Chlamydia pneumoniae Binds to Platelets and Triggers P-Selectin Expression and Aggregation
A Causal Role in Cardiovascular Disease?
Objective— Evidence linking Chlamydia pneumoniae to atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction between C pneumoniae and platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis.
Methods and Results— The effects of C pneumoniae on platelet aggregation and secretion were assessed with lumiaggregometry, and the ability of C pneumoniae to bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found that C pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reduced C pneumoniae-induced platelet aggregation. Exposure of C pneumoniae to polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions.
Conclusion— Our findings demonstrate a new concept of how C pneumoniae activates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.
Atherosclerosis and related diseases are a major cause of death in the industrialized world. Differences in the occurrence of typical cardiovascular risk factors, such as smoking, hypertension, and abnormalities of lipid and sugar metabolism, do not explain thoroughly temporal and geographical variations in the prevalence or severity of coronary artery disease.1 Over the last decade, the potential role of infectious agents in the pathogenesis and progression of atherosclerosis has attracted much attention.2,3⇓ Among different microorganisms suspected, Chlamydia pneumoniae has arisen as the most plausible pathogen having a causal role.4
Plaque rupture and thrombosis are the main mechanisms of acute arterial occlusion, leading to myocardial infarction. After taking into account conventional risk factors and genetic predisposition, at least one third of thrombotic incidents remain unclear. Several studies show an association between C pneumoniae and thrombosis in patients with carotid artery disease and venous thromboembolic disease.5,6⇓
C pneumoniae is an obligate intracellular bacterium responsible for a number of upper and lower respiratory tract diseases in humans, and ≈50% of adults worldwide have antibody evidence of previous infection by this bacterium.7 An association between C pneumoniae infections and atherosclerosis has been demonstrated in a number of epidemiological, serological, immunohistochemical, and molecular biological investigations.2,8,9⇓⇓ Furthermore, in vitro studies have revealed that C pneumoniae is able to infect and replicate in the major cell types found within the atherosclerotic lesion, such as macrophages, endothelial cells, and smooth muscle cells.10 However, the interaction between C pneumoniae and platelets, other important actors in atherogenesis, has to our knowledge not been investigated.
Platelets are specialized in the processes of hemostasis and thrombus formation after an endothelial injury but are also considered to be involved in inflammatory reactions and the development of atherosclerosis.11,12⇓ We have previously reported inflammatory properties of platelets by demonstrating their regulatory effects on leukocyte function.13–15⇓⇓ The α-granule constituent P-selectin is considered as a reliable marker of platelet activation and as a predictor of acute coronary heart disease.16 Several studies indicate that P-selectin mediates adhesion and recruitment of leukocytes to activated platelets, exerts procoagulant activity, and facilitates atherosclerotic development.14,17⇓
In this study, we evaluated the interaction between C pneumoniae and human platelets and found that C pneumoniae binds to platelets and effectively stimulates aggregation, secretion, and surface expression of P-selectin. These observations introduce new possible mechanisms involved in the development of cardiovascular diseases.
Cells and Bacteria
C pneumoniae (strain T45) was cultured in HEp2 cells essentially as described by Redecke et al.18 The bacteria and cells were tested for mycoplasma contamination by using mycoplasma-specific polymerase chain reaction essentially according to van Kuppeveld et al.19 Platelets and neutrophils were isolated from human blood as previously described.13 For a detailed description of the preparation of cells and bacteria, please see http://atvb.ahajournals.org.
Platelet Aggregation and ATP Secretion
Aggregation and ATP secretion were analyzed under stirring conditions by using a calibrated two-sample Lumi-Aggregometer model 560 (ChronoLog Corp). Aggregation was measured as the change in light transmission, where the unstimulated platelet suspension was set to 0% and the buffer (KRG) to 100%. ATP secretion was measured in parallel as change in bioluminescence when ATP interacts with a luciferin–luciferase mixture (1.6 μg/mL luciferin and 176 U/mL luciferase; ChronoLog Corp). Calibration was performed for each test by adding a known amount of ATP.
C pneumoniae–Platelet Interaction
Platelets (2×108/mL) were preincubated for 5 minutes at 37°C under stirring conditions in a 24-well plate (Nunc) before being mixed with various concentrations of C pneumoniae. Samples were taken immediately before, and 1 minute, 5 minutes, 10 minutes, and 20 minutes after adding C pneumoniae to the platelet suspension. In some experiments, viable C pneumoniae was replaced with heat-inactivated (70°C, 30 minutes) C pneumoniae, HEp2 debris, or collagen (2 μg/mL). The role of platelet adhesion proteins was tested by preincubating the platelets with blocking antibodies against CD42b (3.6 μg/mL) and CD41 (4.3 μg/mL). The involvement of chlamydial lipopolysaccharide (LPS) was evaluated by treating C pneumoniae with polymyxin B (100 μg/mL) for 30 minutes at room temperature. Unspecific effects of polymyxin B on platelet activity were tested during collagen-induced activation.
Immunofluorescence staining of platelets and C pneumoniae was performed by incubation with saturating concentrations of monoclonal fluorescein isothiocyanate (FITC)-conjugated anti P-selectin (CD62p; BD Biosciences, Pharmigen) or phycoerythrin-conjugated anti-GpIb (CD42b; Dakopatts), and monoclonal FITC-labeled anti-chlamydia LPS (Boule Diagnostics) at room temperature for 10 minutes in the dark. The samples were then fixed with Optilyse (with 2.5% formaldehyde; Immunotech) under the same conditions and diluted in distilled H2O. Phycoerythrin- or FITC-labeled irrelevant isotype-matched monoclonal antibodies were used as controls for nonspecific staining. Immediately after staining, the samples were analyzed with flow cytometry in a Becton Dickinson FACS Calibur. The platelet population was identified by means of its light-scatter characteristics and by confirming that more than 99% of analyzed particles in each sample were GpIb positive. Events stained positive for both platelet and C pneumoniae antigens (GpIb and LPS, respectively) were considered to represent platelet–chlamydiae complexes and were distinguishable from events stained positive for GpIb alone. The extent of platelet activation was assessed by analyzing anti–P-selectin FITC fluorescence in the platelet gate. The mean fluorescence value of each sample was determined from cells counted during a time period of 20 seconds or from 500 000 counted cells at most.
Binding of C pneumoniae to Neutrophils
Neutrophils were preincubated for 5 minutes at 37°C and then mixed with C pneumoniae at a C pneumoniae:neutrophil ratio of 1:5. After 5 minutes of coincubation, the samples were stained with monoclonal phycoerythrin–conjugated anti-CD11b (Dakopatts) and monoclonal FITC–labeled antichlamydia LPS antibodies. Neutrophil events stained positive for C pneumoniae LPS were considered to represent neutrophil–chlamydiae complexes. Unspecific binding of the FITC-labeled antichlamydia LPS antibody to the neutrophil control was subtracted from the fluorescence value of neutrophils incubated with C pneumoniae.
C pneumoniae Triggers Platelet Aggregation and Secretion
The interaction between C pneumoniae and platelets was assessed by using lumiaggregometry, which enables a simultaneous analysis of platelet aggregation and ATP secretion. We found that the addition of C pneumoniae to a pure platelet suspension (2×108 platelets/mL) induced aggregation (Fig. 1A and 1B) and ATP secretion (Fig. 1C) in a dose- and time-dependent manner. The responses were triggered at an infection forming unit chlamydia:platelet (C/p) ratio of 1:30. The chlamydia-induced platelet aggregation occurred in an all-or-nothing manner and increases in the bacteria concentration affected the lag period (time between addition of bacteria and onset of aggregation) but not the extent of aggregation (Fig. 1A and 1B). Once begun, the aggregation proceeded at about the same rate when using different C/p ratios, as indicated by the slope of the tracings (Fig. 1A). The C pneumoniae-induced platelet aggregation and secretion were comparable with the responses triggered by collagen (Fig. 1C). No aggregation or ATP secretion was observed in stirred, unstimulated platelet suspensions or in samples with platelets incubated with cell debris of uninfected HEp2 cells (data not shown).
C pneumoniae Binds to Platelets
The capacity of C pneumoniae to bind and activate platelets was studied by using flow cytometry. Platelet events positive for FITC-conjugated antichlamydia LPS represent platelets with bound chlamydiae. We found that exposing platelets to C pneumoniae caused a rapid and pronounced increase in platelet-associated FITC fluorescence. Scatterplots on platelets (Fig. 2A) with or without chlamydiae illustrated a change in size distribution, proposing the formation of platelet–chlamydia complexes. Figure 2B illustrates a time-dependent increase in bound chlamydiae as revealed by elevated FITC fluorescence in the platelet gate. Approximately 10% of the platelet population bound to C pneumoniae (C/p 1:10) after 1 minute, 20% after 5 minutes, 40% after 10 minutes, and 45% after 20 minutes of coincubation (Fig. 2B). Control experiments did not reveal any unspecific binding of anti-LPS antibodies to platelets and only a negligible binding of irrelevant isotype control antibodies. The wide distribution in size and fluorescence intensity indicates that platelets and platelet aggregates bound varying numbers of chlamydiae. The chlamydiae themselves did not form aggregates during incubation at 37°C under stirring conditions (data not shown).
Binding of C pneumoniae to Neutrophils
Binding of C pneumoniae to neutrophils was studied with flow cytometry. Neutrophil events stained positive for FITC-conjugated antichlamydia LPS were calculated. Incubation of C pneumoniae with neutrophils at a ratio of 1:5 for 5 minutes resulted in a considerably lower binding of C pneumoniae to the neutrophils compared with the binding of C pneumoniae to platelets (Fig. 2C).
C pneumoniae Increases Platelet Expression of P-Selectin
The effects of C pneumoniae on platelet activity were further evaluated by studying P-selectin expression by flow cytometry. We found that C pneumoniae at a C/p ratio of 1:15 markedly increased the expression of P-selectin, whereas no effects were detected on platelets incubated with uninfected HEp2 cell debris or 2-sp buffer. The P-selectin expression increased dramatically already after 1 minute and reached a maximum after 10 minutes of coincubation (Fig. 3A and 3B). We found a chlamydia-induced increase in platelet P-selectin at C/p ratios as low as 1:60, and the effects of C pneumoniae were comparable with those induced by collagen (2 μg/mL; Fig. 3C).
Effects of Other Bacteria on Platelet Activity
The ability of a number of other bacteria to induce platelet aggregation and secretion was tested. Neither Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, nor Escherichia coli stimulated platelet aggregation, ATP secretion, or P-selectin expression when using similar bacteria:platelet ratios as in the experiments of C pneumoniae. An irreversible, incomplete aggregation was triggered by S. aureus at a considerable higher bacteria:platelet ratio (2:1).
Role of Chlamydial LPS in Platelet Activation
To determine whether platelet activation required viable bacteria, an active release of chlamydial cell components, and/or binding to heat-labile chlamydial surface structures, experiments by using heat-inactivated C pneumoniae were performed. We found that heat treatment of C pneumoniae did not change the platelet binding capacity nor the stimulatory effects on platelet aggregation and P-selectin expression. To study the role of LPS in the interaction between C pneumoniae and platelets, C pneumoniae was preincubated with polymyxin B (100 μg/mL) for 30 minutes at room temperature. Figure 4 shows that polymyxin B–treated C pneumoniae was unable to stimulate platelet P-selectin expression (C/p 1:20). To elucidate whether polymyxin B unspecifically affected platelet activation, we studied the surface expression of P-selectin on platelets exposed to a mixture of polymyxin B (100 μg/mL) and collagen (2 μg/mL). We found that polymyxin B only slightly reduced the collagen-triggered increase of P-selectin (not shown).
Role of Platelet Surface Structures in the Interaction with C pneumoniae
Experiments were also performed to search for the platelet surface components involved in the interaction with C. pneumoniae. Neither monoclonal antibodies directed against GpIb or P-selectin nor the peptide glycocalicin (blocking the von Willenbrand factor binding site on GpIb) antagonized the effects of C pneumoniae on platelet activity (data not shown). However, preincubation of platelets with Arg-Gly-Asp-Ser (RGDS; 1 mg/mL) or the monoclonal Gp IIb/IIIa antibody F(ab)2 fragment abciximab (Reopro; 40 μg/mL), significantly inhibited platelet aggregation triggered by C pneumoniae (Fig. 5)
A growing amount of evidence suggests that C pneumoniae has a role in the development of atherosclerosis.1,2⇓ However, it is uncertain whether a C pneumoniae infection is a triggering event of atherosclerosis or a secondary infectious complication of an already-formed atherosclerotic plaque. Several studies have investigated C pneumoniae interaction with different cell types involved in the atherosclerotic process, for example, monocytes/macrophages, smooth muscle cells, and endothelial cells.10,18⇓ However, it is not known whether C pneumoniae affects platelets. In this study, we evaluated C pneumoniae interaction with platelets by studying binding, aggregation, secretion, and surface expression of P-selectin.
We found that C pneumoniae was highly adhesive to platelets and triggered aggregation and secretion in a time- and concentration-dependent manner. An extensive C pneumoniae-platelet binding was observed already after 1 minute of coincubation and increased significantly during 10 to 25 minutes, whereupon an irreversible complete aggregation was obtained. Flow cytometric analysis shows a continuous increase in the size distribution of the platelet microaggregates during the lag period. The low number of C pneumoniae elementary bodies in relation to the platelet concentration (C/p 1:20) and the kinetics of the chlamydia-induced lumiaggregometry response with a lag period of 20 to 25 minutes suggest a cascade effect, where the chlamydiae initially stimulate few platelets, which activate neighboring cells through paracrine-signaling mechanisms. The ability of C pneumoniae to cross-link platelets and support formation of microaggregates may constitute a mechanism by which C pneumoniae relocalizes from the infected lung epithelium into the circulation. Earlier studies have demonstrated that bacteria can survive inside platelet aggregates, which protect the bacteria from the host defense and spread the bacteria in the circulation.20
Several studies implicate an important role of P-selectin in atherosclerosis and thrombosis, shown by elevated levels of P-selectin in patients with congestive heart failure, stroke, peripheral artery disease, and acute coronary syndromes.21,22⇓ Furthermore, ongoing C pneumoniae infection and the occurrence of myocardial infarction is related to increased plasma levels of soluble P-selectin.23 In this study, we demonstrated that C pneumoniae rapidly increases the surface expression of platelet P-selectin. The degree of P-selectin expression triggered by C pneumoniae was comparable with the effects of collagen, which is a potent platelet activator. P-selectin mediates the interaction of activated platelets with neutrophils and monocytes, which may be important in the pathophysiology of cardiovascular disease.21 Several observations suggest that platelets deposited at sites of thrombosis and vascular injury serve as surrogates for endothelium by recruiting circulating leukocytes.14,24⇓ Tsuji et al25 demonstrated that platelet P-selectin directly triggers oxygen radical production in neutrophils, and we have recently shown that collagen-activated platelets stimulate, via P-selectin, leukocyte reactive oxygen species production in whole blood.26 In addition, raised expression of P-selectin reflects platelet activation involving secretion of a broad range of adhesive proteins, procoagulants, cytokines, and growth factors stored in the α-granules.12
After percutaneous coronary intervention, arterial damage takes place that can lead to restenosis. Adhesion and aggregation of platelets on the damaged arterial wall and expression of P-selectin represent the first steps in these pathophysiological reactions.27 C pneumoniae is suggested to be involved in the progress of restenosis,28 which is supported by this study showing that C pneumoniae stimulates both P-selectin expression and platelet aggregation.
Activated platelets can alter the chemotactic and adhesive properties of endothelial cells by stimulating release of the chemotactic monocyte chemoattractant protein 1 and a surface expression of intercellular adhesion molecule-1. Both monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 have been detected in high concentrations in atherosclerotic lesions. Platelets also stimulate, through a platelet-derived growth factor–dependent mechanism, the migration and proliferation of smooth muscle cells and fibroblasts.29 In this way, platelets may have a central role in the occurrence of atherosclerotic reconstruction processes of the vessel wall.
We found that neither S. epidermidis, S. typhimurium, nor E. coli activated platelets. S. aureus, which has been shown to have a role in infective endocarditis by activating platelets,30 caused in our study an incomplete aggregation at a considerably higher bacteria:platelet ratio (2:1) compared with the low number of C pneumoniae needed for a complete platelet aggregation. The fact that C pneumoniae most effectively activates platelets underlies the tropism of chlamydial infections and strengthens the pathogenic property linking C pneumoniae with atherosclerosis.
Our finding that heat treatment did not change the stimulatory effects of C pneumoniae on platelet activation suggests an involvement of a heat-stable surface structure. Exposure of C pneumoniae to polymyxin B abolished the effects on platelets, which indicates that LPS has a crucial role in platelet activation. The low number of elementary bodies of C pneumoniae required for platelet activation may be caused by an extensive release of chlamydial LPS, which activates the major part of the platelet population in an aggregatory and secretory response. The finding that C pneumoniae but no other tested Gram-negative bacteria (E. coli and S. typhimurium) activates platelets suggests that differences in the chemical structure of LPS are essential. Chlamydial LPS contains a unique lipid A, lacks an O-chain, and exposes a genus-specific highly immunogenic epitope on the polysaccharide core.31 Similar LPS has also been identified in Porphyromonas gingivalis.32 Interestingly, it has been shown that platelets are directly stimulated by lipid A through an activation of protein kinase C and that bacteria with modified LPS, for example, P. gingivalis, are much more potent activators of platelets than classic Gram-negative bacteria.33,34⇓ P. gingivalis is a major pathogen of periodontal diseases and has also been associated with atherosclerosis.35 A role for chlamydial LPS in atherogenesis has previously been reported by its ability to induce foam cell formation.36
C pneumoniae has considerably larger affinity to platelets than to neutrophils, which suggests recognition of specific receptors on the platelet surface. The counter receptors on the platelet surface involved in the interaction with C pneumoniae were studied by using specific blocking antibodies and peptides. We found that inhibition of Gp IIb/IIIa, with RGDS or with the monoclonal fab fragment abciximab, significantly lowered the aggregation induced by C pneumoniae, whereas blocking of GpIb or P-selectin had no effects. Abciximab is used worldwide in patients with acute coronary syndromes and in those undergoing percutaneous coronary intervention.37 The rapid upregulation of platelet P-selectin induced by C pneumoniae reflects an early release of α-granule constituents, including fibrinogen. We suggest that the extensive delay (15 to 20 minutes) between α-granule secretion and platelet aggregation constitutes a period of transformation of GpIIb/IIIa into a competent fibrinogen receptor. This process, supported by generation of intercellular mediators (eg, eicosaniods), engages more and more platelets forming microaggregates and leads finally to a complete aggregation.
C pneumoniae infection is very common among the human population, occurs early and several times in life, and the bacteria persist for long periods in tissues. Thus, there are probably several opportunities for bacteria–platelet interaction, which may stimulate both the early proliferative phases of atherosclerosis and the late thrombotic vascular occlusion. A crucial role for C pneumoniae-induced platelet aggregation in atherogenesis is supported by findings suggesting that recurrent thrombus incorporation into atherosclerotic lesions is fundamental in the pathogenesis and progression of atherosclerotic plaques.38
This study supports the concept that C pneumoniae plays a major causative role in atherosclerosis and suggests that platelets are susceptible target cells. Antibiotics and vaccines against C pneumoniae infections might in the future be complementary to, or even replace, classic antiplatelet and antiatherogenic drugs. We believe that an approach to specifically prevent C pneumoniae binding to platelets and C pneumoniae-induced activation of platelets can be a novel therapeutic tool for cardiovascular diseases.
This study was supported by grants from the King Gustav V 80-year Foundation, the Swedish Medical Research Council (grant no. 12668), the Swedish Society of Medicine, and the Swedish Foundation for Strategic Research. We thank Kristina Orselius and Marie Högdahl for technical assistance and support and Olle Stendahl and Magnus Grenegård for advice and support.
- Received April 29, 2003.
- Accepted June 10, 2003.
- ↵Nieminen MS, Mattila K, Valtonen V. Infection and inflammatory risk factors for myocardial infarction. Eur Heart J. 1993; 14: 12–16.
- ↵Vainas T, Kurvers HAJM, Mess WH, de Graaf R, Rajaa E, Tordoid JHM, Schurink GH, Bruggeman CA, Kitslaar PJEHM. Chlamydia pneumoniae serology is associated with thrombosis-related but not with plaque related microembolization during carotid endarterectomy. Stroke. 2002; 33: 1249–1254.
- ↵Kuo CC, Gown AM, Benditt EP Grayston JT. Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical staining. Arterioscler Thromb. 1993; 13: 1501–1504.
- ↵Gaydos, CA. Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle. Infect Immunol. 1996; 64: 1614–1620.
- ↵Marcus AJ. Platelets: their role in hemostasis, thrombosis, and inflammation. In: Gallin JI, Snyderman R, eds. Inflammation: Basic Principles and Clinical Correlates. Philadelphia, Pa: Lippincott Williams & Wilkins; 1999: 77–95.
- ↵Bengtsson T, Zalavary S, Stendahl O, Grenegård M. Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization. Blood. 1996; 87: 4411–4423.
- ↵Zalavary S, Grenegård M, Stendahl O, Bengtsson T. Platelets enhance Fc γ receptor mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization. J Leukoc Biol. 1996; 60: 58–68.
- ↵Burger P, Wagner D. Platelet P-selectin facilitates atherosclerotic lesion development. Blood. 2003; 101: 2661–2666.
- ↵van Kuppeveld FJ, van der Logt JT, Angulo AF, van Zoest MJ, Quint WG, Niesters HG, Galama JM, Melchers WJ. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992; 58: 2606–2615.
- ↵Clawson CC, White JG. Platelet interaction with bacteria. Am J Pahtol. 1971; 65: 381–395.
- ↵Massberg S, Enders G, Leiderer R, Eisenmenger S, Vestweber D, Krombach F, Messmer K. Platelet endothelial cell interactions during ischemia/reperfusion—the role of P-selectin. Blood. 1998; 92: 507–551.
- ↵Kupatt C, Wichels R, Horstkotte J, Krombach F, Habazettl H, Boekstegers P. Molecular mechanisms of platelet-mediated leukocyte recruitment during myocardial reperfusion. J Leukoc Biol. 2002; 72: 455–461.
- ↵Diacovo TG, Roth SJ, Boccola JM, Bainton DF. Springer T. A. Neutrophil rolling, arrest and transmigration across activated surface adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18. Blood. 1996; 88: 146–157.
- ↵Tsuji T, Nagata K, Kioke J, Todoroki N, Irimura T. Induction of superoxide anion production from monocytes and neutrophils by activated platelets though the P-selectin sialyl Lewis X interaction. J Leukoc Biol. 1994; 56: 583–587.
- ↵Weyrich AS, Prescott SM, Zimmerman GA. Platelets, endothelial cells, inflammatory chemokines, and restenosis: complex signaling in the vascular play book. Circulation. 2002; 106: 1433–1435.
- ↵Hayashida K, Tanaka M, Morita H, Hayashi F, Inada T, Suzuki H, Sakamoto T, Katsuragawa M, Hibino H, Kambara H. Chlamydia pneumoniae seropositivity predicts the risk of restenosis after percutaneous transluminal coronary angioplasty. Heart Vessels. 2002; 6: 137–145.
- ↵Gawaz M. Platelets and the Pathophysiology of Coronary Heart Disease. In: Blood Platelets, Physiology, Pathophysiology, Membrane Receptor, Antiplatelet Principles, and Therapy for Atherothrombotic Diseases. Stuttgart, Germany: Georg Thieme Verlag; 2001: 97–118.
- ↵O’Brien L, Kerrigan SW, Kaw G, Hogan M, Penades J, Litt D, Fitzgerald DJ, Foster TJ, Cox D. Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB, the serine-aspartate repeat protein SdrE and protein A. Mol Microbiol. 2002; 44: 1033–1044.
- ↵Rund S, Idner B, Brade H, Holst O. Structural analyses of the lipopolysaccharide from Chlamydia trachomatis serotype L2. J Biol Chem. 1999; 274: 16819–16824.
- ↵Ogawa T. Chemical structure of lipid A from Porphyromonas (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett. 1993; 32: 197–201.
- ↵Endo Y, Shibazaki M, Nakamura M, Takada H. Contrasting effects of lipopolysaccharides (endotoxins) from oral black-pigmented bacteria and Enterobacteriaceae on platelets, a major source of serotonin, and on histamine-forming enzyme in mice. J Infect Dis. 1997; 175: 1404–1412.
- ↵Grabarek J, Timmons S, Hawiger J. Modulation of human platelet protein kinase C by endotoxic lipid A. J Clin Invest. 1988; 82: 964–971.
- ↵Kalayoglu MV, Byrne GI. A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide. Infect Immunol. 1988; 66: 5067–5072.
- ↵Chronos N, Vahnian A, Betriu A, Emanuelsson H, Goldberg S, Gulba D, van Hout BA. Use of abciximab in interventional cardiology. Eur Heart J. 1998; 19: D31–D39.
- ↵Cimminiello C, Toschi V. Atherothrombosis: the role of platelets. Eur Heart J Suppl. 1999; 1: A8–A13.