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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-01-0069.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Section for Microbiology, Immunology and
Glycobiology, the Institute of Laboratory Medicine, University of Lund,
Lund, Sweden, and Roon Research Center for
Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis
and Thrombosis, Departments of Molecular and Experimental Medicine and
of Vascular Biology, Scripps Research Institute, La Jolla, CA.
We have characterized 2 distinct mechanisms through which
infectious agents may promote platelet adhesion and thrombus formation in flowing blood, thus contributing to the progression of disease. In
one case, the process initiates when the integrin
Platelet adhesion and aggregation at sites of
tissue trauma involve interactions of membrane receptors with
constituents of extracellular matrices, such as collagen, and
circulating macromolecules, such as von Willebrand factor (VWF) and
fibrinogen,1 which contribute to arrest bleeding during
hemostasis. Bacteria, too, can induce platelet
aggregation2-4 or uncontrolled clotting with disseminated
intravascular coagulation (DIC),5,6 which may become
disease mechanisms when the causative agent intermittently invades the
bloodstream. For example, coagulation and hemostasis are activated in
some localized infections, such as necrotizing fasciitis, resulting in
extensive thrombosis of arterioles and veins in and around
lesions.7,8 Moreover, experimental and clinical
observations have demonstrated that platelets play a key pathogenetic
role when certain microorganisms establish infection in the
bloodstream, as in the case of bacterial endocarditis.4,9 In particular, platelet aggregates may allow bacteria to settle and
remain at the site of infection withstanding the shear forces of
flowing arterial blood. In experimental models of this disease, early
vegetations grow by accretion of layers of fibrin and platelets with
bacterial colonies sandwiched between them.10 Similar
mechanisms may facilitate the establishment of bacteria on artificial
devices, such as arterial grafts.11 The number of strains
that can settle in the arterial circulation is limited, but an array of
different species can cause septic venous thrombosis,12,13
a condition in which platelet activation may be involved.
In these studies, we have used 2 invasive species, Streptococcus
pyogenes (also designated group A streptococcus) and
Staphylococcus aureus, as models to examine the mechanisms
involved in bacteria-induced thrombus formation under conditions
mimicking the macromolecular, cellular, and hemodynamic complexity of
blood circulating in different vessels. Only bacteria capable of
binding a platelet-reactive factor from blood, such as fibrinogen,
could initiate adhesion on a surface not intrinsically conducive to
platelet deposition. This step, however, was not required on substrates
that could directly support platelet adhesion, such as immobilized
fibrinogen, fibronectin, or subendothelial matrix. In either case,
specific antibodies bound to surface-immobilized bacterial antigens
were necessary to induce platelet aggregation into thrombi through an
interaction with the Fc Blood and blood cells
Bacterial strains and culture conditions
Proteins To generate the M5 B-encoding construct, the parts of the
emm5 gene located 5' and 3' of the B-repeats were amplified
by polymerase chain reaction (PCR) and then cloned into the shuttle
vector pLZ12(spec). The resulting construct,
emm5B, was used to express rM5B in E coli
and in M5 streptococci.19 To generate the chimeric
proteins M4/M1 and M4/M5, the regions encoding the fibrinogen-binding
B-repeats in emm1 and emm5 were amplified by PCR
and cloned into emm4 in the shuttle plasmid pJRS264, a
derivative of pLZ12(spec).19 Cloning of M1 in
the E coli expression vector pHD389, of M5 in pLZ12(spec), and of the fibrinogen-binding region of
clumping factor (rClfA) in pQE30 have been described
previously.17,20,21 Recombinant M1, M5, and M5B were
purified on agarose coupled with human serum albumin from lysates of
overnight cultures of E coli transformed with the
corresponding genes. Human serum albumin was coupled to agarose columns
activated with N-hydroxysuccinimide ester (Amersham
Pharmacia Biotech, Uppsala, Sweden). Proteins bound to the affinity
columns were eluted using 0.1 M glycine, pH 2.0, and dialyzed against
PBS. The synthetic oligopeptide N23 (AVTRGTINDPQRAKEALDKYELE)
corresponds to the unique N-terminal end of the M5 protein and was
synthesized using fluorenyl-methoxycarbonyl (F-moc) chemistry. The
peptide was purified by high-performance liquid chromatography (HPLC)
and characterized by mass spectrometry. rClfA expressed in pQE30
contains an N-terminal extension of 6 His residues. The fusion protein
was purified using chelating Ni-Sepharose Fast Flow (Amersham Pharmacia
Biotech). Bound proteins were eluted with imidazole (200 mM) and
dialyzed against PBS. Fibrinogen was purified from blood collected in
acid-citrate-dextrose anticoagulant containing 0.1 M (final
concentration)  -aminocaproic acid, using the glycine precipitation
method22 as previously reported.23
High-molecular-weight contaminants were removed by gel permeation
chromatography through a Sepharose CL-4B column. Human VWF was purified
and characterized using methods previously described in
detail.24 Absence of fibronectin and fibrinogen contamination was verified by immunoblotting with monospecific antibodies. Human plasma fibronectin was generously provided by Dr Mark
Ginsberg (Scripps Research Institute). Bovine fibrillar type I collagen
was purchased from Sigma (St Louis, MO).
Antibodies The monoclonal antibodies IV.3 (American Type Culture Collection no. HB-217, Manassas, VA), BIIG2 and P5D2 (both obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA), LJ-CP823 and LJ-Ib125 were purified on protein A agarose. IgG and M5-specific antibodies from human plasma were purified on protein A agarose and agarose coupled with rM5, respectively. Anti-M antibody titers in plasma against 3 different recombinant M proteins (M1, M5, and M53) were determined by enzyme-linked immunosorbent assay (ELISA) using microtiter plates coated with 2.5 µg purified protein. After adding blocking buffer (PBS containing 1% bovine serum albumin [BSA]) overnight, plasma diluted in the same buffer was added to the wells. Following incubation for 1 hour at room temperature and subsequent washing, horseradish peroxidase-conjugated goat antihuman immunoglobulin (Zymed Laboratories, San Francisco, CA), diluted 1:2000, was added. After incubation at room temperature for 2 hours, the wells were washed repeatedly with blocking buffer. Finally 200 µL of a developing buffer containing 1 M citrate, pH 4.0, 0.3% H2O2 and 1/10 ABTS (2,2-azino-di (3-ethylbenzthiazoline) sulfonic acid; Zymed Laboratories) was added. The optical density was measured at 405 nm and plotted versus the dilution. The presence of M5-specific antibodies in plasma was determined by ELISA, using the peptide N23 (see above) as antigen.Preparation of glass coverslips Bacterial proteins were used at a concentration of 100 µg/mL in PBS, and 300 µL of the solution was applied on a glass coverslip (24 × 50 mm, Corning, Vineland, NJ) as previously described.26 Bovine fibrillar type I collagen was used at 200 µg/mL. Human serum albumin, fibrinogen, and fibronectin were used at 50 µg/mL. Bacteria, 300 µL of 108 cfu/mL in PBS, were added either directly to the coverslip or after coating the glass surface with various human plasma proteins or extracellular matrix (ECM). Bacteria were allowed to attach for 30 minutes at room temperature, and after thorough rinses the coverslip was placed in the flow chamber. For coating with ECM, pooled human umbilical vein endothelial cells (Clonetics, San Diego, CA) were cultured as previously described.1 After reaching confluence in T-75 tissue culture flasks, cells were plated and cultured on sterile glass coverslips. Confluent monolayers were washed repeatedly with PBS and detached by incubation in 20 mM NH4OH and 0.5% Triton X-100 in PBS for 3 minutes at 37°. The matrix remaining attached to the glass slides was washed with PBS. The matrix-coated glass slides were assembled as the base of a parallel plate perfusion chamber and used immediately in blood flow studies.Epifluorescence videomicroscopy Platelet interaction with immobilized proteins, ECM, or bacteria under various flow conditions was studied using a modification of a parallel flow chamber as previously described.1,26,27 A syringe pump (Harvard Bioscience, South Natick, MA) was used to aspirate blood through the flow chamber. Platelets were labeled in whole blood by direct incubation with the fluorescent dye mepacrine (quinacrine dihydrochloride, 10 µM). The flow chamber, mounted on an epifluorescence microscope (Axiovert 135 M inverted microscope; Carl Zeiss, Thornwood, NY), allowed direct visualization in real time of platelet adhesion and thrombus formation, which were recorded on videotape. The total volume occupied by thrombi in a given area was measured while blood was flowing from a series of confocal sections at 1.0-µm intervals in the z axis (LSM confocal microscope; Carl Zeiss), and calculated as described previously1,14 using Metamorph (Universal Imaging Corporation, Downingtown, PA) for image analysis.
S aureus and S pyogenes cause platelet thrombus formation under flow conditions The ability of bacteria to induce aggregation of flowing platelets was analyzed using 2 different invasive species: S aureus, which causes endovascular infections, and S pyogenes, which can cause derangement of hemostasis following transient invasion of the bloodstream. The bacteria were allowed to adhere to a glass slide and then exposed to human blood perfused at wall shear rates as high as 2000 s 1 to mimic the hemodynamic conditions of the
arterial circulation. Both strains were able to induce platelet
thrombus formation (Figure 1).
Fibrinogen binds to streptococcal M proteins and supports platelet adhesion and thrombus formation Initial studies on bacteria-induced platelet aggregation were focused on S pyogenes, which expresses on its surface a class of type-specific virulence factors designated M proteins.28 Most M proteins bind fibrinogen, and all group A streptococcal strains express at least one fibrinogen-binding M protein (Figure 2A-B). After immobilization onto a glass surface, recombinant M1 and M5 proteins, which bind fibrinogen, supported platelet adhesion when exposed to whole blood perfused at a wall shear rate between 600 and 1500 s1 (Figure 2C-D). Platelet aggregates began to form in 3 to 4 minutes and became progressively larger as perfusion continued.
After 7 to 9 minutes, the area covered by platelets was similar on
surfaces coated with either rM1 or rM5. In contrast, there was no
platelet interaction with rM5B, which lacks the fibrinogen-binding B
repeats of the wild-type protein (Figure 2).19
Analysis of blood from different individuals revealed a considerable
variability in the extent of platelet adhesion to and thrombus
formation on immobilized rM1 and rM5 (Figure
3A). In some cases there was no response
at the higher shear rates tested, but some degree of platelet
deposition always occurred at the lower shear rates. Table
1A presents a summary of the results obtained in different donors. Platelet adhesion to and aggregation on
rM5 were completely abolished by the monoclonal antibody LJ-CP8, which
blocks ligand binding to the platelet integrin
Antibodies bound to streptococcal proteins contribute to thrombus formation by interacting with the IgG Fc receptor independently of fibrinogen binding As shown above (Figure 2), immobilized rM5B by itself
failed to support platelet adhesion, indicating that streptococcal M
proteins depend on bound fibrinogen to initiate platelet tethering when
they are the only substrate presented to flowing blood. In contrast,
rM5B elicited thrombus formation when immobilized on a surface with
fibrinogen (Figure 3B), although the latter by itself supports adhesion
of single platelets without inducing aggregation.26
Furthermore, the removal of plasma proteins from a blood cell
suspension reduced the interaction of platelets with immobilized rM5,
and the addition of soluble fibrinogen enhanced adhesion but was not
sufficient to restore thrombus formation (Figure 3C). Together, these
findings suggest that the role of M proteins in eliciting platelet
aggregation cannot be explained solely by their ability to bind
fibrinogen. Given the variable response observed in different
individuals, and considering that Fc RIIA (the predominant Fc
receptor on platelets29,30) has been implicated in the
induction of aggregation by anti-platelet IgG,31 we
hypothesized that antibacterial antibodies were involved in the
activation of platelets exposed to immobilized M proteins. Indeed, the
size of platelet thrombi formed on immobilized rM5 was directly related
to the anti-M5 antibody titer in the perfused blood (Figure 3A). Of
note, these antibodies were directed against conserved epitopes of M
proteins and, except in one donor (donor 4; Figure 3A), failed to react
against a specific sequence of M5 (anti-N23; Figure 3A).
To confirm the role of anti-M IgG and Fc
Whole streptococci induce platelet thrombus formation with the same mechanism as isolated M proteins The results obtained with recombinant proteins were confirmed by experiments with S pyogenes, immobilized on glass slides coated with human serum albumin. M5 streptococci induced thrombus formation with all blood samples tested. When the anti-M5 antibody titer was low (Figure 5A), both total surface coverage by platelets (Figure 5B) and thrombus volume (Figure 5C) were less than in samples with high antibody titer. Platelet adhesion and aggregation occurred on or in the immediate vicinity of streptococci, both at the highest (1600 s1) and lowest
(140 s1) shear rates tested (data not shown). Thrombi
formed faster on M5 streptococci (2-3 minutes) than on rM5 protein
(6-8 minutes).
Similar to M5, streptococci expressing the fibrinogen-binding M1 and M3
proteins supported thrombus formation, as did strains expressing
chimerical proteins in which the fibrinogen-binding B repeats from M1
or M5 had been grafted into the M4 protein that cannot bind fibrinogen
(Figure 6A-B). In contrast, strains
expressing M4, M5
A mechanism for bacterial induction of platelet thrombus formation shared by streptococci and staphylococci Experiments were carried out to evaluate whether thrombus formation on S aureus is induced by mechanisms similar to those that determine a platelet response to streptococci. S aureus produces several molecules capable of interacting with fibrinogen. The best known is clumping factor, a surface-associated protein that interacts with the same regions in fibrinogen that bind to IIb 3.18,26,33 Exposure to
flowing blood of an immobilized recombinant 35-kDa fragment of clumping
factor containing the fibrinogen-binding region (Figure
7A)20 resulted in the
formation of thrombi, which after 6 to 8 minutes were comparable in
size to those induced by rM5 (Figure 7B). As with the latter, the
extent of aggregation was donor dependent (data not shown), and both
the anti-IIb3 antibody, LJ-CP8, and the
anti-FcRIIA antibody, IV.3, inhibited the response (Figure 7B). We
then tested a wild-type S aureus strain (Newman) or its
isogenic derivative, ClfA, which contains an inactivated clumping
factor gene.18 Before blood perfusion, the strains were
immobilized on slides coated with either fibrinogen or fibronectin
that, as noted above, by themselves support single-platelet adhesion
but no significant thrombus formation in flowing blood. Both the
wild-type S aureus strain and the ClfA strain induced platelet adhesion and aggregation (Figure 7C). Incubation of blood with
the antibody IV.3 before perfusion markedly reduced aggregation induced
by the wild-type S aureus strain (Figure 7C). Together, these data suggest that platelet aggregation by S aureus and
S pyogenes involve similar mechanisms, including the
bridging of platelets to bacteria through adsorbed fibrinogen if the
substrate cannot tether platelets directly, and activation through the
IgG-FcRIIA interaction.
Our studies define the sequential steps of an immune
system-mediated mechanism of platelet thrombus formation that may be relevant to understand the pathogenic properties of microbes that invade the bloodstream transiently, such as S pyogenes, or
persist in specific locations within the vascular system, such as
S aureus. In the first step of this process, initial
platelet arrest at the site of bacterial presentation can be mediated
directly by surface-immobilized microorganisms that bind plasma
fibrinogen, to which platelets adhere through the integrin
The concepts discussed here must be viewed in relation to established mechanisms that explain the potential pathogenetic role played by hemostatic alterations during invasive bacterial infections. Complications such as DIC are attributed to highly toxic species of lipopolysaccharide (endotoxin), a cell wall constituent that is released by gram-negative pathogenic bacteria such as E coli and Neisseria meningitidis.36 The role of endotoxin in DIC caused by gram-negative organisms can be mimicked in experimental animal models by injection of small quantities of the purified bacterial product.37 The effects of endotoxin on the host organism are multiple, and some are mediated through activation of hematopoietic cells or cells in the vessel lining. A key element in these processes is tissue factor released from monocytes and endothelial cells in response to endotoxin.38 It is also apparent that platelets can play an important role in endotoxin-induced DIC, not only as a catalytic surface for the generation of procoagulant species such as thrombin, but also as enhancers of tissue factor release and presentation by monocytes.39,40 Gram-positive bacteria such as S pyogenes and S aureus do not express endotoxin, but can still give rise to DIC.41 Although these organisms produce substances that may mimic the effects of endotoxin, our findings demonstrate an alternative mechanism through which bacteria may interfere with the normal processes of hemostasis. The pathogenetic relevance of platelet activation by antibacterial antibodies remains to be verified in an appropriate animal model. It should be noted that S pyogenes, the focus of this study, is a strict human pathogen and the symptoms produced in experimental models may not be relevant for the human forms of disease. In fact, lethality is usually the only parameter measured following injection of very large inocula (108-1010 bacteria).15,42 At the molecular level, the inability of S pyogenes to cause infections in species other than human parallels the failure to interact with specific ligands of nonhuman origin. Studies in nonhuman primates, limited by their complexity, have been performed with S aureus41 and group A streptococci43 and, along with the results obtained in dogs exposed to S aureus,44 have typically demonstrated a complex pathology but quite clearly distinct from a typical endotoxin-induced DIC. Our present findings, therefore, provide background information that underlines the need for future experiments addressed at establishing the pathogenic role of direct platelet interactions with bacteria in vivo. As shown here, circulating antibodies and the IgG receptor,
Fc The ability of platelets to become activated by invading
pathogens targeted by the immune system may be a relevant host defense mechanism, because platelets release antimicrobial
proteins.48,49 It is now apparent, however, that this
homeostatic finality may turn into a pathogenic process because any
invasive microorganism that becomes immobilized, transiently or
permanently, onto a platelet-reactive surface exposed to blood and is
recognized by specific IgG may induce or amplify thrombus formation.
Even more important, we show here that the presence of intact organisms
is not necessary to induce platelet thrombi, because isolated microbial
proteins against which the host has mounted an immune response are
sufficient to support thrombus formation. In fact, a single antigen
without direct platelet-binding capacity, here exemplified by rM5
We thank Dr Tim Foster, Trinity College, Dublin, Ireland, for
supplying the bacterial strains Newman, Newman
Submitted January 10, 2002; accepted July 17, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-01-0069.
Supported by grants HL-31950, HL-42846, and HL-48728 from the National Institutes of Health; RR0833 to the General Clinical Research Institute; and by the Stein Endowment Fund (Z.M.R.); the Swedish Medical Research Council (grant no. 9926); the 80 years' Trust of King Gustaf V; the Swedish Society for Medical Research; and the Swedish Heart and Lung Association (U.S.).
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: Zaverio M. Ruggeri, Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Departments of Molecular and Experimental Medicine and of Vascular Biology, MEM 175, Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037; e-mail: ruggeri{at}scripps.edu; or Ulf Sjöbring, MIG, Institute of Laboratory Medicine, BMC B14, Tornav. 10, S-221 84 Lund, Sweden; e-mail: ulf.sjobring{at}mig.lu.se.
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© 2002 by The American Society of Hematology.
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  S. W. Kerrigan, N. Clarke, A. Loughman, G. Meade, T. J. Foster, and D. Cox Molecular Basis for Staphylococcus aureus-Mediated Platelet Aggregate Formation Under Arterial Shear In Vitro Arterioscler Thromb Vasc Biol, February 1, 2008; 28(2): 335 - 340. [Abstract] [Full Text] [PDF]
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 C. Rennemeier, S. Hammerschmidt, S. Niemann, S. Inamura, U. Zahringer, and B. E. Kehrel Thrombospondin-1 promotes cellular adherence of Gram-positive pathogens via recognition of peptidoglycan FASEB J, October 1, 2007; 21(12): 3118 - 3132. [Abstract] [Full Text] [PDF]
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G. Pietrocola, A. Schubert, L. Visai, M. Torti, J. R. Fitzgerald, T. J. Foster, D. J. Reinscheid, and P. Speziale FbsA, a fibrinogen-binding protein from Streptococcus agalactiae, mediates platelet aggregation Blood, February 1, 2005; 105(3): 1052 - 1059. [Abstract] [Full Text] [PDF]
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 J.-S. Chia, Y.-L. Lin, H.-T. Lien, and J.-Y. Chen Platelet Aggregation Induced by Serotype Polysaccharides from Streptococcus mutans Infect. Immun., May 1, 2004; 72(5): 2605 - 2617. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
toward a distinction between uncompensated overt DIC and compensated non-overt DIC.
Thromb Haemost.
2001;86:1489-1494