Download (CD32) Phenotype and Level of Expression

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Response of Human Platelets to Activating Monoclonal Antibodies: Importance
of FcyRII (CD32)Phenotype and Level of Expression
By Yoshiaki Tomiyama, Thomas J. Kunicki, Theodore F. Zipf, Sheila 6.Ford, and Richard H. Aster
Certain monoclonal antibodies (MoAbs) specific for platelet
membrane glycoproteins are known t o be capable of activating platelets, and it is generally thought that platelets from
normal subjects are equally susceptible t o stimulation by
such MoAbs. We found that platelets from 20 normal donors
varied significantly in their sensitivity t o three lgGl murine
MoAbs specific for membrane glycoproteins CD9, GPIV
(CD36). and the GPIIb/ llla complex (CD41). respectively. The
response of platelets t o these MoAbs was blocked by prior
addition of MoAb IV.3 specific for the FcyRll receptor, indicating that activation was Fc receptor mediated. Platelets that
responded poorly t o these MoAbs failed t o bind the MoAb
41H.16, specific for the ”responder“ form of FcyRII, but
platelets that responded well reacted with this MoAb. The
average number of FcyRll receptors on platelets from
“responders” and “non-responders“ was approximately the
same. However, the number of FcyRll receptors expressed
influenced sensitivity of a subgroup of ”responder” platelets
t o the anti-CD41 MoAb. These platelets were judged on the
basis of MoAb binding studies t o be heterozygous for the
two alleles of FcyRIIA. In contrast t o their varying sensitivity
t o lgGl MoAbs, members of the platelet panel responded
equally well t o 50H.19, an lgGzaMoAb specific for CD9, and
these responses could not be blocked by MoAb IV.3 in the
presence of plasma. This appears t o be because of dual
actions of 50H.19 on platelets: one FcR-dependent and the
other complement-dependent. Our findings confirm previous
reports that certain lgG1 MoAbs activate platelets through
binding of their Fc domains t o FcyRll receptors and demonstrate that this response is influenced both by F h p l l phenotype and (in the case of the anti-CD41 MoAb) by the number
of FcyRll receptors expressed. The failure of nonresponding
platelets t o bind detectable amounts of MoAb 41H.16, which
is thought t o recognize all FcyRll receptors except for one
allele of the FcyRllA gene, is consistent with the possibility
that FcyRllA gene products, but not FcyRllB or FcyRllC gene
products, are expressed on platelets.
o 1992b y The American Society of Hematology.
C
extracellular and transmembrane domains of the mature
proteins determined by these three genes differ only by a
small number of amino acids, but the FcyRIIB gene
products differ extensively from those of FcyRIIA and
FcyRIIC in their cytoplasmic domains. Presumably, this
variety of receptors allows for the induction of different
signals on binding of ligands to FcyRII. Which FcyRII gene
products are expressed on platelets has not yet been
defined.
The FcyRIIA gene appears to be diallelic. Monocytes
homozygous for one of these alleles are unable to induce
proliferation of T cells sensitized with murine IgGl MoAbs
specific for CD3,22-24
apparently because of their failure to
interact with murine IgGl Fc sufficiently well to induce
clustering of CD3. Monocytes with this phenotype are
designated “low responders” (LR). Monocytes bearing the
second FcyRIIA gene product produce good stimulation of
sensitized T cells and are designated “high responders”
(HR).= Monocytes and neutrophils from LR and H R
ERTAIN MURINE monoclonal antibodies (MoAbs)
induce release and aggregation on binding to human
platelets.l-ll Some MoAbs specific for glycoproteins IIb or
IIIa act directly on the GPIIb/IIIa complex to induce
conformational changes leading to expression of the fibrinogen receptor.3psJ2 More often, MoAbs specific for these
or other membrane glycoproteins trigger activation and
release through conventional signal transduction pathFor reasons not yet clear, MoAbs specific for
CD9 or GPIV (CD36) are the most consistent platelet
a c t i v a t ~ r s . ~InJ ~general, MoAb-induced transmembrane
signalling requires an intact IgG Fc domain and can be
blocked by MoAb IV.3 specific for the FcyRII receptor
(CD32), implying that binding of monoclonal Fc to that
receptor is important in the process of a~tivation!,~-~~
It has
been observed that platelets from normal subjects differ in
their susceptibility to activation by murine M o A ~ sbut
, ~ the
basis for this has not been established.
Three families of receptors with specificity for the Fc
region of human IgG have been described in humans.lsJ6
FcyRI receptors bind monomeric IgG Fc with high affinity.
FcyRII and FcyRIII receptors bind monomeric IgG Fc
weakly, but bind multimeric IgG Fc and IgG-containing
immune complexes tightly. FcyRII molecules have the
widest tissue distribution of the three FcyR families, being
expressed on all FcyR-bearing blood cells except NK
cells.lS Only FcyRII receptors have been demonstrated
unequivocally on platelets.
FcyRII receptors in humans are the products of at least
three different genes, designated A, B, and C, each coding
for mature transmembrane proteins of about 40 Kd molecular mass.1s-21At least six different mRNA transcripts and
four mature proteins are derived from these three genes.
The FcyRIIAgene produces two transcripts as the result of
alternative polyadenylation, the FcyRIIB gene produces
three or four transcripts by alternative splicing of exons
coding for cytoplasmic and signal sequences, and the
FcyRIIC gene appears to produce a single transcript. The
Blood, Vol80, No 9 (November l),
1992: pp 2261-2268
From The Blood Research Institute, The Blood Center of Southeastem Wisconsin, Inc, Milwaukee; the Departments of Anatomy, Cell
Biology, Medicine, Microbiology, and Pathology, the Medical College
of Wisconsin; and the Divisions of Pediatrics and Laboratory Medicine, The University of T m s , M.D. Anderson Cancer Center, Houston.
Submitted December 18, 1991; accepted June 30, 1992.
Supported by Grants HL-13629 and POI-HL-44612 from the
National Heart, Lung, and Blood Institute and by The Kelcie Margaret
Kana Fund.
Address reprint requests to Richard H. Aster, MD, President, The
Blood Center of Southeastem Wuconsin, 1701 W Wisconsin Ave,
Milwaukee, WI 53233.
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 1992 by The American Society of Hematology.
0006-4971/92/8009-0021$3.00~0
2261
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TOMIYAMA ET AL
2262
Platelets were separated from freshly collected blood anticoaguindividuals can also be differentiated by their ability to form
lated with EDTA and were washed three times in platelet
rosettes and mediate antibody-dependent cellular cytotoxicsuspension buffer (PSB) (0.01 mol/L phosphate-buffered isotonic
ity (ADCC) against target cells sensitized with murine
saline, 1% bovine serum albumin, 4.0 mmol/L EDTA, pH 6.5).
IgG126and by the isoelectric focusing patterns of their
Final preparations contained fewer than one white blood cell per
FcyRII receptor^.^' IEF patterns of monocytes and plate10,000 platelets. Platelets, 1 x lo*, were suspended in 300 pL of
lets from the same individuals are concordant.28
buffer containing 2 bg of lz5I-Fab fragments. Preliminary studies
Two well-studied MoAbs, IV.3 and 41H.16, appear to
demonstrated that this quantity of Fab was in excess of the amount
bind to the products of all three FcyRII genes.15 41H.16
needed to achieve saturation binding. After incubation at room
binds strongly to the HR allele of FcyRIIA, but weakly or
temperature for 1 hour, 50-pL aliquots were layered in triplicate
not at all to the product of the LR allele.25Strong binding of
on 200 FL of 20% Percoll in 400 pL polypropylene microcentrifuge
41H.16 to the H R gene product was recently shown to be
tubes. The tubes were centrifuged at 10,000g for 4 minutes to allow
determined by an arginine, rather than a histidine, residue
pelleting of platelets and platelet-bound Fab. The tubes were then
at position 131 in the second extracellular d ~ m a i n . ~ ~ - clamped,
~l
the tips cut off, and the pelleted radioactivity was
measured in a gamma scintillation counter. Molecules of Fab
Monocytes from about 25% of the general population carry
bound were calculated from the formula:
the form of FcyRIIA that fails to bind 41H.16, ie, has a
histidine residue at position 131.
(cpm bound) x
xA
In a number of clinical disorders, platelet destruction is
SA x No. of Platelets x MW
thought to be mediated by IgG Fc-dependent mechawhere A = Avogadro's number, SA = specific activity of Fab (cpm
nism~.~
A *fuller
~ ~ ~understanding of platelet Fc receptors
per microgram), and MW = 50,000.
could provide clues to the pathogenesis of some of these
Determination of IK3 and 41H.16 binding by flow cytometry.
conditions. Therefore, we characterized the FcyRII recepPlatelet-rich plasma was prepared from freshly collected, citrated
tors of platelets using MoAbs IV.3 and 41H.16 and studied
whole blood using excess acid-citrate-dextrose (ACD) anticoaguthe relationship between FcyRII phenotype and level of
lant to achieve a pH of about 6.4. The platelets were pelleted at
expression and susceptibility of platelets to MoAb-induced
3,OOOg and washed twice in Ringer's citrate dextrose (RCD) buffer
activation.
containing 2 mmol/L EDTA and 50 ng/mL prostaglandin E1
MATERIALS AND METHODS
Murine MoAbs. Characteristics of the murine MoAbs used are
summarized in Table 1. ALB-6 (a-CD9) was obtained from
AMAC, Inc (Westbrook, ME); 8A6 (a GPIV, subclone D9) from
Dr John Barnwell (New York University); 50H.19 (a-CD9) from
Dr A.R.E. Shaw (University of Alberta, Canada); and IV.3
(a-FcyRII) from Dr Clark Anderson (Ohio State University).
Literature citations are provided in Table 1. Monoclonal IgG was
purified from ascites fluid by affinity chromatography on protein A
Sepharose CL4B (Pharmacia, Piscataway, NJ).
Preparation and radiolabeling of Fab fragments. Fab fragments
of MoAb IV.3 were produced by papain digestion of purified IV.3
IgG followed by chromatographic purification on Protein A
Sepharose CL-4B." The final preparation yielded bands of molecular weight (MW) approximately 50 Kd (nonreduced) and 25 Kd
(reduced) on sodium dodecyl sulfate (SDS) gel electrophoresis.
The Fab fragments were labeled with lZ5Iby the lactoperoxidase/
glucose oxidase procedure using Enzymobeads (BioRad, Richmond, CA). Specific activity of the radiolabeled Fab was determined by comparing protein content with counts per minute (cpm)
determined in a gamma scintillation counter. By trichloroacetic
acid precipitation, it was found that more than 95% of the
radioactivity was protein-bound.
Binding of radiolabeled IV3 Fab toplatelets. Binding of lZ51-Fab
from MoAb IV.3 to washed platelets was determined by the
~ ~ minor modification^.^^
method of Court and L o B ~ g l i owith
Table 1. MoAbs Used
MoAb
Target GP
OP-G2
ALE-6
50H.19
8A6
IV.3
41H.16
CD41 (GPllb/llla)
CD9
CD9
CD36 (GPIV)
CD32 (FcyRII)
CD32 (FcyRII)
Subclass
lgGi
lgG,
W z a
lgGi
IgGzb
W z a
Reference
34
4.6
3, 35
36
22,27
28,37
(Sigma Chemical Co, St Louis, MO), pH 6.5. Platelets, 2 x lo7,
were resuspended in 100 p L of RCD buffer containing 1 pg/mL of
MoAb IV.3 or 41H.16 and incubated for 60 minutes at room
temperature. These quantities were more than 10-fold the amounts
required to achieve saturation binding. After two washes in RCD
buffer, the platelets were incubated with a 1:40 dilution of
affinity-purified F(ab')z fragments of fluorescein isothiocyanate
(F1TC)-conjugated goat antimouse IgG (Jackson Immunoresearch, West Grove, PA) for 30 minutes. The labeled platelets
were washed twice and fixed with 1% paraformaldehyde. Bound
FITC was analyzed by flow cytometry (FACStar-Plus; Becton
Dickinson, Mountain View, CA). Antibody binding was expressed
as mean fluorescence intensity in the linear mode.@ Background
values determined with an irrelevant monoclonal (HB-57, an IgGl
MoAb specific for IgM) were subtracted from experimental values
to obtain net fluorescence intensity.
Source of platelets. Platelets were obtained from normal unrelated persons ranging in age from 21 to 60 years with informed
consent. Donors had not ingested aspirin or other drugs known to
inhibit platelet function for 1week before study.
Platelet aggregation. Platelet-rich plasma was prepared from
freshly collected blood anticoagulated with one-tenth part of 3%
sodium citrate. Donors had not ingested aspirin or other drugs
known to inhibit platelet function for 1 week before testing. The
concentration of platelets was adjusted to about 300,00O/pL by
adding platelet-poor plasma from the same donor. The response to
MoAbs and other platelet agonists was measured using a Bio/Data
PAP-4 aggregometer (Bio/Data Corp, Hatboro, PA) at 37°C with a
stirring rate of 1,000 rpm. Reactions were terminated after 20
minutes when platelets failed to aggregate. For reasons to be
described (Results), we found it useful to measure the lag time
required for platelets to respond to activating monoclonals. This
was defined as the time required for light transmission to reach one
half of its maximum value.
Release of 14C-serotoninfrom labeled platelets. Release of 14Cserotonin from platelets in plasma-free medium was determined by
a modification of the method of Sheridan et aL41Serotonin-labeled
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PLATELET ACTIVATING MONOCLONAL ANTIBODIES
platelets were suspended in albumin-free Tyrode's buffer at a
concentration of 3 x 105/pL and 75-pL aliquotswere pipetted into
the wells of a polystyrene microtiter tray (Dynatech, Chantilly,
VA). Twenty-five microliters of test material or buffer was then
added, and the trays were sealed and agitated gently on a
horizontal rotator for 60 minutes at room temperature. Phosphatebuffered isotonic saline, 0.1 mL, pH 7.4 containing 0.5% EDTA
was then added and the trays were spun at 2,500 rpm to pellet the
platelets. Radioactive content of 100-pLaliquots of the supernatants was determined in a beta scintillation counter, and results
were expressed as percent of total serotonin (determined from a
detergent-lysed aliquot of labeled platelets). Release in control
tubes containing labeled platelets and an irrelevant MoAb was less
than 5%. Release of 14C-serotoninfrom platelets in platelet-rich
plasma was determined according to Griffith et al?5
RESULTS
MoAb-induced platelet aggregation. In preliminary studies, it was found that maximum aggregation of normal
platelets could be induced by 10 kg/mL of monoclonal
OP-G2 (a GPIIb/IIIa), 20 pg/mL of ALB-6 (a-CD9), 5
kg/mL of 8A6 (aGPIV), and 5 pg/mL of 50H.19 (a-CD9).
Higher incubations of MoAbs failed to further shorten the
lag time of individual platelet preparations. The response
of platelets to various amounts of MoAb 8A6 is shown in
Fig 1A. Qualitatively similar dose-response curves were
obtained with MoAbs OP-G2, ALB-6, and 50H.19. The
response of platelets to each of the IgGl MoAb (8A6,
ALB-6, OP-G2) was totally inhibited by prior addition of
MoAb IV.3 or Fab from IV.3 (shown for MoAb OP-G2 in
Fig 1B). The response to the IgGP, MoAb 50H.19 (a-CD9)
was not inhibited by IV.3 (see below).
Platelets from different normal subjects varied significantly in their response to the three activating MoAbs of
the IgGl subclass (Fig 2). Platelets from some donors
responded within 1or 2 minutes; others never responded or
-
iaal
lmin
I
100
\
1
Fig 1. (A) Aggregation of platelets in response to different concentrations of MoAb 8A6 (a-GPIV). (6) Aggregation induced by MoAb
OP-G2 (anti-GPllb/llla) at concentration 20 pg/mL was inhibited by
Fab fragmentsfrom MoAb IV.3 (crFcyRII).
2263
crpz
crP3
Fig 2. Variation in the aggregation response of platelets from
three normalsubjects (designated Grp 1, Grp 2, Grp 3) to MoAb OP-G2
at 10 pg/mL.
aggregated only after a delay of 5 or 10 minutes. The extent
of aggregation and the maximum slope of the aggregation
curve were generally less in platelets requiring a longer time
to respond. Further studies were conducted to determine
the basis for these differences.
Quantitation of FcyRII receptors on platelets. Measurement of the binding of MoAb IV.3 Fab fragments to
platelets of 20 unrelated normal subjects (10 male, 10
female) demonstrated a range of binding sites from 1,660to
4,610 per platelet. The specificity of binding was confirmed
by showing that platelet-associated radioactivity was reduced by more than 99% when platelets were preincubated
with a 50-fold excess of nonlabeled IV.3 Fab. The number
of IV.3 Fab binding sites was stable (212% about the
mean) in four individuals, two with relatively high and two
with relatively low FcyRII expression, who were studied on
four occasions over a 6-month period. Platelets from males
and females did not differ significantly in the quantities of
IV.3 Fab bound.
Comparison of ZK.3 and 4IH.16 binding. Binding of the
a-FcyRII MoAbs IV.3 and 41H.16 to platelets of the same
group of normal subjects was measured by flow cytometry.
Platelets from different donors varied considerably in the
quantity of IV.3 bound as expected from the IV.3 Fab
binding studies (see above). MoAb 41H.16 exhibited a
different binding pattern in that no detectable amounts
were bound to platelets from five members of the panel,
while platelets from the remaining 15 donors bound variable amounts. When the ratio of 41H.16 fluorescence to
IV.3 fluorescence was determined, the value was approximately zero for the five platelet preparations that failed to
bind 41H.16. The binding ratios for the remaining 15
donors allowed them to be segregated into two additional
groups with ratios ranging from 0.19 to 0.39 (10 donors) and
from 0.58 to 0.63 (five donors) (Fig 3). The three groups will
be referred to as groups 1, 2, and 3, respectively, in the
following discussion. Varying ratios of 41H.16:IV.3 binding
to platelets were also observed by Gosselin et a125 in six
selected normal subjects.
Response of platelets to the &GI MoAbs OP-G2, 8A6, and
ALB-6. Platelets in the three groups defined by the
41H.16:IV.3 binding ratios (Fig 3) differed in respect to the
time required for MoAbs OP-G2 (a-GPIIb/IIIa), 8A6
(a-GPIV), and ALB-6 (a-CD9) to induce an aggregation
response (lag time) as shown in Fig 4. Platelets from donors
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TOMIYAMA ET AL
0.8
Grp 3
0.6
.
.. .
.
m
Grp 2
m .
Grpl
!
0.2
0
I
I
10
20
Platelet Donor Panel
Fig 3. Ratio of 41H.16:IV.3 binding in platelets from 20 normal
subjects. MoAb binding at saturation was determined by flow cytometry using FITC-labeledFlab'), specific for mouse IgG as a secondary
antibody. Values shown are the average of t w o or three independent
measurements with each MoAb on platelets of each donor. Donors
were divided into groups 1, 2, and 3 on the basis of 41H.16:IV.3
binding ratios.
in group 1 responded slowly, or not at all, while those in
group 3 all responded rapidly. The response of platelets
from donors in group 2 varied between the two extremes.
The magnitude of the aggregation responses and the
maximum slopes of the aggregation curves were also significantly lower with group 1 than with group 3 platelets (not
shown). However, the distinction between groups 1 and 3
was clearest when lag times were compared. Aggregation
responses to each MoAb were completely inhibited by prior
addition of MoAb IV.3 at 10 kg/mL (not shown). Platelets
in the three groups responded equally well to the conventional agonists adenosine diphosphate (ADP) and collagen.
The absolute number of FcyRII molecules per platelet,
determined by binding of IV.3 Fab, was slightly higher in
group 3 (average 3,510 ? 570 molecules) than in group 1
platelets (average 2,830 2 910 molecules) and was intermediate in group 2 platelets (2,940 2 560 molecules). However, these differences were not statistically significant.
Among platelets in group 2, sensitivity to MoAb OP-G2
(a-GPIIb/IIIa) correlated with the number of FcyRII
receptors expressed (3 = .756, P < .01) (Fig 5). No relationship was found between FcyRII expression and the response of group 2 platelets to MoAbs 8A6 and ALB-6.
Thus, responsiveness of platelets in group 2 to OP-G2, but
not to 8A6 or ALB-6, is influenced by the number of
FcyRII receptors.
Response of platelets to the ZgGr, MoAb, 50H.19. In
contrast to their differing sensitivity to the IgGl MoAbs
OP-G2, ALB-6, and 8A6, platelets in groups 1, 2, and 3
aggregated equally well in response to the IgG2, MoAb
50H.19 which, like ALB-6, is specific for CD9. The responses to 50H.19 could not be inhibited by prior addition
of MoAb IV.3 at concentrations as high as 50 yg/mL.
.
..
.. .
.
.
_L
9
.
a0-
"
I
-
-
--
~,-pi 0rp2 G-3
OP-02
Owl
OW2 Grp3
ALE-6
O W
OW2 OW3
8A6
Fig 4. Aggregation responses of platelets from 20 normal subjects
t o platelet-activating MoAbs OP-G2 (10 pg/mL), ALB-6 (20 pg/mL),
and 8A6 (5 pg/mL). Platelets were assigned t o groups 1, 2, and 3 on
the basis of MoAb 41H.16:IV.3 binding ratios (see Fig 3 and text).
Ordinate depicts inverse lag time of aggregation response in minutes-' x 102. A value of 0 indicates an "infinite" lag time, ie, no
response within 20 minutes. Values shown are the average of two or
three independent measurements on platelets from each donor.
Horizontal bars indicate average values for each group. Groups 1and
3 differed significantly from each other for each of the three MoAbs
(P < .Ol).
0-r
wa
flab) Blndlng SIIOS p r
maem
Fig 5. Aggregation responses of platelets from group 2 donors t o
MoAb OP-G2 (a-GPllb/llla) at 10 pg/mL. Ordinate depicts inverse lag
time of aggregation response in minutes-' x 102. Response was
roughly proportional t o the number of binding sites for IV.3 Fab, ie,
the number of FcyRll receptors (P e .01) (y = .02x - 34.8, f l = .756).
Similar, but less striking relationships were observed between FoyRll
expression and response t o MoAbs 8A6 (a-GPIV) and ALB-6 (a-CDS)
(not shown).
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2265
PLATELET ACTIVATING MONOCLONAL ANTIBODIES
Scrotonin relcasc was also induccd by MoAb 50H.19
(IgGl, a-CD9). This response was completely blockcd by
MoAb IV.3, in contrast to aggregation induced by this
MoAb (Fig 7A). Bccausc the scrotonin rcleasc studies were
pcrformcd in thc absence and thc aggrcgation studics in thc
prcscnce of plasma, it sccmcd possible that plasma affcctcd
the ability of IV.3 to inhibit activation by this MoAb. This
was confirmcd by dcmonstrating that IV.3 failed to inhibit
scrotonin rclcasc triggcred by 50H.19 in platelct-rich plasma
(Fig 7B). In contrast, IV.3 completely inhibited scrotonin
rclcasc induced by the IgG, MoAbs ALB-6, 8A6, and
OP-G2 in platelet-rich plasma (not shown). Plasma and
serum hcated to 56°C for 30 minutes and unheated plasma
containing 10 mmol/L EDTA failed to support serotonin
...
...
_
I
I
_
6
Group 3 PImlelels
-1
75
..
..
T
6
1
-r*m
0,s
o(
0.26
ALe4
1
1
0.5
0.5
1
1
0.5
0.5
0
10
0
10
0
10
0
10
0.126
(ughn~)
Fig 6. Release of lT-serotonin from platelets of a donor from
group 3 (r;) and a donor from group 1
in response to different
amounts of M o A k OPG2 (A) and ALB-6 (B) (average of three
determinations). Error bars indicate 21 SD. Significanceof differences
is *** <.001, ** e.01, <.M.
Results shown are typical of three paired
studies in which responses of group 1 and group 3 platelets were
compared. Similar results were obtained with MoAb 8A6 (not shown).
(a)
Induction of serotonin release hy MoAbs. t4C-scrotonin
relcasc from platclcts was induccd by about one tenth the
amount of MoAb rcquircd to induce an aggregation rcsponsc. As shown in Figs 6, A and B, at low conccntrations,
thc IgGl MoAbs caused relcase of I4C-scrotonin from
group 3 platclcts morc readily than from group 1 platelets.
At highcr conccntrations of MoAb, group 1 platclcts also
rclcascd scrotonin. As with aggregation, scrotonin rclcase
was blocked by prior addition of MoAb IV.3 (a FcyRII) at
10 wg/mL (not shown).
54(.19(UPrmO
S
s
1
1
5
5
$
1
w.3 cuo"
0
IO
0
10
0
10
0
10
Fig 7. Serotonin release induced by MoAb 50H.19 In group 1 and
group 3 platelets in plasma-free (A) and plasma-containing media (B).
Release was inhibited by MoAb IV.3 in buffer (A), but not in plasma
le).
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2266
TOMIYAMA ET AL
release in response to 50H.19 in the presence of IV.3.
These findings suggest that complement activation by
50H.19 accounts for its ability to induce the release reaction
in platelets whose FcyRII receptors are blocked by IV.3.
DISCUSSION
Our studies show that platelets from different normal
subjects vary greatly in their responsiveness to IgGl MoAbs
specific for membrane glycoproteins CD9, GPIV, and
GPIIb/IIIa. Inhibition of the action of these MoAbs by
MoAb IV.3 specific for the FcyRII receptor is confirmatory
of previous report^^.^-^^ and consistent with the view that the
Fc domains of activating MoAbs react with platelet FcyRII
receptors to induce release and aggregation. Recent reports indicate that this occurs both on an intra-platelet and
inter-platelet b a s i ~ . ~ J ~
Our quantitative studies of FcyRII expression on platelets using radiolabeled Fab from MoAb IV.3 showed stable
differences in the number of FcyRII expressed on platelets
of normal individuals, in confirmation of the findings of
Rosenfeld et al,42who used intact IV.3. Platelets from the
20 subjects studied bound 1,660 to 4,610 IV.3 Fab (average
3,060), about twice the value (1,350) obtained by Karas et al
using a dimeric IgG pr0be.4~With intact IV.3, we obtained
values for FcyRII expression about half as great as those
found with Fab (data not shown). King et a P also found
that IV.3 Fab binding to platelets was about twice that of
intact IV.3. The total number of binding sites per platelet
they identified using labeled IV.3 Fab (4,700) was about
twice the number we measured. However, their binding
studies were performed at low ionic strength, which they
found doubled the apparent number of Fc receptor sites.
Lower values for Fc receptors obtained with intact MoAbs
than with Fab may reflect bivalent binding of the intact
molecule to FcyRII.
On the basis of the 41H.16:1V.3 binding ratios, it was
possible to divide our panel into three groups (Fig 3). Using
nomenclature applied to monocyte^,^^-^^ these appear to
correspond to homozygotes for the LR form of FcyRII
(group l), homozygotes for the HR form (group 3), and
LR/HR heterozygotes (group 2). Platelets judged to be HR
homozygous on this basis all responded well to activating
MoAbs of the IgGl subclass, and those judged to be LR
homozygous all responded poorly (Fig 4). However, platelets from apparent heterozygotes (group 2) varied widely in
their sensitivity to MoAbs. Responsiveness of group 2
platelets to OP-G2 correlated fairly well with the number of
FcyRII molecules expressed (Fig 5). No such relationship
was observed with 8A6 or ALB-6. These findings indicate
that both FcyRII phenotype and the number of FcyRII
molecules carried on the platelet surface influence the
responsiveness of platelets from heterozygous (group 2)
individuals. The variation in response of group 2 platelets to
8A6 and ALB-6 implies that other, unrecognized factors
are also important. Rosenfeld et a142found that the response of washed platelets to heat-aggregated IgG (HAIgG)
correlated with the number of FcyRII receptors expressed.
We confirmed this finding with our platelet panel (data not
shown). However, platelets from donors in groups 1 and 3
responded similarly to HAIgG, indicating that this particular response is not affected by FcyRII phenotype. Similar
observations were made by Looney et alez8
Target molecules recognized by the three IgGl MoAbs
used in our study are expressed in different numbers on the
platelet surface (CD36 about 20,000, CD41 about 40,000 to
50,000, and CD9 about 65,000 molecules per platelet).
However, MoAb 8A6 specific for CD36 produced a maximum platelet response at a concentration lower than that
required for OP-G2 or ALB-6. Therefore, within the range
from 20,000 to 65,000, the number of target molecules per
platelet does not appear to be an important determinant of
response. CD9, CD36, and CD41 are not known to vary
significantly on platelets of normal subjects except in rare
individuals with genetically determined deficiencies of
CD3645 or CD41 (type I Glanzmann’s thrombasthenia).
The concordant behavior of group 1platelets in response to
all three of the IgGl MoAbs essentially rules out the
possibility that their poor responses were caused by a
deficiency of target protein.
As noted, the FcyRII gene family encodes a number of
mature proteins expressed differently in various cell types.
41H.16 is thought to bind to all FcyRII gene products
except the allelic form of FcyRIIA that contains a histidine,
rather than an arginine residue, at position 131.15 Therefore, the failure of group 1 platelets to bind detectable
amounts of 41H.16 is consistent with the possibility that
only products of the FcyRII gene and not those of the
FcyRII B and C genes are expressed on platelets.
Activation of platelets by the IgG2, MoAb 50H.19 specific for CD9 requires special comment because this MoAb,
in contrast to OP-G2, ALB-6, and 8A6, failed to differentiate between HR and LR platelets and because, in a plasma
medium, activation by 50H.19 could not be blocked by IV.3,
even at concentrations as high as 50 pg/mL. However, IV.3
readily blocked the action of 50H.19 on platelets in a
plasma-free medium (Fig 7). The IgG2, subclass of murine
Ig is a potent activator of the classical complement pathway,
in contrast to the IgGl subclass,46 and it is known that
release can be triggered by assembly of the terminal
complement components in the platelet membrane.47These
facts, together with our finding that heated plasma and
serum and plasma containing 10 mmol/L EDTA were
unable to support 5OH.19-induced release in the presence
of IV.3, strongly suggest that 50H.19 activates platelets by
two mechanisms: one complement-dependent and the other
dependent on binding of its Fc region to FcyRII. Griffith et
a135 have recently shown that immobilized F(ab’)z from
50H.19, but not soluble F(ab’)z, can activate platelets by a
direct action on CD-9.35
Together with previous reports, our findings indicate that
MoAbs activate platelets by several different mechanisms:
(1) Fc-mediated activation of FcyRII; (2) complement
activation; and (3) in the case of certain MoAbs,1,s.12direct
activation via the target protein to which they bind. Platelets from normal subjects vary greatly in their susceptibility
to Fc-mediated activation by IgGl MoAbs, and failure to
respond to these MoAbs is associated with homozygosity
for one of the two defined alleles of the FcyRIIA gene (Figs
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2267
PLATELET ACTIVATING MONOCLONAL ANTIBODIES
studies of platelet FcR may be relevant to platelet destruc3 and 4). In the absence of plasma, “non-responder”
tion mediated by alloantibodies reactive with Class I HLA
platelets can be induced to release serotonin by increasing
alloantigens. A number of reports of platelet activation and
the concentration of stimulating MoAbs (Fig 6). However,
thrombosis induced by platelet-reactive autoantibodies have
high concentrations of MoAbs did not cause group 1
appeared,52-ssand it seems possible that platelet activation
platelets to aggregate in platelet-rich plasma. These obseroccurs via FcR in some of these patients. In one study, a
vations suggest that the two alleles of FcyRII differ in their
mild hemostatic defect was observed in three patients
affinity for murine IgGl Fc and that high levels of monowhose platelets responded poorly to an MoAb specific for
meric IgG in human plasma modulate the response to these
CD9.56Finally, heparin-induced thrombocytopenia/ thromMoAbs by competing with their Fc domains for binding to
bosis appears to be mediated by immune complexes reacFcyRII. Thus, in the presence of plasma, only the allele of
tive with platelet F c R and
~ ~ platelets from different normal
FcyRII with higher affinity for IgGl Fc is activated.
subjects differ widely in their sensitivity to heparinAlthough FcR of classes I, 11, and I11 appear to be
dependent a n t i b o d i e ~ . ~Further
~ , ~ ~ studies to determine
redundant in their functions, it has been argued that
whether the FcyRII receptor phenotype of platelets predisFcyRII has special functional
Whether the
poses to any of these conditions appear warranted.
FcyRII phenotype of platelets is relevant to platelet physiology or pathophysiology in humans therefore deserves
consideration. A recent report showed that the LR form of
ACKNOWLEDGMENT
FcyRIIA preferentially binds multimers of human I ~ G z , ~ ~We are indebted to Drs Clark Anderson, A.R.E. Shaw, and John
the IgG subtype mainly involved in the human immune
Barnwell for generously supplying monoclonal antibodies used in
response to saccharide antigens. Alloantibodies specific for
these studies; to Kurt Wolfmeyer for excellent technical assistance;
class I HLA antigens are capable of inducing platelet
to Dr Peter Newman for helpful advice; and to the Word
activation and a g g r e g a t i ~ nand
~ ~ ,may
~ ~ bind to platelets at a
Processing Department of The Blood Center for preparation of
this manuscript.
site close to the FcyRII receptor.s* Therefore, further
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1992 80: 2261-2268
Response of human platelets to activating monoclonal antibodies:
importance of Fc gamma RII (CD32) phenotype and level of expression
Y Tomiyama, TJ Kunicki, TF Zipf, SB Ford and RH Aster
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