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Dynamics of Leukocyte-Platelet Adhesion in Whole Blood
By Henry M. Rinder, Jayne L. Bonan, Christine S. Rinder, Kenneth A. Auk, and Brian R. Smith
The dynamics of leukocyte-platelet adhesion and plateletplatelet interaction in whole blood are not well understood.
Using different platelet agonists, we have studied the whole
blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets t o
participate in platelet adhesion, and the ligands responsible
for adhesion. When platelet aggregation was inhibited by the
Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of
whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked
increase in the percentage of monocytes and neutrophils
(PMN) binding platelets, as well as an increase in the number
of platelets bound per monocyte and PMN. Lymphocytes
were unaffected. Monocytes bound more platelets and at an
initially faster rate than PMN. This increase in monocyte and
PMN adhesion t o platelets was completely inhibited by the
blocking monoclonal antibody (MoAb), G1, t o GMP-140.
When the combination of epinephrine and adenosine diphosphate (epi1ADP) was used as a less potent agonist in the
presence of RGDS, GMP-140 expression per platelet was
less, and while monocyte-platelet conjugates formed, PMN-
platelet conjugates did not. With epiIADP in the absence of
RGDS, there was an immediate, marked decrease in the
percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet
aggregates. As these platelet aggregates dissociated, the
percentage of monocytes and PMN with adherent platelets
increased, with monocytes again binding at a faster initial
rate than PMN. This recovery of monocyte and PMN adhesion t o platelets was also inhibited by the G1 MoAb. We
conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a
competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used;
and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets t o readhere t o
monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and
possible links between coagulative and inflammatory processes.
8 1991by The American Society of Hematology.
I
of blood cell adhesion and the possible alterations in
adhesion that may occur in pathologic states.
T HAS BEEN previously demonstrated that thrombinactivated platelets adhere to isolated fractions of monocytes and neutrophils (PMN).’ This adhesion is mediated
primarily through expression of platelet activation dependent granule external membrane/granule membrane protein 140 (PADGEM/GMP-140),2,3also designated CD62,
on the platelet surface. Using a newly designed sensitive
assay, we have also demonstrated that even “unactivated”
platelets (not expressing GMP-140) are capable of binding
to isolated monocyte and PMN fractions at relatively low
numbers of platelets per le~kocyte.~
However, the dynamics
and interaction of homotypic (platelet-platelet) and heterotypic (leukocyte-platelet) adhesion in whole blood are not
well understood. In addition, the relative affinities of
competing leukocyte subsets (monocytes, PMN, and lymphocytes) for adhesion to platelets in whole blood is not
defined. Such investigations can clarify the normal physiology of adhesion between inflammatory and hemostatic cells
and allow future studies of both the functional significance
From the Departments of Laboratory Medicine, Internal Medicine
(Hematology), and Anesthesiology, Yale University School of Medicine, New Haven, CT; and the Maine Cytomehy Research Institute,
South Portland, ME.
Submitted March 29, 1991; accepted June 3,1991.
Supported by grants from the National Institutes of Health
(HL08226) and the American Association of Blood Banks. B A S . is a
Scholar of the Leukemia Sociew ofAmerica.
Address reprint requests to Henry M. Rinder, MD, Department of
Laboratory Medicine, Yale University School of Medicine, PO Box
3333,333 Cedar St, New Haven, CT 06510.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191 / 7807-0004$3.00/0
1730
MATERIALS AND METHODS
Antibodies. All monoclonal antibodies (MoAbs) were used as
purified whole IgG, or IgM, except for the anti-GMP-140 MoAb
designated “Gl,” which was used both whole and as an Fab,
fragment. All experiments included irrelevant isotype-specific
mouse MoAbs as negative controls. The MoAbs 1E3’ and G1’ (the
latter kindly donated by Dr R. McEver, University of Oklahoma,
Oklahoma City) are both specific for GMP-l40/PADGEM, but
only G1 inhibits adhesion of stimulated platelets to neutrophils and
monocytes. SZ26and P2’ (AMAC, Inc, Westbrook, ME) recognize
GPIb and GPIIb/IIIa, respectively. Anti-CD45 (HLE; BectonDickinson ImmunocytometrySystems, San Jose, CA) recognizes a
CD45 isoform present on PMN, monocytes, and lymphocytes but
neither erythroid cells nor platelets? The MoAb MMA (Leu-M1,
Becton-Dickinson) recognizes CD15.9
Thrombin stimulation of whole blood. Blood was drawn from
normal human volunteers into a final concentration of sodium
citrate, 0.38%. To prevent clotting through fibrinogen-GPIIb/IIIa
binding, blood was then incubated with Arg-Gly-Asp-Ser(RGDS)’”
peptide (Peninsula Laboratories, Inc, Belmont, CA) at a final
concentration of 1 mg/mL. The sample was stimulated (time 0)
with diluent or bovine thrombin (Sigma, St Louis, MO) at a final
concentration of 0.1 U/mL at 37°C. The blood was kept at 37°C for
the entire assay. Preliminary studies demonstrated no differences
between samples that were agitated or stationary during the assay.
For blocking studies with MoAbs, G1 Fab,, 1E3, or isotypematched control MoAb were preincubated with whole blood for 5
minutes before the addition of agonist. The concentrations of
MoAbs used were found to be saturable at 1 KglmL.
Epinephrine-adenosine diphosphate (epi/ADP)stimulation of whole
blood. Blood was drawn into porcine heparin at a final concentration of 14 U/mL or citrate as described previously. In some
experiments, RGDS was added at a final concentration of 1
mg/mL. One milliliter of blood was then incubated at 37°C with
diluent or epinephrine HCI (Parke-Davis, Morris Plains, NJ) at a
final concentration of 1Kmol/L for 10 minutes, followed by diluent
or ADP (Sigma) at a final concentration of 5 pmol/L at 37°C.
Blood, Vol78, No 7 (October 1). 1991: pp 1730-1737
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1731
WBC-PLATELET ADHESION IN WHOLE BLOOD
Addition of ADP was designated as time 0. Blood samples were
kept at 37°C throughout the experiment. Studies performed at
22°C demonstrated similar results. MoAb blocking experiments
were performed as above.
Sample preparation. At each sampling time point, 100 pL of
blood was removed and incubated with paraformaldehyde (final
concentration 1%) for 60 minutes, followed by addition of 1/8:vol/
vol Tris-glycine solution (250 mmol/L Tris and 500 mmol/L
glycine), as described previously? Preliminary experiments using
isolated cell fractions demonstrated that paraformaldehyde h a tion did not significantly change the percentages of leukocytes
binding platelets. After 15 minutes, samples were washed three
times in Tyrodes-HEPES (TH) buffer” (HEPES 5 mmol/L, NaCl
140 mmol/L, KC12.7 mmol/L, dextrose 5.5 mmol/L, NaH,PO, 0.42
mmol/L, and NaHCO, 12 mmol/L, pH 7.4). Samples were then
resuspended in 200 pL of TH buffer. Each sample was divided in
half, one part for fluorescent labeling of leukocyte-platelet conjugates and the other half for labeling of platelets for GMP-140 and
measurement of platelet aggregates.
Fluorescence labeling. For determination of the percentage of
platelets expressing GMP-140 and the percentage of platelet
aggregates, 100 pL of sample was incubated with fluorescein
isothiocyanate (F1TC)-anti-GPIb (SZ2; AMAC) or FITC-antiGPIIb/IIIa (P2; M A C ) and biotinylated anti-GMP-140 (1E3)
MoAbs for 20 minutes, then washed and resuspended in 100 pL
TH buffer. The sample was then incubated with saturating concentrations of phycoerythrin (PE)-avidin (Becton-Dickinson) for 20
minutes, washed, and resuspended in 300 pL TH buffer for
fluorescence-activated cell sorter (FACS) analysis.12
For leukocyte-plateletconjugate analysis, 100 pL of sample was
incubated with saturating concentrations of FITC-conjugatedantiCD45 and biotinylated anti-GPIb or anti-GPIIb/IIIa MoAbs for 20
minutes, then washed and resuspended in 100 pL TH buffer.
PE-avidin labeling and preparation for FACS analysis was performed as described previously.
Flow cytometry. Samples were analyzed on a FACScan flow
cytometer (Becton-Dickinson)with data stored in list mode files.
The determination of the percentage of platelets expressing
GMP-140 and the percentage of platelet aggregateswas performed
as previously de~cribed.’~”
The determination of leukocyte-platelet conjugates was performed as recently described.‘ In brief, live gating on leukocytesized events was performed to exclude single platelets using a
combination of forward and side scatter and positive anti-CD45
fluorescence.Leukocyte subsets, ie, monocytes, PMN, and lymphocytes, were distinguished from one another on the basis of these
characteristics. An isotype-matched control antibody was used to
set a threshold (99% of events below threshold) for positive
platelet marker fluorescence. The percentage of platelet-markerpositive conjugates represents the percentage of leukocyteswith at
least one bound platelet? In addition, the mean platelet marker
fluorescence per bound leukocyte was determined. This has been
previously demonstrated to correspond in a semi-quantitative
fashion to the number of platelets bound per le~kocyte.~
Previous
studies with fractionated cell populations have demonstrated that
this fluorescence assay detects leukocyteswith only a single platelet
bound per leukocyte? Using either GPIb or GPIIb/IIIa as the
platelet marker was found to yield equivalent results for both
percentages of leukocyte-platelet binding and relative platelet
fluorescence per bound leukocyte. Inert bead and image analysis
studies have also established that measured leukocyte-platelet
conjugates are not the result of simultaneous passage of unbound
platelets and leukocytes through the detection chamber, confirmingthat this assay measures only platelets bound to leukocytes.
A n ACAS 570 microscope-based laser cytometer (Meridian Instru-
ments, East Lansing, MI) was also used to analyze whole blood
preparations and confirmed that dual fluorescent events represent
leukocyte-plateletconjugates in stimulated and unstimulated samples.
All experimentswere performed at least three times. Each of the
figurespresents data from a single representative experiment.
RESULTS
Studies of unstimulated whole blood. Heparinized and
citrated samples showed identical results t o one another
and were analyzed together. In both heparinized and
citrated whole blood transferred immediately to fixative
after drawing, a significant portion of leukocytes at
“baseline,” ie, before any manipulation, have at least one
adherent platelet associated with them when assessed by
the flow cytometric assay. This “baseline” percentage of
leukocytes binding a t least one platelet was averaged for 10
experiments, as shown in Table 1. Monocytes had the
largest percentage of unactivated “baseline” binding, followed by PMN and lymphocytes. These numbers compare
favorably with previously reported results of binding of
unstimulated isolated cell fractions, except for monocytes,
which showed a lesser bound percentage in whole blood
compared with recombination of isolated cell fraction^.^
These percentages were confirmed using dual immunofluorescence and direct visualization with the ACAS 570 laser
cytometer. When unstimulated whole blood samples (in
heparin or citrate) were incubated with 1mg/mL of RGDS,
the percentage of leukocytes with bound platelets was
halved. Using platelet fluorescence as an estimate of the
number of platelets bound per leukocyte, we found that the
number of unactivated platelets per leukocyte a t baseline in
whole blood averaged one platelet per white blood cell,
similar t o previous studies with isolated cell fractions.
Thrombin stimulation of whole blood. Citrated whole
blood was incubated with RGDS peptide at a final concentration of 1 mg/mL t o prevent formation of platelet-platelet
aggregates. After addition of diluent or thrombin (0.1
U/mL a t time 0), samples were studied a t various time
points (Fig 1) while incubating a t 37°C. Fig 1A demonstrates the percentage of monocytes, PMN, and lymphocytes binding platelets over time after addition of thrombin.
Compared with the unactivated sample in Fig 1B (diluent
alone added a t time 0), thrombin stimulation caused a
significant increase in the percentage of monocytes and
PMN with adherent platelets but no change in lymphocyteplatelet adhesion. The initial slope of the curve over the
Table 1. Leukocyte-Platelet Adhesion in Whole Blood
Baseline % of Leukocytes Binding Platelets*
Anticoagulant
Heparin or
citrate
alone
Heparin +
RGDS or
citrate
RGDS
Monocytes
Neutrophils
Lymphocytes
45.25 2 3.20
38.75 2 2.19
36.50 2 2.20
22.29 2 5.23
17.43 2 2.50
15.29 2 5.20
+
*Mean
2
standard deviation of the mean.
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1732
RINDER ET AL
80
80
Table 2. Leukocyte CD15 FluorescenceAfter Agonist
70
Agonist
Monocytes
PMN
Lymph
60
Diluent
ThrombinO.l U/mL
10.43 f 0.16
10.01 f 0.20
373 f 1.50
380 f 1.71
1.10 2 0.12
1.08 2 0.10
Mean CD15 Fluorescence'
70
. .....
::
,
.
60
W
_I
c3
2
m
K
50
+
40
40
8
30
30
4a
IQ
z
0
Q
20
20 K
10
10
0
80
'Ratio to control fluorescence 2 standard error of the mean.
W
50
I
I
I
0
1
2
I
I
I
6
11
TIME (min)
3
I
I
I
16
31
80
...=..
B
70
% GMP-140 t PLTS
70 -
+
% MONO BINDING PLTS
...+..
60 -
0
60
W
% PMN BINDING PLTS
1
" 501
z
-
?h.Pi ti
_I
I
BIND PLTS
50
40
u
4a
8
c
30
z
Q
0
Q
20
(unbound) GMP-140-positive platelets. The percentage of
free platelets expressing GMP-140 increased immediately
after thrombin and corresponds with the initial increase in
the percentage of monocytes and PMN with bound platelets. The percentage of free, activated platelets then decreased over the next 30 minutes while the percentage of
monocytes and PMN with bound platelets continued to
increase. The decrease in free activated platelets was not
due to aggregate formation because RGDS completely
inhibited platelet aggregation. In the unactivated sample
(Fig lB), a slight increase (7% to 18%) in the percentage of
activated platelets late in the assay corresponded to a
minimal rise in the percentage of monocytes binding
platelets without any increase in the percentage of PMN or
lymphocytes bound. Because CD15 has been postulated by
one group of investigators to function as a receptor on
monocytes and PMN for GMP-140 on activated platelet^,'^
we measured surface CD15 fluorescence on leukocyte
subsets in this whole blood system. Thrombin stimulation
had no effect on the surface density of leukocyte CD15, as
measured by its mean fluorescence, shown in Table 2.
Figure 2 demonstrates an experiment similar to that of
Fig 1 in which citrated whole blood with 1 mg/mL RGDS
was preincubated with the Fab, fragment of the anti-GMP140 MoAb G1, at a concentration of 1 pg/mL. Thrombin
stimulation of this sample showed no increase in the
percentage of monocytes or PMN with adherent platelets,
10
01
I
I
4
0
1
2
I
,
I
3
6
11
TIME (min)
I
I
80
I
t
% MONO BINDING PLTS
0
Fig 1. Whole blood, anticoagulatedwith citrate, was preincubated
with RGDS peptide (1 mg/mL) and stimulated with thrombin 0.1
U/mL (A) or diluent (B)at time 0. The blood was kept at 37°C. Samples
were obtained at the indicated time points and examined for the
percentage of monocytes (W), neutrophils (+), and lymphocytes (A)
with adherent platelets and for the percentage of unbound platelets
expressing GMP-140 (0).
-
...+...
1 6 3 1
60 -
% PMN BINDING PLTS
.A-.
% LYMPH BIND PLTS
0
50 -
z
4m 4 0 R
30 -
##"-.."
*"""
.........
'
first 6 minutes for the percentage of leukocytes binding
thrombin-activated platelets was 14.l%/min for monocytes, S.l%/min for PMN, and 0.7%/min for lymphocytes,
indicating that monocytes initially bound platelets at nearly
three times the rate compared with PMN-platelet conjugate formation.
In addition to the percentages of monocytes and PMN
with bound platelets after thrombin stimulation, Fig 1A
also shows the simultaneous percentages of circulating
*OI
10
0
0
1
2
.
.... 3-.-----zt.........-----..--+-...... +
....... ........
A-%:::
3
6
TIME (min)
11
",,,d
~
A
1 6 3 1
Fig 2. Whole blood in citrate and RGDS (as in Fig 1) was preincubated with a saturating concentration of the G1 MoAb to GMP-140.
Thrombin stimulation (at time 0) was performed identically to Fig 1A
and the percentage of leukocytes with adherent platelets measured.
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1733
WBC-PLATELET ADHESION IN WHOLE BLOOD
80
unlike the study in Fig 1A. Thrombin stimulation of a
control sample preincubated with the MoAb 1E3, which
recognizes GMP-140 but has been shown not to block
adhesion, resulted in increases in the percentage of monocytes and PMN with adherent platelets, similar to Fig 1A.
In Fig 3, the mean platelet fluorescence per bound
leukocyte was measured for the same experiment as Fig 1.
This fluorescence corresponds in a relative fashion to the
number of platelets bound to each leukocyte. Thrombin
stimulation (Fig 3A) resulted in a gradual increase in the
mean platelet fluorescence of monocytes and PMN with
bound platelets; the final values for monocytes were more
than twice as high as for PMN, indicating that more
platelets bound per monocyte than per PMN. The platelet
fluorescence per lymphocyte after thrombin and for all
,
2000
i
A
70 -
60 c3
% MONO BINDING PLTS
50 -
z
4m 4 0 -
o
u
% PMN BlNDlNG PLTS
/
l0
0
2000
,
% LYMPH BIND PLTS
5
7
6
[
1800
IBoor
1600
iB
MONO-PLT CONJUGATES
1400
1200w
w
M
E 10003
800-
2
600-
35
20
PMN-PLT CONJUGATES
W
LL
15
TIME (min)
p 1400-
z
10
1200
400 200 -
'*...w,..
...A..."".&...--.A.-...___
A
0 '
I
0
I
1
2
3
6
11
16
31
TIME (min)
2000
I
-m-
1800
I_
MONO-PLT CONJUGATES
1600
WN-PCT CONJUGATES
p 1400
I
W
0
1
0.
5
6
7
10
TIME (min)
15
20
35
Fig 4. Whole blood anticoagulatedwith heparin was preincubated
with RGDS (1 mg/mL). The sample was then incubated with epi 1
pmol/L for 10 minutes followed by ADP 5 pmol/L at time 0. (A) The
percentage of leukocyteswith bound plateletsover the course of the
assay. (B) The mean platelet fluorescence per bound leukocyte, an
estimate of the number of plateletsbound per leukocyte.
g 1200
w
8 1000
3
LL
z
4
9
800
600
400
200
0
0
1
2
3
6
TIME (min)
1 1 1 6 3 1
Fig 3. From the experiment detailed in Fig 1, the mean platelet
fluorescence of each leukocyte-platelet conjugate was measured over
time after thrombin (A) or diluent (8) stimulation. This fluorescence
provides a semi-quantitative estimate of the number of platelets
bound per leukocyte.
leukocytes in the control sample (diluent stimulation only,
Fig 3B) did not change over time.
epilADP stimulation of whole blood with RGDS peptide.
Formation of platelet aggregates was again inhibited by
preincubating heparinized whole blood with RGDS (1
mgImL). The sample was incubated with epi for 10 minutes, followed by ADP stimulation at time 0 (Fig 4). Over
the next 30 minutes, the percentage of monocytes with
adherent platelets more than doubled (30% to 72%, Fig
4A), and the number of platelets bound per monocyte (as
estimated by fluorescence in Fig 4B) also increased (200 to
850). However, unlike after thrombin, PMN-platelet conjugate formation did not increase either with respect to the
percentage of cells binding platelets (21% to 24%, Fig 4A)
or the number of platelets bound per cell (relative fluorescence 440 to 450, Fig 4B). Lymphocytes were again unaf-
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1734
RINDER ET AL
fected. Although thrombin and epi/ADP resulted in similar
percentages (72% v 69%) of free platelets expressing
GMP-140 (Table 3), thrombin stimulation resulted in a
fourfold higher surface expression of GMP-140 per platelet, as measured by fluorescence, when compared with the
weaker agonist epi/ADP. Experiments performed using
citrated whole blood yielded similar results.
epi/ADP stimulation of whole blood without RGDS peptide.
Figure 5 illustrates a representative study in which heparinized whole blood (without RGDS) at 37°C was incubated
with 1 kmol/L epi followed by 5 ymol/L ADP at time 0 (Fig
5A) or no agonist (Fig 5B). RGDS peptide was not used to
allow the formation of platelet-platelet aggregates. Because
the combination of epi and ADP is a less potent platelet
agonist than thrombin and there is little fibrin formation
with epi/ADP, platelet-platelet interaction is reversible,
and whole blood clotting does not occur despite the
absence of RGDS. Figure 5A demonstrates the percentage
of leukocytes with bound platelets over time after epi/ADP.
The percentage of all leukocytes with bound platelets
decreased markedly after ADP. The percentage of monocytes with adherent platelets subsequently increased over
time, reaching a peak similar to that seen in Fig 4A.
However, the percentage of PMN and lymphocytes with
adherent platelets increased only slightly and did not fully
recover to the preagonist level of binding. Figure 5B
demonstrates that, in the absence of agonist, leukocyteplatelet binding changes minimally over the time of the
assay, similar to Fig 1B. Experiments in which citrate was
substituted for heparin as the anticoagulant and then
stimulated with epi/ADP showed an identical pattern of
leukocyte-platelet adhesion.
To further study this dynamic pattern of leukocyteplatelet adhesion after the addition of epi/ADP in a
situation where platelet aggregate formation occurs, we
simultaneously measured the percentages of (1) free (not
bound to leukocytes) GMP-140-positive platelets; (2) free
platelet aggregates; and (3) leukocytes with bound platelets
over time (Fig 6). In this experiment, 1 pmol/L epi was
added to citrated whole blood 10 minutes before 5 p.mol/L
of ADP was added at time 0. Figure 6A demonstrates a
slight decrease and a later increase in leukocyte-platelet
binding after epi, and then a rapid decrease (like the
experiment in Fig 5A) in the percentage of leukocytes with
bound platelets after addition of ADP. The identical
pattern of recovery of binding as observed in Fig 5 occurred
in this representative experiment as well. Figure 6B demonstrates that immediately after ADP, the percentages of both
free platelet aggregates and GMP-140-positive platelets
increased dramatically. Furthermore, the peak percentages
Table 3. Expression of GMP-140 on Unbound Platelets
After Agonist
Platelet GMP-140 Expression
Agonist
EpilADP
Thrombin
Peak % of GMP-140
Positive Platelets
69
72
Mean GMP-140'
Fluorescence
1.10k 0.05
4.23f 0.42
*Ratio to control fluorescence f standard error of the mean.
80
A
70
60
0
1
50 -
I
2m 4 0 -
'3 0 20 10-
0
0
5
6
7
10
TIME (min)
15
20
35
80
c
% MONO BINDING PLTS
70
...+...
% PMN BINDING PLTS
.A..
60
% LYMPH BIND PLTS
0
50
z
4m 40
'30
2oi
10
0 '
,
0
5
6
7
10
TIME (min)
15
20
35
Fig 5. Whole blood was anticoagulated with heparin in the absence of RGDS and stimulated with epi and ADP (A) in an identical
fashion to Fig 4A to determine the percentage of leukocytes with
adherent platelets over time. ( 6 )The changes in the percentage of
leukocyte-plateletadhesion in response to diluent alone.
of platelet aggregates and GMP-140-positive platelets corresponded to the nadir of leukocyte-platelet binding. After
this point, the percentage of free platelet aggregates and
GMP-140-positive platelets then decreased as the simultaneous percentage of monocytes (and to a lesser extent
PMN) with bound platelets increased.
In a separate experiment (Fig 7A), the inhibition of the
recovery of leukocyte-platelet adhesion after epi/ADP was
examined by preincubating whole blood (without RGDS)
with the G1 MoAb to GMP-140 before stimulation with epi
and ADP. In the presence of the G1-blocking MoAb, the
preagonist binding of leukocytes to platelets was unaffected. There was also no change in the immediate decrease
in leukocyte-platelet adhesion after ADP. However, the
recovery phase for leukocyte-platelet conjugates after the
initial decrease in binding was nearly completely abolished.
There was only a slight increase in the percentage of
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1735
WBC-PLATELET ADHESION IN WHOLE BLOOD
80
70
I*
metabolic cooperation between platelets and heterotypic
blood cells has been postulated. Studies have demonstrated
that platelets supply free cholesterol to monocytes for
cholesteryl ester ~ynthesis'~;such monocytes may then
participate in foam cell generation seen in atherosclerotic
lesions.16Platelets have also been shown to provide prostaglandin endoperoxide H, to endothelial cells and lymphocytes for production of prostacyclin (PGI,), which is a
potent vasodilator and platelet inhibit~r.",'~
Recent work
has demonstrated that platelets synthesize leukotriene C,
(LTC,) from PMN-derived L T h ; this cell-cell interaction
for eicosanoid synthesis is aspirin-insensitiveand independent of platelet acti~ation.'~
To further understand these
interactions, model systems are necessary in which heterotypic and homotypic conjugate formation can occur together under circumstancessimilar to those found in vivo.
1
I
100 ' 1
-13
I
-8
I
-3
I
0
I
!
!
2
3
4
TIME (min)
I
I
7
12
. '
I
17 32
50
80
70 -
I
% GW-140 t PLTS
i T L T AGGREGATES
c
A
B
II .-+-
% MONO BlNOlNG PLTS
70
70-1
60
60 -
I
% PMN BNOING PLTS
% LYMPH BND PLTS
Q.
t
50 -
50
50
0
0
40
I
2
w
1
L
m
bp
30
40
30 -
4
_I
20
;
20 -
'
10
0
0
0
5
6
Fig 6. An experiment in which citrated whole blood in the absence
of RGDS was stimulated with epi/ADP. Epinephrine was added at
time -10 minutes followed by ADP stimulation at time 0. (A) The
percentage of leukocytes with bound platelets. (B) The simultaneous
measurements of unbound GMP-140-positive platelets (B) and free
platelet aggregates (0).
6A.
DISCUSSION
Leukocyte-plateletadhesion is likely of physiologic importance not only for the targeting of both cell types to
appropriate inflammatory and/or hemostatic sites but also
for functional alterations in these cells. For example,
10
15
20
35
15
20
35
TIME (min)
TIME (min)
monocytes with adherent platelets, but this remained well
below the preagonist levels. Platelet aggregate formation
and dissociation was identical to that shown in Fig 6B,
indicating that the G1 MoAb only blocked the GMP-140dependent recovery of leukocyte-platelet adhesion and had
no effect on platelet aggregates. In Fig 7B, the sample was
preincubated with the nonblocking anti-GMP-140 MoAb
1E3. This demonstrated a pattern of binding in which there
was no inhibition of the recovery of leukocyte-platelet
adhesion over time, ie, identical to the experiment of Fig
7
Y"
8
70 60 0
50 -
I
9m 4 0 bp
30 -
20 10-
0 '
I
0
5
6
7
10
TIME (mi.)
Fig 7. Whole blood was anticoagulated with heparin in the absence of RGDS and then incubated with a saturating concentration of
the 01 MoAb to GMP-140 (A) or the lE3 MoAb (6). epi/ADP stimulation was then performed in both samples identically to the study in
Fig 5A and the percentage of leukocytes with adherent platelets was
measured over time.
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1736
Isolated PMN and monocytes have been shown to bind
activated and, to a lesser degree, unactivated platelet
fractions. The kinetics of this adhesion and some responsible receptor-ligand pairs have been determined in
However, the interactions between platelets and leukocytes
have not previously been examined in whole blood. The
current studies have determined the conditions under
which heterotypic cell-platelet adhesion occurs in whole
blood, the dynamic interaction between homotypic (plateletplatelet) and heterotypic (leukocyte-platelet) adhesion in
whole blood, and the relative affinities of different leukocyte subsets for activated (GMP-140-positive) platelets.
Baseline studies demonstrated that unstimulated whole
blood samples show both a minority of leukocytes with
bound platelets and a low level of platelets bound per white
blood cell. These results were similar to those observed
when unstimulated isolated cell fractions were allowed to
recombine in vitro; this included a similar percentage of
both neutrophils and peripheral blood lymphocytes that
bound resting platelets. However, monocytes in whole
blood had a lower percentage of platelet binding (45%)
compared with isolated cell fractions (87%) in which
monocytes and platelets are recombined in the absence of
red blood cells, plasma, and other leukocytes. In the whole
blood assay, the presence and much higher concentration of
other leukocytes that bind unactivated platelets, albeit less
effectively than monocytes, would be expected to decrease
monocyte-platelet adhesion when compared with the isolated fraction assay. It is also interesting to note that, in
whole blood, RGDS peptide reduced the percentages of all
leukocytes with adherent unactivated platelets by more
than 50%, whereas in isolated cell fraction experiments,
RGDS had no effect on unactivated platelet binding to
leukocytes. One important difference between isolated cell
fraction studies and these whole blood experiments is the
lack of plasma fibrinogen in the former. Because RGDS
inhibits fibrinogen binding to the GPIIb/IIIa integrin receptor,” it is possible that fibrinogen-integrin interaction plays
a role in unactivated platelet adhesion to leukocytes in
whole blood. When isolated cell fractions are studied, only
platelet and leukocyte-associated fibrinogen is available for
such an interaction and thus the effect of added RGDS in
isolated fractions is minimal. It is also possible that RGDS
may lower the baseline percentage of leukocyte-platelet
binding through inhibition of platelet-platelet interaction.
If platelet-platelet conjugates are present on the leukocyte
surface at baseline, their aggregate platelet marker fluorescence will determine the lower limits of detection of
platelet binding, especially since there may be some fluorescent quenching by adjacent erythrocytes in this whole blood
assay, If RGDS dissociates these platelet conjugates on the
cell surface, then the aggregate fluorescence is decreased,
lowering the measured percentage of leukocyte binding.
When platelets were stimulated with thrombin in whole
blood under conditions that preclude formation of platelet
aggregates (RGDS)? our studies have shown that: (1)
platelets rapidly express GMP-140; ( 2 ) monocytes rapidly
bind platelets and continue to increase their cumulative
platelet binding as the number of available circulating
activated platelets decreases; (3) PMN bind platelets at
RINDER ET AL
about one third the rate of monocytes and in far fewer
numbers of platelets per PMN; (4) lymphocyte-platelet
adhesion is not changed by thrombin; and (5) all of the
increased monocyte and PMN adhesion of platelets after
thrombin is dependent on the epitope of platelet GMP-140,
which is recognized by the G1 MoAb but not the epitope
recognized by 1E3. These findings are consistent with data
reported in isolated fraction experiments2a4and present
further evidence for a competitive advantage of monocytes
over PMN in both the initial binding of GMP-140-positive
platelets and the cumulative binding of platelets, ie, the
eventual number of platelets bound per leukocyte. This
advantage of monocytes over PMN is particularly striking in
view of the log-higher concentration of PMN in whole
blood. The advantage of monocytes over PMN is further
supported by the increase in monocyte-platelet conjugate
formation observed after epi/ADP with no increase for
PMN-platelet adhesion. This competitive advantage of
monocytes over PMN for platelet conjugate formation may
be exaggerated when platelets are stimulated to express
relatively less surface GMP-140 per platelet compared with
the expression of this receptor on platelets after thrombin
stimulation (Table 3).
The advantage of monocytes over PMN in binding
activated platelets cannot be currently explained by data
regarding the postulated receptor on PMN and monocytes
for binding GMP-140. The identity of this receptor is
controversial. Larsen et all4 have found evidence that
CD15; a nonsialated penta-sugar not present on resting
lymphocytes, is a leukocyte ligand for PADGEM/GMP140. However, other investigators have not found conclusive evidence that CD15 is the GMP-140 receptor and have
additionally shown that the leukocyte receptor for GMP140 is likely a sialated glycoprotein, unlike CD15.22,23
Our
studies suggest a competitive advantage of monocytes over
PMN for adhesion to activated platelets, an intriguing
finding in this regard because there is a 30-fold higher
expression of CD15 per PMN compared with monocytes.
Our data suggest either that CD15 is not the receptor for
GMP-140-positive platelets or that qualitative differences
between monocyte and PMN CD15 exist with respect to
GMP-140 binding (a less likely alternative in view of the
small size of the CD15 sugar).
To determine the competitive dynamics of plateletplatelet versus leukocyte-platelet adhesion in whole blood,
epi/ADP in the absence of RGDS was used to cause
reversible platelet aggregati~n.’~
Our studies demonstrated
that: (1) initial platelet activation resulted in preferential
platelet-platelet adhesion with a simultaneous decrease in
the percentage of all leukocytes with bound platelets; ( 2 ) as
the platelet aggregates spontaneously dissociated, the activated platelets returned to heterotypic cell adhesion over
time; (3) monocytes again had the advantage over PMN for
this recovery of platelet adhesion; and (4) the G1 (blocking)
MoAb, but not the 1E3 (nonblocking) MoAb, to GMP-140
prevented the recovery of leukocyte-platelet adhesion at a
time when platelet aggregates were dissociating. These
studies suggest that early after the addition of agonist,
adherent platelets dissociate from monocytes, PMN, and
lymphocytes to undergo homotypic binding (aggregation)
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
1737
WBC-PLATELET ADHESION IN WHOLE BLOOD
mediated by an RGDS binding site revealed by the agonist.
As platelet aggregates dissociate, the GMP-140-positive
platelets readhere to monocytes and to a lesser degree to
PMN, but not to lymphocytes. This is consistent with
studies demonstrating that resting lymphocytes do not bind
GMP-140 or activated platelets?*
It is important to note that the setting in which the
dynamics of leukocyte-platelet adhesion are determined in
this study differs from in vivo conditions by several factors.
Because these experiments were performed under relatively static conditions, the crucial effects of blood flow and
shear rate are not taken into account when cell-cell interactions are measured. In addition, perturbations introduced
by endothelial injuq or inflammation are not accounted for
in this study and may significantly affect the interactions
between blood cells described. It will be instructive for
future studies to determine similar leukocyte-platelet and
homotypic platelet interactions under more physiologic
conditions.
As noted earlier, metabolic cooperation has been shown
to occur between platelets and heterotypic blood cells;
presumably these interactions require some ability for these
cells to adhere or at least remain in close proximity for a
time?5s26These whole blood studies have demonstrated that
monocytes have a competitive advantage over PMN for
binding to activated platelets and that platelet-platelet
adhesion is initially favored over platelet-leukocyte adhesion after addition of a platelet agonist. Subsequent platelet
aggregate dissociation allows GMP-14kxpressing platelets to readhere to monocytes and, to a lesser extent, to
PMN. The functional consequences of such adhesion are
not yet known, but the dynamics of interaction between
hemostatic and inflammatory cells provide a basis for
studying relevant pathophysiologies.
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1991 78: 1730-1737
Dynamics of leukocyte-platelet adhesion in whole blood
HM Rinder, JL Bonan, CS Rinder, KA Ault and BR Smith
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