For personal use only. by guest on July 10, 2011. ..... stoned at-. 70 #{176}Cin aliquots. Specific activity was. 50,000 to 70,000 cpm/g;. 3H-Fn was always.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
1986 67: 629-636
Platelet-leukocyte interaction: selective binding of thrombin- stimulated platelets to human monocytes, polymorphonuclear leukocytes, and related cell lines TW Jungi, MO Spycher, UE Nydegger and S Barandun
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From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
Platelet-Leukocyte of Thrombin-Stimulated Polymorphonuclear By T.W. The
association
gated.
of
using
and
then
that
stimulation
trations sion
fixed
surface and
weakly
binding more
(0.01
U/mI)
determinants
HE
cells,
platelets but
associate the
interaction
gations
of
has
not
pointed
with
leukocytes,
elucidated.
of platelets investigations
with
37
and
was
effect
monothis
investi-
of platelets
in adhen-
requirements of More recently, and monocytes properties2 and
metabolic activity5 of these cells, and recommendations were made for obtaining platelet-free lymphocyte2 on monocyte35 preparations. In addition, an increased association of platelets with monocytes and (PMNs) (so-called satellitism) particularly
in disorders
association
tiDn
with
only
and
which
pnovided
study
stimulated
In the
of the
platelets,
purified
suited
fibronectin ing
the
and binding
present
which
lymphocyte
are
collected within minutes part
at 4 #{176}C. Three of citrate
mmol/L
fuged
after buffer
of Na-citrate,
then
a
defined
insights
and
are
thrombin-
populations
and
stimulation in the presplatelets acquire surface manner
to
human cell an involvement
lines of
as ligands
mediat-
AND Citrated
blood
donations
METHODS
67.
thrombin-stimulated
macrophages
lines
kept
reduced fibronectin
T
platein
binding
myris-
(B cells. culture
of
could
for
thrombin-
be ruled
out
that mediate binding. Grune & Stratton. Inc.
temperature
without washed pended
to
of
18 parts
1.3%
formaldehyde
in
MTB,
pH
BSA, and chilled for one hour at 4 #{176}C. Fixed platelets twice with MTB, pH 6.5, without BSA and finally in MTB,
pH
7.4,
ofa
stored in the presence Prolonged storage reduced platelets
to associate
Preparation serving
with
of 0.05%
for
the
of
capacity The
for up
of
resus-
x l09/mL.
They
to two
days.
thrombin-stimulated
pellets
of the
same
buffy
coats
(see above) were the starting
preparation
preparation
Na-azide
6.5, were
monocytes.
of monocytes.
for platelet
of 1.1
concentration
were
monocytes.
They
were
washed
mate-
twice
with
buffer and centrifuged at low speed (ten minutes, 200 g) to remove most of the platelets. The pellets were then resuspended in washing solution [phosphate-buffered saline (PBS) containing 13 mmol/L of EDTA, 0.1% wt/vol sodium azide, 1% wt/vol modified gelatin (Physiogel, SRK)J and subjected to centnifugation on Ficoll-Hypaque for isolation of mononuclear cells.9 These were chilled to 4 #{176}C and washed two more times in the cold for thorough removal of platelets. Mononuclear cells (up to 50 x 106) were layered on top of discontinuous density gradients of Percoll with densities ranging from 1.066 to 1.060. After centnifugation (600 g for 30 minutes at 4 #{176}C), the cell bands of a density of < I .060 and I .062
were
From
individual
bank were used. Buffy coats were obtained collection by centnifugation at I ,200 g for five parts (102 4.8
of buffy mmol/L mmol/L
coats
were
of NaC1, of glucose,
at room
Vol
cell
U
days
[MES], 3 mmol/LofKCI,4.8 mmol/Lofglucose, 130 mmol/L of NaCI, and 2 mg/mL bovine serum albumin (BSA), pH 6.5). Gel-filtered platelets were counted on a TOA platelet counter (TOA Medical Electronics Co. Ltd. Japan) and adjusted to 2.5 x l0 platelets per milliliter. For activation ofplatelets with bovine thrombin (61 NIH-U/mg; Hoffmann La Roche, Basel, Switzerland), the pH of platelets was adjusted to 7.4. One part of platelets was then incubated with four parts of modified Tyrode’s buffer (MTB; Tyrode’s buffer containing 2 mmol/L of MgCI2, and 2 mg/mL BSA but no CaCI2) (pH 7.4) and one pant of thrombin in 0.1 5 mol/L of NaCI for 30 minutes at 37 #{176}C without stirring. The incubation mixture was then added at room
95% cells excluded Trypan blue. Culture of mononuclear phagocytes. Mononuclear cells were isolated from buffy coats as described, except that sterile conditions were used and washing solution was replaced by PBS containing 0.3 mmol/L of EDTA.’3 The cells were resuspended in HEPESbuffered RPMI 1640 containing 2% vt/vol pooled heat-inactivated (30 minutes at 56 #{176}C) human group AB serum and placed in 75-cm2 culture flasks ( Falcon, Oxnard, Calif). After one hour of incubation, nonadherent cells were removed by washing with RPMI 1640, and adherent cells (‘80% to 95% monocytes) were cultured exactly as described.’4 Following an overnight culture with RPMI 1640 contaming 15% heat-inactivated group AB serum, the cells were dislodged from the flask and put in bags made of teflon foil (type 100 A; Angst & Pfisten, Zurich, Switzerland). The sealed bags were kept at 37 #{176}C in an atmosphere of 5% CO2 for one week, by which time monocytes had matured to macrophages. Culture
ofestablished
cell lines.
Established
human
cell
lines
collection
Berne,
Research,
Platelet-leukocyte
interaction
test.
Fifty
microliters
of
the
suspension were admixed to 50 il of RHA and 100 ol of cell suspension adjusted to 3 to 5 x 10’ cells/mL in 12 x 75 mm Minisorp tubes (Nunc, Roskilde, Denmark). These were incubated for 30 minutes (unless otherwise indicated) at 37 #{176}C or 0 #{176}C under platelet
rocking
conditions.
Aliquots
were
then
placed
in a Neubauer
cham-
bar and immediately scored for the association of more than two platelets per cell. Another aliquot was mixed with Turk’s solution and then evaluated for the platelet-cell association. In each case, 200 to 400 viable single cells were scored. In some experiments, 51Crlabeled platelets were allowed to interact with monocytes, and the incubate
was
separated
into
free
bound platelets, using zonal rate procedure allowed determination associating with leukocytes. Assessment lets. gelatin
was
Fn
of binding was
coupled pure,
as
platelets
and
centnifugation of the mean
offibronectin
leukocytes
Interaction
which
carrying
over sucrose.’7 The number of platelets to gel-filtered
band,
representing
plate-
purified by affinity chromatography on modified to sepharose 4B,is lyophilized, and stored at 4 #{176}C. It determined by gel electrophoresis under reducing
a
molecular
of Fn
with
gelatinized
weight
erythrocytes.
of
Erythrocytes,
were tanned and gelatinized essentially were incubated with graded amounts
T-G), conditions
used,
6%
of the
added
Fn
as descnibed,#{176}(E’of Fn. Under the
became
bound
to E’-T-G
as
radiometrically. E’-T-G charged with subagglutinating Fn doses ( 10 g/mL) were used in rosette formation assays. Agglutinating doses were used in hemagglutination inhibition tests to determine the active titer of goat anti-human Fn antibodies (punassessed
chased
from
Calbiochem,
Behning
Diagnostics,
La Jolla,
Calif).
RESULTS
Interaction
of
monocytes.
thrombin-stimulated,
Human
fixed
stimulated
platelets
platelets
with
with
thrombin
and
then fixed with formaldehyde were assessed for their capacity to adhere to purified, platelet-poor, autologous monocytes. Although unstimulated platelets had little tendency to associate with monocytes, the platelets acquired the capacity to adhere amounts
to monocytes upon stimulation with increasing of thrombin (Fig I ). This adherence was observed microscopically whether the platelet-monocyte mixtures were evaluated unstained immediately after incubation (30 minutes and then proportion
at
37 #{176}C), stained
with
stained. At 0.02 of platelet-bearing
U/mL
additional monocyte
numbers of platelets when the thrombin
Turk’s
solution,
of thrombin, monocytes was were observed concentration
or chilled the maximal reached, but
per responsive was further
increased.
The
time
course
of the
platelet-monocyte
interaction
at
0 #{176}C and at 37 #{176}C, using formaldehyde-fixed viously stimulated with 0.02 U/mL of thrombin,
platelets preis shown in
Fig was
monocytes proportion of
2; a maximal proportion observed after I 0 to
platelet-bearing the interaction mmol/L) influence Role
(Fn)
one
AL
22Q,000, was observed. Fn was labeled with 3H by reductive methylation’9 and stoned at - 70 #{176}C in aliquots. Specific activity was 50,000 to 70,000 cpm/g; 3H-Fn was always centrifuged for five minutes at I I ,000 g and at room temperature before use. To 50 zL ofgel-filtered platelets (2.5 x 109/mL), I 50 zL of MTB pH 7.4, 50 ML of 3H-Fn in MTB, and 50 sL of thrombin in 0.15 mol/L of NaCI were added, and the tubes were incubated at 37 #{176}C for 30 minutes without stirring. Then, I 50 sL of a mixture of dinonylphthalate/dibutylphthalate (I :2.56) was underlayered, and the tubes were centrifuged at I 1,000 g for 30 seconds, separating the platelets from the water phase through the phthalate mixture. Aliquots of the supernatants were counted, and the tubes were inverted. After removing traces of supennatant using absorbent paper, 150 ML of 0.5% Triton X-l00 and two drops of xylol were added to the sediments prior to solubilization with a sonifier (Branson Sonic Co. Danbury, Conn). Solubilized platelets were transfenred to a counting vial, and radioactivity was measured in a Packard TniCarb 2450 Liquid Scintillation Spectrometer (Downers Grove, Ill).
of
of the Institute for Clinical and Experimental Cancer Switzerland, were used. These included T cell lines MOLT 4 and Jurkat, B cell lines Raji and Namalva, myeloid line 1(562, and myelomonocytoid lines U 937 and HL 60. They were cultured in RPMI 1640 supplemented with HEPES (10 mmol/L), glutamine (2 mmol/L), penicillin (100 lU/mL), streptomycin (100 .tg/mL), sodium pyruvate (I mmol/L), and 20% fetal calf serum (FCS) for obtaining rapid growth and 10% FCS for maintenance and slow proliferation, respectively. For induction of terminal differentiation of U 937 and HL 60 cells, 4$-phorbol 12-mynistate I 3-acetate (PMA) was used.iSi6 Two and a half million cells in 25-cm2 area flasks (in 10 mL) were treated with PMA (16 mmol/L); 20 to 44 hours later, cells were harvested for use in the platelet interaction test. the
Only
ET
monocytes was reproducibly higher when occurred in the cold (0 #{176}C). Sodium azide (20
added
to the
monocyte-platelet
monocyte-plate[et of
of platelet-bearing I 5 minutes. The
plasma
mixtures
did
not
association.
fibronectin
(Fn)
in
platelet-monocyte
previous findings that Fn binds to stimulated platelets222 and that monocytes express cell surface receptors for this protein,20’23 a role of plasma Fn in the platelet-monocyte interaction was sought. interaction.
In
view
of
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
PLAThLET-LEUKOCYTE
INTERACTION
631
so
0 U,
a,
40
0 0I
S
C
S 0.
Ca
a,
93 a,
a,
0
15
35
45
60
Ca
0.
Twne (mm)
‘0
1O
Thrombin Fig
1
fixed. than
two
(U/mI)
platelets.
to monocytes. platelets
10-0
Concentration
Thrombin-stimulated
.
bind
10.
10’
The
(means
then
percentage i SD).
±
as
Fig 2. Kinetics of the binding of thrombin-stimulated and control platelets to monocytes in suspension. Incubation of monocytes and stimulated platelets was performed at 37 ‘C (#{149}) and 0 ‘C (U); incubation of monocytes and control platelets was performed at 37 ‘C (0). formaldehyde-
of cells assessed
bearing
more
microscopically.
3H-Fn
after 60-minute incubation in medium RHA. is plotted as a function of the thrombin concentration used for platelet stimulation. In addition. the degree of platelet association per single monocyte was scored arbitrarily from - to + + + and is indicated in the top row
panel.
stained
Left
with
Turk’s
microscopic
First, stimulated
unstained
solution;
preparations;
right
panel.
middle
tubes
chilled
panel.
stimulation between exogenous
cells
in ice before
examination.
the
agreement mum when mmol/L with 2.5
panel.
of exogenous Fn amounts ofthrombin
with previous work,2’ 0.2 U/mL thrombin
maximal
bound to platelets was estimated. In
Fn binding reached a maxiwas used in the presence of 2
while 1 S mg/mL of Fn was allowed to react platelets per milliliter. Under conditions of
platelet-monocyte
association
thrombin-stimulated platelets rather
Table
platelets native
than
1 . Influence
of Fibronectin
(0.02
and then platelets,
U/mL
formaldehydesaturation
(Fn) and Anti-Fn-Ant
Thrombin
1
of
bodies
platelets
on during
of
ibodies
tination
on Association
-
0.1 U/mL
-
0.1
Fibronectin -
300jg/mL -
3O0zg/mL
8g/mL
-
-
8 og/mL
U/mL
8ig/mL
0. 1 U/mL 2
Platelets
-
0.1 U/mL
300 300
8 g/mL
thrombin
concentra-
the
thrombin
interaction
was assessed. the proportion
Fn (data
were
not shown).
anti-Fn
antibodies were co-incubated platelets and monocytes. The
nonreactive
with
monocyte
of ESTG
mediated
of Stimulated
and Monocytes Anti-Fn
by Fn, yet they
Gel-Filtered
of Formaldehyde-Fixed
Antiserum -
Fc receptors,
phagocytosis shown to block
Platelets
failed
and Monocytes Cells
After Incubation (30 mm at 37
(%) ‘C)
1 SD
±
9±3
-
12±4 51
±
10
-
49
±
11
7±0
tg/mL
-
10
±
3
-
-
41
±
6
g/mL
-
42
±
3
-
-
-
-
-
-
to inhibit
-
-
-
1:320
13±5 9±2
U/mL
-
-
-
43
±
14
0.02
U/mL
-
-
1:320
44
±
6
0.02U/mL
-
-
1:640
48±1k
0.1U/mL
-
-
-
-
1:320
42
±
1
-
-
1:640
46
±
8
U/mL
0.1 U/mL from
control
(first
line)
(P
90%. Using fixed
was
bearing monocytes (Table when platelet-monocyte
amount by various
MgCI2 x l0
binding
tion, but binding was diminished by 40% to 60%. Second, the effect of Fn added either during
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
JUNGI
632
the association I , experiment Role
ofstimulated 2).
of
IgG
in
the
possibility
that
IgG
monocyte
Fc receptors
bin-stimulated trations and
platelets
was
tested
and
monocytes
platelets
IgG,
25%
cation-chelating
serum
as
well
(not
requirementsfor
agents
minutes monocyte
prior to fixation adhesiveness in
the
absence
preincubated then stimulated
manner
as did stimulated
contrast, interaction
formation presence
bound
largely In
asso-
by including stimulation
phase. thrombin
EDTA
(Table
2, experiment
platelets
ifeither EDTA of stimulated
certain
to
treatment
of experiments,
platelets
platelet-PMN in the presence
I).
to EDTA.
that
the
optimally
Binding
of
with
isolated by means
In
macnophages despite their macrophages
were included during the with monocytes, even
EDTA.
capacity
adhesive
of
purified
tested. PMNs platelets, but were required
to PMNs
was also observed (Table 3).
thrombin-stimulated
Freshly macnophages
10%) did so however, was
(Fig
of platelets pen PMN was number binding to monocytes.
association of EDTA
cells.
lympho-
to bind stimulated platelets. (except Percoll density-gradient bound stimulated platelets, only
to a subsequent set
In addition, the number tently smaller than the
in 5 mmol/L of behaved in the same
not exposed
or EGTA platelets
capacity fractions monocytes
resistant another
for rendering
Simultafor 30
yielded no significant reduction in as compared with platelets stimufor 30 minutes with thrombin,
possible
PMNs to bind stimulated platelets was appeared to be capable of binding stimulated higher thrombin concentrations (0.1 U/mL)
shown).
was tested thrombin
it was
AL
absent from the tested density fractions to bind stimulated platelets. Therefore, cell fractions were also washed and
a small subpopulation of lymphocytes (5% (Fig 3). Such lymphocyte-platelet interaction,
of 8
platelets,
of IgG. Likewise, in the presence of
interaction to EDTA and
of
Platelets EDTA,
tested for their Although in all fraction II), most
at concen-
platelet-monocyte
cations either during
or during the platelet-monocyte neous exposure of platelets
lated
cells
The role ofdivalent
ciation.
the
± 14% in the absence association occurred
heat-inactivated cation
In
7% mononuclear
±
with
throm-
IgG
rosette
completely.
to 43%
Divalent
with
preparation,
cyte subpopulations also had the capacity lymphocyte-enriched
The
interacts
by co-incubating
Fc-receptor-mediated
54%
as compared platelet-monocyte
platelets24
monocyte
(Table
interaction.
with
enythrophagocytosis
mg/mL
monocytes
platelet-monocyte
associating
blocking
with
ET
at 0 #{176}C, but not
platelets
monocytes of teflon
had little tendency increased surface eagerly ingested
were bag
4).
consisThe
to
cultured
further matured to cultures.’4 Mature
to bind stimulated platelets, area (Table 4). The same erythrocytes sensitized with
fewer monocytes bound such platelets than occurred if monocytes were allowed to interact with unstimulated platelets in Ca2-sufficient and Mg2-sufficient medium (Table 2, experiment 2), suggesting that the interaction of stimulated
IgG or with 1gM and complement (not shown). Established cell lines of various origin were also tested for their capacity to bind thrombin-stimulated platelets. Table 4 shows that the
platelets
had
stimulated platelets. The tendency to bind platelets was also Ca2 dependent and restricted to thrombin-stimulated platelets. Preferential binding was observed with cells in exponen-
the
tial
with
Binding
of
types
derived
little
tendency
monocytes
is strictly
Ca2
thrombin-stimulated from
human
to bind Table
platelets blood.
Effect
to
of Divalent
Cations
Second t
-
2
3
*5
mmol/L
(experiments
cell lines
when
. .
-
-
Thrombin
-
EDTA
+ Thrombin
(0.02
EDTA
+ Thrombin
(0.02
-
(0.02
to
Platelet-Monocyte Incubate at 37 ‘C
-
U/mL)
HL 60) bound
differentiation
Platelets Addition
EDTA
(U 937 and
terminal
of Thrombin-Stimulated
30 mm 37 ‘C
-
EDTA*
tSignificantly SignificantIy
on Association
growth;
of Platelets
First 30 mm at 37 ‘c
1
cell
contaminating
Pretreatment
Exp.
various
if platelets
Even
to lymphocytes 2.
monocyte-like
dependent.
thrombin-
of U 937
and Monocytes Platelet-Bearing
.
15±4 16±2
43
±
14t
U/mL)
-
47
±
6t
U/mL)
-
47
±
4t
-
33
±
8f
42 53
±
12t
±
4t
Thrombin
(0. 1 U/mL)
-
EDTA
+ Thrombin
(0.1
U/mL)
-
EDTA
EDTA
+ Thrombin
(0.1
U/mL)
-
-
-
-
-
EDTA
-
-
-
EGTA
9±3 6±2 13±3
l0
-
Thrombin
(0.1
-
Thrombin
(0. 1 U/mL)
EDTA
11 ± 5
-
Thrombin
(0. 1 U/mL)
EGTA
6 ± 2
-
Thrombin
(0.02
-
Thrombin
(0.02 U/mL)
-
Thrombin
(0. 1 U/mL)
-
52±16
-
Thrombin
(0. 1 U/mL)
EGTA
2
1 and
higher than negative lower than positive
3) or 4 mmol/L
(experiment
U/mL)
-
U/mL)
-
EGTA
2).
control (first line of this experiment) control (first line of this experiment)
(P < .0 1). (P < .0 1).
Cells
After 30 mm (%) (Mean ± 1 SD)
-
-
and
51
±
49±1 9 ±
±
2* 1*
HL
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
PLATELET-LEUKOCYTE
%
INTERACTION
633
% Lymphocytes
MonoCytes 60
80
20
40
20
40
60
± 80
+
.
±±
100
Mo
++,++
+
PMN
+4+
100
U
0
bH
0-
0 a,
E
a,
a,
NF
a
:
40 .,,,,,..,,,7
a
iJ
0.
#
:V
20
V/////////7/iW/////i
P1
0
10’
10-’
Thrombm
P2 a
P3
Fig
P4
monocytes,
:
Fig
3.
Binding
lymphocytes
stippled
of
stimulated
in unseparated
mononuclear ered from
show
the
platelets
mononuclear
cells (NF). and a discontinuous
bars
in
different
Percoll
proportion
by cells
monocytes
(U).
and
in nonadherent
density
fractions
gradient.
The
of monocytes
and
recov-
hatched
and
lymphocytes.
respectively. in each fraction; the open bars show the proportion of monocytes and lymphocytes. respectively. within the whole fraction binding more than two platelets in the absence (a) or presence (b) of 5 mmol/L of EDTA.
60 was
induced
stimulated toid cell platelets.
by PMA,
the capacity
of these
cells
to bind
platelets gradually waned. No other nonmonocyline was capable of binding thrombin-stimulated Thus, the interaction of stimulated platelets with
cellular surfaces is restricted phagocytes in certain defined that
of established
cell
lines
to surfaces of professional stages of differentiation and closely
related
to
to phagocytes.
between
platelets
ill-defined. Although platelets are to get involved in areas in which brane has been exposed, phagocytes essentially determine the cellular of inflamed tissue, and platelets inflammation arthritis
such patients.25
platelets,26 producing
and and
arachidonic receptor
acid spectra
common
with
often
contaminate
as
the
stimulated
phagocytes
soon follow.’ molecular too are found
generate
tissue
(U/mI)
platelets.
then
formaldehyde-
mediators
metabolism.5’27’28 of phagocytes
Furthermore, and platelets
other.2#{176}24293#{176} In the preparations
of phagocytes,
procedures
are
necessary
monocytes
and
PMNs
lead to aggregation hyde. This fixative
in a specific
and
to prevent
not unexpected associated with
reproducible
with gel-filtered under conditions
and that were then fixed agent, which cross-links
determinants, leaves various including antigenic properties,32
fashion. platelets that that did not
with formaldecertain surface
platelet properties unaffected, the capacity to bind myxovi-
ruses,33 reactivity in lectin-induced involvement in aggregation when
aggregation tests,34 and precoated with fibrinogen
and co-incubated with native platelets and thrombin.35 Yet formaldehyde-fixed platelets fail to aggregate upon stimulation with thrombin34 and thus are suitable for studying their interaction Platelets
as single acquired
3.
.
Incubation Prior
of
rheumatoid affecting
the surface have much in
laboratory,
special
Binding
particles with leukocytes. adhesiveness to monocytes
of Stimulated
Gel-Filtered
when
Platelets
at 37 ‘C and at 0 ‘C. but Not in the Presence
stimu-
to PMNs of EDTA
are
Phagocytes composition in areas
of
and
The finding is based on studies were stimulated with thrombin
cellular elements basement mem-
and
synovial
Phagocytes
and
the first vascular
both phagocytes and platelets are capable of responding to a variety of substances of the
each
concentration
of platelets with monocytes.3’5’3’ of these considerations, it was found that, in vitro, platelets
occurs
interactions
of
10’
10
association In view when we
Table
DISCUSSION
The
Binding
iF’
fixed. to PMNs as a function of the thrombin concentration used for stimulation. Purified PMN were incubated for 30 minutes at 37 ‘C and then assessed after being stained with Turks solution (#{149}). For comparison. the proportion of platelet-bearing monocytes in a parallel experiment with the same platelets is shown (0). The degree of platelet-association per single PMN or monocyte. respectively. is expressed in arbitrary scores from - to + + and is indicated in the top panel.
W1711A
P5
4.
1
.
‘
a
platelets particularly
of Platelets to Fixation
Buffer Thrombin
Thrombin
0.1 U/mL 0. 1 U/mL
Buffer
ThrombinO.1 U/mL Thrombin 0. 1 U/mL PMNs,
Conditions During Platelet- PMN Interaction EDTA
Significantly respectively).
±
O’C
-
0 ‘C
5 mmol/L
37’C
-
37
5 mmol/L
Unstained
Turk-stained
5±2 54
0 #{176}C 8
-
±
6±3
18t
41
± 0
14±4 ‘C
61
37 ‘C
5
±
7*
17 ± 7 14±
10
±
4t
40
±
4
15 ±
±
14t
1
leukocytes.
1 SD of triplicate different
Temp.
-
polymorphonuclear
Means
Platelet-Bearing Cells 1%)
(P
evaluations
