|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Clinical Pharmacology, Royal
College of Surgeons in Ireland, Dublin, Ireland, and Department of Oral
Pathology, School of Clinical Dentistry, University of Sheffield,
Sheffield, United Kingdom.
Numerous studies have implicated bacteria in cardiovascular
disease, but there is a paucity of information on the mechanism involved. In this study we show how the common oral bacterium Streptococcus sanguis can directly interact with platelets,
resulting in activation and aggregate formation. Platelet aggregation
was dependent on glycoprotein IIb/IIIa (GPIIb/IIIa) and thromboxane. Platelets could also directly bind to S sanguis, but this
interaction was not inhibited by GPIIb/IIIa antagonists. Antibodies to
GPIb could inhibit both platelet aggregation and platelet adhesion to
bacteria. This suggested a direct interaction between GPIb and S
sanguis; however, this interaction did not require von Willebrand factor, the normal ligand for GPIb. By use of a range of monoclonal antibodies to GPIb and the enzyme mocharagin, which cleaves GPIb at
amino acid 282, the interaction was localized to a region within the
N-terminal 1-225 portion of GPIb Recent reports suggest a role for infectious
agents in cardiovascular disease. Much of this work is in the form of
clinical evidence of infection1-6 or the effect of
antibiotics on the incidence of cardiovascular disease.7,8
Although studies have found evidence of bacteria in atherosclerotic
plaques,1,2,4,5,9-13 their role in the etiology of
cardiovascular disease is uncertain. In contrast, the role of bacteria
in infective endocarditis is well established and the molecular
mechanisms involved may also occur in other forms of
cardiovascular disease.
Infective endocarditis involves inflammation of the heart valves
due to infection and if untreated can lead to valve failure and death.
In most cases there is one or more predisposing factors, which results
in damage to the endothelium on or adjacent to the valves. This area of
damage becomes covered with a platelet-fibrin vegetation and these can
become colonized by bacteria that gain access to the blood. The 2 species most commonly involved are Streptococcus
sanguis14 and Staphylococcus
aureus.15 Historically oral streptococci have been
referred to as Streptococcus viridans, but this name was
never accepted as a recognized taxon because of the biochemical and
serologic heterogeneity among isolates. Subsequent detailed biochemical
and genetic studies allowed the definition of at least 17 taxa within
what was originally called S viridans. However, the term
viridans has survived but is now used as viridans group of streptococci
to recognize the existence of various taxa and S sanguis is
one taxon within this group of organisms. Studies using animal models
of endocarditis caused by S sanguis have shown that the
severity of the disease is associated with an ability of the infecting
organism to adhere to and cause aggregation of
platelets.16,17
Platelets are anucleated cellular fragments of megakaryocytes,
which, when activated, aggregate to form a thrombus. Activation can be
mediated by many different agonists that lead to fibrinogen binding to
its receptor, glycoprotein (GP) IIb/IIIa, and cross-linking of
platelets into aggregates.18,19 When the endothelium is damaged, platelet GPIb can bind to von Willebrand factor (VWF) in the
subendothelial matrix resulting in platelet activation20 and deposition of platelets on the area of damage. Thrombus formation on ruptured atherosclerotic plaques is also important in myocardial infarction21 and has led to the use of antiplatelet agents
for treatment and prevention.22
Streptococcus sanguis has been shown to induce
platelet aggregation in a thromboxane-dependent manner requiring
adenosine diphosphate (ADP) secretion.23 It has also been
shown to be complement and Fc-receptor
(Fc In this study, we have further investigated the roles of GPIb and
Fc Materials
Bacterial strains and growth
Platelet preparation For routine assays, blood was drawn (using a 19-gauge butterfly needle) from the antecubital vein of healthy human volunteers who had not taken any nonsteroidal anti-inflammatory drugs during the previous 10 days. Nine volumes of blood were added to 1 volume of 3.8% sodium citrate or acid-citrate-dextrose (ACD). Platelet-rich plasma (PRP) was prepared by centrifugation of anticoagulated whole blood at room temperature at 150g for 10 minutes. The blood remaining after removing the PRP was centrifuged at 630g for 10 minutes at room temperature to yield platelet-poor plasma (PPP).In some experiments platelets were collected from patients with specific bleeding disorders. Whole blood collected from a well-characterized patient with Bernard-Soulier syndrome30 was allowed sediment at room temperature for 3 hours to obtain PRP. Platelets were tested for normal responses to arachidonic acid (0.5 mg/mL) or ADP (2 mM). Changes in light transmission were recorded against autologous PPP (100% light transmission). Blood was also collected from 2 patients with Glanzmann thrombasthenia, who lack normal levels of GPIIb/IIIa on their platelets. PRP was prepared by the normal procedure described above and gel-filtered platelets were prepared as described below. Glanzmann thrombasthenia was diagnosed by immunofluorescence using kits from BioCytex. Whole blood was diluted 1:4 with kit buffer and 20 µL of this added to 20 µL antibody. Three antibodies were used: anti-GPIb, anti-GPIIIa, and an isotype control. After 20 minutes' incubation, 20 µL FITC-labeled secondary antibody was added. At the same time 20 µL of the secondary was also added to 40 µL of calibration beads (4 populations of 2-µm beads containing known quantities of antibody). Samples were then analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Oxford, United Kingdom). The relationship between fluorescence and antibody molecules bound was determined allowing the number of platelet glycoprotein molecules to be determined. Gel-filtered platelets Platelets were separated from plasma proteins by gel filtration. PRP (collected in ACD) was adjusted to pH 6.5 with ACD and apyrase (1 U/mL) and prostaglandin-E1 (2 µM) were added prior to centrifugation at room temperature at 630g for 10 minutes. The PPP was removed and the platelet pellet was resuspended in 2 mL modified Hepes-Tyrode buffer (JNL; 6 mM dextrose, 130 mM NaCl, 9 mM NaCl2, 10 mM Na citrate, 10 mM Tris base, 3 mM KCl, 0.8 mM KH2PO4, and 0.9 mM MgCl2). The platelet suspension was then applied to a chromatography column containing 5 mL packed Sepharose 2B-300, which was previously equilibrated with JNL buffer. The resultant platelet fractions were pooled. The platelet concentration was determined using a Sysmex-100 particle counter (Sysmex, Kobe, Japan).Albumin-gradient platelets For preparation of highly purified platelets free from platelet-associated proteins an albumin gradient technique was used. A 2-mL aliquot of PRP was layered on a gradient of high-grade BSA (50%, 25%, 17%, 12%, and 10%) in a 15-mL tube. The tubes were then centrifuged at 1200g for 15 minutes. The resultant platelet layer (lying on top of the 50% solution) was removed using a Pasteur pipette.Platelet aggregation Platelet aggregation was assayed by light transmission at 37°C using a PAP-4 aggregometer (Bio-Data). The gel-filtered and albumin-gradient platelets were adjusted to a final concentration of 2 × 108 platelets/mL. Physiologic concentrations of CaCl2 (1.8 mM) and fibrinogen (1 mg/mL) were added to the platelets prior to aggregation studies.Platelet adhesion assay The 96-well microtiter plates were coated with 100 µL bacteria (OD 1.0), fibrinogen (20 µg/mL), or BSA (20 µg/mL). The plate was incubated at 37°C for 2 hours. Following this, the plate was washed and blocked with 1% BSA for a further 1 hour at 37°C. The plate was washed 3 times in JNL buffer to remove any unbound protein. Then 50 µL gel-filtered platelets (2 × 108 platelets/mL) was added to each well and allowed to adhere for 30 minutes at 37°C. Each well was gently washed 3 times with 100 µL JNL to remove any nonadhered platelets. Adherent platelets were then lysed with 100 µL lysis buffer containing a substrate for acid phosphatase (0.1 M Na acetate pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenol phosphate) and incubated for 2 hours at 37°C. The reaction was stopped by the addition of 1 M NaOH and the resultant color was read at 410 nm in a microtiter plate reader (Wallac Victor2, Perkin Elmer, Cambridge, United Kingdom). Adhesion data were expressed as the color produced by platelets adherent to bacteria as a percentage of that produced by platelets adherent to fibrinogen.Transient transfection The CHO /IX cells were grown to 30% confluence in a
tissue-culture Petri dish and transfected as previously
described.30 pcI-neoGPIb or unligated pcDNA for the
control (9 mg) was mixed with 60 µL lipofectamine and 1.6 mL optiMEM
for 15 minutes in the dark. This transfection mixture was added to the
CHO cells and incubated at 37°C with 5% CO2 for 5 hours.
Fresh media was added and the CHO cells incubated for 60 hours at
37°C with 5% CO2. After 60 hours, the cells were
harvested by scraping from the tissue culture Petri dish, washed twice
in -minimal essential medium (MEM) and finally resuspended in Hanks
buffered saline solution. To examine for transfection efficiency, the
transfected cells were incubated with 10 µg/mL primary antibody
(mouse-anti-GPIb antibody, clone SZ-2) or isotype control antibody
for 20 minutes at room temperature. Antibody binding was determined
using 10 µg/mL specific goat antimouse FITC-labeled secondary
antibody for 20 minutes at room temperature. The samples were then
diluted in 2 mL PBS and analyzed on a FACSCalibur flow cytometer
(Becton Dickinson).
Immunofluorescent microscopy For binding studies, stably transfected CHO/IX cells
transiently transfected with GPIb were mixed with 100 µL S
sanguis (OD 1.0) and gently agitated for 10 minutes at room
temperature. The cells were then applied to the slides. CHO cells were
incubated with a "primary" antibody raised against GPIb (5 µg/mL) for 45 minutes at room temperature. Unbound antibody was
removed by gentle washing in Tris (hydroxymethyl)aminomethane-buffered
saline (TBS). The CHO cells were finally incubated with
fluorescent 546-labeled goat antimouse antibody (4 µg/mL) or no
antibody for 10 minutes in the dark. Slides were mounted for
fluorescent antibody visualization using a confocal microscope (LSM
510; Zeiss, Herts, United Kingdom). Images of stained cells were
acquired using an argon laser at 488 nm, whereas nonstained cells
images were acquired using the differential interference contrast mode.
Mocarhagin-treated platelets Platelet-rich plasma was pretreated with a proteinase-disintegrin, mocharagin (5 µg/mL)31 for 45 minutes at 37°C. Treated platelets were tested for their ability to aggregate in response to ADP (20 µM), ristocetin (1.5 mg/mL), and S sanguis (OD 1.6).Thromboxane ELISA Production of TXB2 following platelet stimulation by the agonists arachidonic acid (1.5 mM), S sanguis (OD 1.6), and ristocetin (1.5 mg/mL) was measured using a commercial ELISA kit according to the manufacturer's instructions. Aliquots (50 µL) of each reaction were snap frozen in liquid nitrogen for analysis of TXB2 levels.Preparation of glycocalicin Glycocalicin, the soluble cleavage fragment of GPIb, was prepared according to the method of Loscalzo and Handin,32 which involved incubation of platelets in 3 M KCl at 37°C to allow calpain cleavage of GPIb, wheat germ agglutinin affinity chromatography followed by column chromatography of the eluted material on Sepharose CL-6B. Positive column fractions were identified by reaction with an anti-GPIb antibody (PM6/40) and stored at 20°C until required.
Ligand blotting Purified glycocalicin was run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, blotted onto nitrocellulose and after blocking in 5% (wt/vol) hemoglobin, incubated with biotin-labeled S sanguis cells overnight at room temperature. Blots were then washed in PBS, incubated in avidin-conjugated horseradish peroxidase (Sigma), washed, and developed in 4-chloro-1-napthol (Sigma; 0.6 mg/mL in 100 mM Tris-HCl buffer, pH 7.5) and H2O2 (5 µL/mL).Adhesion to glycocalicin Aliquots of glycocalicin were diluted to 10 µg/mL in 50 mM carbonate-bicarbonate buffer, pH 9.6, and coated onto ELISA wells overnight at 4°C. After washing and blocking wells with 2% (wt/vol) BSA, FITC-labeled S sanguis cells were added to coated wells and incubated at 37°C for 90 minutes. Wells were then washed with PBS several times and adherent bacteria detected using a fluorescence plate reader (POLARStar Galaxy; BMG Lab Technologies, Aylesbury, United Kingdom) at 490exc nm and 540emm nm.
S sanguis-induced platelet aggregation A number of strains of S sanguis were tested for their ability to induce platelet aggregation and typical examples of the traces obtained are shown in Figure 1. Aggregation occurred in an all-or-nothing manner, and altering the concentration of bacteria affected the lag time but not the maximum aggregation. Lag time is defined as the time taken from addition of bacteria to PRP until the first recognizable signs of aggregation. A range of lag times was observed with the strains used, but the lag time was consistent for each strain. Comparison of the lag times of strains with that of NCTC 7863, the type strain of the species and which has previously been shown to induce aggregation,24 3 significantly different groups could be recognized (P < .001, analysis of variance [ANOVA]). One group had relatively short lag times (around 5 minutes) and included strain 133-79. A second group had longer lag times (around 16 minutes), similar to NCTC 7863, and finally one strain (SK96) failed to induce aggregation within 30 minutes (Figure 2).
S sanguis-platelet adhesion The adhesion assay used relied on platelets binding to bacteria that had been immobilized on polystyrene wells and made use of the intracellular platelet enzyme, acid phosphatase, for quantification. The method proved to be sensitive and reproducible and the level of acid phosphatase in platelets was much higher than the levels in the streptococci (data not shown). The assay had the advantage that the adhesion event could be studied in the absence of any surface activation of the platelets, which would have occurred if the platelets had been bound to the polystyrene wells. In early experiments we used an assay that measured adhesion of radiolabeled bacteria to immobilized platelets. This yielded similar data to that reported here but had the disadvantage of requiring the use of radioisotopes to enumerate the adherent bacteria (data not shown).Strains differed in their ability to bind platelets using the above
assay (P < .0001, ANOVA). Certain strains supported high levels of platelet adhesion, whereas others were less adhesive or did
not support adhesion at all (Figure 3).
The extent of platelet attachment to the adhesive strains was usually
similar to the level of adhesion seen to a fibrinogen-coated surface.
For example, the adhesion of strain 133-79 was 139% ± 28%
(n = 5) of the adhesion of platelets to fibrinogen-coated wells. In
addition, adhesion to strain 133-79 did not require activation of
platelets, as it was not inhibited by pretreating platelets with
aspirin or PGE1 (7.5% ± 6% inhibition, n = 4).
Strain 133-79 was selected as a representative of the adherent,
fast-aggregating strains and used for further studies.
Platelet thromboxane production Platelet aggregation by strain 133-79 was inhibited by aspirin (100% inhibition, n = 3), a cyclooxygenase inhibitor, suggesting a role for thromboxane-A2 in the aggregation response. Platelets exposed to S sanguis (133-79) produced thromboxane (248 ± 11 ng/mL; n = 3) at levels that were intermediate between that produced with arachidonic acid (441 ± 21 ng/mL) and ristocetin (48 ± 24 ng/mL). However, aggregation was not dependent on the release reaction because apyrase (10 U/mL) failed to inhibit 133-79-induced aggregation (51% ± 2% versus 48% ± 4% with apyrase, n = 3, P = NS), whereas it completely inhibited ADP-induced aggregation (61% ± 3% versus 4% ± 1% with apyrase, n = 3, P = .003).Platelet GPIIb/IIIa Platelet aggregation induced by S sanguis was inhibited by PGE1 (100% inhibition, n = 3), which inhibits platelet activation. The GPIIb/IIIa receptor antagonists eptifibatide and abciximab also inhibited (98% ± 1% inhibition, n = 3 and 98% ± 0.5% inhibition, n = 4, respectively). However, the specific GPIIb/IIIa antagonists, eptifibatide and abciximab, did not inhibit adhesion of platelets to S sanguis (13% ± 13% inhibition n = 3; 12% ± 4% inhibition n = 7, respectively). Platelets from 2 patients with Glanzmann thrombasthenia (absence of GPIIb/IIIa on the platelet surface) also adhered to bacteria as effectively as control platelets (82% of control adhesion, n = 2). These patients had less than 2000 GPIIb/IIIa receptors/platelet (as determined by flow cytometry), whereas normal platelets have about 50 000 receptors/platelet.Platelet GPIb The anti-GPIb antibody AN51 inhibited platelet aggregation induced by all strains of S sanguis (88% ± 2% inhibition, n = 6) suggesting a role for GPIb. To further confirm this we tested a panel of GPIb antibodies directed against different regions of the GPIb molecule (Figure 4). Antibodies that recognize the extreme N-terminal portion of GPIb between amino acids
1 and 225 inhibited aggregation. Furthermore, cleavage of GPIb from platelets with mocarhagin, an enzyme specific for GPIb at amino acid
282/28331 resulted in 97.5% ± 2.5% inhibition of
aggregation (n = 2), confirming the involvement of the N-terminal
region. This was further confirmed by the failure of S
sanguis to induce aggregation (2% ± 1% aggregation, n = 2)
of platelets from a patient with Bernard-Soulier syndrome (with no
detectable GPIb30) even though they responded to ADP
(34% ± 2% aggregation, n = 2).
The S sanguis strain 133-79 was able to interact
directly with GPIb in a number of experiments. First, platelet adhesion
to strain 133-79 was inhibited by the anti-GPIb antibody AN51
(46% ± 9% inhibition) at concentrations that completely inhibited
aggregation, whereas antibodies to GPIIb/IIIa were without effect.
Second, S sanguis 133-79 was able to bind to CHO
The role of the Fc receptor NCTC 7863-mediated aggregation was previously shown to involve binding of antibody and to be dependent on the platelet Fc RIIA receptor.24 The antibody IV3, which
is directed to the FcRIIA receptor inhibited aggregation
induced by 133-79 (100% inhibition, n = 3). However, it had no
effect on platelet adhesion to 133-79 (5% inhibition).
A role for plasma proteins To determine whether any plasma components were required for aggregation by S sanguis, platelets purified on an albumin gradient were used because this method has been shown to be the most effective at separating platelets from plasma proteins.33 Albumin gradient platelets did not aggregate with S sanguis 133-79 unless fibrinogen was added (47% ± 7% aggregation, n = 4), suggesting that neither antibody nor other plasma factors were required. Also, gel-filtered platelets adhered to S sanguis 133-79 (139% ± 28%, n = 5) showing that plasma factors were not required for adhesion.Role of VWF VWF is one of the natural ligands for GPIb.34 Because it is possible that VWF may contaminate the purified platelet preparations, we investigated whether VWF played a role in platelet aggregation by S sanguis. An antibody to VWF that inhibited ristocetin-induced aggregation (AVW-3) had no effect on S sanguis-induced aggregation (60% ± 2% aggregation, n = 3).
The strains of S sanguis tested here showed 3 phenotypes with regard to the nature of their interaction with platelets. Type I induced rapid aggregation and were highly adhesive to platelets (eg, strain 133-79), type II induced aggregation with longer lag times and were not adhesive to platelets (eg, NCTC 7863), and type III neither induced aggregation nor supported adhesion of platelets (eg, SK96). Similar phenotypes have been described by Herzberg et al.35 Platelet aggregation induced by types I and II S
sanguis strains was dependent on activation and required
fibrinogen binding to its receptor, GPIIb/IIIa. This was true
aggregation and bacteria did not bind directly to GPIIb/IIIa because
adhesion was not inhibited by GPIIb/IIIa antagonists. Rapidly
aggregating strains (type I) did not, however, require ADP or VWF
secretion. Because bacteria can cross-link platelets, it is surprising
that there is no evidence of agglutination. This is likely due to the
fact that platelet aggregometry only detects large aggregates and that
the strength of the bacterial-platelet interaction may only support the
formation of microaggregates. We have previously shown that platelet
aggregation induced by NCTC 7863 (type II) depends on IgG binding to
the Fc Because we had previously shown that the anti-GPIb antibody, AN51, inhibited NCTC-7863-induced platelet aggregation,24 we turned our attention to GPIb. After GPIIb/IIIa it is the most abundant glycoprotein on platelets and it plays an essential role in platelet adhesion in vivo.20 However, here we have extended that work to include a panel of monoclonal antibodies against GPIb and studied their effect on strain 133-79. S sanguis-induced platelet aggregation was inhibited by antibodies that inhibit ristocetin-induced aggregation (AN51, MB45, and AP-1) but not by antibodies that inhibited thrombin binding or botrocetin-induced aggregation (Vm16-d and SZ2).36,37 The latter result differs from that reported by Sullam and coworkers38 who found that SZ2 inhibited S sanguis-induced platelet aggregation, but this is likely to be due to the use of different strains. Platelet aggregation by S sanguis (type I and type II) was
also inhibited by antibody to the Fc Although our data apparently provide evidence for a role of GPIb and
Fc Our data and those of others then suggest that strains of S
sanguis vary in the mechanisms by which they interact with
platelets but all may well involve GPIb, the Fc Although nothing is currently known about the S sanguis
GPIb-binding molecule, it is likely to be protein in nature because protease treatment of the bacteria reduced adhesion to platelets (data
not shown). This suggests that there may be a prothrombotic virulence
factor on some strains of S sanguis and it is tempting to
speculate that it may be a VWF-like molecule on the surface of the
bacteria. Previously, a collagenlike protein termed platelet aggregation-associated protein (PAAP) on 133-79 was implicated in
aggregation.28,29 Whereas some collagens can bind to GPIb via VWF,42 our data would suggest that a protein other
than PAAP is involved due to the absence of a role for VWF, although it
has been reported that the snake venom lectin aggretin is able to bind
to both the Binding of GPIb to VWF only occurs under shear conditions20 or with chemical modification of VWF by ristocetin or botrocetin and is accompanied by a weak thromboxane signal. However, the interaction of S sanguis with GPIb is unusual in that it requires neither shear nor chemical modification to facilitate binding and appears to induce a thromboxane-dependent activation signal leading to GPIIb/IIIa-mediated aggregation. Previous work has shown that engagement of GPIb can lead to activation of GPIIb/IIIa,45-47 but this is the first direct evidence of activation of GPIIb/IIIa that is mediated by thromboxane production due to a ligand binding to GPIb. Bacterial polymorphisms are known to be important factors in
disease48 and presence of the GPIb-binding protein in some strains may explain the strong association between S sanguis
and infective endocarditis. Despite this, S sanguis is not
unique in its ability to interact with GPIb and may be a more general mechanism for bacterial-platelet interactions. Recently we have shown
that Helicobacter pylori binds to GPIb and induces
aggregation, although via a different mechanism.49 Also,
Staphylococcus aureus protein A has been shown to interact
with platelets via VWF-GPIb and IgG-Fc Our data suggest that the use of antiplatelet agents may be useful in both the prevention and treatment of infective endocarditis. It also has implications for conditions other than infective endocarditis. Recent reports have suggested that periodontal disease is an independent risk factor for myocardial infarction51 and S sanguis has been identified in atherosclerotic plaque.9 Another oral bacterium Porphyromonas gingivalis has recently been shown to activate platelets via a secreted enzyme acting on the protease activated receptor 1 (thrombin receptor) on platelets52 and to increase atherosclerosis in an animal model.53 It is known that oral bacteria can gain access to the circulation as a result of periodontal disease. In patients with damaged heart valves this can lead to infective endocarditis. However, in patients with unstable atherosclerotic plaques this may aggravate existing unstable angina by increasing platelet activation. The interaction of bacteria with platelets may therefore play an important role in cardiovascular diseases.
We would like to thank Ms Teresa Keane for her assistance in the confocal microscopy experiments. We would also like to thank Dr José Lopez, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; Dr Michael Berndt, Baker Research Medical Institute, Praham, Victoria, Australia; and Dr Dermot Kenny, Department of Clinical Pharmacology, Royal College of Surgeons in Ireland for gifts of reagents.
Submitted December 5, 2001; accepted March 5, 2002.
Supported by grants from the Royal College of Surgeons in Ireland, the University of Sheffield, and the British Heart Foundation.
This work was a poster presentation at the American Society of Hematology meeting in 1999.54
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dermot Cox, Department of Clinical Pharmacology, Royal College of Surgeons, 123 St Stephens Green, Dublin 2, Ireland; e-mail: dcox{at}rcsi.ie.
1.
Gupta S, Leatham E, Carrington D, Mendall M, Kaski J, Camm A.
Elevated Chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction.
Circulation.
1997;96:404-407
2.
Danesh J, Youngman L, Clark S, et al.
Helicobacter pylori infection and early onset myocardial infarction: case-control and sibling pairs study.
Br Med J.
1999;319:1157-1162
3.
Mayer M, Kiechl S, Willeit J, Wick G, Xu Q.
Infections, immunity and atherosclerosis. Associations of antibodies to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus with immune reactions to heat-shock protein 60 and carotid or femoral atherosclerosis.
Circulation.
2000;102:833-839
4.
Kaul R, Uphoff J, Wiedeman J, Yadlapalli S, Wenman W.
Detection of Chlamydia pneumoniae DNA in CD3+ lymphocytes from healthy blood donors and patients with coronary artery disease.
Circulation.
2000;102:2341-2346
5.
Siscovick D, Schwartz S, Corey L, et al.
Chlamydia pneumoniae, Herpes simplex virus type 1, and cytomegalovirus and incident myocardial infarction and coronary heart disease death in older adults: The Cardiovascular Health Study.
Circulation.
2000;102:2335-2340
6.
Rupprecht HJ, Blankenberg S, Bickel C, et al.
Impact of viral and bacterial infectious burden on long-term prognosis in patients with coronary artery disease.
Circulation.
2001;104:25-31
7.
Meier C, Derby L, Jick S, Vasilakis C, Jick H.
Antibiotics and risk of subsequent first-time acute myocardial infarction.
JAMA.
1999;281:427-431
8.
Muhlestein J, Anderson J, Carlquist J, et al.
Randomized secondary prevention trial of azithromycin in patients with coronary artery disease. Primary clinical results of the ACADEMIC study.
Circulation.
2000;102:1755-1760 9. Chiu B. Multiple infections in carotid atherosclerotic plaques. Am Heart J. 1999;138:S534-S536[CrossRef][Medline] [Order article via Infotrieve].
10.
Anderson J, Muhlestein J, Carlquist J, et al.
Randomized secondary prevention trial of azithromycin in patients with coronary artery disease and serological evidence for Chlamydia pneumoniae infection: The Azithromycin in Coronary Artery Disease: Elimination of Myocardial Infection with Chlamydia (ACADEMIC) study.
Circulation.
1999;99:1540-1547
11.
Farsak B, Yildirir A, Akyon Y, et al.
Detection of Chlamydia pneumoniae and Helicobacter pylori DNA in human atherosclerotic plaques by PCR.
J Clin Microbiol.
2000;38:4408-4411
12.
Patel P, Mendall M, Carrington D, et al.
Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors.
Br Med J.
1995;311:711-714
13.
Whincup P, Danesh J, Walker M, et al.
Prospective study of potentially virulent strains of Helicobacter pylori and coronary heart disease in middle-aged men.
Circulation.
2000;101:1647-1652 14. Douglas C, Heath J, Hampton K, Preston F. Identity of viridans streptococci isolated from cases of infective endocarditis. J Med Microbiol. 1993;39:179-182[Abstract]. 15. Kurland S, Enghoff E, Landelius J, Nystrom S, Hambraeus A, Friman G. A 10-year retrospective study of infective endocarditis at a university hospital with special regard to the timing of surgical evaluation in S. viridans endocarditis. Scand J Infect Dis. 1999;31:87-91[CrossRef][Medline] [Order article via Infotrieve].
16.
Herzberg M, MacFarlane G, Gong K, et al.
The platelet interactivity phenotype of Streptococcus sanguis influences the course of experimental endocarditis.
Infect Immun.
1992;60:4809-4818 17. Sullam P, Bayer A, Foss W, Cheung A. Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis. Infect Immun. 1996;64:4915-4921[Abstract].
18.
Siess W.
Molecular mechanisms of platelet activation.
Physiol Rev.
1989;69:58-178 19. George J. Platelets. Lancet. 2000;355:1531-1539[CrossRef][Medline] [Order article via Infotrieve]. 20. Andrews R, Shen Y, Gardiner E, Dong J, Lopez J, Berndt M. The glycoprotein Ib-IX-V complex in platelet adhesion and signaling. Thromb Haemost. 1999;82:357-364[Medline] [Order article via Infotrieve]. 21. vanZanten G, de Graaf S, Slootweg P, et al. Increased platelet deposition on atherosclerotic coronary arteries. J Clin Invest. 1994;93:615-632[Medline] [Order article via Infotrieve]. 22. Harrington R. Overview of clinical trials of glycoprotein IIb-IIIa inhibitors in acute coronary syndromes. Am Heart J. 1999;138:276-286[CrossRef][Medline] [Order article via Infotrieve].
23.
Sullam P, Valone F, Mills J.
Mechanisms of platelet aggregation by viridans group streptococci.
Infect Immun.
1987;55:1743-1750 24. Ford I, Douglas C, Cox D, Rees D, Heath J, Preston F. The role of immunoglobulin G and fibrinogen in platelet aggregation by Streptococcus sanguis. Br J Haematol. 1997;97:737-746[CrossRef][Medline] [Order article via Infotrieve].
25.
Sullam P, Jarvis G, Valone F.
Role of immunoglobulin G in platelet aggregation by viridans group streptococci.
Infect Immun.
1988;56:2907-2911 26. Ford I, Douglas C, Preston F, Lawless A, Hampton K. Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis. Br J Haematol. 1993;84:95-100[Medline] [Order article via Infotrieve]. 27. Gong K, Wen D, Ouyang T, Rao A, Herzberg M. Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies. Infect Immun. 1995;63:3628-3633[Abstract]. 28. Erickson P, Herzberg M. A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation. J Immunol. 1987;138:3360-3366[Abstract].
29.
Erickson P, Herzberg M.
Purification and partial characterization of a 65kDa platelet aggregation-associated protein antigen from the surface of Streptococcus sanguis.
J Biol Chem.
1990;265:14080-14087
30.
Moran N, Morateck P, Deering A, et al.
Surface expression of glycoprotein Ib alpha is dependent on glycoprotein Ib beta: evidence from a novel mutation causing Bernard-Soulier syndrome.
Blood.
2000;96:532-539 31. Ward C, Andrews R, Smith A, Berndt M. Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet vonWillebrand factor receptor, glycopr |
Top
Abstract
Introduction
RIIA)-dependent
