Linköping University Medical Dissertations No. 1176
Platelets in inflammation
Role of complement protein C1q, Creactive protein
and tolllike receptors
Caroline Skoglund
Division of Drug Research
Department of Medical and Health Sciences
Faculty of Health Sciences
Linköping University, SWEDEN
Linköping 2010
© Caroline Skoglund 2010
ISBN 9789173934183
ISSN 03450082
over: Adhering platelets stained for F‐actin and visualized
C
with fluorescence microscopy
During the course of research underlying this thesis, Caroline
Skoglund was enrolled in Forum Scientium. A multidisciplinary
doctoral program at Linköping University, Sweden
Printed by LiUtryck, Linköping, Sweden, 2010
“I find that a great part of the information I have w f as acquired by looking up something and inding something else on the way” (Franklin P. Adams 1881‐1960)
ABSTRACT
Platelets are proven essential in haemostasis, however, they are now also increasingly recognized as cells with important immunomodulatory properties, e.g. through interaction with leukocytes and several species of bacteria and by release inflammatory mediators upon activation. Moreover, platelets express receptors involved in immunity and inflammation such as Fcγ‐receptor IIa, complement protein C1q‐receptors (gC1qR, cC1qR, CD93 and α2β1) and toll‐like receptors (TLR‐1, ‐2, ‐4, ‐6 and ‐9). C1q, C‐reactive protein (CRP) and TLRs are all pattern recognition molecules able to recognize non‐self structures and initiate an immune response. Uncontrolled or misdirected activation of platelets and the immune response is involved in the onset and progress of several conditions with an inflammatory component, such as coronary artery disease and autoimmune diseases. Hence, the aims of the present thesis were to investigate the effects and
q
mechanisms of C1 and CRP on platelet activation, and to clarify the intracellular signaling events provoked by TLR‐2 stimulation of platelets. Platelet interaction with immune complexes is poorly understood, however by utilizing well‐characterized model surfaces with adsorbed IgG and microscopy, we show that both C1q and CRP are able to inhibit FcγR‐ mediated platelet adhesion and spreading. Using isolated platelets in suspension and flow cytometry, we also found that C1q triggers a rapid, moderate and transient up‐regulation of P‐selectin that is sensitive to blockade of gC1qR and protein kinase C (PKC), but not blockade of α2β1. Additionally, subsequent platelet activation by collagen or collagen‐related peptide (GPVI specific) is inhibited by C1q, suggesting a role for GPVI in C1q‐ mediated regulation of collagen‐induced platelet activation. Whole blood studies revealed that C1q inhibits total cell aggregation, formation of platelet‐ leukocyte aggregates, and potentiates the production of reactive oxygen species (ROS), all in a platelet‐dependent manner. Furthermore, using the TLR‐2/1 agonist Pam3CSK4 we found that TLR‐2/1‐activation of platelets is mediated via a P2X1‐dependent increase in intracellular free Ca2+, P2Y1 and P2Y12 –receptor ligation, and activation of cyclooxygenase. We also found that platelets express IRAK‐1, however, without being rapidly phosphorylated upon Pam3CSK4 stimulation and thus probably not involved in the early aggregation/secretion response. Furthermore, TLR‐2/6 stimulation does not lead to platelet activation but instead inhibits TLR‐2/1‐provoked activation.
Taken together, these findings further strengthen the role of platelets as key layers in inflammatory processes.
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POPULÄRVETENSKAPLIG SAMMANFATTNING
Blodplättarna (trombocyterna) har en viktig roll i hemostasen, d.v.s. den process som hjälper till att stoppa en blödning som uppstått då ett blodkärl skadats. När blodplättarna aktiveras blir de större, ändrar form och klumpar ihop sig (aggregerar), allt för att täppa till och förhindra blodförlust. Förutom denna uppgift, så har det på senare år även visat sig att blodplättarna har andra viktiga egenskaper, till exempel så medverkar de i den inflammatoriska processen. Inflammation är en viktig del i kroppens försvar mot framför allt bakterieinfektioner, då immunförsvaret jobbar hårt för att bekämpa de skadliga inkräktarna. Blodplättarna kan bland annat samverka med vita blodkroppar och frisätter ämnen som påverkar såväl dom själva som andra typer av celler. På sin yta uttrycker blodplättarna olika igenkänningsmolekyler, så kallade receptorer, som binder in och reagerar på specifika ämnen, vilket ofta leder till aktivering. Under den tidiga fasen av inflammationen ökar blodets nivåer av C‐reaktivt protein (CRP) snabbt och komplementsystemet, som är en viktig del av vårt medfödda immunförsvar, aktiveras. C1q är ett av proteinerna som ingår i komplementsystemet. Det har också visat sig att blodplättar kan aktiveras av vissa bakterier, t.ex. via receptorer på blodplättarna som kallas toll‐lika receptorer (TLR:er), och som reagerar på olika bakteriestrukturer. Idag vet vi att en okontrollerad eller felriktad aktivering av blodplättar och ett immunförsvar i obalans kan bidra till inflammatoriska sjukdomar som hjärt‐kärlsjukdom och autoimmuna sjukdomar. Det är därför viktigt att förstå de bakomliggande mekanismerna. Syftet med studierna som ingår i den här avhandlingen var således att studera hur C1q och CRP påverkar aktivering av blodplättar samt att klargöra hur aktivering via TLR‐2/1 går till, genom att undersöka vilka signaler som skickas inuti cellen.
Resultaten visar att CRP och C1q minskar blodplättarnas förmåga att binda in till och sprida ut sig på en IgG‐yta, som normalt är en kraftigt blodplättsaktiverande yta. Vidare fann vi att C1q som ges till blodplättar i lösning, ökar uttrycket av P‐selektin på ytan av blodplättarna, och att detta beror på aktivering av receptorn gC1qR och enzymet proteinkinas C. Dessutom så såg vi att C1q hämmar den fortsatta aktiveringen av blodplättarna som uppkommer om man tillsätter kollagen. Kollagen är ett protein som finns rikligt i kärlväggen och som vid en kärlskada exponeras och har aktiverande verkan på blodplättar. Därför är det mycket intressant
om C1q kan fungera som en naturlig regulator för denna process. I helblod fann vi också att C1q hämmar aggregatbildningen mellan blodplättar och vita blodkroppar, och samtidigt ökar produktionen av reaktiva syremetaboliter. Reaktiva syremetaboliter är viktiga för att bekämpa bakterier vid en infektion. Genom att använda Pam3CSK4, en molekyl som specifikt aktiverar blodplättarna via TLR‐2/1‐receptorn, såg vi att aktiveringen är beroende av en ATP‐receptorförmedlad frisättning av kalcium inuti cellen. Dessutom är aktivering av enzymet cyklooxygenas och de båda receptorerna för ADP (P2Y1 and P2Y12) inblandade.
Sammantaget så visar resultaten att C1q, CRP och TLR:er, som alla medverkar i vårt immunförsvar, också har förmåga att reglera blodplättarnas aktivitet. Dessa rön bidrar till en bättre förståelse av blodplättarnas roll och beteende id inflammation och kan i framtiden leda till utveckling av nya läkemedel för tt reglera blodplättarnas aktivitet vid kroniska inflammatoriska sjukdomar. v a
TABLE OF CONTENTS
ABSTRACT...i POPULÄRVETENSKAPLIG SAMMANFATTNING... iii TABLE OF CONTENTS... vi ABBREVIATIONS...8 LIST OF PAPERS ...9 .... on... 11 INTRODUCTION ... spects of inflammati ecognition receptors ... 11 ...11 General a Pattern r Platelets ... 12 Biology, structure and function ... 12 Platelet granules... ... 16 ... 13 Platelet receptors ... ... ction ... 17 ... 14 Platelet‐bacteria interaction ... 17 Platelet‐leukocyte intera ... 18 Neutrophils ... The complement‐system. Complement protein C1q... 20 Structure and synthesis ... 20 C1q receptors .. Cellular effects C‐reactive protein ... 22 ... 21 ... 21 Historical background... 22 Structure, synthesis and ligands ... 22 Complement ac Cellular effects and Toll‐like receptors ... 25 tivation and regulation by CRP ... 23 role in inflammation... 24 Structure, ligands and function ... 25 TLR signaling... 25 TLR2... TLR expres Fcγ receptors... 27 ... 26 sion and function in platelets ... 27 AIMS...29 COMMENTS ON METH Isolation of blood cells ... 31 ODS...31Platelets (Paper I, II and IV)... 31 Neutrophils (Paper II) ... ear cells (PaperIV) ... sorption ... 32 ... 31 Mononucl ... 33 ... 32 Surface methylation and protein ad Ellipsometry... Platelet adhesion and morphology ... 33 Platelet adhesion to coverslips with adsorbed IgG and HSA ... 34 Fluorescence microscopy an te ... 35 d image analysis... 34 Enzymatic detection of pla ... 35 let adhesion to collagencoated microplates... 35 Phosphatidylserine expression ... roduction ... selectin expression... 35 Thromboxane B2 p t ... 36 Flow cytome ry analysis of P‐ ... 36 Soluble P‐selectin... ... secretion ... 37 Cytosolic Ca2+ measurements ... gometry and dense granule whole blood ... 38 Light‐transmission aggre ... 38 Luminol‐dependent ROS‐production in ... te formation ... 38 Whole blood aggregation .. eukocyte aggrega blot experiments ... 39 Platelet‐l Western Statistics ... 39 SUMMARY OF PAPERS ...41 RESULTS & DISCUSSION...43 a‐mediated platelet adhesion 3 Inhibitory effects of C1q and CRP in FcγRII 6 and activation... 4 Adhesion of platelets to ligand‐bound CRP... 4 gulates collagen and . 48 C1q induces P‐selectin expression and re . 51 collagen‐related peptide activation in washed platelets in suspension... Regulatory effects of C1q in whole blood... TLR‐2/1‐activation of platelets is mediated by ATP‐dependent Ca2+ increases, ADP receptors and activation of cyclooxygenase ... 54 CONCLUSIONS...57 ACKNOWLEDGEMENTS ...59 REFERENCES ...63
ABBREVIATIONS
ACD acid‐citrate dextrose solution ADP adenosine diphosphate sphate n 1q ATP adenosine tripho C1q complement protei COX cyclooxygenase CRP C‐reactive protein HSA human serum albumin 1 ciated kinase‐1 IgG immunoglobulin G IRAK‐ interleukin‐1 receptor asso KRG Krebs‐Ringers glucose ex 2 crophage‐activating lipopeptide‐2 MAC membrane attack compl ived ma MALP‐ mycoplasma‐der MBL mannose binding lectin mCRP monomeric CRP SK MyD88 myeloid differentiation factor 88 Pam3C 4 triacylated lipopeptide Pam3Cys‐Ser‐(Lys) rn 4 PAMP pathogen associated molecular patte r C lear cells PAR protease activated recepto nonuc saline PBM peripheral blood mo PBS phosphate buffered PCh phosphorylcholine e ‐3‐kinase PFA paraformaldehyd PI3‐K phosphoinositide PKC protein kinase C PLC phospholipase C PRM pattern recognition molecule PRP platelet rich plasma PRR pattern recognition receptor ‐1 igand‐1 PS phosphatidylserine otein l PSGL P‐selectin glycopr ROS reactive oxygen species r r activating peptide TLR toll‐like recepto P to TRA thrombin recep e A TXA2 thromboxan XB e B2 T 2 thromboxan 2LIST OF PAPERS
This thesis is based on the following Papers, which will be referred to by their roman numerals: I Caroline Skoglund, Jonas Wetterö, Thomas Skogh, Christopher Sjöwall, Pentti Tengvall and Torbjörn Bengtsson. C‐reactive protein and C1q regulate platelet adhesion and activation on adsorbed immunoglobulin G and albumin. Immunology and Cell Biology 2008; 86: 466474 II Caroline Skoglund, Jonas Wetterö, Pentti Tengvall and Torbjörn Bengtsson. C1q induces a rapid up‐regulation of P‐selectin and modulates collagen‐ and collagen‐related peptide‐triggered activation in human platelets. Immunobiology In Press 2010; doi:10.1016/j.imbio. 2009.11.004 III Caroline Skoglund, Jonas Wetterö and Torbjörn Bengtsson. C1q regulates collagen‐dependent production of reactive oxygen species, formation of platelet‐leukocyte aggregates and levels of n whole blood. 201 soluble P‐selectin i 0; Manuscript IV Hanna Kälvegren, Caroline Skoglund, Christian Helldahl, Maria Lerm, Magnus Grenegård and Torbjörn Bengtsson. Toll‐like receptor 2 stimulation of platelets is mediated by purinergic P2X 1‐dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation. Thrombosis and Haemostasis 2010; 103: 398407
Papers are reprinted with permission from Nature Publishing Group (Paper I), Elsevier (Paper II) and Schattauer (Paper IV)
INTRODUCTION
General aspects of inflammation
The skin and epithelial mucosa constitute a first line of defence in order to protect our bodies from injury and infection. However, if a foreign particle/organism e.g. a bacteria, fungus or virus in some way pass these barriers the inflammatory reaction is crucial in order to uphold our defence against infection and tissue injury [1]. The inflammatory response, described already 5000 years ago by Celcus and Galen, is characterized by the five classical cardinal signs: robur (redness), calor (heat), tumor (swelling), dolor (pain) and finally functio laesa (loss of function) [1, 2]. In the acute inflammatory state, e.g. upon intrusion of a pathogen cells present at the site of infection such as macrophages, and neutrophils recruited from the blood stream, quickly react and try to fight off the infection with the help of the complement system. This process is a part of our innate (unspecific/natural) immunity. Later on, the acquired (specific/adaptive) immunity, mainly involving dendritic cells, T‐cells and antibodies produced from B‐cells responds to the ongoing inflammation. During the whole process, inflammatory mediators such as prostaglandins, leukotriens and cytokines are released. Moreover a systemic acute‐phase response which alters the synthesis of certain proteins, e.g. C‐reactive protein (CRP) and serum‐amyloid A is initiated [1, 3, 4]. Today it is known that an inappropriate or misdirected inflammatory reaction is involved in the onset and progression of many different disorders, such as coronary artery disease and autoimmune diseases.
Pattern recognition receptors
Pattern‐recognition molecules (PRMs) or pattern‐recognition receptors (PRRs) as they are denoted when performing as receptors, are molecules involved in innate immunity that recognize foreign structures and initiate an immune‐response. PRMs/PRRs can be cell‐associated, as well as found in the fluid phase, and recognize specific molecular structures known as pathogen associated molecular patterns (PAMPs). Toll‐like receptors (TLRs), CRP and complement protein C1q, are all PRMs/PRRs able to bind to foreign structures. Other PRMs/PRRs include mannose‐binding lectin (MBL), surfactant‐protein A and D (SP‐A and SP‐D), L‐ficolin/P35 (Ficolin‐2) and H‐ ficolin (Ficolin‐3). Apart from TLRs, nod‐like receptors (NODs) and retinoic
acid‐inducible gene‐I like receptors (RLRs) and scavenger receptors are cell‐ associated PRRs. Several recent reviews describing PRMs/PRRs and their role in innate immunity as well as regulators of adaptive immunity are available [4‐7]. Apart from the traditional inflammatory cells, e.g. neutrophils and macrophages, platelets have emerged as potent actors in inflammation. In the present thesis we have examined the effects and mechanisms of platelet interaction with C1q and CRP and clarified some intracellular events upon TLR‐2/1 activation.
Platelets
Biology, structure and function Small undefined particles in blood were first observed and described already 1780 by Hewson and later in the middle of the 19 in by M pl de th century also reported several other researches, e.g. Donné, Beale and Zimmermann, [8]. In 1865, ax Schultze was the first to publish a convincing report on the presence of atelets in blood and in 1882 Giulio Bizzozero described platelets in moretail and also started to elucidate their role in hemostasis [9, 10].
B A Figure 1. A) Differential interference contrast (DIC) microscopy image showing unstimulated isolated blood platelets in suspension. B) Fluorescence microscopy mage showing activated platelets adhered to a protein (fibrinogen) coated
i
surface. Scale bars: 10 µm.
Platelets are non‐nucleated cell fragments derived from megakaryocytes, most often present in the bone marrow. Since megakaryocytes are able to migrate to other tissues, formation of platelets may also occur in the blood stream and in the lungs [11‐13]. However, the exact mechanism by which platelets are formed is still incompletely understood [11]. Interestingly, it
was recently shown that exposure of megakaryocytes to high shear rates lead to increased formation of platelets, possibly indicating that platelets produced from megakaryocytes present in the microvasculature is governed by circulatory forces [14]. The resting platelet (Figure 1A) has a discoid shape, an average size of 2 to 5 µm in diameter and a thickness of about 0.5 µm [15]. Non‐activated platelets circulate for 7 to 10 days at a concentration of 150‐400 x 109 cells/L blood before being degraded in the liver or spleen [16]. The main known function of platelets is to participate in haemostasis. If exposed to subendothelial structures (e.g. collagen) in an injured vessel, circulating platelets respond quickly (within seconds), become activated, adhere and start to form a hemostatic plug, with the purpose to form a temporary shield to stop an ongoing bleeding (reviewed in [17, 18]). In order to get a more permanent repair of the injured vessel, the coagulation‐cascade is activated e.g. by negatively charged phosphatidylserine (PS) expressed on the activated platelet and tissue factor present on damaged cells in the vessel wall.
c i
Activation of the oagualat on cascade leads to formation of a fibrin network and a more stabile plug [19].
Upon activation platelets change shape, going from discoid to a spread morphology, and increase their size considerably (Figure 1B). Their ability to do so is dependent upon rapid reorganization of the actin‐cytoskeleton [20, 21].
Platelet granules
Apart from their important role in haemostasis, it has become evident that platelets also are active players involved in inflammation and immunity. Platelets contain granules of at least three different types, α‐granules, dense granules and lysosomes. The granules store a variety of substances that are secreted when platelets are activated. Released granule components regulate further platelet activation and recruitment of inflammatory cells. The α‐ granules are the most abundant ranging from 40‐100 per platelet [15]. They contain proteins that upon secretion are expressed on the platelet surface e.g. adhesion molecules such as P‐selectin (CD62P) as well as soluble factors that are released into the extracellular space, for example the chemokines CCL5 (RANTES) and CXCL8 (IL‐8). Moreover, they also hold substances involved in growth, angiogenesis and coagulation [22]. It has recently been reported that α‐granules contain 284 different proteins [23] and that the α‐granules are not
as homogenous as was previously considered. The release of granule contents may be differently regulated depending on the stimulus [24]. Platelets contain almost a 10‐fold fewer dense granules than α‐granules, and these contain e.g. adenine nucleotides (ATP, GTP, ADP and GDP), serotonin, divalent cations (Ca2+, Mg2+) and lysosomal membrane proteins (LAMPs). Lysosomes in platelets contain similar substances as lysosomes in other types of cells e.g. LAMPs, hydrolases and cathepsins [22]. Furthermore, despite not having a nucleus, platelets are indeed capable of synthesizing proteins [25]. The ability of platelets to not only release immunomodulatory mediators such as IL‐1β from pre‐loaded granules but also to synthesize this cytokine further support the importance of platelets in inflammation [26].
Platelet receptors
Platelet adhesion to structures revealed in the subendothelial matix upon vessel injury is crucial in order to prevent blood loss [17]. However, uncontrolled platelet activation may cause fatal complications, such as thrombosis. Thrombus formation in coronary arteries due to rupture of an atherosclerotic plaque may lead to myocardial infarction, which is a major cause of death in the western world [27]. The activity of platelets is mediated and regulated via receptors present on the platelet surface. Platelets express many types of receptors, those that are essential for haemostasis as well as receptors implicated in other activities, e.g. inflammation and antimicrobial defence, reviewed in [17, 28, 29]. The following section highlights some selected platelet receptors, including a few that are highly relevant for the present thesis (ADP‐, ATP‐ and collagen‐receptors). Platelet toll‐like
receptors, C1q receptors and Fcγ‐receptors are further discussed in specific sections below.
The vonWillebrand receptor complex (GPIb‐IX‐V) is involved in the initial adhesion of platelets to the injured vessel and belongs to the leucine‐rich repeat family of receptors. GPIb‐IX‐V binds to vonWillebrand factor (vWF) adsorbed from plasma to exposed collagen or endogenous vWF from the endothelium at high shear stress [17, 28, 30]. Firm adhesion of platelets is then initiated due to interaction between platelet integrin α2β1 (GPIa/IIa, originally described as very‐late‐activation antigen VLA‐2) and collagen in the vessel wall. There are around 2000‐4000 α2β1 per platelet [29]. However, there are still some controversies in the current model of α2β1 dependent platelet activation. Initially, it was considered that α2β1 was constitutively expressed on the platelet surface in a high‐affinity state and upon collagen
ligation interaction with some other receptor resulted in platelet activation [17]. However, Jung and Moroi have shown that the ability of α2β1 to bind soluble collagen increases if platelets are first pre‐stimulated with another agonist [31, 32]. As for many integrins, α2β1 is thus also thought to require a conformational change in order to transform to a high‐affinity state and efficiently bind collagen. However, the complete signaling events (“inside‐out signaling”) leading to this conformational change are insufficiently understood [17]. It has also been suggested that there exist an intermediate form of the α2β1, where collagen may bind without the receptor being in a high‐affinity state [33]. The other collagen receptor on platelets is the immunoglobulin superfamily receptor GPVI. GPVI is non‐covalently associated to the FcR‐γ chain of the FcγRIIa (CD32) receptor and thus coupled to the associated ITAM‐signaling [34, 35]. GPVI‐mediated activation leads to an abundant secretion of granule contents and induces integrin activation [17]. Other receptors involved in platelet adhesion are the
ptor)
integrins αvβ3 (vitronectin receptor), α5β1 (fibronectin rece and α6β1 (laminin receptor)[29].
Platelet aggregation is dependent upon fibrinogen binding to the αIIbβ3‐ integrin (GPIIb/IIIa). This fibrinogen receptor is abundantly expressed with approximately 50000‐80000 receptors per platelet. The main ligand is fibrinogen, however, the receptor also recognizes several other ligands containing an RGD sequence (arginine‐glycine‐aspartic acid), e.g. fibronectin, vWf and thrombospondin. Before ligand‐binding the αIIbβ3‐integrin also requires “inside‐out” signaling leading to a conformational switch exposing the RGD‐binding sequence [17, 27].
Platelets express several receptors belonging to the G‐protein coupled family of receptors (7‐transmembrane receptors) e.g. thrombin receptors (protease‐ activated receptors PAR‐1, PAR‐4), thromboxane A2 receptor (TX) and ADP receptors (P2Y1 and P2Y12). Thrombin is formed during the coagulation cascade and binds to PAR‐1 and PAR‐4 leading to a massive platelet activation [36]. PAR activation is unique in that thrombin cleaves the receptor, thus exposing a new N‐terminal that serves as the receptor ligand [37]. Interestingly, other proteases are also able to cleave PARs, inducing activation. For example, proteases from the periodontal pathogen
e 3
Porphyromonas gingivalis are shown to activate plat lets in this way [ 8]. Both PARs couple to Gαq and Gα12/13 [29].
ATP is not only released from dense granules upon platelet activation but also from erythrocytes [39]. Platelets express two types of ADP receptors
P2Y1 and P2Y12 [40, 41]. P2Y1 induces activation via Gαq and P2Y12 is linked to Gαi [27]. By analyzing mRNA levels Wang et al. confirmed the two ADP receptors to be present on platelets, and also found that P2Y12 mRNA levels were higher than levels for P2Y1 [42]. However, P2Y1 is shown to display higher affinity for ADP compared to P2Y12 [43]. The receptor for adenosine triphosphate (ATP) is denoted P2X1 and is also a ligand‐gated calcium channel [44]. Interestingly, when examining mRNA levels at different time points, Wang et al. found markedly reduced levels of P2X1 mRNA over time, whereas the mRNA levels for the ADP receptors remained unchanged or only slightly reduced, indicating that the ATP‐receptor has a much shorter halflife [42]. Both ADP and ATP receptors are important amplifiers of responses provoked by other receptor agonists [45, 46]. They are also easily esensitized, however the mechanism of desensitation is incompletely nderstood [47].
d u
Plateletbacteria interaction
Direct interaction between platelets and bacteria leading to platelet activation has been reported in several studies in vitro and in vivo, recently reviewed by Fitzgerald et al. [48]. Bacteria‐induced platelet activation may cause serious conditions such as endocarditis [49] and immune thrombocytopenic purpura [50]. In the case of Staphylococcus aureus induced
interest g b et a e t
endocarditis, in reports y Nguyen al. [51] nd P erschke e al. [52] show involvement of gC1qR (C1q‐receptor) on the platelet surface. Among the bacterial species known to interact with platelets are Staphylococccus aureus [53], Staphylococccus epidermis [54], Enterococcus spp. [55], Helicobacter pylori [56] and Porphyromonas gingivalis [38]. Moreover, our group have previously shown that Chlamydia pneumoniae, a common respiratory pathogen, binds to platelets and triggers aggregation, P‐selectin expression and lipoxygenase‐dependent production of reactive oxygen species (ROS) [57, 58]. In addition, several species of bacteria are found in atherosclerotic plaques and it is plausible that they are released into the bloodstream during plaque rupture or during angioplasty, thereby activating circulating platelets [59‐61]. However, platelets also possess anti‐ bacterial properties as they hold platelet microbicidal proteins (PMPs) including chemokines, platelet factor 4 and fibrinopeptide B, which are released upon platelet activation [48].
Plateletleukocyte interaction
Interaction between platelets and leukocytes and subsequent formation of aggregates is considered to be clinically relevant [62‐64]. The initial interaction involves P‐selectin, that is abundantly expressed on the surface of activated platelets, and its ligand on leukocytes, P‐selectin glycoprotein ligand 1 (PGSL‐1). Blockade of P‐selectin‐PSGL‐1 interaction inhibits IgG mediated platelet‐neutrophil binding and associated ROS‐production [65]. However, several other adhesion mechanisms are also described including firm adhesion accomplished via fibrinogen binding to GPIIb/IIIa on platelets and Mac‐1 (CD11b/CD18) on leukocytes [66] as well as interaction between platelet intracellular adhesion molecule‐2 (ICAM‐2) and CD11a/CD18 (LFA‐ 1) on neutrophils [67]. Our group and others have reported several cellular consequences upon platelet‐leukocyte interaction e.g. tyrosine phosphorylation and modified production of ROS, chemotaxis and phagocytosis [65, 68‐72].
Neutrophils
Neutrophils, discovered by Schultze in the 1860s [73], belong to the polymorphonuclear granulocytes together with basophils and eosinophils. Mature neutrophils are around 10 µm in diameter and are produced daily in a number of 1.6 x 109/ kg bodyweight from stem cells in the red bone marrow. In healthy individuals, neutrophils have a lifespan in circulation of 10 hours before they migrate into surrounding tissues where they survive for 1 to 2 days [74‐76]. The main function of neutrophils is to participate in our defence against invading microbes. Very simplified, neutrophils in the blood stream are captured and activated by molecules and receptors up‐regulated on the endothelium upon tissue injury and infection. Neutrophils then migrate towards the site of inflammation in response to increased concentrations of released pro‐inflammatory molecules (chemotaxis), whereafter they phagocytose and kill intruding microbes. An important part of the microbicidal mechanism in neutrophils is the oxidative burst, leading to formation of ROS. The production of ROS is accomplished via a multicomponent nicotinamide‐adenine‐dinucleotide‐phosphate (NADPH) oxidase enzyme system. Since the NADPH‐oxidase may be located both in the plasma membrane and in membranes of granules/phagosomes ROS‐ production may be extracellular as well as intracellular (reviewed in [75, 77]).
Recently described, neutrophils may also form microbe‐capturing
neutrophil extracellular traps (NETs), i.e. extracellular fibers of chromatin and granule proteins [78].
The complementsystem
The complement‐system, outlined in Figure 2, is an important defence and clearance system and a major part of our innate immunity. It is comprised of approximately 30 soluble or membrane‐bound proteins and activation of the complement‐cascade is today known to occur via at least three different pathways, the classical pathway, mannose‐binding lectin pathway and alternative pathway [79, 80].
Most proteins in the cascade are named with a “C” followed by a number (the number in the order in which the respective proteins were discovered) and a letter indicating the fragment. Classical pathway activation is initiated upon binding of C1q to antibody (IgG or IgM)‐antigen complexes. Or, alternatively by gram‐negative bacterial walls, viral envelopes, cytoskeletal filaments, myelin or CRP [80]. Together with serine proteases C1r and C1s, C1q forms the C1‐complex that cleaves C4 and C2, leading to generation of the classical C3‐convertase. The mannose‐binding lectin pathway is activated as mannan‐ binding protein (MBP) recognizes and binds to carbohydrate structures found on a variety of microorganisms. MBP‐associated serine proteases (MASPs) then form a complex with MBP and cleave C4. The alternative pathway is an important amplification loop that is activated by C3b deposited on surfaces on e.g. pathogens, cells or biomaterials/adsorbed proteins upon activation of classical and MBL‐pathways. Furthermore, there is a constant ongoing hydrolysis (“tickover”) of native C3 in plasma leading to formation of C3b that will react and bind to amino‐ or hydroxyl‐groups on surfaces, if present close enough. Together with factor B and the help of factor D, surface bound C3b forms the alternative‐C3 convertase [79, 80]. Even though the three pathways are initiated differently they all lead to cleavage of C3 and C5 followed by formation of a membrane‐attack‐complex (MAC) responsible for making pores in the membrane of an intruding pathogen, often resulting in lysis. However, classical pathway activation by phosphorylcholine‐bound CRP is an exception, where the cascade is halted at the C3 level (further discussed below) [81]. Moreover, all pathways are tightly regulated by several proteins (e.g properdin, C1‐inhibitor, factor I, factor H and DAF (decay accelerating factor)), especially the more “unspecific” alternative pathway where C3b bound to host cells is quickly inhibited by regulatory proteins [79, 82].
Figure 2. Schematic illustration of the three different activation pathways
of complement. The classical pathway is initiated upon binding of C1q to immunoglobulin G or M, or by phosphorylcholinebound Creactive protein (CRP). Mannosebinding lectin (MBL) recognizes sugar residues (mannose) on intruding pathogens, leading to activation of the cascade. The alternative pathway is initiated by surface binding of C3b. Indifferent of which pathway that serves as initiator, the cascade leads to cleavage of C3, C5, formation of membrane attackcomplex (MAC) and formation of inflammatory mediators. All pathways are tightly regulated by several proteins (in italic) of which properdin is the only known positive regulator. Classical pathway Antibody-antigen complexes C-reactive protein Mannose-binding lectin pathway Sugar residues Alternative pathway
Surface of e.g. a bacteria or a biomaterial C3-convertases C3-convertases MBL MASPs C4 C2 C3 C3b C3a C5-convertases C5-convertases C5 C5b C6 C7 C8 C9 C5a C1q C1r C1s C4 C2 C3b Factor B Factor D Properdin C5b-9 (MAC) C5b-9 (MAC) Lysis Regulators: C1-INH factor I factor H CR1 MCP C4bp DAF Classical pathway Antibody-antigen complexes C-reactive protein Classical pathway Antibody-antigen complexes C-reactive protein Mannose-binding lectin pathway Sugar residues Mannose-binding lectin pathway Sugar residues Alternative pathway
Surface of e.g. a bacteria or a biomaterial
Alternative pathway
Surface of e.g. a bacteria or a biomaterial C3-convertases C3-convertases MBL MASPs C4 C2 C3 C3b C3a C3 C3b C3a C5-convertases C5-convertases C5 C5b C6 C7 C8 C9 C5a C5 C5b C6 C7 C8 C9 C5a C1q C1r C1s C4 C2 C3b Factor B Factor D Properdin C5b-9 (MAC) C5b-9 (MAC) Lysis Regulators: C1-INH factor I factor H CR1 MCP C4bp DAF Regulators: C1-INH factor I factor H CR1 MCP C4bp DAF
Several protein fragments formed during cleavage of proteins throughout the cascade do not participate in the subsequent steps of the cascade. These fragments are instead involved in other parts of the inflammatory process, for example C3a and C5a which are called anaphylatoxins and are potent chemotactic substances, and iC3b that is an important opsonin. Many celltypes also express receptors for complement proteins and cleavage products, e.g. complement receptor 1 (CR1, CD35) which binds C3b. Complement is also considered to bridge the innate and the adaptive immune
systems. For example, C3 degradation products (C3d and C3dg) enhances antigen presentation by dendritic cells and the production of antibodies from B‐cells [80].
Complement protein C1q
Structure and synthesis C1q is the target recognition molecule of the classical pathway, as described above. C1q has a hexameric structure and consists of 18 polypeptide chains (called A, B and C‐chains) with an amino‐terminal collagen‐like region and a carboxyl‐rich terminal globular region [83, 84]. The protein structure has been described as a “bundle of tulips”, schematically shown in Figure 3. The collagenous part of the C1q molecule resembles collagen molecules which are haracterized by repeating sequences with Glycine‐X‐Y, where X and Y often re proline, hydroxyproline or hydroxyllysine residues [84, 85]. c aStructurally, C1q is also closely related to the proteins in a family called collectins (collagen‐like lectins), to which MBP, conglutinin, collectin‐43 and SP‐A, and SP‐D belong. However, in contrast to the collectins, C1q does not have a carbohydrate recognition domain [79]. Whereas most complement proteins are produced in the liver, C1q is continuously synthesized by macrophages and dendritic cells [79, 86, 87] and the production is regulated by e.g. lipopolysaccharide (LPS), interleukin‐6 (IL‐6), interferon‐γ (IFN‐γ) and anti‐inflammatory steroids (dexamethasone and prednisone) [85, 88, 89]. C1q circulates in plasma at concentrations of 80‐180 µg/mL but is also present in tissues [79, 85]. Collagenlike part Globular heads
Figure 3. Schematic structure of C1q
with the globular heads and the collagenlike stalk.
C1q receptors
Many cell types are reported to bind C1q, including endothelial cells [90], monocytes and granulocytes [91], fibroblasts [92], epithelial and smooth muscle cells [93], platelets [94], dendritic cells [95] and lymphocytes [96]. However, the literature regarding receptors responsible for C1q binding is still somewhat inconclusive. For example Steinberger et al. [97] recognized CD93 to be identical to the previously described C1qRp, shown to enhance phagocytosis in monocytes. In contradiction, McGreal et al. [98] have demonstrated that CD93 is indeed equal to C1qRp, however they found that it does not bind C1q. There are also conflicting results regarding the described receptor for the globular heads of the C1q molecule, gC1qR/p33. As reviewed by Ghebrehiwet et al. [99], gC1qR/p33 is expressed on the cell surface leading to cellular responses upon C1q binding. On the other hand, van den Berg et al. [100] did not detect any gC1qR/p33 on the surface of monocytes, neutrophils or Raji cells unless cells were first treated with saponin indicating that gC1qR/p33 is an intracellular protein, and apparently not expressed on the extracellular surface of cells. One receptor that several authors agree on as capable of binding C1q as well as to be present on cell surfaces is the cC1qR (also denoted calreticulin in the literature). cC1qR binds the collagenous part of C1q [94, 101]. Interestingly, Edelson et al. [102] reported that the α2β1‐integrin is a C1q binding receptor on mast cells and ligation with C1q induced mast cell activation and cytokine secretion. Other receptors suggested to bind C1q are CD91 (low‐density lipoprotein receptor‐
related protein 1 or alfa‐2‐macroglobulin receptor) [103] and CD35 (CR1) [104].
Platelets express both gC1qR and cC1qR [94, 105, 106]. In agreement with the findings by van den Berg et al., where gC1qR is found as an intracellular protein, gC1qR‐expression on platelets is shown to be activation dependent [107]. The somewhat controversial CD93 is also present on platelets [108]. Moreover, the α2β1‐integrin is one of the collagen binding receptors on platelets [29]. Receptor‐independent binding of C1q to the platelet surface (via chondroitin sulphate) has also been reported recently [109]. Cellular effects The most apparent function of C1q is to recognize and mediate clearance of potential harmful pathogens by initiation of the classical complement cascade via binding to antigen‐bound IgG or PCh‐bound CRP. However, several direct cellular effects are also attributed to C1q. For example C1q induces cytokine
release from mast cells [102], increases chemotaxis of dendritic cells [95] stimulates IgG production from Staphylococcus aureus activated B‐cells [96] and enhances Fcγ‐receptor mediated phagocytosis in macrophages and monocytes [110]. Direct effects of C1q on platelets include massive P‐selectin expression accompanied with increase in cytosolic inositol‐1,4,5‐ triphosphate (IP3) [111], enhancement of aggregation in response to sub‐ optimal concentrations of IgG‐aggregates [112], and inhibition of
mmunecomplex‐induced aggregation [113, 114]. Moreover, platelet ggregation induced by collagen is inhibited by C1q [115, 116]. i a
Creactive protein
Historical background C‐reactive protein (CRP) was discovered in 1930 by Tillet and Francis as they studied sera from patients suffering from pneumonia. They found a non‐ antibody serum component in sera from acute ill patients that precipitated with a “fraction C” derived from pneumococcus bacteria. When patients recovered, the “C‐reaction” was diminished [117, 118]. Bacterial C‐ polysaccharide was later shown to be responsible for the C‐reaction, hence the name C‐reactive protein [118, 119].Structure, synthesis and ligands
CRP is a part of the evolutionary conserved family of proteins called pentraxins and is together with serum amyloid P (SAP) denoted a short pentraxin, whereas pentraxin 3 is regarded as a long pentraxin. CRP is an acute phase protein, meaning that the plasma concentration increases rapidly (within 48 hours) and potently (up to a 1000 times in the case of CRP) in response to infection and inflammation, and is normalized again as the infection is cleared [4, 120]. The CRP molecule is made up of 5 identical non‐ covalently associated 23 kDa subunits that are symmetrically arranged around a central pore [121]. CRP has affinity for phosphorylcholine (PCh), present on microorgansims, damaged cells and oxidized low‐density lipoproteins to which it binds in a Ca2+ dependent manner [122‐124]. On the opposite side of the molecule, CRP displays binding sites for C1q [125] and Fcγ‐receptors [126]. Moreover, CRP is described to bind nuclear antigens [127]. The pentameric CRP molecule may irreversibly dissociate into monomers called mCRP (sometimes also denoted modified CRP or neo‐CRP). Dissociation occurs when CRP encounters an acidic environment and in
absence of Ca2+ and also if exposed to high urea concentrations [128, 129]. However, other alternative mechanisms for mCRP generation may certainly be involved in vivo e.g. in the inflammatory environment of an atherosclerotic plaque. Most CRP‐production takes place in the liver in response to IL‐6 [130], but extrahepatic production is also described [3, 131, 132]. Furthermore, regarding the regulation of CRP‐production, Enocsson et al. recently showed that CRP synthesis induced by IL‐6 is inhibited by IFN‐α. Possibly, this is an important clue to why the concentration of CRP is not raised during viral infections or in systemus lupus erythrematosus (SLE) patients, where IFN‐α levels are increased [133]. Indeed, the fact that the plasma level of CRP is not elevated upon a viral infection is used clinically to distinguish between bacterial and viral infections.
Complement activation and regulation by CRP
CRP bound to PCh on microorgansims or damaged cells activates the classical complement cascade via binding to C1q. However, the cascade does not yield much complement activity past the C3 level, indicating that activation is mainly restricted to the first part of the cascade and does not give rise to an abundant MAC formation [81, 134]. The restricted complement activation by CRP is suggested to be due to interaction between CRP and factor H, of which the latter is an important regulator of the alternative pathway. Thus, interaction between factor H and CRP inhibits the amplification loop of the alternative pathway leading to less activation past the C3 level [134‐136]. A binding motif for native CRP on complement factor H‐related protein 4 has also been reported [137]. Recently mCRP was also shown to bind factor H in the fluid‐phase, leading to increased inactivation of C3b [138]. Furthermore, interaction between C4‐binding protein, which is another complement regulatory protein, and CRP has also been described [139]. In addition, our group has previously shown that complement activation at PCh model surfaces is down‐regulated by fluid‐phase interaction between CRP and C1q at high CRP‐levels (>150 mg/mL) [140]. The CRP mediated complement activation leads to sufficient opsonization, but yields no complement induced lysis, suggesting that CRP has both host defence and anti‐inflammatory roles [134].
Cellular effects role in inflammation
Elevated levels of CRP have been implicated in conditions with a chronic inflammatory component, such as coronary artery disease. A minor, but still elevated level of CRP is shown to predict cardiovascular and thrombotic events, initially described by Ridker et al. [141] and then confirmed in
tu h d
and
numerous s dies [142, 143]. This as lead to the evelopment of high sensitive‐assays to accurately detect moderate increases in CRP.
Apart from mediating complement activation, CRP also affects various cellular responses in many different cell types. Recent reviews summarizing these effects and role the of CRP in inflammation are available [122, 134, 144‐ 147]. Both pro‐ and anti‐inflammatory effects are described, for example, CRP promotes proliferation of smooth muscle and endothelial cells [148], formation of monocyte‐platelet aggregates [149] as well as inhibits neutrophil ROS‐production and chemotactic response [150, 151]. To further complicate the scenario, native and mCRP elicit different and sometimes opposite responses [152‐154]. Only a few studies have investigated the effect of CRP on platelets. Monomeric CRP is shown to induce an aggregatory response whereas the native CRP molecule does not [155]. On the contrary, enzymatically digested CRP inhibits platelet aggregation [156].
Pentameric CRP binds to immunoglobulin G receptors FcγRIIa (CD32) [157] and FcγRI (CD64) [158] and monomeric but not native CRP is capable of binding to FcγRIII (CD16) [153]. CRP ligation of FcγRs is presumed to have the same effects as the binding of IgG, thus leading to activation upon ligation to the stimulatory FcγRI and FcγRIIa‐receptors and inhibition when binding to FcγRIIIa [122]. CRP interaction with FcγRs is thus considered to contribute to the opsonizing effects of CRP. Indeed, binding of CRP to FcγRIIa results in increased phagocytosis [159]. Despite numerous studies during the past decades, the question is still if CRP is an innocent bystander, a protective olecule, a risk marker and/or an active contributor to the inflammatory rocess. m p
Tolllike receptors
Structure, ligands and function
Initially, toll‐protein was discovered in the Drosophila fly (fruit fly) where it participates in the protection against fungal infection [160]. Later, a whole family of receptors that resembled the toll‐protein were found in mammals, hence the name toll‐like receptors (TLRs) [6, 161]. In 1998, the findings by Poltorak et al. [162] lead to the conclusion that TLRs serve as recognition molecules and inducers of intracellular signaling. These authors showed that signal transduction was inhibited in mice with a mutation in the TLR‐4 gene, making them resistant to LPS but still sensitive to infection by gram‐negative bacteria. TLRs are expressed in cells participating in the immune response as well as in cells considered as non‐immune, e.g. B‐cells, natural killer (NK) cells, macrophages, dendritic cells, platelets, fibroblasts, endothelial cells and epithelial cells [6]. So far 12 TLRs have been discovered [163]. They are found both on the cell surface (TLR‐1, 2, 4, 5, 6, 11) and as intracellular receptors (TLR‐3, 7, 8, 9) and are responsible for recognizing a diverse range of PAMPs, (Figure 4) thus triggering an inflammatory response. Recognized PAMPs include bacterial cell wall components, LPS, single and double stranded‐RNA and DNA, viral envelope proteins and flagellin [163]. During the past years, TLRs have attracted a lot of interest and several extensive reviews are available [6, 163‐166].
TLR signaling
Upon binding of PAMPs to TLRs, various TIR‐domain containing adaptor‐ proteins are recruited i.e. MyD88 (myeloid differentiation factor 88), TIRAP (TIR‐containing adaptor protein), TRIF (TIR‐containing adaptor including IFN‐β) and TRAM (TRIF‐related adaptor protein). Different TLRs recruit one or a combination of adaptors, and the signaling can be divided into MyD88‐ dependent and TRIF‐dependent pathways [6, 163]. In the MyD88‐dependent pathway IRAKs (interleukin‐1 receptor‐associated kinases) 1, 2 and 4 are recruited leading to a signal‐cascade including IRAK‐phosphorylation and MAP kinases (mitogen‐activated protein kinase). Finally, transcription factors NFκB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) and IRFs (interferon regulatory factors) are activated leading to production of inflammatory cytokines, chemokines and interferons [6, 163, 164]. Relevant to the present thesis, C1q (possibly via both gC1qR and cC1qR) was recently shown to down‐regulate TLR‐4 induced production of IL‐12, but not IL‐6 or
TNF [167, 168]. Furthermore, in a study by Fraser et al. [169] C1q and MBL inhibited IL‐1α and IL‐1β, and enhanced IL‐10, IL‐1 receptor antagonist, monocyte chemoattractant protein‐1, and IL‐6 secretion. Additionally, in a recent study by Lood et al. [170], C1q inhibited IFN‐α secretion from immune‐complex, herpes simplex virus and CpG‐DNA stimulated PBMC peripheral blood mononuclear cells) and immune‐complex, CpG‐DNA timulated plasmacytoid dendritic cells. ( s TLR-2 TLR-4 TLR-2/1 TLR-2/6 TLR-5 TLR-11 Hemaglutinin Peptidoglycan LAM ospholipomannan poarabinomannan Porins Glycosylphosphophatidyl-Inositol Mucin LPS Mannan Glycoinositolphospholipids
Virial envelope proteins Triacyl lipopeptides
Zymosan Lipoteichoic acid Diacyl Lipopeptides
Figure 4. Tolllike receptors (TLRs) are expressed on the membrane and
intracellularly on a wide range of cells where they recognize conserved microbial molecules, collectively named pathogenassociated molecular patterns (PAMPs). Recognition of PAMPs by TLRs is an important part of the innate immune system. In the present thesis we have investigated the intracellular signaling upon TLR2/1 stimulation of platelets, using a triacylated lipopeptide as ligand.
TLR2
Of all TLRs, TLR‐2 is able to recognize the highest number of PAMPs, e.g. bacterial lipoproteins [171, 172], peptidoglycans [173] whole Chlamydia pneumoniae bacteria [174], and LPS from certain bacteria such as Porphyromonas gingivalis [175]. TLR‐2 forms heterodimers with TLR‐1 or TLR‐6 and the dimerization is crucial for ligand‐binding and further
Ph Li CD36 Flagellin CD14 MD2 LBP Uropathogenic bacteria Profillin like molecule
TLR-3 TLR-9 TRL-7 TLR-8 Endosome dsRNA ssRNA CpG-DNA Hemozonin ds-DNA viruses ssRNA virus ssRNA virus
TLR-2 TLR-4 TLR-2/1 TLR-2/6 TLR-5 TLR-11 Hemaglutinin Peptidoglycan LAM ospholipomannan poarabinomannan Porins Glycosylphosphophatidyl-Inositol Mucin LPS Mannan Glycoinositolphospholipids
Virial envelope proteins Triacyl lipopeptides
Zymosan Lipoteichoic acid Diacyl Lipopeptides Ph Li CD36 Flagellin CD14 MD2 LBP Uropathogenic bacteria Profillin like molecule
TLR-3 TLR-9 TRL-7 TLR-8 Endosome dsRNA ssRNA CpG-DNA Hemozonin ds-DNA viruses ssRNA virus ssRNA virus
TLR-3 TLR-9 TRL-7 TLR-8 Endosome dsRNA ssRNA CpG-DNA Hemozonin ds-DNA viruses ssRNA virus ssRNA virus
intracellular signaling [163, 176, 177]. TLR‐2/TLR‐1 recognizes triacylated lipopeptides (e.g. Pam3Cys‐Ser‐(Lys)4, Pam3CSK4), whereas TLR‐2/TLR‐6 binds diacylated lipopeptides (e.g. Mycoplasma‐derived macrophage‐ activating lipopeptide 2, MALP‐2) [178]. Furthermore, CD36 is found to be a co‐receptor for TLR‐2/TLR‐6 [179, 180]. CD36 is a scavenger receptor present on platelets, monocytes, adipocytes, hepatocytes and epithelial cells that for example oxidized low density lipoproteins and thrombospondin‐1 [181]. TLR expression and function in platelets Using flow cytometry, Cognasse et al [182] identified TLR‐2, 4 and 9 on platelets and megakaryocytes and by analysis of mRNA and protein levels, Shiraki et al. [183] also demonstrated TLR‐1 and 6 in platelets. The functional consequences of TLR activation in platelets are at the moment under intense investigation. It has previously been shown that stimulation of TLR‐4 increases platelet adhesion to fibrinogen under flow and that it causes thrombocytopenia in TLR‐4 deficient mice, whereas wild type mice remain unaffected [184]. Activation of TLR‐2/TLR‐6 with the synthetic lipopeptide Pam3CSK4 induces platelet aggregation, secretion and mobilization of Ca2+ in washed platelets [185, 186], however, has no detectable stimulatory effects on platelets in plasma [187].
Fcγ receptors
Fcγ receptors recognize the constant part of the immunoglobulin molecule, mediating cellular effects such as immune complex elimination, cytokine production and phagocytosis. At present four types of FcγRs are described, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV. FcγRII and FcγRIII are also further divided into subclasses denoted a and b. All receptors, except for FcγRIIb mediate a cellular activating signal, reviewed in [188‐190]. Platelets express FcγRIIa and ligation induces platelet activation mediated via phospholipase C and increased levels of intracellular free Ca2+ [191].
AIMS
The role of platelets in inflammation and immunity is an expanding field of research. The overall aim of this thesis was to investigate the effects of the attern‐recognition molecules/receptors C1q, CRP and TLR‐2/1 on platelet p activation. he sp T ecific aims of the enclosed studies were to: • elucidate the effects of C1q and C‐reactive protein on platelet adhesion and activation utilizing well‐characterized model surfaces with adsorbed human immunoglobulin G and human serum albumin (Paper I).
• clarify the role of C1q in platelet activation including the involvement
of receptors and intracellular signaling events (Paper II).
• investigate the regulatory role of C1q on collagen‐induced platelet activation on isolated platelets and in whole blood, respectively (Papers II‐III). • study the effects of C1q on production of reactive‐oxygen species and formation of platelet‐leukocyte aggregates in whole blood. (Paper III). • reveal the intracellular signaling events provoked by toll‐like receptor‐ 2 stimulation of platelets (Paper VI).
COMMENTS ON METHODS
F m
or further experimental details and source of chemicals and buffers, see aterials and methods sections of Paper I‐IV, respectively.
Isolation of blood cells
The blood used in the present thesis was heparinized whole blood collected from apparently healthy, non‐medicating consenting donors at the blood bank, University Hospital, Linköping, Sweden.
Platelets (P per I, II and IV)
There are several methods available for isolating platelets involving centrifugation [192] and gel filtration [193]. In the present studies platelets were isolated by centrifugation and several washing steps according to the protocol by Bengtsson and Grenegård [194]. To avoid activation during the isolation procedure all buffers were prepared without Ca
a
2+ and only plastic utensils were used. Briefly, 5 parts of whole blood was mixed with 1 part of an acid‐dextrose solution (ACD) pH 4.6 and centrifuged at 220 x g for 20 minutes. Platelet rich plasma (PRP) was collected and centrifuged at 480 x g for 20 minutes. In order to investigate changes in cytosolic Ca2+ (Paper II and IV) platelets in PRP were loaded with 4 µM Fura‐2‐acetoxymethylester (FURA‐2‐AM) during 45 minutes of incubation before centrifugation. Furthermore, in Paper VI, some platelets were isolated in the presence of platelet inhibitors apyrase (0.5 U/ml) and aspirin (100 µM). The platelet pellet was washed three times by careful replacement of the buffer in direct contact with the pellet using Krebs Ringers Phosphate buffer (KRG, pH 7.4). Platelets were then carefully re‐suspended in KRG and the cell count determined using a Bürker chamber. Morphological examination by light microscopy showed that the isolated platelets were solitary and appeared non‐activated. Platelets were diluted in KRG and kept at room temperature for up to 2 hours until experiments. Immediately before each experiment, the extracellular concentration of Ca2+ was restored to 1 mM.
Neutrophils (Paper II)
Neutrophils were isolated according to Böyum and others [195, 196]. In short, whole blood was layered onto Lymphoprep and Polymorphprep and centrifuged at 480 x g for 40 minutes at room temperature. The fraction
