Vol.
Platelet Aggregation Yukio
USUI,*
Immunol. 942, 1991
by Strains of Enterococci
Yoshitoshi and
Microbiol. 35 (11), 933
ICHIMAN, Kosaku
Masaru
SUGANUMA,
YOSHIDA
Departmentof Microbiology,St. Marianna UniversitySchool of Medicine, Kawasaki, Kanagawa 216, Japan (Accepted
for publication,
August 21, 1991)
Abstract The platelet aggregation capability of whole cells of Enterococcusfaecalis, E. faecium and E. avium was tested. The optimum ratios of bacteria to platelets in E. faecalis (strain SMU-37), E. faecium (strain SMU-138) and E. avium (strain SMU197) were 1.0, 1.2 and 2.0, respectively. During the platelet aggregation induced by the three strains of enterococci, 65-69% of total serotonin was released. The aggregation was totally inhibited by ethylenediaminetetraacetate (10 mM) and apyrase (1 mg/ml), while no effect was shown by aspirin (10 mM), indomethacin (10 mM) and quinacrine (1 mM). By pretreatment of platelet-poor plasma with heat (56 C, 30 min) or zymosan, the reactivities with platelets of each strain of species were markedly diminished. These results suggest that enterococci-induced platelet aggregation was an ion-dependent, cyclooxygenase-insensitive event, and plasma component(s) was (were) required for the reaction.
Platelets play an important role in the process of hemostasis and vascular integrity under normal physiological conditions. In hemostasis, platelets respond to several extracellular stimuli including thrombin, adenosine diphosphate (ADP), collagen, epinephrine, arachidonate, etc., resulting in platelet shape change, aggregation, and serotonin secretion (19). The mechanisms of platelet aggregation by these stimuli have been studied in detail (16). In contrast to these effects favorable for hemostasis, platelets also possess a pathogenic significance in disseminated intravascular coagulation (DI C) during sepsis or infective endocarditis (3, 7, 22, 30) and observations have been made concerning the relationship between the platelet and several species of microorganisms, Staphylococcus aureus (8, 18, 28, 40), coagulasenegative staphylococci (CNS) (41), streptococci (24, 35, 36, 39, 43), Listeria monocytogenes(9), Fusobacteriumnecrophorum(15), Aspergillusfumigatus (31), Candida albicans (33), Histoplasmacapsulatum(10). Enterococci are one of the opportunistic pathogenic organisms of endocarditis (23, 26, 27, 42) and other acute inflammatory infectious disease, urinary tract infection (17) and meningitis (1). Enterococci are regarded as the third most common cause
of infective
endocarditis,
after
viridans
group
Streptococcus
and
Staphylococcus
aureus. According to Kaye (21), 5 to 15% of all cases of infective endocarditis were caused by enterococci, although few efforts have been made to elucidate the detailed processes involved in this disease. In this regard, the bacteria-platelet interac933
v.
934
USI:1
Fl AL
tion has been suggested to be an important step in the induction of endocarditis (7, 22, 30), indicating the necessity of investigations concerning the interactions between platelets and enterococci. This paper demonstrates that platelet aggregation by enterococci was not inhibited by the addition of arachidonate cascade inhibitors, that adenosine diphosphate release component(s)
from platelets was required for the aggregation to proceed, was (were) shown to participate in the reaction. MATERIALS
Bacterial SMU-197
of
strains.
Strains
E.
were
avium
Marianna
University
chemically
by
Culture. Difco,
Detroit,
They
were
by
the
cells
U.S.A.) three
saline,
the
described
pH
reference
to
a
by
were times
7.4.
E.
laeealis,
C
with
by
saline
curve
light
faecium
and St.
identified
bio-
(14).
in
Todd-Hewitt
centrifugation
broth
at
resuspended
were
of
hr
E.
Laboratory, were
Carey
20
and
numbers
standard
for
of Clinical
species
and
37
collected
Bacterial
the
Their
Facklam at
SMU-138 from
Hospital.
grown
plasma
MEITIODS
obtained
School
bacterial
Mich.,
of
isolates
method
washed
buffered
SMU-37 fresh
Medical
The
AND
and
7,000•~
in
estimated
g
0.01
m
(BHI.
for
30min.
phosphate-
nephelometrically
scattering
versus
and
viable
count.
Chemicals. Human fibrinogen (grade L; AB KABI, Stockholm, Sweden) was further purified using the DEAE-cellulose method reported by Lawrie et al (25). Ethylenediaminetetraacetic acid-disodium salt (EDT,A), indomethacin, and acetylsalicylic acid were obtained from \Vako Pure Chemical Industries (Tokyo), quinacrine dihydrochioride, human fibronectin (fin), ADP, apyrase (grade III from potato), zymosan (ZYMOSAN A from Saccharornycescererisiae, and human im munoglobulin G (IgG) were purchased from Sigma (St. Louis, Mo., U.S.A.), and Sepharose-2B was purchased from Pharmacia (Uppsala, Sweden). Collagen was purchased from Niko Bioscience (Horm: Bovine Achilles tendon, Tokyo). Other chemicals were of reagent grade. Preparation from as
the
these
source
10
min
platelet
of
at
platelets.
was
stored the
assay
were
equilibrated 8 glliter, liter, Timmons
with KCl NaH2PO4 and
method
plastic
tubes
of
HEPES
buffer,
0.2g/liter,
Hawiger
at
room
PRP pH
(38
and
3 •~
105:/ƒÊl PRP by
obtained
g
until platelets
g/liter, by
this
at
of 10
(GFP) a
final 1.0
150•~ and
Platelet (4).
PRP
determinations were
Sepharose-2B
prepared column
concentration g/liter,
of albumin
according
to
method
were
the
platelet-poor min.
Cronkite and
used
throughout
collected
for
use,
through
dextrose 0.9
was
and
a
used
addition
Brecher
containing
0.2g/liter,
Platelets
the
citrate was
centrifuged
1,640 •~
Gel-filtered
7.35,
HEPES
then
by
sodium Tokyo)
were
(PRP)
at
preparation
MgCl2-H2O giliter
mixed,
the
(w/v) Farm,
pipettes
temperature
4 hr.
the
3.8% Clea
plasma
described
within
portions
0.45
to
vol.
and
gently
centrifuging the
completed ml
tubes
was
adjusted
by
0.1 Nihon
Platelet-rich
was
capped
2.0
blood
by
into rabbit,
plastic
temperature.
counted
in
passing
Only
obtained
were
collected white
Citrated room
(PPP)
numbers
Blood ( Japanese
concentration
plasma
by
platelets.
rabbits
experiments.
for
of
of
drug-free
the not
NaCl 3.5
method responsive
g/ of
PLATELET
to
ADP
unless
rabbit
were
adjusted
platelets For
the
by
to
4
centrifugation IgG
ponents
the
concentration
for
90
then
Platelet
4A,
periment,
GFP
was
to
stirred
at
1,000
of
the
expressed
the
preparation at
suspension
and
final
for used
was
4
C
37
C
removed Fn,
plasma
added
37
C
com-
(34).
to
with
PPP
at
agitating
The
supernatant
a
adjust
to
pl
to
in
37
C
suspension to
reaction
p.m
or
either
In
the
the for
zero.
to
actual
10
min
ex-
prior
added,
procceed,
or
aggregometer to
and
the
units
whether
10 ƒÊg/ml,
100% PRP
Aggregation
examine
ex-
to
of
was
1
each
instrument 200
placed
warm
the
Tracer In
aggregation).
To
1.5
the of
cuvette
bacterial
for
Hema
aggregation.
solution
readjusted
of
of
(NKK
(zero to
the
adequate
7.35, at
Fibrinogen,
at
at
to
allowed
concentration
hr
then
effect
zymosan
platelet
A
was
were
the
incubated min
aggregation
normal a
PPP,
30
added
of
1
(Z-PPP).
used
being
22 ƒÊl
maximum
was
added
for
were
were
while
Then
see
pH
for
obtained.
to
was
transmission
GFP
rpm,
bacterial
at
in
aggregation.
0% or
cells
aggregometer
buffer
buffer, kept
of
aggregation.
mixture
was
Numbers
HEPES
was
above
PPP
100%
PRP
serum
added.
was Blood
and
tetra-channel
HEPES
of rabbits
noted
9,000 •~g
Tokyo)
testing.
density
was
A
represent
200 ƒÊl
aggregation
min,
The at
represent
used
addition from
platelet
mg/ml.
was
contraction.
zymosan-treated
and to
periments, and
as
the
buffer
935
fibrinogen,
components
PAT-4M,
PPP
transmission
20
HEPES
aggregation.
Pat
by drawn
clot
centrifuged
used
of
105/ƒÊl
complement
was
model
source
enterococci-induced
5
BY ENTEROCOCCI
blood
for
in
of
min,
obtained
a
facilitate
1,500 •~g
inactivate
the
to
dissolved
on To
3 •~
scrum,
C
at
were
as
to of
transfer
and
plasma,
preparation
before
AGGREGATION
the ADP
were platelet
or
collagen
respectively.
Original acetylsalicylic acid solution (100 mm) was dissolved in PBS containing 1( ETOH. EDTA and quinacrine2HCl were dissolved in 0.01 m-PBS, pH 7.4. Original indomethacin solution (100 mm) was dissolved using 0.1 N NaOH. The final pH of the PRP mixed with either EDTA, acetylsalicylic acid, indomethacin, or quinacrine was the same as those of original PRP. To minimize the dilution of PRP by the addition of the inhibitors, the volume of the inhibitors was one hundredth of PRP. In or
the
was
transfer
to
plasma at
inhibition
EDTA
the
perature.
After
From
a separate
bath,
300 ƒÊl mm ice.
natants Triton
release PRP
with
the
addition
tube
were
tube
obtained X-100
of
2
vol.
one
min
(37
of
GFP
several
KBq)
51
mm
were
of
for
the of
EDTA
in for
added 0.4%,
in
same 1
min
1.5
ml
2 min, to w/v,
5
was
min
at
DPO),
in
duplicate
and
min
labeled
room
tem-
recorded.
a 37
added
centrifuge
scintillation
of
10
(10 ƒÊl).
was
and
and
for
platelets 30
effects
kept
mixture
ml
before the
aggregation
intervals
12,000 •~g were
observe
components
serotonin
quinacrine,
temperature
ml)
plasma
[3H]
volume at
containing
(0.2
suspension,
up
acid,
room
Serotonin
bacterial
at
at
To
dehvdrogenase.
centrifugation toluene
10
aggregometer. the
a larger taken
centrifuged
after and
1 ƒÊCi
containing was
the
lactate
containing
portions
acetylsalicylic for
aggregation, with
and
PRP
in
platelet
formaldehyde The
the
environment
together
of
indomethacin, with
on
of serotonin
incubation
933
C
temperature
Assay
in
37
components
room
by
experiments,
preincubated
to tubes 50 ƒÊl fluid
radioactivity
C
water
50 ƒÊl
of
placed super(1
vol. was
936
V. USUI
measured
by
trols
used
were
serotonin
an
was
Aloka for the
Liquid
calculated %
Scintillation
determination from
ET AL
System
of total
the
LSL-753.
radioactivity.
Uncentrifuged Release
con-
of radiolabeled
formula:
Release= (S test-S
control/T control - S control)
100,
where S equals the radioactivity in the supernatant fluid and T equals the total radioactivity measured as disintegrations per minute (20). Lactate dehydrogenase (LDH) released from platelets during these experiments was determined by the method reported by Bergmeyer et al (2). A solution of Triton X-100 was used to lyse the platelets to determine the total amount of intracellular LDH. RESULTS
OptimumRatios betweenPlatelets and Enterococci In cases induced by strains SMU-37 of E. faecalis, SMU-138 of E. faecium,and SMU-197 of E. avium, maximal activities of platelet aggregation were obtained when the logarithmic numbers of bacteria were more than 8.49, 8.56, and 8.79 colonyforming units (c.f.u.)/ml, respectively (Fig. 1). In these results, optimum ratios were determined to be at 1.0, 1.2, and 2.0 in strains SMU-37 of E. faecalis, SMU-138 of E. faecium, and SMU-197 of E. avium, respectively. Thereafter, these ratios were used for platelet aggregation by each species of enterococci. Patterns of Platelet AggregationInducedby Enterococci The platelet aggregation induced by strain SMU-37 of E. ja ecaliv,SMU-138 of E. faecium, and SMU-197 of E. avium required approximately 1 min lag time and
Fig.
1.
Percentage
concentrations
aggregation of three
different
of platelets species
in platelet-rich of strains
plasma
of enterococci
upon
exposure
after
5 mm
E. faecalis SMU-37: 111,E. faecium SMU-138: H, E. az,iuntSMU-197.
to different incubation.
PLATELET
AGGREGATION
BY ENTEROCOCCI
937
(A)
(B)
(C)
Fig.
2. tions) of
E.
Aggregation of
patterns
rabbit
filecium
SMU-37
and of
3.6 •~
108,
2.7
108/ml.
platelets
and
E.
(C)
SMU-197
faecaliss, 6.1 •~
and exposed
SMU-138 10
c.f.u./ml,
of
serotonin
release
to
(A)
strains
E. of
aviuin. E.
(•œ, SMU-37
Bacterial faecium
respectively.
bars
indicate of
numbers and The
SMU-197 final
S.D.
Enterococcus (final
of
three
faecalis,
determina(B)
SMU-138
concentration) of
concentration
E.
in
avium
were of
platelets
3.2 •~
strains 108, was
938
Y.
then
were
mostly
enterococci, with
5.2%,
from
the
68.5 •}
platelets
E. faecium, To
of
periment. 282.3 •}
4.3
and
and
of
1 mm these
totally jected
of
cases
the
E.
E.
avium,
was
by
avium,
During
platelet
detected
after
strains,
of
the
intracellular
strains
SMU-37
respectively
platelets
were
10
mmn.after
measured
2).
enterococcal 7.0%
induced of
(Fig. platelets
by
of
the
min
incubation was
released SMU-138
2).
of
346.5 gb•}5.3 U/ml, of E. faecalis, total
by
incubation
E. faecalis,
action
commencement
The
10
serotonin
(Fig.
lysed
amounts of by SMU-37
respectively.
after
aggregation 1 min
amount
of
enterococci,
the
the
aggregation
ex-
500.0 •}9.4 SMU-138 of
LDH
of
in
U/ml, and E. faecium,
platelets
was
U/ml.
on
the
Platelet
of
strains
no
of
quinacrine
as
10
in
mm
Table
sufficient
cascade
platelet
10
mm the
ADP
(10 ƒÊM)-
either
aggregation
Indomethacin,
10
caused
were
aggregation
Incidentally,
inhibit
Acid,
and
Enterococci
indomethacin,
However,
platelet
by
arachidonate of
1. to
aggregation. the
on
reduction at
shown
Acetylsalicylic
Induced
inhibitors
marked
were
inhibited the
Aggregation
enterococci
examinations platelet
Apyrase,
various
experiments,
to
of
64.6 •}
was released in in cases induced
effects
induced
the
from
Ethylenediaminetetraacetate,
The
these
and
SMU-197
LDH U/ml
Quinacrine
these
each
6.2%,
was
1,103.3 •}21.9
Effects
With
ET AL
3 min
serotonin
whether
LDH
SMU-197
within
of
in
determine
activity
at
release
enterococci.
66.3 •}
of
terminated
the
USUI
by
mm
tested.
was
acetylsalicylic doses and
of
inhibitors
or
strain
by
acid,
collagen
EDTA
each
With
observed
used
and in
(100ƒÊg/ml)1 mg
of
apyrase/ml
enterococci
sub-
examinations.
Requirementsof Plasma Factors Using GFP, the requirement of plasma cofactors in the platelet aggregation caused by species of 3 strains of enterococci was examined (Table 2). Three of the strains of enterococci were not capable of inducing platelet aggregation with GFP alone. The addition of several plasma proteins, fibrinogen, Fn, IgG and heatTable 1. Effects of the addition of ethylenediaminetetraacetate, indomethacin, acetylsalicylic acid, quinacrine on the platelet aggregation by strains of Enterococcusfaecalis, E. faecium and E. avium
EDTA,
results
The
final
SMU-138 108
ethylenediaminetetraacetate;
The
c.f.u./ml,
are
ASA,
mean •}S.D.
of three
concentrations
of E. faecium, respectively.
of and
the
SMU-197 The
final
acetylsalicylic
acid.
determinations.
bacterial of E.
cells avium
concentration
of
were
strains 3.1 •~
of platelets
SMU-37 108
of
, 3.6 •~ was
2.7 •~
E. faecalis,
108,
and
108/ml.
6.1 •~
PLATELET
AGGREGATION
BY ENTEROCOCCI
939
Table 2. Effects of supplementation of plasma components on the aggregation of gel-filtered platelet by strains of Enterococcusfaecalis, E. faecium, and E. avium
The
results
H-serum treated were
mean±S.D. Z-PPP
platelet-poor as follows:
heated-serum (20% cells
are and
avium centration
plasma, fibrinogen
(20%
v/v), of
were
v/v),
3.1 •~
of
108,
of platelets
determinations.
respectively.
serum plasma
Enterococcus
three
heat-inactivated
(0.3%
platelet-poor
strains
of
mean
w/v),
(20%
3.6 •~
108,
was
2.7 •~
v/v), v/v).
6.1 •~
C,
30
(0.01%
The
108
final of c.f.u./ml,
min)
of the w/v),
zymosan-treated
SMU-138
and
(56
concentration
fibronectin
(20%
faecalis,
serum Final
IgG
zymosanproteins
(0.124%
platelet-poor
concentrations E.
and plasma
faecium,
of the and
respectively.
w/v), plasma bacterial
SMU-197 The
of E. final
con-
108/ml.
inactivated serum into GFP, was unsuccessful in producing complete The species of 3 strains were able to induce platelet aggregation with mented with PPP, but not with GFP supplemented with Z-PPP.
aggregation. GFP supple-
DISCUSSION
Among microorganisms possessing the ability to induce platelet aggregation in PRP, Gram-positive cocci, S. aureus, S. sanguis, and group A streptococci have been studied in detail (8, 18, 24, 28, 35, 36, 39, 43). Concerning the interaction between enterococci and platelets, more detailed investigations have been needed for comparison with other bacteria-platelets interaction, although Clawson et al (8) reported some properties of the platelet aggregation. Enterococci-induced platelet aggregation was not inhibited by the addition of the inhibitors to arachidonate cascade. Similar results were reported in strains of CNS (41), Streptococcussanguis (35, 36), Listeria monocytogenes(9), and Aspergillus fumigatus (31). Additionally, apyrase, which hydrolyzed ADP (29), has been reported to prevent platelet aggregation induced by viridans group streptococci (35). Similarly, our studies indicated that preincubation of platelets with 1 mg apyrase per ml completely inhibited the aggregation, suggesting that platelet aggregation by enterococci was mediated by the extracellular release of ADP from dense granules of platelet. Requirements of the plasma proteins in the bacteria-platelet interactions are reported in several bacteria. In group A streptococci (24)- and S. aureus (8, 28,40)induced platelet aggregation, fibrinogen was required. Also, in S. sanguis (35, 36) and Histoplasma capsulatum (10), both fibrinogen and IgG were required and complement components were reported to be essential for the aggregation by L. monocytogenes(9), A. fumigatus (31), or Candida albicans (33). From the present experimental results, complement components would possibly be required in the case of
940
Y. USUI
ET AL
enterococci, since GFP supplemented with zymosan-treated PPP lost its reactivity with these organisms remarkably. Similar results were obtained in the case of various species of CNS (41). Further, supplementation of fibrinogen, Fn, or IgG into GFP had little effect in the platelet aggregation by enterococci. This indicated that the mechanism of enterococci-induced platelet aggregation would be different from those by S. aureus, Group A streptococci, or S. sanguis. In addition, the amount of fibrinogen added to GFP in this study was sufficient for the platelet aggregation by S. aureus (laboratory investigations), which is known to induce platelet aggregation in the presence of fibrinogen in the reaction system (8, 28). A majority of the experimental data concerning interaction between bacteria and platelets were collected from strains isolated from infectious foci (35, 36) and popular stock strains (8, 24, 28). Not only the strains used in these experiments, but also other strains randomly collected from fresh isolates induced platelet aggregation (data not shown). Additionally, it is known that there are species differencies between human and rabbit platelets (32). However, since rabbits are often used for the induction of experimental infective endocarditis (5, 6, 11-13, 37), these data obtained by rabbit PRP would be useful to explain how enterococcal endocarditis was induced. REFERENCES
1) Bayer, A.S., Seidel, J.S., Yoshikawa, T.T., Anthony, B.F., and Gum., L.B. 1976. Group 1) enterococcal meningitis. Arch. Intern. Med. 136: 883-886. 2) Bergmeyer, H.U., Bernt, E., and Hess, B. 1965. Lactic dehydrogenase, p. 736-741. In Bergmeyer, H.U. (ed), Methods of enzymatic analysis, Academic Press, New York. 3) Bick, R.L. 1978. Disseminated intravascular coagulation and related syndromes: etiology, pat hophysiology, diagnosis, and management. Am. J. Hematol. 5: 265-282. 4) Brecher, G., and Cronkite, E.P. 1950. Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3 : 365-377. 5) Carrizosa, J., and Kaye, D. 1976. Antibiotic synergism in enterococcal endocarditis ill rabbits. J. Lab. Clin. Med. 88: 132-141. 6) Carrizosa, J., and Kaye, D. 1977. Antibiotic concentrations in serum bactericidal activity, and results of therapy of streptococcal endocarditis in rabbits. Antirnicrobiol. Agents Chemother. 12: 479-483. 7) Clawson, C.C. 1979. The role of platelets in the pathogenesis of endocarditis. Am. Heart Assoc. Monogr. 52 : 24-27. 8) Clawson, C.C., White, J.G., and Herzberg, M.C. 1980. Platelet interaction with bacteria. VI. Contrasting the role of fibrinogen and fibronectin. Am. j. Hematol. 9: 43-53. 9) Czuprynsky, C. J., and Balish, E. 1981. Interaction of rat platelets with Listeria monoologenes. Infect. Immun. 33 : 103 108. 10) Des Prez, R.M., Steckley, S., Stroud, R.M., and Hawiger, J. 1980. Interaction of Ili.sloplasma capsulatum with human platelets. J. Infect. Dis. 142: 32-39. 11) Durack, D.T., and Beeson, P.B. 1972. Experimental bacterial endocarditis. I. Colonization of a sterile vegetation. Br. J. Exp. Pathol. 53: 44-49. 12) Durack, D.T., and Petersdorf, R.G. 1973. Chemotherapy of experimental streptococcal endocarditis. I. Comparison of commonly recommended prophylactic regimens..j. Clin. Invest. 52 : 592-598. 13) Egert, J., Carrizosa, J., and Kaye, D. 1977. Comparison of metliicillin, nafcillin, and oxacillin in therapy of Staphylococcusaureus endocarditis in rabbits. J. Lab. Clin. Med. 89: 1262-1268.
PLATELET
AGGREGATION
BY ENTEROLOCCI
941
14)
Facklam, R.R., and Carey, R.B. 1985. Streptococci and aerococci, p. 154-175. In Lennette, E.H., Balows, A., Hausler, W. J., Jr., and Shadomy, H.J. (eds), Manual of clinical microbiology, 4th ed, American Society for Microbiology, Washington, D.C. 15) Forrester, L.J., Campbell, B.J., Berg, J.N., and Barret, J.T. 1985. Aggregation of platelets by Fusobacteriumnecrophorwn.J. Clin. Microbiol. 22: 245-249. 16) Gordon, ,J.L. 1981. p. 1-17. In Gordon, J.L. (ed), Platelets in biology and pathology, Elsevier/ North-Holland, Amsterdam, New York. 17)
Gross, P.A., Harkavy, L.M., Barden, G.E., comial enterococcal urinary tract infection.
and Flower, M.F. 1976. The epidemiology Am. J. Med. Sci. 272: 75-81.
of noso-
18)
Hawiger, J., Steckley, S., Hammond, D., Cheng, C., Timmons, S., Glick, A.D., and Des Prez, R.M. 1979. Staphylococci-induced human platelet injury mediated by protein A and immunoglobulin G Fc fragment receptor. 1. Clin. Invest. 64: 931-937. 19) Holmsen, H., Day, H. J., and Stormorken, H. 1969. The blood platelet release reaction. Scand. J. Haematol. (Suppl.) 8: 3 26. 20)
21) 22)
Holmsen, H., and Dangelmaier, (.A.S. 1977. Adenine nucleotide metabolism of blood platelets. X. Formaldehyde stops centrifugation-induced secretion after A23187-stimulation and causes breakdown of metabolic ATP. Biochim. Biophys. Acta 497: 46-61. Kaye, 1). 1982. Enterococci-biological and epidemiologic characteristics and in vitro susceptibility. Arch. Intern. Med. 142: 2006-2009. Kessler, C.M., Nussbaum, E., and Tuazon, C.U. 1987. In vitro correlation of platelet aggregation with occurrence of disseminated intravascular coagulation and subacute bacterial endocarditis. A. Lab. Clin. Med. 109: 647-652.
23)
Koenig, M.G., and Kaye, D. 1961. Enterococcal endocarditis. Report of nineteen cases with long-term follow-up data. N. Engl. J. Med. 264: 257-264. 24) Kurpicwski, G.E., Forrester, L.J., Campbell, B.J., and Barrett, J.T. 1983. Platelet aggregation by Streptococcus pyogenes.Infect. Immun. 39: 704-708. 25) Lawrie, IS., Ross, J., and Kemp, G.D. 1979. Purification of fibrinogen and the separation of its degradation products in the presence of calcium ions. Biochem. Soc. Trans. 7: 693-694. 26)
Mandell, An
G.L.,
analysis
Intern.
Med.
Kaye,
D.,
of 38 patients 125:
Levision, observed
M.E., at
the
and New
Hook, York
1970.
Enterococcal
Hospital-Cornell
E.W.
Medical
endocarditis. Center.
Arch.
258-264.
27)
Moellering, R.C., Watson, B.K., and Kunz, L.J. 1974. Endocarditis due to group D streptococci. Comparison of disease caused by Streptococcusbovis with that produced by the enterococci. Am. J. Med. 57: 239-250. 28) Pfueller, S.L., and Cosgrove, L.J. 1980. Staphylococci-induced human platelet injury. Thromb. Res. 19: 733-735. 29)
Rozenberg, M.C., and Holmsen, IV. Platelet aggregation response 280-288.
30)
Scheid, W.M., Valone, J.A., and Sande, M.A. 1978. Bacterial adherence in the pathogenesis of endocarditis. Interaction of bacterial dextran, platelets, and fibrin. J. Clin. Invest. 61: 1394-1404. Sheth, N.K., Kurup, and Barron, B.A. 1980. The role of Aspergillusfumigatus antigens in blood coagulation and platelet function. Microbiols 28: 91-96.
31) 32) 33) 34)
35) 36)
Sinakos,
Z., and Caen,
J.P.
H. 1968. Adenine nucleotide metabolism to exogeneous ATP and ADP. Biochim.
1967. Platelet
aggregation
in mammalians
of blood platelets. Biophys. Acta 157:
(human,
rat,
rabbit,
guinea-pig, horse, dog). Comparative study. Thromb. Diath. Haemorrh. 17: 99-111. Skerl, K.G., Calderone, R.A., and Sreevalsan, T. 1981. Platelet interactions with Candida albicans. Infect. Immun. 34: 938-943. Spilvoget, A.R. 1967. An ultrastructural study of the mechanisms of platelet-endotoxin interaction. J. Exp. Med. 126: 235-267.
Sullam, P.M., Valone, F.H., and Mills, J. 1987. Mechanisms of platelet aggregation by viridans group streptococci. Infect. Immun. 55: 1743-1750. Sullam, P.M., ,Jarvis, G.A., and Valone, F.H. 1988. Role of immunoglobulin G in platelet aggregation by viridans group streptococci. Infect. Immun. 56: 2907-2911.
942
37)
38)
39) 40)
41)
42) 43)
Y. USUI
ET AL
Tanphaichitra, D., Ries, K., and Levison, M.E. 1974. Susceptibility to Streptococcusviridans endocarditis in rabbits with intracardiac pacemaker electrodes or polyethylene tubing. J. Lab. Clin. Med. 84: 726-730. Timmons, S., and Hawiger, J. 1978. Separation of human platelets from plasma proteins including factor VIII by a combined albumin gradient-gel filtration method using HEPES buffer. Thromb. Res. 12: 297-306. Usui, Y., Ohshima, Y., and Yoshida, K. 1987. Platelet aggregation by group B streptococci. J. Gen. Microbiol. 133: 1593-1600. Usui, Y., Ohtomo, T., Suganuma, M., and Yoshida, K. 1990. Fibrinogen, immunoglobulin G, and fibronectin binding to interacting Staphylococcusaureusand platelets. Med. Sci. Res. 18: 687688. Usui, Y., Ohshima, Y., Ichiman, Y., Ohtomo, T., Suganuma, M., and Yoshida, K. 1991. Platelet aggregation induced by strains of various species of coagulase-negative staphylococci. Microbiol. Immunol. 35: 15-26. Uwaydah, M.M., and Weinberg, A.N. 1965. Bacterial endocarditis-a changing pattern. N. Engl. J. Med. 273: 1231-1235. Wood, E.G., and Gray, B.M. 1986. Type-specific antibody prevents platelet aggregation induced by group B streptococci type III. J. Lab. Clin. Med. 107: 322-326. (Received
for publication,
May 2.1991)
Platelet Aggregation Yukio
USUI,*
Immunol. 942, 1991
by Strains of Enterococci
Yoshitoshi and
Microbiol. 35 (11), 933
ICHIMAN, Kosaku
Masaru
SUGANUMA,
YOSHIDA
Departmentof Microbiology,St. Marianna UniversitySchool of Medicine, Kawasaki, Kanagawa 216, Japan (Accepted
for publication,
August 21, 1991)
Abstract The platelet aggregation capability of whole cells of Enterococcusfaecalis, E. faecium and E. avium was tested. The optimum ratios of bacteria to platelets in E. faecalis (strain SMU-37), E. faecium (strain SMU-138) and E. avium (strain SMU197) were 1.0, 1.2 and 2.0, respectively. During the platelet aggregation induced by the three strains of enterococci, 65-69% of total serotonin was released. The aggregation was totally inhibited by ethylenediaminetetraacetate (10 mM) and apyrase (1 mg/ml), while no effect was shown by aspirin (10 mM), indomethacin (10 mM) and quinacrine (1 mM). By pretreatment of platelet-poor plasma with heat (56 C, 30 min) or zymosan, the reactivities with platelets of each strain of species were markedly diminished. These results suggest that enterococci-induced platelet aggregation was an ion-dependent, cyclooxygenase-insensitive event, and plasma component(s) was (were) required for the reaction.
Platelets play an important role in the process of hemostasis and vascular integrity under normal physiological conditions. In hemostasis, platelets respond to several extracellular stimuli including thrombin, adenosine diphosphate (ADP), collagen, epinephrine, arachidonate, etc., resulting in platelet shape change, aggregation, and serotonin secretion (19). The mechanisms of platelet aggregation by these stimuli have been studied in detail (16). In contrast to these effects favorable for hemostasis, platelets also possess a pathogenic significance in disseminated intravascular coagulation (DI C) during sepsis or infective endocarditis (3, 7, 22, 30) and observations have been made concerning the relationship between the platelet and several species of microorganisms, Staphylococcus aureus (8, 18, 28, 40), coagulasenegative staphylococci (CNS) (41), streptococci (24, 35, 36, 39, 43), Listeria monocytogenes(9), Fusobacteriumnecrophorum(15), Aspergillusfumigatus (31), Candida albicans (33), Histoplasmacapsulatum(10). Enterococci are one of the opportunistic pathogenic organisms of endocarditis (23, 26, 27, 42) and other acute inflammatory infectious disease, urinary tract infection (17) and meningitis (1). Enterococci are regarded as the third most common cause
of infective
endocarditis,
after
viridans
group
Streptococcus
and
Staphylococcus
aureus. According to Kaye (21), 5 to 15% of all cases of infective endocarditis were caused by enterococci, although few efforts have been made to elucidate the detailed processes involved in this disease. In this regard, the bacteria-platelet interac933
v.
934
USI:1
Fl AL
tion has been suggested to be an important step in the induction of endocarditis (7, 22, 30), indicating the necessity of investigations concerning the interactions between platelets and enterococci. This paper demonstrates that platelet aggregation by enterococci was not inhibited by the addition of arachidonate cascade inhibitors, that adenosine diphosphate release component(s)
from platelets was required for the aggregation to proceed, was (were) shown to participate in the reaction. MATERIALS
Bacterial SMU-197
of
strains.
Strains
E.
were
avium
Marianna
University
chemically
by
Culture. Difco,
Detroit,
They
were
by
the
cells
U.S.A.) three
saline,
the
described
pH
reference
to
a
by
were times
7.4.
E.
laeealis,
C
with
by
saline
curve
light
faecium
and St.
identified
bio-
(14).
in
Todd-Hewitt
centrifugation
broth
at
resuspended
were
of
hr
E.
Laboratory, were
Carey
20
and
numbers
standard
for
of Clinical
species
and
37
collected
Bacterial
the
Their
Facklam at
SMU-138 from
Hospital.
grown
plasma
MEITIODS
obtained
School
bacterial
Mich.,
of
isolates
method
washed
buffered
SMU-37 fresh
Medical
The
AND
and
7,000•~
in
estimated
g
0.01
m
(BHI.
for
30min.
phosphate-
nephelometrically
scattering
versus
and
viable
count.
Chemicals. Human fibrinogen (grade L; AB KABI, Stockholm, Sweden) was further purified using the DEAE-cellulose method reported by Lawrie et al (25). Ethylenediaminetetraacetic acid-disodium salt (EDT,A), indomethacin, and acetylsalicylic acid were obtained from \Vako Pure Chemical Industries (Tokyo), quinacrine dihydrochioride, human fibronectin (fin), ADP, apyrase (grade III from potato), zymosan (ZYMOSAN A from Saccharornycescererisiae, and human im munoglobulin G (IgG) were purchased from Sigma (St. Louis, Mo., U.S.A.), and Sepharose-2B was purchased from Pharmacia (Uppsala, Sweden). Collagen was purchased from Niko Bioscience (Horm: Bovine Achilles tendon, Tokyo). Other chemicals were of reagent grade. Preparation from as
the
these
source
10
min
platelet
of
at
platelets.
was
stored the
assay
were
equilibrated 8 glliter, liter, Timmons
with KCl NaH2PO4 and
method
plastic
tubes
of
HEPES
buffer,
0.2g/liter,
Hawiger
at
room
PRP pH
(38
and
3 •~
105:/ƒÊl PRP by
obtained
g
until platelets
g/liter, by
this
at
of 10
(GFP) a
final 1.0
150•~ and
Platelet (4).
PRP
determinations were
Sepharose-2B
prepared column
concentration g/liter,
of albumin
according
to
method
were
the
platelet-poor min.
Cronkite and
used
throughout
collected
for
use,
through
dextrose 0.9
was
and
a
used
addition
Brecher
containing
0.2g/liter,
Platelets
the
citrate was
centrifuged
1,640 •~
Gel-filtered
7.35,
HEPES
then
by
sodium Tokyo)
were
(PRP)
at
preparation
MgCl2-H2O giliter
mixed,
the
(w/v) Farm,
pipettes
temperature
4 hr.
the
3.8% Clea
plasma
described
within
portions
0.45
to
vol.
and
gently
centrifuging the
completed ml
tubes
was
adjusted
by
0.1 Nihon
Platelet-rich
was
capped
2.0
blood
by
into rabbit,
plastic
temperature.
counted
in
passing
Only
obtained
were
collected white
Citrated room
(PPP)
numbers
Blood ( Japanese
concentration
plasma
by
platelets.
rabbits
experiments.
for
of
of
drug-free
the not
NaCl 3.5
method responsive
g/ of
PLATELET
to
ADP
unless
rabbit
were
adjusted
platelets For
the
by
to
4
centrifugation IgG
ponents
the
concentration
for
90
then
Platelet
4A,
periment,
GFP
was
to
stirred
at
1,000
of
the
expressed
the
preparation at
suspension
and
final
for used
was
4
C
37
C
removed Fn,
plasma
added
37
C
com-
(34).
to
with
PPP
at
agitating
The
supernatant
a
adjust
to
pl
to
in
37
C
suspension to
reaction
p.m
or
either
In
the
the for
zero.
to
actual
10
min
ex-
prior
added,
procceed,
or
aggregometer to
and
the
units
whether
10 ƒÊg/ml,
100% PRP
Aggregation
examine
ex-
to
of
was
1
each
instrument 200
placed
warm
the
Tracer In
aggregation).
To
1.5
the of
cuvette
bacterial
for
Hema
aggregation.
solution
readjusted
of
of
(NKK
(zero to
the
adequate
7.35, at
Fibrinogen,
at
at
to
allowed
concentration
hr
then
effect
zymosan
platelet
A
was
were
the
incubated min
aggregation
normal a
PPP,
30
added
of
1
(Z-PPP).
used
being
22 ƒÊl
maximum
was
added
for
were
were
while
Then
see
pH
for
obtained.
to
was
transmission
GFP
rpm,
bacterial
at
in
aggregation.
0% or
cells
aggregometer
buffer
buffer, kept
of
aggregation.
mixture
was
Numbers
HEPES
was
above
PPP
100%
PRP
serum
added.
was Blood
and
tetra-channel
HEPES
of rabbits
noted
9,000 •~g
Tokyo)
testing.
density
was
A
represent
200 ƒÊl
aggregation
min,
The at
represent
used
addition from
platelet
mg/ml.
was
contraction.
zymosan-treated
and to
periments, and
as
the
buffer
935
fibrinogen,
components
PAT-4M,
PPP
transmission
20
HEPES
aggregation.
Pat
by drawn
clot
centrifuged
used
of
105/ƒÊl
complement
was
model
source
enterococci-induced
5
BY ENTEROCOCCI
blood
for
in
of
min,
obtained
a
facilitate
1,500 •~g
inactivate
the
to
dissolved
on To
3 •~
scrum,
C
at
were
as
to of
transfer
and
plasma,
preparation
before
AGGREGATION
the ADP
were platelet
or
collagen
respectively.
Original acetylsalicylic acid solution (100 mm) was dissolved in PBS containing 1( ETOH. EDTA and quinacrine2HCl were dissolved in 0.01 m-PBS, pH 7.4. Original indomethacin solution (100 mm) was dissolved using 0.1 N NaOH. The final pH of the PRP mixed with either EDTA, acetylsalicylic acid, indomethacin, or quinacrine was the same as those of original PRP. To minimize the dilution of PRP by the addition of the inhibitors, the volume of the inhibitors was one hundredth of PRP. In or
the
was
transfer
to
plasma at
inhibition
EDTA
the
perature.
After
From
a separate
bath,
300 ƒÊl mm ice.
natants Triton
release PRP
with
the
addition
tube
were
tube
obtained X-100
of
2
vol.
one
min
(37
of
GFP
several
KBq)
51
mm
were
of
for
the of
EDTA
in for
added 0.4%,
in
same 1
min
1.5
ml
2 min, to w/v,
5
was
min
at
DPO),
in
duplicate
and
min
labeled
room
tem-
recorded.
a 37
added
centrifuge
scintillation
of
10
(10 ƒÊl).
was
and
and
for
platelets 30
effects
kept
mixture
ml
before the
aggregation
intervals
12,000 •~g were
observe
components
serotonin
quinacrine,
temperature
ml)
plasma
[3H]
volume at
containing
(0.2
suspension,
up
acid,
room
Serotonin
bacterial
at
at
To
dehvdrogenase.
centrifugation toluene
10
aggregometer. the
a larger taken
centrifuged
after and
1 ƒÊCi
containing was
the
lactate
containing
portions
acetylsalicylic for
aggregation, with
and
PRP
in
platelet
formaldehyde The
the
environment
together
of
indomethacin, with
on
of serotonin
incubation
933
C
temperature
Assay
in
37
components
room
by
experiments,
preincubated
to tubes 50 ƒÊl fluid
radioactivity
C
water
50 ƒÊl
of
placed super(1
vol. was
936
V. USUI
measured
by
trols
used
were
serotonin
an
was
Aloka for the
Liquid
calculated %
Scintillation
determination from
ET AL
System
of total
the
LSL-753.
radioactivity.
Uncentrifuged Release
con-
of radiolabeled
formula:
Release= (S test-S
control/T control - S control)
100,
where S equals the radioactivity in the supernatant fluid and T equals the total radioactivity measured as disintegrations per minute (20). Lactate dehydrogenase (LDH) released from platelets during these experiments was determined by the method reported by Bergmeyer et al (2). A solution of Triton X-100 was used to lyse the platelets to determine the total amount of intracellular LDH. RESULTS
OptimumRatios betweenPlatelets and Enterococci In cases induced by strains SMU-37 of E. faecalis, SMU-138 of E. faecium,and SMU-197 of E. avium, maximal activities of platelet aggregation were obtained when the logarithmic numbers of bacteria were more than 8.49, 8.56, and 8.79 colonyforming units (c.f.u.)/ml, respectively (Fig. 1). In these results, optimum ratios were determined to be at 1.0, 1.2, and 2.0 in strains SMU-37 of E. faecalis, SMU-138 of E. faecium, and SMU-197 of E. avium, respectively. Thereafter, these ratios were used for platelet aggregation by each species of enterococci. Patterns of Platelet AggregationInducedby Enterococci The platelet aggregation induced by strain SMU-37 of E. ja ecaliv,SMU-138 of E. faecium, and SMU-197 of E. avium required approximately 1 min lag time and
Fig.
1.
Percentage
concentrations
aggregation of three
different
of platelets species
in platelet-rich of strains
plasma
of enterococci
upon
exposure
after
5 mm
E. faecalis SMU-37: 111,E. faecium SMU-138: H, E. az,iuntSMU-197.
to different incubation.
PLATELET
AGGREGATION
BY ENTEROCOCCI
937
(A)
(B)
(C)
Fig.
2. tions) of
E.
Aggregation of
patterns
rabbit
filecium
SMU-37
and of
3.6 •~
108,
2.7
108/ml.
platelets
and
E.
(C)
SMU-197
faecaliss, 6.1 •~
and exposed
SMU-138 10
c.f.u./ml,
of
serotonin
release
to
(A)
strains
E. of
aviuin. E.
(•œ, SMU-37
Bacterial faecium
respectively.
bars
indicate of
numbers and The
SMU-197 final
S.D.
Enterococcus (final
of
three
faecalis,
determina(B)
SMU-138
concentration) of
concentration
E.
in
avium
were of
platelets
3.2 •~
strains 108, was
938
Y.
then
were
mostly
enterococci, with
5.2%,
from
the
68.5 •}
platelets
E. faecium, To
of
periment. 282.3 •}
4.3
and
and
of
1 mm these
totally jected
of
cases
the
E.
E.
avium,
was
by
avium,
During
platelet
detected
after
strains,
of
the
intracellular
strains
SMU-37
respectively
platelets
were
10
mmn.after
measured
2).
enterococcal 7.0%
induced of
(Fig. platelets
by
of
the
min
incubation was
released SMU-138
2).
of
346.5 gb•}5.3 U/ml, of E. faecalis, total
by
incubation
E. faecalis,
action
commencement
The
10
serotonin
(Fig.
lysed
amounts of by SMU-37
respectively.
after
aggregation 1 min
amount
of
enterococci,
the
the
aggregation
ex-
500.0 •}9.4 SMU-138 of
LDH
of
in
U/ml, and E. faecium,
platelets
was
U/ml.
on
the
Platelet
of
strains
no
of
quinacrine
as
10
in
mm
Table
sufficient
cascade
platelet
10
mm the
ADP
(10 ƒÊM)-
either
aggregation
Indomethacin,
10
caused
were
aggregation
Incidentally,
inhibit
Acid,
and
Enterococci
indomethacin,
However,
platelet
by
arachidonate of
1. to
aggregation. the
on
reduction at
shown
Acetylsalicylic
Induced
inhibitors
marked
were
inhibited the
Aggregation
enterococci
examinations platelet
Apyrase,
various
experiments,
to
of
64.6 •}
was released in in cases induced
effects
induced
the
from
Ethylenediaminetetraacetate,
The
these
and
SMU-197
LDH U/ml
Quinacrine
these
each
6.2%,
was
1,103.3 •}21.9
Effects
With
ET AL
3 min
serotonin
whether
LDH
SMU-197
within
of
in
determine
activity
at
release
enterococci.
66.3 •}
of
terminated
the
USUI
by
mm
tested.
was
acetylsalicylic doses and
of
inhibitors
or
strain
by
acid,
collagen
EDTA
each
With
observed
used
and in
(100ƒÊg/ml)1 mg
of
apyrase/ml
enterococci
sub-
examinations.
Requirementsof Plasma Factors Using GFP, the requirement of plasma cofactors in the platelet aggregation caused by species of 3 strains of enterococci was examined (Table 2). Three of the strains of enterococci were not capable of inducing platelet aggregation with GFP alone. The addition of several plasma proteins, fibrinogen, Fn, IgG and heatTable 1. Effects of the addition of ethylenediaminetetraacetate, indomethacin, acetylsalicylic acid, quinacrine on the platelet aggregation by strains of Enterococcusfaecalis, E. faecium and E. avium
EDTA,
results
The
final
SMU-138 108
ethylenediaminetetraacetate;
The
c.f.u./ml,
are
ASA,
mean •}S.D.
of three
concentrations
of E. faecium, respectively.
of and
the
SMU-197 The
final
acetylsalicylic
acid.
determinations.
bacterial of E.
cells avium
concentration
of
were
strains 3.1 •~
of platelets
SMU-37 108
of
, 3.6 •~ was
2.7 •~
E. faecalis,
108,
and
108/ml.
6.1 •~
PLATELET
AGGREGATION
BY ENTEROCOCCI
939
Table 2. Effects of supplementation of plasma components on the aggregation of gel-filtered platelet by strains of Enterococcusfaecalis, E. faecium, and E. avium
The
results
H-serum treated were
mean±S.D. Z-PPP
platelet-poor as follows:
heated-serum (20% cells
are and
avium centration
plasma, fibrinogen
(20%
v/v), of
were
v/v),
3.1 •~
of
108,
of platelets
determinations.
respectively.
serum plasma
Enterococcus
three
heat-inactivated
(0.3%
platelet-poor
strains
of
mean
w/v),
(20%
3.6 •~
108,
was
2.7 •~
v/v), v/v).
6.1 •~
C,
30
(0.01%
The
108
final of c.f.u./ml,
min)
of the w/v),
zymosan-treated
SMU-138
and
(56
concentration
fibronectin
(20%
faecalis,
serum Final
IgG
zymosanproteins
(0.124%
platelet-poor
concentrations E.
and plasma
faecium,
of the and
respectively.
w/v), plasma bacterial
SMU-197 The
of E. final
con-
108/ml.
inactivated serum into GFP, was unsuccessful in producing complete The species of 3 strains were able to induce platelet aggregation with mented with PPP, but not with GFP supplemented with Z-PPP.
aggregation. GFP supple-
DISCUSSION
Among microorganisms possessing the ability to induce platelet aggregation in PRP, Gram-positive cocci, S. aureus, S. sanguis, and group A streptococci have been studied in detail (8, 18, 24, 28, 35, 36, 39, 43). Concerning the interaction between enterococci and platelets, more detailed investigations have been needed for comparison with other bacteria-platelets interaction, although Clawson et al (8) reported some properties of the platelet aggregation. Enterococci-induced platelet aggregation was not inhibited by the addition of the inhibitors to arachidonate cascade. Similar results were reported in strains of CNS (41), Streptococcussanguis (35, 36), Listeria monocytogenes(9), and Aspergillus fumigatus (31). Additionally, apyrase, which hydrolyzed ADP (29), has been reported to prevent platelet aggregation induced by viridans group streptococci (35). Similarly, our studies indicated that preincubation of platelets with 1 mg apyrase per ml completely inhibited the aggregation, suggesting that platelet aggregation by enterococci was mediated by the extracellular release of ADP from dense granules of platelet. Requirements of the plasma proteins in the bacteria-platelet interactions are reported in several bacteria. In group A streptococci (24)- and S. aureus (8, 28,40)induced platelet aggregation, fibrinogen was required. Also, in S. sanguis (35, 36) and Histoplasma capsulatum (10), both fibrinogen and IgG were required and complement components were reported to be essential for the aggregation by L. monocytogenes(9), A. fumigatus (31), or Candida albicans (33). From the present experimental results, complement components would possibly be required in the case of
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enterococci, since GFP supplemented with zymosan-treated PPP lost its reactivity with these organisms remarkably. Similar results were obtained in the case of various species of CNS (41). Further, supplementation of fibrinogen, Fn, or IgG into GFP had little effect in the platelet aggregation by enterococci. This indicated that the mechanism of enterococci-induced platelet aggregation would be different from those by S. aureus, Group A streptococci, or S. sanguis. In addition, the amount of fibrinogen added to GFP in this study was sufficient for the platelet aggregation by S. aureus (laboratory investigations), which is known to induce platelet aggregation in the presence of fibrinogen in the reaction system (8, 28). A majority of the experimental data concerning interaction between bacteria and platelets were collected from strains isolated from infectious foci (35, 36) and popular stock strains (8, 24, 28). Not only the strains used in these experiments, but also other strains randomly collected from fresh isolates induced platelet aggregation (data not shown). Additionally, it is known that there are species differencies between human and rabbit platelets (32). However, since rabbits are often used for the induction of experimental infective endocarditis (5, 6, 11-13, 37), these data obtained by rabbit PRP would be useful to explain how enterococcal endocarditis was induced. REFERENCES
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