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Effects of OKM5, a Monoclonal Antibody to Glycoprotein IV, on Platelet
Aggregation and Thrombospondin Surface Expression
By Martha L. Aiken, Mark H. Ginsberg, Vicky Byers-Ward, and Edward F. Plow
The monoclonal antibody, OKM5. recognizes an 88-Kd
monocyte membrane protein and also binds to the platelet
membrane protein, GPIV (GPlllb, CD36). In this study, we
have found that the OKM5 target epitope is present at
approximately 12.000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory
effects on platelet function. In the absence of other stimuli.
OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses
required intact antibody as F(ab’), fragments were not
active but blocked the stimulatory activity of the intact
antibody. In contrast, exposure of platelets to OKM5
followed by another strong stimulus such as thrombin
resulted in a marked suppression of fibrinogen, fibronectin.
and von Willebrand factor binding to the cells. This effect
was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations
that interfered with fibrinogen binding to thrombinstimulated platelets by 80% to 90%. platelet binding of
exogenous thrombospondin, or surface expression of endogenous thrombospondinwas not affected. The inhibitory
effect of OKM5 on fibrinogen binding t o thrombinstimulated platelets was related to the formation of massive platelet aggregates in the samples. These results
show that interaction of OKM5 with its target antigen on
platelets can elicit diverse functional responses from the
cells.
0 1990 by The American Society of Hematology.
M
responses of platelets and other cells. In the present study, we
have examined the stoichiometry and functional consequences of the interaction of OKM5 with platelets. The
results indicate that this MoAb exerts multiple effects on
platelets, suggesting a multifunctional role of its target
antigen in platelet responses.
ONOCLONAL antibodies (MoAbs) have been used
extensively to examine the structure and function of
platelet membrane glycoproteins.’.’ The predominant targets
of such MoAbs have been GPIb and GPIIb-IIIa. While the
vast majority of described MoAbs to these or other platelet
membrane proteins have inhibited platelet responses, a small
number of reports have also described antibodies that activate platelet^.^" The target antigens of these activating
MoAbs have been either the light chain of GPIIb’ or a 23-Kd
protein:’
unrelated to GPIIb. In the present report, we
describe an MoAb that activates platelets by reacting with a
different target antigen. In addition, this same MoAb that
initiates the classical platelet responses of aggregation and
secretion, can also, under certain circumstances, inhibit
other platelet functions. The MoAb exhibiting these properties is OKMS.
OKMS was initially described by Talle et a16 as an MoAb
reacting with monocytes; and it was shown by immunoprecipitation that the MoAb recognized an 88-Kd membrane
protein. OKMS was also reported to react with platelets,
endothelial cells, and certain tumor cells.6-*On platelets, the
target antigen exhibits the properties of GPIV:-” also
designated CD36 or GPIIIb. Additional studies have identified several interesting functions for the OKM5 antigen. One
intriguing set of data has indicated that the OKMS antigen is
involved in mediating the adherence of malarial-infected
erythrocytes to endothelial cells, as well as other cell
A second function has been attributed to the
OKM5 antigen on platelets; namely, GPIV has been implicated as a collagen receptor on platelets.” Still a third set of
data has suggested that GPIV is the platelet receptor for
thrombospondin (TSP)?” a member of the platelet-adhesive
protein family. This proposed function for GPIV originally
was based on the observation that OKMS inhibited the
platelet surface expression of TSP as reported by a radiolabeled polyclonal anti-TSP Fab fragment, and TSP interacted
with immunopurified OKMS antigen.gSupport for this latter
function of GPIV was subsequently obtained in studies
involving the purified membrane protein.” However, it is of
note that when CD36 was expressed in COS cells, these cells
did not acquire TSP binding proper tie^.'^ Based on these
analyses, the OKMS antigen appears to function in adhesive
Blood, Vol76, No 12 (December 15). 1990 pp 2501-2509
MATERIALS AND METHODS
OKM5 purification and characterization. OKMS ascites was
obtained from Ortho Diagnostics Systems, Inc (Raritan, NJ), and
three separate batches of this reagent were used during the course of
these studies, including 10 mL of ascites (3503-15)provided by Dr
Peter Kramer of Johnson and Johnson (Raritan, NJ). The MoAb
was purified essentially as described by Ey et a1.I6In brief, 3 mL of
ascites fluid, diluted 1:l with 0.1 mol/L phosphate buffer, pH 8.0,
was incubated for 18 hours with 6 mL of Protein-A-Sepharose
(Pharmacia Fine Chemicals, Piscataway, NJ) with gentle agitation
at 4OC. The resin was then washed with phosphate-buffered saline
(PBS) at pH 7.3 to remove unbound material. The MoAb was eluted
with 0.1 mol/L Na citrate, pH 4.5, and collected in 4-mL aliquots
into 0.5 mL of 1.0 mol/L Tris, pH 8.0.The fractions containing the
MoAb were pooled, treated with 0.5 mmol/L pamidinophenylmethyl-sulfonyl fluoride (Calbiochem, La Jolla, CA), and dialyzed
against PBS. It was our experience that purified OKM5 frequently
lost activity during storage due to aggregation and that a high
concentration of bovine serum albumin (BSA) helped to stabilize the
From the Department of Biochemistry, University of Texas
Health Science Center at Tyler; and the Committee on Vascular
Biology, Research Institute of Scripps Clinic, La Jolla, CA.
Submitted December 21,1989; accepted August 17,1990.
Supported by National Institutes of Health Grant Nos. HL16411
and HL39702. This is publication No. 4804 of the Research
Institute of Scripps Clinic. “Blood drawing was performed in the
GCRC of Scripps Clinic. supported by Mol RR00833.”
Address reprint requests to Edward F. Plow, PhD, Committee on
Vascular Biology, Research Institute of Scripps Clinic. 10666 N
Torrey Pines Rd, La Jolla, CA 92037.
Thepublication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordancewith 18 U.S.C. section 1734 solely to
indicate thisfact.
6 1990 by The American Society of Hematology.
OOO6-4971/90/7612-0010$3.00/0
2501
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2502
reagent. Therefore, most of the antibody was stored at -8OOC in
1.0% BSA, and only a portion was frozen without BSA for
radiolabeling. The activity of OKM5 was routinely tested on the day
of its use in a solid phase radiometric assay in which binding of
OKM5 to platelets coated onto microwells was assessed as previously
described for other platelet MoAbs.I7 OKM5 was detected by the
binding of 1251-antimouseimmunoglobulin G (IgG) at 0.5 pg/mL.
Active OKM5 gave a signal of at least threefold above background
in this assay at concentrations of 20 fmol/L.
F(ab’), fragments of OKM5 were prepared by pepsin digestion of
the antibody. OKM5, at 140 to 220 pg/mL in 1% BSA (spiked with
a small amount of ‘Z51-OKM5to permit polyacrylamide analysis of
the preparations), was dialyzed overnight into 0.2 mol/L NaCI, 0.2
mol/L sodium acetate, pH 4.0, and the pH was then adjusted to 4.0
with acetic acid. Pepsin (Sigma Chemical Co, St Louis, MO) was
added to a final concentration of 30 pg/mL, and the mixture was
incubated for 3 hours at 37OC, at which time the pH was adjusted to
7.0.
Other proteins. Fibrinogen (Fg), fibronectin (Fn), and von
Willebrand factor (vWF) were purified from fresh human plasma.
The purifications involved differential ethanol or ether precipitation
for Fg, gelatin-Sepharose affinity chromatography for Fn, and gel
filtration of cryo-precipitates for vWF. TSP was purified from fresh
platelet concentrates. The physical and platelet binding characteristics of the radioiodinated and nonlabeled forms of each of these
proteins as prepared by our laboratories have been previously
described.I8-” The preparation of the polyclonal anti-Fg and the
production and purification of F(ab’), fragments of this antibody
have also been previously reported in detail.” The MoAbs, TSPI-1
(a TSP antibody), and PMI-1 (a GPIIb antibody), were produced
and isolated as previously reported.22TSPI-3, an MoAb to TSP that
does not cross-compete with TSPI-1, is of the IgG, subclass and was
derived from the same fusion as TSPI- 1. These MoAbs were purified
by the same protocol as described for OKM5, but they did not
exhibit the instability of OKM5, even in the absence of BSA.
Radiolabeling of proteins. Proteins were radiolabeled by a
modified chloramine-T procedure to specific activities of 0.5 to 1.5
pCi/pg. Residual free iodine was removed from the adhesive
proteins by dialysis against PBS. To remove the residual free iodine
from the radiolabeled antibodies, including OKM5, the samples
were gel filtered on PD-10 columns (Pharmacia), equilibrated in and
eluted with PBS. The protein concentrations of the radiolabeled
antibodies was determined from the original protein concentration of
the unlabeled protein, based on Lowry protein determinations or by
their absorbance at 280 nm, and the precipitability of the radioactivity in 15% trichloroacetic acid. The precipitability in 15% trichloroacetic acid of radiolabeled OKM5, as well as of the other radiolabeled proteins, exceeded 95%, and protein recoveries were typically
greater than 90%.
Platelets. Washed human platelets were isolated from the blood
of healthy adult donors with the approval of our local Human
Investigations Committee and the Department of Health and
Human Services. In brief, 90 mL of fresh human blood, collected
into acid-citrate-dextrose, was centrifuged in two steps, first to
obtain platelet-rich plasma (PRP) and then to obtain a platelet
pellet. The cells were resuspended and gel filtered on Sepharose
CL-2B (2.5 x 8 cm) (Pharmacia). The washed platelets were
collected, and the cells were counted electronically in a Coulter
Counter Model Zf (Coulter Electronics, Inc, Hialeah, FL) or
microscopically in a hemacytometer. In some experiments, the cells
were fixed with paraformaldehyde before microscopic counting. For
this purpose, paraformaldehyde was added to the platelet suspension
at a final concentration of 0.5%. After 30 minutes at 22OC, an equal
volume of 0.02 mol/L NH,Cl in 0.15 mol/L Tris, pH 7.2, was
AIKEN ET AL
Ligand binding assays. 1251-radiolabeled
antibodies or adhesive
proteins were incubated with washed human platelets at cell
concentrations ranging from 1 x lo7 to 4 x 108/mL in Tyrode’sBSA buffer, pH 7.4, supplemented with selected divalent ions as
previously
Following 30 to 60 minutes at 22OC, 50-pL
aliquots of the cell suspension were layered on 20% sucrose, and the
platelets were pelleted by a 2.5-minute centrifugation in a Beckman
microfuge B (Beckman, Palo Alto, CA). The tips of the tubes were
amputated, and the molecules associated per cell calculated based on
the determined specific activities of the ligands. Binding isotherms
were analyzed with the Ligand Computer Program26as previously
described in studies from our
Platelet aggregation. Washed human platelets at a final concentration of 1 x 108/mLin Tyrode’s-BSA buffer containing 1 mmol/L
Ca2+and Mg2+were incubated at 37OC at a constant stirring rate of
1,000 rpm in a dual channel platelet aggregometer (Scienco, Inc,
Morrisen, CO). Exogenous Fg was added at 0.1 mg/mL. Aggregation was initiated by addition of the stimulus. Aggregation studies
were also performed in a I/* dilution of PRP with cells at 1 to 2 x
1O8/mL.
Platelet secretion. To quantitate the release of serotonin, PRP
(-20 mL) was incubated with 150 pCi of ’H-serotonin (27.3
Ci/mmol/L; NEN Research Products, Boston, MA) for 60 minutes
at 37OC. The platelets were then isolated by gel filtration and
suspended at 1 x 108/mL in Tyrode’s buffer containing 2 mmol/L
Ca2+, 2 mmol/L imipramine, 300 nmol/L Fg, and the selected
stimulus. After 30 minutes at 22OC, conditions which paralleled the
ligand binding assays used in this study, aliquots were centrifuged
through 20% sucrose. The pelleted cells were solubilized with 10 mL
of Aquafluor (NEN) and counted with a Beckman LS 7500
B-counter (Beckman Instruments, Inc, Fullerton, CA). j3-thromboglobulin secretion was measured with a commercial radioimmunoassay (Amersham, Arlington Heights, IL). Washed human platelets at
2 x 108/mL were incubated with or without 20 nmol/L OKM5 for 5
minutes followed by the addition of other platelet stimuli to the
samples. After 5 minutes, D-phenylalanyl-L-prolyl-L-arginine
chloromethylketone (PPACK from Calbiochem), was added to the cells
stimulated with thrombin. Following a 10-minute incubation at
22OC, the cells were placed on ice and fixed with cold 2% paraformaldehyde. After 30 minutes, the paraformaldehyde was neutralized
with NH4C1, the platelets were pelleted by centrifugation, and the
supernatants assayed for j3-thromboglobulin.
Analytical procedures. Polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS-PAGE) was performed in the buffer
system of LaemmLz7 When required for sample reduction, 1%
2-mercaptoethanol was used. Molecular weights were estimated
from the electrophoretic mobilities of the test samples relative to
those of protein standards. For quantitation of the intensity of
autoradiographic bands, a Zeineh soft laser scanning densitometer
equipped with an integrator was used. For molecular exclusion
chromatography by high performance liquid chromatography
(HPLC), a TSK 4000 column (0.75-X60 cm) was used. The column
was equilibrated in PBS at pH 6.75 and run at 0.9 mL/min. Elution
profiles were monitored at 280 nm or by collecting 1-minute
fractions and testing them for the platelet aggregating activity. For
OKM5, 45 pg of the antibody was applied to the column and the
aggregating activity of the fractions was detected by added IO-pL
samples to PRP.
RESULTS
Binding of OKMS to platelets. To assess the direct
interaction of OKM5 with platelets, purified OKM5 was
radioiodinated to a specific activity of approximately 1.5
pCi/pg. The radiolabeled antibody was characterized by
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2503
OKM5 AND PLATELET FUNCTION
15r
--0
X
A
flesring
A
Fig 1. Bindingof OKM5 to platelets. Thrombintheophstimulated (0.26 U/mL) or resting (PGE,
ylline at 1 pg/mL and 1 mmol/L, respectively)
washed human plateletswere incubated with varying concentrations of OKM5. After 30 minutes at
22°C. the number of antibody molecules bound per
cell was determined. The data are not corrected
for nonspecific binding, which was estimated to be
less than 0.1% by the Ligand Computer Program?'
+
SDS-PAGE. Under both reducing and nonreducing conditions, '251-OKMSand nonlabeled OKM5 exhibited the same
electrophoretic properties. In preliminary experiments, the
radiolabeled antibody was found to bind rapidly to platelets;
apparent equilibrium binding was observed within 5 minutes,
and the extent of binding did not change for at least 60
minutes. When varying quantities of '251-OKMSwere added
to washed human platelets, saturable binding of OKMS to
both resting and stimulated platelets was observed at approximately 10 nmol/L (Fig 1). At a saturating concentration of
'251-OKMS,a fivefold excess of nonlabeled OKM5 inhibited
binding of the radiolabeled ligand by greater than 80%. Data
such as those shown in Fig 1 were analyzed with the Ligand
Computer Program,26 and binding parameters derived for
nonstimulated and thrombin-stimulated platelets in the presence or absence of divalent ions are summarized in Table 1.
The nonsaturable binding, N l , was extremely low; and, as it
did not exceed 0.1% of total added 1251-OKMSradioactive
under all conditions, subtraction of nonspecific binding was
not necessary to see clear evidence of saturation of binding
(see Fig 1). For example, at a IO-nmol/L input concentration, 8,500 molecules of 1251-OKMSwere bound per platelet
of which 320 molecules were nonspecifically associated. The
mean number of OKM5 binding sites per platelet under all
experimental conditions was 11,900 f 1,800. Platelet stimulation and/or divalent ion conditions had only a minimal
Table 1. OKM5 Binding Parameters
Platelet Stimulation
None
None
Thrombin
Thrombin
Divalent Ions
kd (nmdILI
+
Ca2+ Mg2+ 0.73 f 0.31
EDTA
0 . 6 2 f 0.25
Ca2+ Mg2+ 1.70 k 0 . 5 0
EDTA
2.80 f 0.20
+
No. of Sites
13,200 f 3,100
10,800 f 5 0 0
13,600 f 1,100
10,000 2 400
Varying quantities of '251-OKM5 were added to platelets at 2 x
108/mL in the presence of CaZ+ Mg2+ (2 mmol/L each) or EDTA (5
mmol/L). The cells were either stimulatedwith 0.25 U/mL of thrombin or
maintained in PGE, and theophylline at 1 pg/mL and 1 mmol/L,
respectively, to prevent stimulation. Binding was measured after 30
minutes at 22OC. The data were subjected to Scatchard plot analyses,
using the Ligand Computer Program,26 and the results are summarized
above.
+
influence on the number of binding sites. From the apparent
kd values listed in Table 1, it is clear that OKMS bound to
both stimulated and resting platelets with high affinity, and
the interaction was not affected by divalent ion availability.
Thrombin stimulation increased the apparent kd of the
interaction slightly. The binding of '251-OKMSto platelets
was not inhibited by large excesses of TSPI-3 or PMI- 1, two
MoAbs of the same subclass as OKMS, whereas it was
inhibited by nonlabeled OKM5 derived from three different
lots of the antibody.
OKM5 as a platelet stimulus. OKMS, at 20 nmol/L,
was added to platelets at 1 x 108/mL in the presence of 300
nmol/L Fg and 1 mmol/L Ca2+and Mg2+.After a short lag
phase of approximately 2 minutes, an increase in light
transmittance was observed in a platelet aggregometer,
consistent with an aggregation or agglutination response. A
classical platelet stimulus, adenosine diphosphate (ADP),
induced aggregation of the same preparation of platelets
without a detectable lag phase (Fig 2). The response induced
by 20 nmol/L OKMS was completely blocked by addition of
EDTA (5 mmol/L), PGE, (1 pg/mL) and theophylline (1
mmol/L), the arginyl-glycyl-aspartyl-serinetetrapeptide (0.5
mmol/L), and the ADP scavenger system of creatine phosphate (12.7 mg/mL)/creatine phosphokinase (SO pg/mL).
All of these agents inhibited Fg-dependent platelet aggregation induced by ADP and low concentrations of other platelet
agonists such as epinephrine and t h r ~ m b i n , ~ but
~ . * ~are
known not to effect platelet agglutination such as in response
to ristocetin. OKMS also aggregated platelets in plasma in
the same dose range as it was active with washed platelets.
However, in plasma the inhibition by ADP scavengers could
be overcome by a further increase (fivefold) in OKM5
concentration. The existence of ADP-dependent and independent pathways for platelet aggregation is also noted for many
platelet agonists.29
The capacity of OKM5 to induce a platelet secretory
response was also examined. Serotonin and B-thromboglobulin were used to monitor dense and a granule secretion,
respectively. As shown in Table 2, OKMS induced both
serotonin and B-thromboglobulin release. The extent of the
secretion of the a and dense granule markers induced by
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2504
AIKEN ET AL
Table 2. Effect of OKM5 on Platelet Secretion
% Secretion
Platelet Stimulation
+
theophylline)
None (PGE,
OKM5
Thrombin
Thrombin
OKM5
ADP
OKM5
ADP
+
+
I-
r
Y
CJ)
.2
Serotonin
&Thromboglobulin
0
51
57
89
29
75
<9
53
50
ND
16
46
PRP was incubated with 3H-serotonin for 1 hour at 37°C and the
platelets were then isolated by gel filtration. The washed cells were
incubated with prostaglandin E, (PGE,) and theophylline, at 1 pg/mL and
1 mmol/L, respectively, 20 nmol/L OKM5, 0.25 U/mL thrombin, 10
pmol/L ADP, or a combination of these reagents in the presence of 2
mmol/L Ca’+ and 2 pmol/L imipramine. After 10 minutes, the cells were
centrifuged through 20% sucrose, and the cell pellet was solubilized and
counted to determine the percent serotonin release. j3-thromboglobulin
secretion was measured by radioimmunoassay. Washed human platelets
at 2 x 108/mLwere incubated with the same reagents indicated above.
After 10 minutes, the cells were placed on ice and fixed with 2%
paraformaldehyde for 3 0 minutes (seeMaterials and Methods). The cells
were then pelleted by centrifugation, and the supernatants were assayed
for 8-thromboglobulin by radioimmunoassay. Percent secretion is calculated relative to a sample of lysed platelets, which contained 26.4 p g
8-thromboglobulinll O9 cells.
Abbreviation: ND, not determined in this experiment. (In a separate
thrombin produced 8% greater
experiment, the combination of OKM5
j3-thromboglobulin release than OKM5 alone, and 27% greater release
than thrombin alone).
+
Fig 2. Effect of OKM5 on platelet aggregation. Washed human
platelets in the presence of 1 mmol/L Ca2+were stirred at 1,OOO
rpm in an aggregometer at 37°C. Fg was present at 0.1 mg/mL.
ADP (10 pmol/L) or OKM5 (20 nmol/L) was added to the cells.
OKM5 was similar to that obtained with 0.25 U/mL
thrombin. ADP caused only modest a and dense granule
secretion; but, when added in combination with OKM5,
extensive secretion was observed.
To further assess the ability of OKM5 to stimulate
platelets, its capacity to induce Fg receptors was examined.
Addition of 20 nmol/L OKM5 to resting platelets resulted in
an increase in 12’I-Fg binding to the cells (Table 3). The
extent of Fg binding induced by OKM5 was similar to that
supported by ADP and thrombin. PGE, and theophylline
inhibited Fg binding induced by OKM5.
To investigate the basis for the stimulatory activity of
OKM5, F(ab’), fragments of the antibody were prepared.
Densitometric scanning of SDS-PAGE established that
greater than 75% of the intact antibody was digested to a
105-Kd F(ab’)* fragment. While 20 nmol/L intact OKM5
induced aggregation of washed platelets, a fivefold higher
concentration of the F(ab’)* fragments did not (Fig 3).
Moreover, the antibody fragments blocked the capacity of
the intact antibody to induce platelet aggregation. This effect
was specific as the platelets retained their capacity to respond
to ADP. BSA in buffer, exposed to the same protocol used to
prepare the OKM5 F(ab’), fragments, did not block OKM5-
induced platelet aggregation. When OKM5 was subjected to
the protocol, except that the pepsin was omitted, the aggregating activity of the antibody was retained. The requirement
for intact antibody was also shown for induction of Fg
binding. In an experiment in which 5 nmol/L intact OKM5
induced the binding of 4,600 Fg molecules per platelet, 100
nmol/L F(ab’), fragments supported the binding of only 900
Fg molecules, very similar to the level of 700 Fg molecules
bound per platelet without any added stimulus.
To determine if the stimulatory activity of intact OKM5
was a function of the monomeric antibody, OKM5 was
subjected to HPLC analysis on a TSK 4000 molecular
exclusion column and 1.O-minute fractions were collected.
Table 3. Induction of Fg Receptors by OKM5 and Other
Platelet Stimuli
StimuIus
None
OKM5
ADP
Thrombin
PGE,/theo
Fibrinogen Bound
imolecules/platelet)
550
17,230
14,230
25,800
480
+ OKM5
Washed human platelets were incubated in Tyrode’s-BSA buffer
containing 1 mmol/L Ca2+with OKM5 (20 nmol/L), ADP (10 pmol/L),
PGE, and theophylline (1
thrombin (0.25 U/mL), or 20 nmol/L OKM5
pg/mL and 1 mmol/L, respectively). After 5 minutes, ’‘%Fg was added
to a final concentration of 300 nmol/L. Followinga 30-minute incubation
at 22°C. the molecules of Fg bound per platelet were determined. The
data are the means of triplicate determinations from within a single
experiment and are representative of eight similar experiments.
+
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2505
OKM5 AND PLATELET FUNCTION
Fig 3. Effects of intact OKM5 and its F(ab’), fragments on
platelet aggregation. Aggregation was performed as in Fig 2, and
the tracings have been followed for 6 minutes after addition of the
antibody or its F(ab’), fragments. Samples were tested at the
following concentrations: OKM5 IgG, 20 nmol/L; OKM5 F(ab’),.
100 nmol/L: ADP, 10pmol/L.
Platelet aggregatory activity eluted in the fractions from
19.0 to 23.0 minutes, coinciding to the elution position of a
control MoAb, TSPI-1 (protein peak at 20.2 minutes). No
platelet aggregation activity was detected in fractions eluting
before 18.0 minutes. Fg, with a molecular weight of 340 Kd,
eluted with a peak at 14.7 minutes, establishing that the
19.0- to 23.0-minute range was not the void volume of the
column. Thus, the platelet stimulatory activity of OKM5 was
not dependent on aggregates of the antibody.
Efects of OKM5 on the interaction of adhesive proteins
with the platelet surface. In the course of experiments to
assess the effects of OKM5 in combination with other
platelet stimuli on 1251-Fg
binding to platelets, unanticipated
results were obtained. Namely, Fg binding did not occur
when both OKM5 and thrombin were added to platelets. In
these studies, OKM5 was preincubated with washed platelets before addition of thrombin. As shown in Fig 4, Fg
binding to thrombin-stimulated platelets was suppressed by
OKM5; in a dose-dependent manner; and, at concentrations
as low as 5 nmol/L, the inhibition exceeded 90%. Indeed, it
should be noted that the inhibitory effect of OKM5 occurred
in a dose range consistent with the estimated affinity of the
antibody for platelets. The estimated kd of OKM5 for
thrombin-stimulated platelets was 1.7 nmol/L (Table 1) and
12’I-Fg binding to thrombin-stimulated platelets was 50%
inhibited by approximately 1 nmol/L OKM5 (Fig 4),
showing a concordance between the binding of OKM5 to
platelets and its inhibitory activity. Thus, the combination of
OKM5 and thrombin abrogated the component of Fg binding induced by thrombin alone or by the antibody alone. In
contrast, OKM5 concentrations as high as 22 nmol/L had no
effect on ADP-induced Fg binding. With phorbol myristate
acetate (PMA) as the platelet stimulus, intermediate inhibition was observed, which reached 48% at 22 nmol/L OKM5.
Under the same experimental conditions that resulted in
effective inhibition of Fg binding to thrombin-stimulated
platelets, OKM5 (4.5 nmol/L) also suppressed the binding
of Fn by 77% and vWF by 93%. As with Fg, a partial
inhibition of binding was noted for PMA-stimulated platelets; Fn and vWF binding are inhibited by 50% and 51%,
respectively, under conditions where Fg binding was inhibited by 48%. In contrast, TSP binding to thrombinstimulated platelets was not inhibited by OKM5. With
’”I-TSP added at 90 nmol/L, no inhibition was observed
either in the presence of 1 mmol/L Ca2+ + Mg2+or in the
presence of 1 mmol/L EDTA. At this TSP input concentration, 72% of the binding was divalent ion dependent. Thus,
neither the divalent ion dependent nor independent component of TSP binding was affected by OKM5.
As inhibition of TSP binding by OKM5 is predicted from
the data of Asch et
the capacity of the MoAb to influence
the surface expression of endogenous TSP was also evaluated. In this analysis, the surface expression of TSP on
thrombin-stimulated platelets was monitored with 12’I-TSPI1,22and 1251-Fgbinding was measured in parallel. Divalent
ion conditions were used to attain maximal surface expression for both ligands. Under these conditions, the stimulated
cells bound 13,900 molecules of TSPI-1 (the divalent iondependent component of the measured TSP surface expression accounted for 68% of the total TSPI-1 binding22)and
29,000 molecules of Fg. As shown in Fig 5, OKM5 did not
inhibit the surface expression of TSP at a concentration of 9
.-
fOKM51x 1P M
Fig 4. Effect of OKM5 on the binding of Fg to stimulated
platelets. OKM5 was added at varying concentrations to washed
human platelets at 1 x 108/mL in the presence of 2 mmol/L Caz+.
The cells were stimulated with either ADP (10 pmol/L), thrombin
(0.25 U/mL). or PMA (200nmol/L) before the addition of ’*‘I-Fg
(300 nmol/L). After 30 minutes, Fg binding was determined.
Percent inhibition was calculated by dividing the molecules of
ligand bound per cell in the presence of the antibody by the number
of molecules bound, in the absence of OKM5.
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2506
AIKEN ET AL
Table 4. Relationship Between the Effect of OKM5 on Fg Binding
and the Extent of Platelet Aggregation
Platelet Stimulation
None
OKM5
Thrombin
OKM5
thrombin
PMA
OKM5
PMA
ADP
OKM5
ADP
+
-
40
20!
+
E
0
..e
+
c
c
Platelet Amegation
(single platelets/grid)
Fibrinogen Bound
(molecules/platelet)
113
44
61
12
52
600
7,000
16,200
2,100
20,100
8,900
5,200
6,500
33
45
40
Washed human platelets were incubatedfor 5 minutes with 20 nmol/L
OKM5 before the addition of a second stimulus (thrombinat 0.25 U/mL,
PMA at 0.2 pmol/L or ADP at 10 pmol/L) or buffer. Aliquots of the fresh
cells were used to determine the extent of lz51-Fgbinding. The rest of the
cells were fixed with paraformaldehyde and resuspended in Tyrode's
buffer. After diluting the cells 1/10 with isotonic saline, the single
platelets were counted on a hemacytometer. These results sre representative of at least four experiments.
tool
I
I
I
I
J
2
4
6
8
10
IOKM51 x 10-gM
Fig 5. Effect of OKM5 on TSP surface expression. Conditions
were used t o obtain maximal TSP surface expression" or Fg
binding?' For TSP surface expression, washed human platelets, at
2 x 108/mLin the presence of 2 mmol/L Ca2+and 2 mmol/L Mg2+,
were incubated with varying concentrations of OKM5 in the
presence of PGE, and theophylline (1 pg/mL and 1 mmol/L,
respectively) t o prevent OKM5 induced secretion. After the
bminute preincubation, thrombin was added at 0.75 U/mL followed by a saturating concentration of '261-TSPI-1 (1.1 j"l/L).
After 60 minutes, the molecules of TSPI-1 bound per cell was
determined. For Fg binding, washed human cells at 1 x 108/mL
were incubated with varying concentrations of OKM5 in the
presence of 2 mmollL Ca. After 5 minutes, the cells were
stimulated with 0.25 U/mL thrombin. The molecules of Fg bound
per cell were determined after 30 minutes at 22°C.
nmol/L, whereas Fg binding was fully inhibited by OKM5 in
the same cell preparation. In similar experiments, but with
fourfold higher platelet concentrations, TSP surface expression was variably inhibited by saturating concentrations of
OKM5 (20 to 100 nmol/L). When inhibition was observed,
it did not exceed 50%, and visible platelet aggregates were
apparent. Under these circumstances, surface expression of
platelet Fg, measured with "'I-F(ab'), fragments of an
anti-Fg,*' was also inhibited.
The above observations suggest that platelet aggregation
contributes to the effects of OKM5 on the interaction of
exogenous or endogenous adhesive proteins with the cell
surface. Therefore, we determined if aggregate formation
paralleled the differential effects of the MoAb on Fg binding
induced by thrombin, PMA, and ADP. Platelets were
incubated with 20 nmol/L OKM5 or buffer for 5 minutes
before addition of thrombin, ADP, or PMA. Nonlabeled Fg
was present at 300 nmol/L to duplicate the experiments in
which the binding of 12'I-Fg was measured. After 30 minutes,
the cells were fixed with paraformaldehyde and observed
microscopically to determine the number of single versus
aggregated platelets per sample (Table 4). Cell suspensions
stimulated with thrombin in the presence of OKM5 exhibited a significant decrease in single platelets as compared
with cells stimulated with thrombin alone. In addition to this
marked decrease in free platelets, the aggregates themselves
were massive (Fig 6), containing large numbers of platelets.
These changes were not observed with ADP-stimulated cells,
and an intermediate response was observed with PMA. In
parallel with nonfixed cells, OKM5 inhibited the binding of
Fg by 87% to thrombin-stimulated cells, 0% to ADPstimulated cells, and 55% to PMA-stimulated cells. Thus, in
all samples exhibiting an inhibition of "'I-Fg binding by
OKM5, there was also a marked increase in aggregate
formation as reflected by a decrease in the number of single
platelets. Other combinations of agonists did not have the
Fig 6. Formation of massive aggregates in the presence of
OKM5
thrombin. Platelets in the left tube were unstimulated.
Platelets in the middle tube were identical except for the addition
of thrombin (0.25 UlmL) followed by PPACK (3pmol/L) before the
addition of Fg. In the tube on the right the cells were exposed t o 20
nmol/L OKM5 before the addition of thrombin. The concentrations
of cells (4 x 108/mL), divalent ions (Ca2+2 mmollL), and Fg (300
nmol/L final) was the same in all tubes. The arrow indicates the
massive aggregates formed when platelets were exposed t o both
OKM5 and thrombin.
+
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2507
OKM5 AND PLATELET FUNCTION
same effect as OKM5 and thrombin. Stimulating the cells
with thrombin and PMA resulted in 44 free cells per grid and
18,500 molecules of Fg bound per cell. If OKM5 was also
added with the thrombin and PMA, the number of free cells
per grid was reduced to 7 and the molecules of Fg bound per
cell was reduced to 1,700.
Because platelet aggregation should be less extensive at
low cell concentrations, the ability of OKM5 to inhibit
thrombin-induced Fg binding was assessed as a function of
cell concentration. The results of these analyses are summarized in Table 5 . The molecules of Fg bound per thrombinstimulated platelet in the absence of OKM5 remained
relatively constant, varying from 27,400 to 32,600 over the
range of cell concentrations tested in one experiment, and
from 14,200 to 15,200 in the second experiment. However,
the inhibition of Fg binding induced by OKM5 varied
considerably with cell concentration. The inhibition was only
14% at the lowest platelet concentration tested in contrast to
83% at the highest concentration tested.
Additional evidence for the role of platelet aggregation in
the inhibitory effects of OKM5 was obtained in experiments
altering the timed additions of thrombin, OKM5, and 1251-Fg
to the platelets. Maximal inhibition of Fg binding by OKM5
required that the cells be preincubated with the antibody
before the addition of the adhesive protein. A saturating
concentration of OKM5 (20 nmol/L) inhibited Fg binding
by 92% (19,500 molecules/platelet to 1,500 molecules/
platelet) when the antibody was preincubated with the cells
for 5 minutes before thrombin stimulation. Similar inhibition
was observed when OKM5 and thrombin were added simultaneously to the cells but still before Fg. In contrast, when the
same concentration of OKM5 was added after thrombin and
simultaneously with the '251-Fg,only 42% inhibition of Fg
binding was observed. These changes in inhibition were
paralleled by changes in platelet aggregation in the samples.
When incubated for 30 minutes in the absence of either
thrombin or OKM5, most of the platelets (85%) remained as
single, nonaggregated cells. OKM5 alone or thrombin alone
caused some platelet clumping during the 30-minute incubation, but single cells still predominated in the suspension and
the size of the platelet aggregates was small, usually containing approximately two to five platelets. When the platelets
Table 5. Effect of Platelet Concentration on the Inhibition of Fg
Binding by OKM6
Platelet Concentration
(platelets/mL)
Fibrinogen Bound
(% inhibition)
14.5
62
78
83
Washed human platelets, at varying concentrations, were incubated
with or without 20 nmol/L OKM5 for 5 minutes before thrombin
stimulation (0.25 U/mL). After a 5-minute incubation, the thrombin was
inactivated with PPACK and lZ51-Fgadded (300 nmol/LI. The molecules
of Fg bound per cell were determined after 30 minutes at 22OC. Percent
inhibition was calculated by dividing the Fg molecules bound in the
presence of OKM5 by the number bound in the absence of the MoAb and
is the mean of two experiments.
were treated first with OKM5 and then with thrombin, the
condition that resulted in greater than 90% inhibition of Fg
binding, most of the platelets were aggregated, and the
aggregates were massive. In contrast, when the platelets were
treated first with thrombin followed by the simultaneous
addition of OKM5 and Fg, the condition at which the
inhibition of Fg binding was less extensive, platelet aggregation was also considersbly less extensive.
The effect on Fg binding of F(ab'), fragments of OKM5,
which, as shown above, do not induce platelet aggregation,
was tested. With thrombin-stimulated (0.1 U/mL) platelets,
32,900 + 1,000 Fg molecules were bound per platelet at an
input concentration of 300 nmol/L Fg. OKM5, at a 20
nmol/L concentration reduced Fg binding to 4,700 * 100, an
86% inhibition. In contrast, the F(ab'), fragments, at a
concentration as high as 100 nmol/L, did not inhibit Fg
binding (36,700 5 1,050 Fg molecules/platelet).
DISCUSSION
The OKM5 antigen is expressed by a variety of cell^^-^;
and, on platelets, the antibody reacts with the membrane
protein designated GPIV.9 Our analysis of the interaction of
OKM5 with platelets provides a quantitative estimate of the
density of GPIV. The number of OKM5 epitopes per platelet
was estimated to be approximately 12,000. Assuming one
epitope per GPIV, this membrane protein is present at to
Y2 the abundance of GPIIb-IIIa and GPIb.
OKM5 can act as platelet stimulus. The antibody induced
platelet aggregation, secretion, and expression of Fg binding
sites, causing a response pattern similar to that induced by
thrombin. That these effects were due directly to the antibody, and not a contaminating component within the OKM5
preparations, is shown by: (1) the low concentrations of
OKM5 required to induce these effects; (2) the failure of
other MoAbs, isolated by an identical protocol, to induce
these effects; (3) the failure of protease inhibitors, including
PPACK and p-amidinophenylmethyl-sulfonylfluoride, to
abrogate these effects; and (4) the capacity of F(ab'),
fragments of OKM5 to selectively block the stimulatory
activity of intact OKM5. The later results show that the Fc
region of antibody is required for induction of platelet
responses. As many antibodies to platelet antigens, including
those of the same subclass as OKM5, such as PMI-I, do not
stimulate platelets, engagement of the target antigen by the
antibody combining site, as well as secondary Fc-dependent
interactions, are required for platelet stimulation.
When thrombin-stimulated platelets were preincubated
with OKM5, the capacity of these cells to bind "'I-Fg was
markedly decreased. Thus, the combination of these two
platelet agonists suppressed Fg binding induced by either
agonist individually. The effect was observed at OKM5
concentrations consistent with the measured affinity of the
antibody for the platelet. This effect was not due to neutralization of the thrombin as shown by the observations that: (1)
thrombin retained its capacity to induce platelet secretion in
the presence of OKM5 (Table 2); (2) thrombin activity
measured with the synthetic substrate S2238 (Ortho Diagnostics) was not affected by OKM5 (data not shown); and (3) Fg
binding induced by PMA was also inhibited by OKM5,
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2508
AIKEN ET AL
although less extensively than with thrombin. OKM5 was
not a general inhibitor of platelet stimulation as Fg binding
to ADP-stimulated platelets was unaffected and the antibody
itself could induce Fg binding in the absence of other stimuli.
Moreover, these effects were not restricted to Fg binding, as
vWF and Fn binding to thrombin or PMA-stimulated
platelets were also affected. By direct examination, it was
found that conditions under which OKM5 inhibited Fg
binding to platelets also resulted in extensive platelet aggregate formation. Furthermore, when aggregation was less
extensive, at lower platelet counts or by altering the order of
thrombin and OKM5 addition, OKM5 produced minimal
inhibition of Fg binding. Taken together, these observations
suggest a strong correlation between the inhibition of Fg
binding by OKM5 and its induction of aggregates within the
platelet suspension. However, cell-cell contact cannot account entirely for the inhibition of Fg binding by OKM5.
Platelet suspensions stimulated with thrombin also showed a
reduced number of free platelets, but the extent of Fg
binding to thrombin-stimulated platelets was independent of
cell concentration. One difference in the platelets stimulated
with thrombin alone or OKM5 alone and those treated with
OKM5 + thrombin may reside in the massive size of the
platelet aggregates formed. The massive aggregates may
prevent access of Fg to its receptors or unoccupied Fg
receptors may be downregulated on aggregated platelets.
The present study has shown that an MoAb reactive with a
target antigen other than GPIIb-IIIa can inhibit the binding
of adhesive proteins to platelets. We have previously demonstrated that MoAbs to GPIIb-IIIa can inhibit the binding of
an adhesive protein to a receptor independent of GPIIb-IIIa;
ie, binding of TSP to platelets does not appear to be mediated
by GPIIb-IIIa.30-32Nevertheless, certain MoAbs to GPIIbIIIa inhibit TSP binding to platelets.20s33
These observations
suggest a close proximal relationship between the TSP
receptor and GPIIb-IIIa, as suggested by L e ~ n g and
,~~
further emphasize the need for considerable caution in
interpreting antibodies inhibition of receptor-ligand interactions. Finally, the differences in the data presented in this
study with those presented by Asch et a19 require comment.
In contrast to our results, these investigators found that
OKM5 inhibited surface expression of TSP and concluded
that the OKM5 antigen was the TSP receptor on platelets.
We have previously found that TSP binds to platelets
through two distinct mechanisms, a divalent ion-dependent
and a divalent ion-independent pathway.20,22The OKM5
antibody did not appear to interfere with either of these two
pathways, as measured by binding of added TSP or surface
expression of endogenous TSP. Other pathways for TSP
surface expression may exist. TSP interacts with Fg with
high affinity.35Based on our data, such a pathway could be
inhibited by OKM5 if the antibody induced extensive platelet aggregation. Finally, while our data raise questions
regarding the mechanism by which OKM5 might inhibit
TSP surface expression on platelets, they do not exclude a
role of GPIV as a TSP receptor. A potential role of GPIV in
TSP binding to platelets has recently been
although TSP receptors unrelated to GPIV have now been
identified on other cell type^.^^.^^
ACKNOWLEDGMENT
We thank Judy Taylor and Shari Olsen for the preparation of this
manuscript.
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OKM5 AND PLATELET FUNCTION
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1990 76: 2501-2509
Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet
aggregation and thrombospondin surface expression [see comments]
ML Aiken, MH Ginsberg, V Byers-Ward and EF Plow
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