages ensures that the AT-binding pentasaccharide sequence of heparin is left intact. The CHS is stable with no leakage of heparin into the blood. An extracorporeal closed loop system modified with CHS used during ECC for twenty-four hours in pigs without any systemic dose of heparin had no effect on the clotting time (data submitted to the Swedish Medical Products Agency).
18 In paper III the CHS was applied in two different concentrations to PVC tubing of the same kind as used during cardiopulmonary bypass. Different surface concentrations of heparin and AT-binding capacity are achieved by repeating the application of polymeric amine and heparin conjugate. The surface concentration of heparin is 0.5 µg/cm2 in the single layered surface and 0,9 µg/cm2 in the double layered surface and the AT binding capacity is 6 pmol/cm2 and 12 pmol/cm2 in the single and double layers, respectively155.
Oxygenator (Papers I and II) The Quadrox hollow fibre membrane oxygenator (Jostra) was used. In paper II the oxygenators were collected at the end of surgery, washed and adhered cells were retrieved by recirculation of a weak EDTA-solution according to Kappelmayer et al.26. The retrieved cells were then counted and divided into two parts; one was labeled for flow cytometry (see below) and the other part was separated on a Ficoll-Paque gradient (Amersham Biosciences). The purity of mononuclear cells was 49 %. Total RNA was prepared and further analysed with real-time quantification PCR (see below).
Cardiopulmonary bypass (Papers I and II) Standard anesthesia was used. Techniques and equipment for CPB was similar in all patients. The standard set consisted of a Stöckert roller-pump (Stöckert Instrumente GMBH) with a Quadrox hollow fibre oxygenator and a hard-shell venous reservoir (Jostra). In the groups with heparinised CPB equipment the tubing, including cannula, was modified with the CHS from tip-to-tip. Surgery was performed with moderate hypothermia (32-35oC) and the blood flow was non-pulsatile. Before weaning from CPB the patients were rewarmed to 36oC rectal temperature.
Anticoagulation and operative procedures (Papers I and II) Patients in the groups with standard systemic heparin dose were anticoagulated by intravenous administration of heparin given as a bolus of 300 IU/kg body weight (b.w.) after completed dissection of the internal thoracic artery and prior to cannulation for CPB. In the groups with low and high systemic heparin dose, the bolus doses of heparin were 200 and 400 IU/kg b.w., respectively. If necessary, additional doses of heparin were given to maintain the desired activated clotting time (ACT). After weaning from CPB, heparin was reversed using protamine chloride in a 1:1 proportion to the administrated heparin dose. Cardioplegic arrest was achieved with antegrade infusion of modified St. Thomas’ cardioplegic solution at 4oC through the aortic root. During the CPB procedure cardiotomy suction was used. Blood remaining in the CPB circuit after decannulation was collected
19 in an infusion bag and immediately retransfused. Postoperatively, shed blood was collected in the reservoir but no retransfusion was performed.
Blood sampling and analysis (Papers I and II) In papers I and II blood samples were drawn from a catheter in the radial artery before and after CPB. In paper I were samples also drawn from the patients during surgery and performed from the pericardial cavity at the end of CPB and from the drainage-reservoir (after gentle mixing of the reservoir) three hours after CPB. For coagulation and flow cytometry assays samples were drawn into citrate tubes whereas EDTA tubes were used for cytokine determinations. One citrated sample was immediately brought to the laboratory for cell count and analysis of cellular surface antigens by flow cytometry. The other samples were immediately centrifuged and plasma was stored in aliquots at -70oC until analysed. Platelet and leukocyte differential counts were determined using an automatic cell counter, Coulter STKS (Coulter Electronics). Plasma levels of coagulation, fibrinolysis and inflammation markers and cytokines were quantified by sandwich ELISA techniques (se below). No correction for hemodilution was made.
The Chandler loop model and sampling (Paper III) The Chandler loop model is used to mimic extracorporeal circulation156. Fresh whole blood was drawn from healthy volunteers into a heparinised Falcon tube using heparinised equipment. An unmodified PVC tubing was compared with a single and a double layered CHS tubing. A low dose of unfractionated heparin was added to the blood destined for the uncoated surface and the single layered CHS loops to avoid clot formation. Blood destined for the double layered loops had no additives. The tubings were filled with fresh whole blood leaving a gas volume so the blood could circulate passively and closed into loops using surface heparinised connectors of thin walled steel. The Chandler loops were rotated vertically in a 37oC water bath for up to four hours. After incubation, the samples were citrated and aliquoted for RNA preparation, real-time quantification PCR, flow cytometry or centrifuged and stored at -70oC for analysis of activation of inflammation and coagulation in plasma. The blood, tubings and connectors were inspected for visible clots.
Preparation of mononuclear cells, culture and stimulation (Paper IV) Briefly, whole blood drawn from healthy volunteers was separated on a Ficoll-Paque gradient157. The mononuclear cells were suspended in cell culture medium, RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% glutamine (Gibco).
20 The purified human mononuclear cells were cultured with or without 10 ng/mL LPS (Sigma) in 37oC in a humidified chamber containing 95% air and 5% CO2 for three hours, subsequently the conditioned medium, containing lymphocytes and non-adhered monocytes, were harvested and centrifuged and the supernatants stored at -70oC. For the chemotaxis assay the adhered cells were harvested. To investigate the influence of G-protein- mediated signal transduction, the LPS-stimulated cells were incubated two more hours with 500 ng/mL pertussis toxin (Sigma) in 37oC in a cell culture incubator before chemotaxis. For RNA and flow cytometry assays, instead new cell culture medium was added, after washing, and the cells were further incubated with or without 10 ng/mL BrefeldinA (BFA) and 100nM FVIIa for another three hours. The extra-cellular expression of TF was assayed by flow cytometry before and after incubation. The supernatant from the cultured monocytes was centrifuged and stored at -70oC until analysis.
RNA preparation and cDNA synthesis (Papers II-IV) In paper II total RNA was isolated from the mononuclear cells retrived from the oxygenators by EDTA/SDS/sodium acetate method described earlier158. The RNA concentration was measured by a spectrophotometer at 260 nm and the integrity of 28S and 18S ribosomal RNA was checked in ethidium bromide-stained agarose gel. Isolation of RNA from whole blood in paper III was performed with QIAmp RNA Blood Mini Kit® (Qiagen). The RNA concentration was measured spectrophotometrically. In paper IV RNA was isolated from the cultured monocytes with TriZol reagent (Life Technologies) and the concentration was determined. After preparation of total RNA annealing of oligo dT primer (Invitrogen) was performed and total RNA was subject to a reversed transcription polymerase chain reaction (RT-PCR).
Real-time quantification PCR TFmRNA (Papers II-IV) The relative amount of reverse transcribed TFmRNA was quantified with the TaqMan real-time PCR assay on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems)159,160. The method is based on 5’-3’ nuclease activity of the Taq polymerase in which the enzyme degrades the dual-labelled fluorogenic target-specific TaqMan probe. One fluorescent dye, FAM (6-carboxy-fluorescein), serves as reporter and its emission spectra is quenched by the second fluorescent dye, TAMRA (6-carboxy- tetramethyl-rhodamine). On cleavage of the probe by the Taq polymerase, the quenching of FAM fluorescent emission is released resulting in an accumulation of fluorescent emission as the reaction proceeds.
21 For each sample two parallel reactions were performed; one for TF primers and probe and the other with the house-keeping gene primers and probe160,161. The PCR was performed in a two-step protocol: initial denaturation of DNA and activation of AmpliTaq Gold followed by 50 cycles of amplification. Each quantitation target was amplified in duplicate samples. In paper II the amount was related to TFmRNA expression detected in 50 ng total RNA from the human fibroblast cell-line AG1523 whereas in papers III and IV a standard in 1, 1:10, 1:100 and 1:1000 dilutions of 50 ng total RNA from human monocytes stimulated with 1 µg/mL LPS for 3 h were included in the experiments.
Flow cytometry The principle of flow cytometry analysis of cell surface antigens is based on detection of fluorochrome-labelled antibodies directed to the structures of interest. In the flow cytometer the cells passes a laser beam in single file. When the light from the laser hits the cells it is scattered in all directions. Detectors placed in a forward and 90o right angle (side scatter) of the incoming laser beam detect the size and the internal structures or granularity of the cells, respectively. Different cell types can be distinguished by their different forward and side scatter properties. For further differentiation between cells with similar forward and side scatter properties, a cell-specific, fluorochrome-labelled antibody, e.g. CD14 for monocytes, can be used. The different fluorochromes used to label antibodies absorb the laser light energy and emit fluorescence of different colours detected at specific wavelengths by the cytometer. Up to four different wavelengths can be detected at the same time, however, in this thesis only two antigens were studied at a time using the fluorochromes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) detected at 525 nm (fluorescence channel 1) and 575 nm (fluorescence channel 2), respectively. The signals from the different detectors are presented in histograms and the cell populations of interest can be marked, or gated, for further processing. The amount of antibodies bound to the gated cells is presented in a histogram of the fluorescence emitted. Two parameters are measured, percentage of positively stained cells in the population and the intensity of the staining, reflecting the amount of antibodies bound per cell. All flow cytometry analyses were performed using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter). In papers I-III whole blood was labelled with fluorescence-conjugated antibodies for detecting the surface expression of TF, CD14 and CD11b on leukocytes and in paper II cells retrieved from the oxygenators were also labelled. After incubation with antibodies on ice in the dark, the cells were washed, erythrocytes were lysed and fixed with Lyse (Beckman Coulter), washed again and suspended in PBS.
22 In papers I and II platelet-rich plasma was used to determine surface expression of P-selectin on platelets. For platelet microparticle analysis the cells were stained with a FITC-conjugated anti-whole platelet-antibody. In paper III instead a whole blood method was used for detection of surface expression of P-selectin (CD62P) and CD40L (CD154) on platelets. After labelling of the platelets with the fluorescence-conjugated antibodies, the samples were diluted and fixed with ice-cold PBS containing 1% paraformaldehyde. No washing steps were included in the platelet methods. Gating of both leukocytes and platelets was carried out using either forward and side scatter parameters or side scatter in combination with a fluorescence channel. The percentage of positive cells and mean fluorescence intensity (MFI) were determined. The flow cytometer also generates information of the relative number of different celltypes in the samples which was utilised. For platelet microparticle analysis, gating was performed using forward scatter and fluorescensce-1 (anti-whole platelet-antibodyFITC). Platelets with fluorescence lower than a pre-set cut-off level were identified as microparticles. The percentage of positive cells was determined. In paper III complexes between platelets and leukocytes were detected in whole blood, described in detail by Li et al.162. Briefly, the samples were
L M G CD45
count A
CD45 SS
monocytes granulocytes count count B C
CD42a CD42a Figure 3. Flow cytometric analysis of platelet-leukocyte complexes in whole blood. CD45-positive cells (leukocytes) are gated by A. These cells are then discriminated by their CD45/side scatter properties; L=lymphocytes, M=monocytes and G=granulocytes. Monocytes and granulocytes are analysed due to their CD42a positivity (platelet-conjugate).
23 incubated with an anti-leukocyte antibody (CD45) in combination with an antibody directed against platelets (CD42a). For analysis, CD45FITC- positive particles (leukocytes) were gated and further discriminated by their CD45 expression and granular characteristics (side scatter). The amount of platelet-conjugated monocytes and granulocytes were then analysed in their individual populations. The number of platelets per leukocyte was detected on the intensity of CD42a-positive cells (Figure 3). In paper IV the cultured monocytes were labelled with a primary, unconjugated, mouse-antihuman TF-antibody. The cells were then washed and incubated with a secondary, flourescence-labelled, rabbit-anti-mouse antibody for detection of TF expression. In paper IV also intracellular cytokines were detected. For staining of intracellular antigens, the cells must be permeabilised to allow entrance of the antibodies into the cell. We used Cytofix/Cytoperm (BD Pharmingen) for permeabilisation and labelled the cells with fluorescence-conjugated antibodies for detection of the cytokines IL-1β, IL-6, IL-8 and TNF-α. Gating and analysing of the cells was performed as described above. Irrelevant antibodies of the same subtypes, labelled with matching fluorocromes, were always included as negative controls in all experiments. A viability assay was also performed by flow cytometry in paper IV. The cells were incubated with flourescein diacetate that is cleaved to fluorescein by viable cells. Fluorescein was then detected in fluorescence channel 1 by the flow cytometer.
Analysis of markers of coagulation, fibrinolysis and inflammation In paper I-III plasma levels of markers for coagulation; soluble TF (sTF), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), fibrinolysis; plasmin-antiplasmin complex (PAP), inflammation; myelo- peroxidase (MPO) and the cytokines interleukin-6 (IL-6), IL-8, IL-10 and soluble IL-2 receptor (sIL-2R) were quantified by commercial sandwich ELISA kits. Activated FVII was analysed by a clotting assay (Staclot VIIa- rTF) using a Thrombolyzer. In paper I determination of activated coagulation factor XI-antithrombin complex (FXIa·AT), activated coagulation factor XII-antithrombin complex (FXIIa·AT) and activated coagulation factor XII-C1-esterase inhibitor complex (FXIIa·C1 INH) were performed using a solid-phase ELISA according to Sanchez et al163. In paper IV levels of the cytokines interleukin-1β (IL-1β), IL-6, IL-8 and TNFα were quantified by sandwich ELISA techniques (R&D) in the supernatants from the cultured monocytes.
24 Chemotaxis assay (Paper IV) Adhered monocytes were washed in PBS, detached with a rubber policeman and suspended in RPMI 1640 supplemented with 2% FBS. The migration of monocytes was assayed in a modified 48-well Boyden µ-chamber (Neuro Probe Inc.) by means of the leading front technique as previously described164 (Figure 4).
nitrocellulose filter migration distance migration
medium+cells gradient
medium + chemoattractant
Figure 4. Boyden chamber method. A schematic drawing of one well of the µ- chamber and a cross section of the nitrocellulose filter showing the migration distance of the two leading cells at focus.
Briefly, the cells migrated in a micropore nitrocellulose/nitroacetate filter, with a pore size of 5 µm (Millipore Corporation). Various concentrations of f-MLP, ZAS, CRP and PDGF-BB were diluted in the assay media and added below the filter in the Boyden chamber. The cells, in the assay media, or cells pre-incubated with either 100nM of FVIIa or FFR-FVIIa were applied above the filter. The chambers were incubated for 1 hour at 37oC in a cell culture incubator. Filters were then fixed, stained, washed and mounted. Migration was measured with the leading front technique, as the distance from the top of the filter to the two furthest migrating cells, in a high-power field at focus. The migration distance in each filter was calculated as the mean of observations of at least three different parts of the filter. Experiments were performed in duplicate for each concentration of the chemoattractants and repeated at least three times. For each set of experiment, the random migration of monocytes towards the assay media served as standard and was calculated as 100% migration.
Statistical analysis Statistica for Windows (StatSoft) was used for all statistical analyses. In papers I and II the results are presented as medians and 25th-75th percentile
25 ranges. The Friedman ANOVA test and the Kruskal-Wallis ANOVA test were used for analysis of time series within each group or for an overall comparison between the groups, respectively. When significant differences were obtained, these were further evaluated using the Wilcoxon matched pairs test for comparison between two samples within the series, or the Mann-Whitney U test for intergroup comparison. No adjustment for multiple testing was done. In paper III and IV the results are presented as mean and standard deviation (SD) or standard error of the mean (SEM). The Kruskal-Wallis ANOVA test was used for analysis of time series within each group and for overall comparison between the groups. When significant differences were obtained, these were further evaluated using the Mann-Whitney U or Wilcoxon matched pairs test test for comparison between samples within the series or groups. In paper IV also student’s t-test was used.
26 RESULTS AND DISCUSSION
TF-expression, coagulation and inflammation during CABG using devices coated with heparin according to a new principle (papers I and II) In these studies we investigated the biocompatibility of a newly developed heparin surface, the Corline Heparin Surface (CHS) used in the CPB during CABG. We also investigated the influence of different systemic heparin levels in combination with the CHS in respect of inflammation, coagulation and fibrinolysis.
Adhesion Modification of the surface of the extracorporeal device with the CHS- coating prevented adhesion of leukocytes and platelets. This is in contrast with two previous studies, using other heparin-coated surfaces, where no difference in cell adhesion to the oxygenator membrane as a result of heparin coating could be detected28,165. However, variation of the systemic heparin level in the CHS-coated groups did not have any influence on the adhesion of cells (Figure 5). The chemical constitution of heparin-coated surfaces varies considerably and thus, the biological effects of heparin surfaces in general are difficult to estimate. The encouraging results on reduced cell adhesion to the CHS recorded in this study confirm that the coating is stable and has an appropriate chemical composition.
Cell activation and TF-expression The number of leukocytes in the patients increased during surgery, with maximum levels three hours after and was still elevated three days after the operation. Also the activation of granulocytes and monocytes, measured as expression of CD11b, increased during the operation, however, least in the heparin-coated group with standard dose of systemic heparin. In granulocytes and monocytes from the pericardial cavity, the surface expression of CD11b was higher than in the blood. Leukocytes retrieved from the oxygenator were activated, with higher expression of CD11b compared with cells from the patients at end of CPB.
27 1600 )
6 1400 1200
1000
800
600
400
200
0 80s ACT 4 oated unc (*10 oxygenator from retrieved cells T 480s ted AC S-coa p CH 0s ly la CT 30 m te ated A m p le HS-co g o ho ts C 0s ra no c CT 60 nul cy yt ated A o te es HS-co c s C yt es
Figure 5. Cells retrieved from the oxygenators. Results are presented as median. ACT=activated clotting time; CHS=Corline Heparin Surface.
The CD11b-expression per cell on granulocytes from the heparin-coated oxygenators, independent of systemic heparin concentration, was higher compared with cells from the uncoated oxygenators, bearing in mind that the number of adhered granulocytes was significantly lower on the heparin modified oxygenators. Thus these results indicate that only the most activated cells adhere when the surface is heparin-coated. The number of platelets, on the other hand, decreased during surgery. The decrease was most pronounced in the uncoated group and in the group with high systemic heparin, probably due to more adhesion and activation in these groups. The activation of platelets, measured as expression of P-selectin, increased in all groups during surgery, however, less in the heparin- coated group with standard systemic heparin level. The expression of TF on the surface of monocytes in the blood was not upregulated until three hours after the operation, however, in monocytes from the pericard the expression was higher already at the end of surgery. The TF-expression on monocytes in circulating blood during CPB did not increase, in agreement with a number of other clinical studies showing no increase in TF of circulating monocytes during CPB28,127,166. The low expression of TF on the cell surface in circulating blood is due to too short time for translation to occur29. Monocytes in samples taken from the pericardial cavity have been shown to express approximately two-fold increased tissue factor on the cell-surface134 in a study performed with an uncoated extracorporeal device. Our results are confirming, with no differences between the groups. The increased TF-expression in monocytes
28 from the pericardium may be explained by a high level of activation due to the surgical trauma or by the formation of TF-expressing microparticles aggregated to the surface the of monocytes. We have, for the first time, analysed TFmRNA in cells retrieved from the oxygenator and show that monocytes collected from the uncoated oxygenators express low levels of TF together with high levels of TFmRNA indicating a potential for high TF surface-expression within some hours. A possible interpretation can be related to the striking adhesion of platelets to the unmodified surface leading to platelet activation and P-selectin expression. In the interaction between platelets and monocytes P-selectin has been shown to induce de novo synthesised tissue factor in monocytes167. The high TFmRNA levels in the uncoated group could also be induced by the adhesion of the monocytes themselves14. Again, the low expression of TF on the cells in this group is due to the kinetic of TF up-regulation in agreement with another study of adherent cells on uncoated oxygenator fibres, used during CPB, where no TF was found on the adhered monocytes166. In comparison with an operation time of less than two hours in this study, a prolonged (2-6 h) in vitro perfusion of blood in an uncoated oxygenator circuit clearly demonstrated up-regulation of monocyte TF and the most activated cells were adhered in the oxygenator26. The coated groups with standard and reduced systemic heparin also showed low levels of TF expression on the surface but rather low levels of TFmRNA. In the heparin- coated group with high level of systemic heparin on the other hand, a high surface-expression of TF was found in combination with low levels of TFmRNA. An increased turnover rate of TF-expression due to activation by heparin can be suggested as one possible interpretation of the TFmRNA/TF- antigen pattern found in this group. Moreover, TF can be expressed on monocytes independent of de novo synthesis through P-selectin coupling of activated platelets13. This very fast expression of TF, within 10-15 minutes, may also be a result of platelet microparticles recorded in the platelet- monocyte complex. Interestingly, a high dose of heparin in combination with a heparin-coated oxygenator surface has resulted in highly activated platelets in vitro168. The release of the pro-inflammatory cytokines IL-6 and IL-8, and the anti-inflammatory cytokine IL-10 increased during surgery with elevated levels still the following morning. sIL-2R fluctuated during the operation with lowest levels at the end of CPB and highest the morning after.
Coagulation activation
Thrombin generation, measured as F1+2 and TAT increased during the operation with maximum levels 30 minutes after CPB in all groups, however, least in the heparin-coated group with standard dose of systemic
29 heparin (Figure 6). The thrombin activity also increased during surgery with remaining high levels the following morning.
14 A uncoated ACT 480s 12 CHS-coated ACT 480s
10 *** 8 (nM)
1+2 *
F 6 ** 4
2 **
0
before sternotomy start of end of 30 min 3 h 8 h 20 h surgery CPB CPB after protamine ¤¤ 14 B ### 12 CHS-coated ACT 300s CHS-coated ACT 600s 10 #
8 ¤ ### (nM) 6 ¤¤¤ 1+2 ## ¤¤¤ F # ¤¤¤ 4 ### * ¤¤¤ ### 2
0
before sternotomy start of end of 30 min 3 h 8h 20 h surgery CPB CPB after protamine
Figure 6. F1+2 in plasma before, during and after the operation in patients. Results are presented as median and 25th-75th percentiles. A:*p=0.05, **p=0.02; ***=0.008. B:*p=0.02, ACT 300s vs ACT 600s; #p=0.03, ACT 300s vs ACT 480s; ##p=0.005; ###p<0.001; ¤p=0.004, ACT 600s vs ACT 480s; ¤¤p=0.008; ¤¤¤p<0.001
Also the fibrinolysis, reflected by PAP, increased in all groups during CPB and a further increase until three hours after surgery with least increase in the heparin-coated group with standard dose of systemic heparin. The morning after surgery, PAP levels were normalized in all groups. The formations of activated FXI and FXII increased during surgery and returned to pre-surgical levels the following morning. Contact activation was reflected distinctly differently as FXIa-AT reached maximum at end of CPB whereas the increase of FXIIa-AT was earlier, starting before CPB. Quite interestingly, the kinetic of FXIIa-AT generation differed between the groups so the uncoated and the heparin-coated group with standard heparin
30 levels reached maximum at start of CPB whereas the heparin-coated groups with high and low systemic heparin increased further during CPB. It has been reported that the capacity of the natural endothelium to inhibit FXIIa is impaired after systemic administration of heparin169 which might explain the early appearance of FXIIa, irrespective of type of extracorporeal circuit, following heparinisation of the patient. As the generation of FXIa is influenced by a thrombin feed-back loop170 it seems reasonable that the time course and relative concentrations of FXIa would coincide with those of thrombin formation. The biocompatibility of the newly developed heparin surface is higher than an uncoated surface. The low dose of systemic heparin may not be sufficient to maintain the anti-thrombotic activity at the same level as a standard heparin dose during this kind of surgery. The high dose, on the other hand, resulted in a direct cell activating situation and upregulated TF on monocytes from oxygenators, rather than generate a further anti- inflammatory and anti-coagulatory effect.
Shed mediastinal blood Autotransfusion of shed blood after cardiac surgery is an often used method to reduce the exposure of patients to homologous blood,142,143 however, a matter off debate144,149,171. Furthermore the quality of shed blood has been questioned171,172 with indications of defective haemostasis148,149,173 reports of systemic blood activation174 and more febrile patients171 after autotransfusion of this blood. We found, in agreement with previous studies, that the SMB after CPB was characterised by activated cells and extraordinary activated coagulation and inflammatory processes. The number of both leukocytes and platelets was lower in all groups compared to circulating blood three hours after surgery. The shed blood contained rather few cells, but with high expressions of CD11b, TF and P-selectin, indicating elevated activation. The low number of cells may be due to adhesion, lysing or dilution by exsudation. The release of myeloperoxidase MPO and pro-inflammatory cytokines was higher than in circulating blood at the same time. However, the level of IL- 10 in SMB was not different from systemic blood three hours after surgery and the level of sIL-2R was lower. The formation of thrombin was dramatic in the SMB, the thrombin activity and fibrinolysis were higher than in circulating blood. The activation of the intrinsic pathway was also increased in the shed blood compared to systemic blood. Thrombin has been shown to induce several cell responses involved in inflammation, among others cytokine release in monocytes and endothelial cells73. In contrast, the levels of IL-10, an anti-inflammatory cytokine and a potent inhibitor of monocyte activation and with anticoagulant effect175, did not differ between systemic
31 blood and shed blood. Consequently, the imbalance between the cytokines in shed blood may reflect ongoing activity with subsequent effects on the coagulation and inflammatory systems. In this study autotransfusion was not performed and the duration of increased inflammatory and coagulant activity appeared to be shorter compared to previous studies in which shed blood was retransfused146,172,173,176.
The influence of different heparin surface concentrations and antithrombin-binding capacity on inflammation and coagulation (paper III)
In the two previous studies the CHS was proved to reduce activation of coagulation, inflammation and cell adhesion induced during CABG. In spite of the reduction achieved by the coated surface, the contact between blood and the artificial surface in combination with the surgical procedure still induced activation of these systems. Photoelectron spectroscopy studies of the molecular structure of this surface demonstrated that a single layer of the heparin surface, equivalent to what was used in the in vivo studies, was thin and did not completely cover the substrate surface177. These data can help to explain the biological results. However, further development of the surface with an increase of the heparin concentration accompanied by an increased capacity to bind AT achieved by formation of an additional layer of immobilised heparin has resulted in a complete coverage of the surface177. We therefore examined the biological effects, i.e. activation of inflammation and coagulation, by different heparin concentrations in the CHS. We used a modified Chandler loop model, established as a sensitive in vitro model of extracorporeal circulation when studying new materials and modified surfaces178.
Cell activation Statistically significant differences were found between the uncoated and the different heparin-coated groups on the activation of both monocytes and granulocytes. However, activation of leukocytes, measured as expression of CD11b, followed different kinetics depending on the surface used in the loops. The activation of monocytes increased continuously in blood circulated in the uncoated loops. In monocytes from the heparin-coated loops no increase was recorded during the first two hours apart from a small elevation the first fifteen minutes in the loops with low heparin concentration. During the following two hours of the experiment, the activation increased in monocytes from the heparin-coated loops, however,
32 only marginally in cells from the double-coated loops. The activation of granulocytes, on the other hand, increased during the first 15 minutes in all groups and remained elevated throughout the experiment, with highest level in the uncoated group and lowest in high surface concentration of heparin. (Figure 7).
¤¤¤ 20 A uncoated single-coated heparin double-coated heparin 15
*** 10 ¤¤¤ * ¤¤ ** CD11b (MFI) CD11b ¤¤¤ ¤¤¤ 5
0 0 30 60 90 120 150 180 210 240 time (min) ¤¤¤* # 40 B * * ¤¤¤ ¤¤¤ ¤¤¤ 35 # # * ## 30 ¤¤¤ ## 25 20 15
CD11b (MFI) CD11b 10 5 0 0 60 120 180 240 time (min)
Figure 7. CD11b expression on monocytes (A) and granulocytes (B) in whole blood from Chandler loops. Results are presented as mean±SEM. MFI=mean fluorescence intensity. *=uncoated vs single-coated; ¤=uncoated vs double-coated; #=single- vs double-coated; one symbol=p<0.05; two symbols=p<0.01; three symbols=p<0.001
In platelets from the loops with high surface concentration of heparin the P-selectin expression was constant during the experiment and after four hours the expression was statistically significantly lower than in the other groups. We observed that the activation of leukocytes and platelets was significantly lower throughout the experiment using the surface with double heparin-coating compared to single. Thus, only slight cell activation was seen when the surface with high heparin surface concentration was used. Importantly, the number of complexes formed between leukocytes and platelets, and particularly the number of platelets per leukocyte, were
33 reduced by the surface with high heparin concentration. Formation of complexes between platelets and monocytes or granulocytes followed, however, different patterns. The formation of aggregates between monocytes and platelets increased in all groups during the first hour of the experiment and remained at the same level during the following hours (Figure 8a). In comparison, granulocyte complexes were formed faster in the unmodified
A B 40 30 ¤¤** ¤ ¤ 25 30 20
20 15
10
uncoated MCA (% positive cells) MCA (% positivecells) 10 single-coated heparin double-coated heparin 5
0 0 0 60 120 180 240 0 60 120 180 240 time (min) time (min)
Figure 8. Formation of cell aggregates between platelets and monocytes (A) and granuocytes (B) in whole blood from Chandler loops. Results are presented as mean±SEM. MCA=mixed cell aggregates. *=uncoated vs single-coated; ¤=uncoated vs double-coated; one symbol=p<0.05; two symbols=p<0.01 loops than in the loops with low concentration of heparin with subsequent dissociation of the complexes (Figure 8b). No significant increase was observed in the loops with high heparin concentration. Moreover, the number of platelets per leukocyte did not increase even though the number of complexes increased during the experiment when the surface with high heparin surface concentration was used. Since the adhesion of platelets to leukocytes can induce TF expression on monocytes and platelet microparticles with subsequent activation of coagulation and inflammation13,29,79, a reduced formation of these complexes is desirable. The activation of inflammation, measured as release of IL-6, increased during the experiment, statistically significant only in the unmodified loops.
34 Monocytic TF-expression and thrombin generation TF did not increase during the experiment in the loops with high concentration of immobilised heparin either on mRNA level, expression of the protein on monocyte surface or release of the soluble protein. In monocytes retrieved from the other loops a significant increase of TFmRNA was recorded during the first two hours and the expression of TF increased throughout the entire experiment but no statistically significant differences were seen between the groups. The release of soluble TF increased slightly only in the uncoated loops. FVIIa decreased during the experiments in all groups with the simultaneous increase of TF expression in the unmodified and the heparin-modified surface with low concentration. The assay detects free FVIIa, and thus these results indicate an increased formation of TF·FVIIa complexes and activation of the coagulation cascade over time. Thrombin generation, reflected by F1+2, increased during the experiment in all groups, however, later and to a lesser degree in the group with high surface concentration of immobilised heparin where the increase was seen only the last two hours in contrast to the other groups with an increased formation already after fifteen minutes. Generation of thrombin measured as TAT showed in principle the same pattern.
The effects of active site-inhibited FVIIa (FFR-FVIIa) In the light of the upregulation of TF and increased thrombin generation in the uncoated and loops with low heparin surface concentration we wanted to further study the importance of the TF-pathway in the Chandler loop model. Instead of adding soluble heparin to the blood we added active site- inhibited FVIIa which acts as an antithrombotic agent through competing with FVIIa for TF binding and thereby blocking thrombin generation by the TF·FVIIa activity179. Also the inflammatory response is decreased by TF blockage with active site-inhibited FVIIa180. We then circulated the blood in uncoated loops or loops with low concentration of the immobilised heparin surface. Upon addition of FFR-FVIIa to the blood no clots were seen in any of the loops during the experiments. When FFR-FVIIa was added to the unmodified loops the production of TFmRNA was lower compared to the same loops without FFR-FVIIa. In the loops with single-coated surface the production of TFmRNA was later and increasing throughout the experiment upon addition of FFR-FVIIa, however, not statistically different from loops without FFR-FVIIa. The results of the surface expression of TF upon addition of FFR-FVIIa showed that the binding of FFR-FVIIa to monocytes in both the unmodified and heparin- modified loops was efficient and constant during the experiment, i.e. no TF was detected.
35 The thrombin generation, reflected by F1+2, was lowered upon the addition of FFR-FVIIa in the loops. The same pattern was observed by TAT- values. Since thrombin is a strong inducer of platelet activation181 we also studied the effects of FFR-FVIIa on formation of cell complexes between platelets and leukocytes. The number of platelets per leukocyte was lower when FFR-FVIIa was added to the blood and circulated in the loops, however significant only in complexes formed with granulocytes. The data from this study clearly demonstrated that increased AT-binding capacity and uniform surface coating of the CHS results in only minor activation of coagulation, inflammation and cell activation. Although the Chandler loop model is a sensitive in vitro system for detecting activation of these processes, care should be exercised in predicting the in vivo performance.
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) The TF·FVIIa proteolytic complex seems, in addition to its role as inducer of coagulation in vivo, to be involved in various non haemostatic functions as wound healing, local inflammation, the formation and progression of the artherosclerotic plaques and tumour metastasis8. During these processes, migration of cells (chemotaxis) is also an important mechanism84. Moreover, a number cytokines and growth factors are involved. The aim of this study was to investigate whether the TF·FVIIa complex also induces molecular signals and biological functions in human monocytes. We used a well characterized model system with freshly isolated human monocytes, first stimulated by a minimal concentration of LPS for induction of TF, to study cytokine production and cell migration, two central biological functions in inflammation.
TF-expression and cytokine production LPS-stimulation for three hours resulted in upregulation of both monocytic TFmRNA and protein expression. The formation of the proteolytically active TF·FVIIa complex on monocytes increased the amount of intracellular TF, caused either by internalization or new production. An increased concentration of TFmRNA, however not significant, in the cells cultured with an addition of FVIIa suggests the latter. Stimulation of the monocytes with LPS for three hours resulted in intracellular production of IL-1β, IL-6, IL-8 and TNF-α (Figure 9a). FVIIa binding to TF did not influence the intracellular amount of these cytokines as measured by flow cytometry. However, significantly higher concentrations
36 of IL-8 and TNF-α, measured with ELISA in the supernatants, was recorded by the formation of the proteolytically active complex TF·FVIIa (Figure 9b).
40 A RPMI 5% FBS 3+3h 30 LPS 3h+BFA 3h LPS 3h+FVIIa/BFA 3h
20
cytokine (MFI) 10
0 IL-1β IL-6 IL-8 TNF-α 7000 * B 60 6000 * 5000 40
4000 (pg/mL) α
3000
2000 20 TNF- interleukins (pg/mL) interleukins 1000 0 0 IL-1β IL-6 IL-8 TNF-α
Figure 9. Cytokines in cultured monocytes. A; intracellular expression of cytokines, B; cytokine concentration in conditioned medium. Results are presented as mean±SD, *=p<0.05. BFA=BrefeldinA; LPS=lipopolysaccharide; MFI=mean fluorescence intensity;
There was no influence of FVIIa-binding on the concentrations in conditioned medium of IL-1β and IL-6. In a human keratinocyte cell-line, binding of FVIIa to TF leads to up-regulation of several genes, one of which is IL-853,54. Our results with human monocytes also indicate induced production of the cytokines. Both IL-8 and TNF-α generation is regulated by transcriptional activation of the gene by NF-κB pathway182,183. IL-8 generation is also regulated by the JNK pathway182 and both cytokines by p38MAPK pathway; IL-8 via post-transcriptional stabilization of the mRNA and TNF-a by regulation of the translation182,184, where JNK and p38MAPK pathways are activated in TF·FVIIa signalling36,37. IL-8 is a chemotactic chemokine produced by stimulated monocytes and macrophages among other cells. Moreover, macrophages from atherosclerotic plaques are shown to have an enhanced capacity to produce IL-8 compared with normal blood monocytes185. We have, for the first time, in repeated experiments shown that TF·FVIIa signalling results in increased production of TNF-α in monocytes. The cytokine exerts pleiotropic effects on a variety of cells involving inflammation, necrosis, apoptosis and immunity. Also, TNF-a
37 promotes thrombotic processes via upregulation of TF and downregulation of TM in endothelial cells186. However, in our hands, none of the cytokines IL-1β, IL-6, IL-8 and TNF-α upregulate the expression of TF in monocytes (unpublished data, Siegbahn et al.). Thus, the increased expression of TF by the TF·FVIIa signalling is probably not due to an autocrine loop by the newly produced cytokines.
Chemotaxis TF-expressing human monocytes migrated towards a concentration gradient of PDGF-BB with a starting effect from >1 ng/mL and a maximum response at 10 ng/mL. The chemotactic response showed an increased sensitivity to PDGF-BB by a 100-fold upon formation of active TF·FVIIa complex. The sensitivity to low concentrations of PDGF-BB was dependent of TF·FVIIa signalling as hyperchemotaxis was not induced in TF-expressing monocytes ligated with FFR-FVIIa (Figure 10a). PDGF-BB is a growth factor known to
monocytes 130 A monocytes+100 nM FVIIa monocytes+100 nM FFR-FVIIa 120 *** *** 110
100
90
00,01 0,1 1 10 100 PDGF BB (ng/mL) 350 300 B monocytes/pertussis toxin (PTX) 250 monocytes/PTX+100 nM FVIIa monocytes 140 monocytes+100 nM FVIIa
cell migration (% of controls) * 120
100
0 0,01 0,1 1 10 100 10 nM fMLP PDGF-BB (ng/mL)
Figure 10. Migration of TF-expressing monocytes stimulated by PDGF-BB. In panel B, cells pretreated with pertussis toxin. Results are presented as mean±SEM; *=p<0.05, ***=p<0.001 induce mitogenic response, chemotaxis and actin re-organization in a number of different cells187. Previously, we reported that when FVIIa is bonded to TF the chemotactic response is increased to these low concentrations of PDGF-BB in fibroblasts, vascular smooth muscle cells52 and endothelial cells transfected with TF and PDGF-β-receptor51. Thus, the
38 data from these studies together indicate that the migration response to PDGF-BB after formation of the TF·FVIIa complex seems to be a general pattern. To investigate if this increased sensitivity to a chemotactic factor upon TF·FVIIa signalling was applicable on other chemotactic molecules we tested a number of known chemotactic inducers for monocytes as MCP-1, CRP, f-MLP and ZAS. However, no alteration of the chemotactic response to any of these chemotactic substances was seen. The significance of this finding is unclear but might suggest a more specific role for PDGF-BB in the recruitment of TF-expressing cells in the presence of FVIIa. TF shows structural and sequence homology with the cytokine receptor superfamily and especially the interferon class of receptors12 and is as such involved in signal transduction. The signalling induced upon binding of FVIIa to TF is suggested to be mediated via a G protein-linked protease activated receptor, most possibly PAR-243,44. Pretreatment of cells with pertussis toxin is known to interfere with signals mediated through Gαi-type nucleotide-binding proteins. Incubation of monocytes with pertussis toxin after LPS stimulation totally abolished the G-protein mediated fMLP- induced chemotaxis. In contrast, the chemotactic response to PDGF-BB was not affected. PDGF-BB induces chemotaxis via tyrosine-phosphorylation signal-transduction pathways187. The TF·FVIIa-signalling inducing PDGF- BB stimulated hyperchemotactic response in monocytes was not sensitive to pertussis toxin treatment (Figure 10b). The results indicate that Gαi-protein was not involved in the signalling still, the hyperchemotactic effect was abolished without formation of the proteolytic complex TF·FVIIa. Thus, our results on monocytes suggest that the intracellular domain of TF, shown to bind actin-binding protein 280 (ABP-280)47, critical for migration48, may be involved in the migration response to PDGF-BB induced by the TF·FVIIa-signalling. Also, ligation of the extracellular domain of TF was necessary for the binding of ABP-280 leading to reorganization of the actin cytoskeleton47. The results with monocyte migration are in line with the idea that the TF·FVIIa signalling involves two different pathways (Figure 11). We could conclude that the TF·FVIIa signal transduction inducing an increased sensitivity to PDGF-BB-stimulated migration and an increased production of IL-8 and TNF-α in monocytes could be important mechanisms for continued recruitment and activation of cells to sites of inflammation.
39 Figure 11. TF·FVIIa proposed signalling and biological functions. .
40 Concluding remarks
Open heart surgery with CPB exposes the body to a massive trauma. The anestesia, surgical trauma, suction of blood, blood circulating in contact with artificial surfaces etc. together contribute to the flora of complications connected to this kind of surgery. Inflammation and imbalance in the coagulation system with blood loss and thrombosis as an effect of surgery could result in post-operative complications with neurological and physiological effects. Nevertheless, an early invasive strategy improves survival without recurrence of myocardial infarction and revascularisation for severe angina188,189. Taken together these results emphasize the importance for improvements connected to the surgical procedure. We have explored the biological effects of a heparin surface prepared according to a new principle used in the CPB during CABG and demonstrated an increased biocompatibility accomplished by the heparin- coated surface in terms of low adherence of cells, decreased activation of coagulation and inflammation. Despite these promising results, the activation of these systems was still substantial. Investigation of the surface by photoelectron spectroscopy using synchrotron radiation excitation demonstrated that the heparin-coating was not completely covering. An improvement of the heparin-coated surface achieved by formation of an additional layer of immobilised heparin resulted in a complete coverage of the substrate surface177. This resulted in a higher surface concentration of heparin accompanied with an enhanced AT-binding capacity. The modification of the heparin-coating proved to further improve the biocompatibility of the surface. Future studies using this improved surface in an in vivo situation will be interesting. TF is, both in its role as inducer of the coagulation in vivo, and as participant in the inflammatory response, an important actor in these processes. Interestingly, our results were we blocked the binding of FVIIa to TF with the active site-inhibited FVIIa in the simulated circulation were comparable with the results with double heparin coating. A future strategy for this kind of surgery with CPB may be single or double coating of the extracorporeal circuit in combination with inhibitors of TF. The mechanisms behind the biological effects of FVIIa binding to TF, in addition to thrombin generation, are important to explore. Our results with
41 induction of two important pro-inflammatory cytokines, IL-8 and TNF-α in monocytes are intriguing. IL-8 is central in activation of leukocytes and cross-talk with granulocytes and TNF-α with pleiotropic effects among which the procoagulant effects are of special interest. Moreover, we demonstrated that the signal transduction of importance for cell migration induced by FVIIa binding to TF is dependent of the cytoplasmic domain but also of the proteolytic activity of the TF·FVIIa complex. Our results support the idea that signalling, resulting in important mechanisms for continued recruitment of cells to sites of inflammation, involves two different pathways.
42 SUMMARY
• The Corline Heparin Surface prevented adhesion of cells to the oxygenators.
• Monocytes retrieved from the uncoated oxygenators had a low TF expression and high TFmRNA levels, indicating a potential for increasing TF expressing over time whereas monocytes from the heparin-coated oxygenators with high systemic heparin, were highly activated with high TF expression.
• The Corline Heparin Surface is more biocompatible than an uncoated surface used during CABG.
• The most successful protocol proved to be the combination of the heparin surface and a standard dose of systemic heparin.
• A reduced dose of systemic heparin in combination with the heparin surface was not be sufficient to maintain this antithrombotic activity.
• A high dose of systemic heparin resulted in a merely cell-activating situation rather than a further anti-inflammatory and anti-coagulatory effect.
• The shed blood was highly activated, both regarding inflammation and coagulation and retransfusion of this blood may be harmful.
• A further development of the Corline Heparin Surface with higher heparin surface concentration and antithrombin-binding capacity proved to increase the biocompatibility of the surface.
• The addition of active-site inhibited FVIIa resulting in low thrombin generation confirms the importance of the TF-induced coagulation during simulated extracorporeal circulation.
43 • TF·FVIIa induced signalling in monocytes leads to increased production of the pro-inflammatory cytokines IL-8 and TNF-α.
• The increased sensitivity to PDGF-BB-stimulated migration in monocytes, induced by FVIIa binding to TF, is an indicator of a general mechanism since the same migration pattern was earlier recorded in several other cell types.
44 SAMMANFATTNING PÅ SVENSKA
Vid hjärtoperation med hjärt-lungmaskin exponeras blodet för vävnadsfaktorn, tissue factor (TF), initierare av koagulationen i kroppen, samt för en stor främmande yta vilket leder till aktivering av inflammation och koagulation. TF finns normalt inte i blodet utan endast i vävnaden för att förhindra stora blödningar vid vävnadsskada. Då blodet kommer i kontakt med TF binds koagulationsfaktor VIIa och koagulationen startar. Ytan i hjärt-lungmaskinen kan modifieras med heparin för att öka biokompatibiliteten, d.v.s. de egenskaper hos ett biomaterial som är nödvändiga för att det ska fungera i en biologisk vävnad. Jag har studerat biokompatibiliteten hos en ny heparinyta, Corline Heparin Surface, använd i hjärt-lungmaskin vid kranskärlsoperation. Jag har även studerat hur biokompatibiliteten förändras i en modifierad, vidareutvecklad Corline-yta i en experimentell studie. TF bildas i celler i blodet vid kontakt med en artificiell yta, och förutom att starta koagulationen har TF även betydelse för inflammation. Jag har därför i det sista delarbetet även studerat betydelsen av bindningen mellan faktor VIIa och TF i monocyter (en sorts vit blodkropp) för produktion av cytokiner (signaleringsmolekyler mellan celler) och migration (cellvandring) vilket är två viktiga mekanismer vid inflammation.
Vi har visat att Corline Heparin Surface använd i hjärt-lungmaskin vid kranskärlsoperation är mer biokompatibel än en obehandlad yta vid standardnivå av heparin i blodbanan under operationen. Heparinytan förhindrar adhesion, vidhäftning, av celler till ytorna i hjärtlungmaskinen samt minskar aktiveringen av trombocyter, leukocyter (vita blodkroppar), koagulation, fibrinolys och inflammation. En minskning av heparinnivå i blodbanan verkade inte vara tillräcklig för att upprätthålla antikoagulerande aktivitet medan en högre heparinnivå resulterade i en direkt cellaktivering och uppreglering av TF i monocyter från oxygenatorn, hjärt-lungmaskinens lunga.
Corline-ytan som användes i hjärt-lungmaskinen vid kranskärlsoperation har karaktäriserats med synkrotronljus, en undersökning på atomnivå, som visade att heparinytan inte är heltäckande. En modifiering av ytan i två eller
45 flera lager ger dock en heltäckande heparinyta. I den experimentella studien av Corline-ytan jämfördes heparinytan i ett och två lager med en obehandlad yta. Färskt, humant helblod cirkulerades i ett slang-loop system. Vi visar att en vidarutveckling av Corline Heparin Surface med dubbelt heparinlager ger en ytterligare ökad biokompatibilitet hos ytan i form av minskad cell-och koagulationsaktivering.
Celler i blodet har normalt inte TF på sin yta men bl.a. då monocyter kommer i kontakt med biomaterial stimuleras de till TF-uttryck och produktion av cytokiner. Koagulationsfaktor VIIa från blodet binder till TF och startar koagulationen i kroppen via aktivering av koagulationsfaktorerna X och IX, vilket leder till trombinbildning och spjälkning av fibrinogen till fibrin, det bildas ett koagel. Det har nyligen visats att då faktor VIIa binder till TF aktiveras, förutom koagulation, även andra processer, såsom inflammation, sårläkning, kärlnybildning och metastasering. Kunskapen om de molekylära signaler i cellen som leder till dessa biologiska funktioner är dock liten. Vi visar att den signalering som induceras i monocyten då faktor VIIa binder till TF ökar känsligheten för en tillväxtfaktor så att lägre koncentrationer behövs för att stimulera till migration. Det intracellulära uttrycket av TF ökar pga nyproduktion. Dessutom ökar koncentrationen av cytokinerna IL-8 och TNF-α. Detta kan vara viktiga mekanismer som försäkrar såväl rekrytering som aktivering av leukocyter vid inflammation.
46 ACKNOWLEDGEMENTS
This thesis was carried out at the Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, Uppsala, Sweden. I wish to express my sincere gratitude to all friend and colleagues, who in different ways, have supported me throughout this study. In particular, I would like to thank;
Agneta Siegbahn, my supervisor, for giving me the opportunity and encouraging me to take this step into the world of science, for sharing your vast knowledge, especially concerning coagulation and inflammation, for your never ending ideas, enthusiasm and constructive criticism, for guidance into the mysteries of scientific writing, for always believing in me, and for your friendship;
Rolf Larsson, my co-supervisor, for introducing me into the field of biomaterials and heparin-coated surfaces, for support and fruitful discussions;
Graciela Elgue, Anders Larsson, Stefan Thelin, my co-authors, for your knowledge and for valuable discussions;
Anita Bertilsson, research nurse during the clinical studies, for your support and invaluable help;
Taavo Tenno, researcher in our group and my room-mate for so many years, for sharing your knowledge, for valuable discussions and friendship;
Birgitta Fahlström, Helena Vretman, Christina Christersson, Anders Mälarstig and Mikael Åberg, my colleagues in the research group, for friendship, excellent and promptly performed technical assistance, for help, blood donations, discussions and for creating a nice and friendly atmosphere;
Lena Carlsson, Kristina Seton, David Carlander, Jonas Byström, Eva Ristoff, Charlotte Woschnagg, Mia Lampinen, Jenny Eriksson and all the rest of my, former and present, fellow PhD-students, for your friendship,
47 support, pleasant pub-nights and for being examples of the feasibility of this kind of project…;
Barbro Bjurhäll, for all help concerning practical matters;
All of my other friends and colleagues at the department for your help, donating blood, baking Friday-cakes and for making the department a nice place to work in;
Barbro Björklund and Stefanoskören, for friendship, for being a source of energy and joy and for being such a euphonious choir. You are the best!;
Jonas, friend, fellow choir-member and colleague, and his wife Elisabet for your warm friendship, for appreciated late-night conversations after good dinners and wine and for being so hearty;
Eva and Daniel, Marita and Pelle, Marie and Åke, Anna-Karin and Jan for being my oldest friends, always supportive and welcoming whenever we drop by;
All my other friends - no one mentioned, no one forgotten, for just being my friends;
My parents, Maud and Hans, for bringing me up, your support and never ending belief in me. I especially would like to thank my father for inspiring me to become a scientist;
My beloved husband, Jörgen, for being such a good golf-partner, for your support, your endurance with my always being late, your patience, and love. I love you!
This work was supported by grants from; the Swedish Foundation for Strategic Research, the Swedish Research Council, the Swedish Heart and Lung Foundation, Konung Gustaf V:s och Drottning Victorias stiftelse and Josef och Linnéa Carlssons minnesfond, Uppsala University.
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65 Acta Universitatis Upsaliensis Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the sum- mary alone is distributed internationally through the series Comprehen- sive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to October, 1985, the series was published under the title “Abstracts of Uppsala Dissertations from the Faculty of Medicine”.)
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