Download Identification of a Platelet-aggregating Factor of

[CANCER RESEARCH 48. 6411 -6416. November 15,1988]
Identification of a Platelet-aggregating Factor of Murine Colon Adonneareinoma 26:
MT 44,000 Membrane Protein as Determined by Monoclonal Antibodies1
Masahiko Watanabe, Etsuko Okochi, Yoshikazu Sugimoto, and Takashi Tsuruo2
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo ¡70,Japan
This material has been reported to be a large complex of
proteins and lipids (4). In spite of these studies, membrane
factors which induce platelet aggregation by their direct inter
action with platelets have not been identified yet.
In previous studies we have established several metastatic
clones derived from a highly metastatic variant of murine colon
adenocarcinoma 26 (24). Among these clones, NL-17 is highly
metastatic after i.v. inoculation and possesses platelet-aggre
gating ability (10, 25, 26). In this study we examined the
mechanism of platelet aggregation induced by NL-17 and found
it to be dependent on a trypsin-sensitive protein present on the
cell surface. To elucidate the factor(s) causing platelet aggre
gation, we generated two mAbs'1 which retarded platelet aggre
gation induced by NL-17 cells. Both antibodies were found to
recognize a single trypsin-sensitive membrane protein of a
molecular weight of 44,000.
ABSTRACT
The interaction between platelets and tumor cells plays an important
role in the hematogenous spread of certain malignant cancers. We found
that metastatic clones of murine colon adenocarcinoma 26 induced plate
let aggregation in a membrane protein-dependent manner. Two mono
clonal antibodies (mAbs) of IgG2a class were generated against a highly
metastatic colon 26 clone, NL-17. These two mAbs, designated 8F11 and
20 M I. showed a two-fold higher level of NL-17 binding than a low
metastatic clone, M -14. which possesses low platelet-aggregating abil
ity. Both mAbs retarded platelet aggregation induced by NL-17. Western
blot analysis showed that both mAbs recognized the same M, 44,000
membrane protein as antigen under reducing conditions. Trypsin treat
ment of NL-17 diminished the ability of the cells to induce plateletaggregation and resulted in a decrease in the reactivity of the cells to
Mil. These results suggest that the M, 44,000 membrane protein
recognized by the two mAbs is a platelet-aggregating factor of colon 26
cells.
MATERIALS
INTRODUCTION
Recent studies on cancer metastasis indicate that platelets
play an important role in the hematogenous spread of certain
tumors based on the following: (a) tumor cells injected i.v. into
experimental animals have been observed to aggregate platelets
and induce thrombocytopenia (1-3); (b) in certain tumor cell
lines, positive correlations have been found between the capacity
of experimental pulmonary metastasis in vivo and the ability of
the tumor cells to induce platelet aggregation in vitro (1, 4-6).
Actually several inhibitors of platelet aggregation have been
reported to retard tumor metastasis in certain animal models
(1, 7-11). Interactions between tumor cells and platelets have
been considered to facilitate the arrest of tumor cell emboli in
the microcirculation with the subsequent formation of experi
mental lung metastasis (1). In spite of these findings, contro
versial results were also reported and some inhibitors of platelet
aggregation of tumor cells could not inhibit tumor metastasis
(8, 12).
The mechanisms of tumor cell-induced platelet aggregation
have been studied following specific enzymatic treatments of
the tumor cell surface or by inhibitors of platelet aggregation.
With various tumor cells, the following mechanisms have been
proposed: (a) the interaction of tumor cells with platelets via a
sialolipoprotein (4, 13-15) and/or a trypsin-sensitive protein
(16); (b) the generation of thrombin through a procoagulant
activity of tumor cells (17-23); (c) the transmembrane efflux of
ADP occurring as a consequence of tumor cell metabolism (15,
23). Among these mechanisms, one factor which generates
thrombin through the activation of Factor X have been purified
(17, 18) and platelet aggregating material has been extracted
from certain virally transformed cells with 1 M urea (4, 13).
AND METHODS
Chemicals and Enzymes. The source of chemicals used in this work
were as follows: Na'25I from Amersham Japan Ltd., Tokyo, Japan;
apyrase (from potato, grade III, 55 U/mg for ATP); hirudin (from
leeches, grade IV, 1860 U/mg); neuraminidase (from Clostridium perfringens, type VI, 1.3 U/mg); phospholipase A: (from bee venom, 760
U/mg); trypsin (from bovine pancreas, type XI, 9200 U/mg); and
soybean trypsin inhibitor (type I-S) from Sigma Chemical Co., St.
Louis, MO; HEPES buffer (1 M solution, pH 7.3); and HBSS from
GIBCO Laboratories, Grand Island, NY; polyethylene glycol 1540
from Wako Ltd., Osaka, Japan; complete or incomplete Freund's
Received I/I 1/88; revised 7/7/88; accepted 8/9/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by grants for Cancer Research from the Ministry of Education,
Science and Culture, Japan, and from the Vehicle Racing Commemorative
Foundation.
2 To whom requests for reprints should be addressed.
adjuvant from Difco Laboratories, Detroit, MI; lodogen from Pierce
Chemical Co., Rockford, IL; and Percoli from Pharmacia Fine Chem
icals AB, Uppsala, Sweden. MD805, a synthetic thrombin-inhibitor
was kindly provided by Mitsubishi Kasei Co., Ltd., Tokyo, Japan. All
other chemicals and reagents were of the highest purity available.
Animals and Tumor Cells. Female SD rats and BALB/c and CD2F,
mice were obtained from Charles River Japan, Inc., Tokyo, Japan, and
female BALB/c-nu/nu athymic nude mice were from Nihon CLEA
Inc., Tokyo, Japan.
Metastatic clones of murine colon adenocarcinoma 26 (24) used in
this study are as follows: high metastatic NL-17; low metastatic NL-4,
NL-14, and NL-44. These clones were maintained in Eagle's minimum
essential medium (Nissui, Tokyo, Japan) supplemented with 10% calf
serum, 2% fetal bovine serum and kanamycin (100 ^g/ml). Mouse
myeloma P3X63-Ag8 •¿
Ul was kindly provided by Hoechst Japan Ltd.,
Tokyo, Japan. The myeloma cells were maintained in RPMI 1640
medium (Nissui) supplemented with 15% fetal bovine serum and kan
amycin (100 Mg/m')- These cultures were incubated at 37"C in a
humidified atmosphere of 5% CO2.
Platelet Aggregation. PRP and PPP were prepared as follows. Fresh
blood was drawn from CD2F, mice by heart puncture with a 22-gauge
needle and a heparin solution (10 U/ml) blood ratio of 1:9 (v/v). Then
blood was diluted with an equal volume of 0.9% NaCl and centrifuged
for 7 min at 400 x g on performed gradients of 70% Percoli containing
0.9% NaCl (27). PPP is a yellowish supernatant and PRP forms a white
band between PPP and Percoli layer. Platelets in PRP were counted by
3The abbreviations used are: mAb, monoclonal antibody; HEPES, N-(2hydroxyethyl)-l-piperazineethanesulfonic
acid; HBSS, Hanks' balanced salt so
lution without calcium and magnesium; PBS. phosphate-buffered saline without
calcium and magnesium; ELISA, enzyme-linked immunosorbent assay; PRP,
platelet-rich plasma; PPP, platelet-poor plasma.
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IDENTIFICATION
OF A PLATELET-AGGREGATING
a Coulter counter (model ZBI) and adjusted to l x l <)"/mni' by adding
PPP.
Cultured tumor cells were harvested after brief treatment with 0.02%
EDTA in HBSS containing 10 mM HEPES (pH 7.3) and washed by
centrifugation. The cells were resuspended in HBSS containing 10 m\i
HEPES (pH 7.3), adjusted to 1 x IO7cells/ml and incubated on ice for
20 min with 10 U/ml of apyrase.
Platelet aggregation was measured turbidometrically by an NKK
H EM A TRACER I (Niko Bioscientific Co., Tokyo, Japan). 200 M>of
PRP was incubated in a cuvet at 37°Cunder constant stirring in the
aggregometer. After 5 min, 10 ß\
of tumor cell suspension was added,
and change in light transmittance was monitored for IS min.
Treatment of NL-17 Cells with Enzymes. Enzymatic modification of
the cell surface of NL-17 was performed by the method of Grignani et
al. (15). NL-17 cells were prepared as described above and suspended
in HBSS containing 10 mM HEPES (pH 7.3) at a density of 2.5 x 10"
cells/ml. The enzyme solutions were prepared in HBSS containing 10
mM HEPES (pH 7.3) at the following concentrations: trypsin, 1000 Na-benzoyl-L-arginine ethyl ester U/ml; phospholipase A2, 280 U/ml;
neuraminidase, 1 U/ml with 1 mM phenylmethylsulfonyl fluoride. The
cell suspensions (400 fil) were incubated with 100 ^ of each enzyme
solution as follows: (a) trypsin; the mixture was incubated for l h at
37"C; thereafter, enzymatic activity was neutralized by adding 100 n\
of soybean trypsin inhibitor solution (60 ^g/ml) in HBSS containing
10 mM HEPES (pH 7.3); (b) phospholipase A;; the mixture was
incubated for 30 min at 37"C; (c) neuraminidase; the mixture was
incubated for l h at 37'C. In control experiment, the cells were
incubated with HBSS containing 10 mM HEPES (pH 7.3) without
respective enzymes. After the treatment, the cells were washed three
times with HBSS containing 10 mM HEPES (pH 7.3), resuspended in
the same solution, adjusted to 5 x Ml'1cells/ml, and incubated on ice
for 20 min with 10 U/ml (final) of apyrase. Then, the tumor cellinduced platelet aggregation was examined as described above.
Isolation of the Crude Membrane Fraction of NL-17. The crude
membrane fraction of NL-17 was isolated as described previously (28).
The logarithmically growing cells were collected as described above,
washed with PBS, suspended in reticulocyte standard buffer (10 mM
NaCl-1.5 mM MgCl2-IO mM Tris-HCl, pH 7.4) containing 1 mM
dithiothreitol, and stood on ice for 1 h. The swollen cells were disrupted
with a tightly fitting Dounce homogenizer. The preparation was centrifuged at 400 x g on a 10-ml layer of 25% sucrose in reticulocyte
standard buffer containing 1 mM dithiothreitol to remove cell nuclei.
The supernatant fluid was centrifuged at 4000 x g for 10 min to
sediment the mitochondria! fraction. The crude membrane was pelleted
by the centrifugation of the supernatant at 35,000 x g for 30 min, and
suspended in PBS. The protein amount was determined by the method
of Lowry et al. (29).
Preparation of Monoclonal Antibody. SD rats (female, 3-4 months
old) were primed i.p. with 0.2 ml of the crude membrane fraction of
NL-17 (500 Mg protein) and an equal volume of Freund's complete
adjuvant. After 2 weeks, the rats were further immunized by an i.p.
injection of 500 ¿igof membrane protein with Freund's incomplete
adjuvant. After 2 weeks, the rats were boosted i.p. with 2 mg of NL-17
membrane protein in saline.
Fusions were performed 3 days after the final immunization by the
method as previously described (30). Briefly, spleen cells obtained from
immunized rats were mixed with mouse P3X63-Ag8 •¿
Ul myeloma at a
ratio of 10:1 and fused with 43% polyethylene glycol and 10% dimethyl
sulfoxide in serum-free RPMI 1640 medium. The cells were selected
in RPMI 1640 medium supplemented with 15% fetal bovine serum
containing 100 (<\i hypoxanthine, 0.4 /<\i aminopterin, and 16 /IM
thymidine in 96-well plates. After 10-12 days, the culture supernatants
of hydridomas were screened for the production of antibodies against
NL-17 and examined the difference in the reactivity between NL-17
and NL-14 using the ELISA assay as described previously (31). The
positive clones were further cultured and the culture supernatants of
positive clones were examined to inhibit platelet aggregation induced
by NL-17 using the method described below. The positive clones were
subcloned two or three times by limiting dilution and propagated by
i.p. injection into pristane-primed nude mice. MAbs were purified from
FACTOR
ascitic fluid by precipitation with 50% saturated ammonium sulfate and
DEAE-sephacel column chromatography (32).
The subclasses of the mAbs were identified by an ELISA kit from
Zymed Laboratories, San Francisco, CA.
Inhibition of Tumor Cell-induced Platelet Aggregation by mAbs. 50
^1 of the NL-17 cell suspension (5 x 10*cells/ml) prepared as described
above was incubated on ice for 20 min with 500 ¿il
of culture superna
tants of hydridomas. After centrifugation, the cell pellets were sus
pended in 50 ¿il
of HBSS containing 10 U/ml of apyrase and 10 mM
HEPES (pH 7.3) and incubated on ice for 20 min. The cell suspensions
were subjected to the platelet-aggregation assay as described above.
Alternatively, the NL-17 cell suspension prepared as described above
was incubated for 30 min on ice with various concentrations of mAbs
purified from the ascites of nude mice and 10 U/ml of apyrase. The
mixture was subjected to the platelet-aggregation assay.
Radioimmunoassay. Radioiodination of the two mAbs was performed
by the lodogen method (33). Briefly, 50 //g of mAb in 100 M!of PBS
was incubated with 0.25 mCi of Na'25I and 10 /¿gof lodogen for 10
min at room temperature. Protein-bound iodine was separated from
free iodine by gel filtration on a PD-10 column (Pharmacia). 12SIlabeled antibodies were stored at 4"C. The specific activity of '"!labeled 8F11 and 20A11 was 3.1 x 10" cpm/^g protein and 2.6 x IO6
cpm/^g protein, respectively.
For the radioimmunoassay, the suspended tumor cells were plated
at a density of 1 x lit cells/well in 96-well filtration plates (Milliliter
SV; Millipore Co., Bedford, MA), and blocked for l h at room temper
ature with 3% bovine serum albumin. The cells were incubated with 50
/jl of the unlabeled mAb or normal rat Igs fraction (each 2 mg/ml) for
l h at room temperature. Fifty ^1 of '"l-labeled mAb (2 ¿ig/ml)was
added to the plates, and the mixture was incubated for 2 h at room
temperature. After several washes, the filters were removed and counted
by a gamma counter. The amount (A) of the labeled mAb specifically
bound to the cells was calculated by the following equation: A = An —¿
Am, where An and Am are the amounts of the labeled mAb bound to
the cells in the presence of normal rat Igs and unlabeled mAb, respec
tively.
Normal rat Igs fraction was prepared from rat serum by three times
precipitation with 50% saturated ammonium sulfate.
Western Blot Analysis. Proteins from the crude membrane fraction
of NL-17 were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis as described by Laemmli using a separating gel of 10%
acrylamide and a stacking gel of 3% acrylamide (34). The proteins in
the gel were transferred to a nitrocellulose filter (Scheicher and Scimeli,
Dassel, W. Germany) electrophoretically. The filter was incubated
overnight at 4*C with 3% bovine serum albumin, 0.1% NaN3 in 150
mM NaCl, 10 mM Tris-HCl (pH 7.5). Then, the filter was incubated
for 2 h at room temperature with 125I-labeledmAbs. After washing with
150 mM NaCl, 10 mM Tris-HCl (pH 7.5), the filter was air dried and
exposed to a Kodak XAR-5 film at -70"C.
RESULTS
Platelet Aggregation Induced by Colon 26 Clones. Fig. 1 shows
the representative tracing of platelet aggregation caused by the
four clones used in the present experiments at a tumor cell
concentration of 1.25 x IO5 cells/ml. At this concentration,
three clones, NL-4, NL-17, and NL-44 induced platelet-aggre
gation within 8 min, but the NL-14 induced only a weak
aggregation after 12 min. Taking the lag time into considera
tion, the platelet-aggregating activities of the four clones ap
peared to be in the order NL-4 > NL-17, NL-44 > NL-14.
Among three clones with high platelet-aggregating activity,
NL-17 was highly metastatic, while NL-4 and NL-44 were
rarely metastatic to the lung when the cells were injected i.v.
These observations indicate that platelet-aggregating activity is
one of the important determinants for successful experimental
metastasis, but metastasis can not be controlled only by plateletaggregating activity of tumor cells. Other factors such as
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IDENTIFICATION OF A PLATELET-AGGREGATING FACTOR
growth-promoting factors also have an important role in tumor
metastasis as described previously (25, 26, 35).
Characteristics of Platelet Aggregation Induced by NL-17.
Tumor cell-induced platelet aggregation has been reported to
be modified by the enzymatic treatment of the tumor cells or
by various pharmacological agents (14-16). Platelet aggrega
tion induced by NL-17 cells was inhibited by pretreatment of
the tumor cells with trypsin (Fig. 2/1), but neither by neuraminidase (Fig. 2B) nor by phospholipase A2 (Fig. 2C). This platelet
aggregation was observed in PRP containing 100 U/ml of
hinulin and 10 MMof MD805 (Fig. 3). These drugs were specific
thrombin inhibitors and the concentrations of these two drugs
were enough to inhibit the platelet aggregation induced by
80
o
60
40
a
20
6
8
Time (min)
Fig. 1. Platelet aggregation induced by various clones of colon 26. 10 n\ of
cell suspensions (2.5 x 10' cells/ml) were added to 200 ii\ of heparinized PRP at
time 0. and change in light transmittance was monitored, a, NL-4; b, M 44; c,
NL-17;</, NL-14.
Time { min )
Fig. 2. Effects of enzymatic treatments of the cell surface on platelet aggre
gation induced by NL-17 cells. The cell suspensions (2 x 10* cells/ml) were
incubated with the following enzymes at 37'C: A, trypsin (200 /V-o-benzoyl-Larginine ethyl ester U/ml); B, neuraminidase (0.2 U/ml); C, phospholipase A2
(56 U/ml). After three washes, platelet aggregation induced by the modified cells
was measured as described in the legend of Fig. 1. a, control cells (no treatment);
b, treated cells.
80
•¿Ã±
60
40
r 20
6
Time
( min
)
Fig. 3. Effect of thrombin-inhibitors on platelet aggregation induced by NL17 cells. Platelet aggregation was measured in PRP containing the following
compounds: a, HBSS-10 mM HEPES; b, HBSS-10 min HEPES-0.1% dimethyl
sulfoxide; c, 100 U/ml hirudin; d, 10 MMMD805-0.1% dimethyl sulfoxide.
thrombin (data not shown). Furthermore, this platelet aggre
gation was not induced by ADP-efflux from the tumor cells,
since the platelet aggregation was always measured in PRPcontaining apyrase (0.5 units/ml), an ADP degrading enzyme.
These data indicate that the platelet aggregation induced by
NL-17 cells is mediated by a trypsin-sensitive, phospholipase
Ai-insensitive protein, but neither by thrombin produced during
procoagulant process nor by ADP-efflux from tumor cells.
Generation of inAhs against the Platelet-aggregating Factor
Present on the Surface of NL-17 Cells. Approximately 7000
hybridomas were screened by ELISA for the production of
mAbs demonstrating differential binding between NL-17 and
NL-14, which showed high and low platelet-aggregating ability,
respectively. 25 hybridomas showed a higher reactivity to NL17 than to NL-14. The culture supernatants of these hybrido
mas were further examined for their ability to inhibit platelet
aggregation induced by NL-17 cells. Two mAbs produced from
two independent hybridomas were found to efficiently inhibit
platelet aggregation induced by NL-17 cells. These mAbs were
designated as 8F11 and 20A11. The immunoglobulin subclass
of both mAbs was IgGja- These mAbs were purified from ascitic
fluids of nude mice, and their properties were further examined.
Reactivity of mAbs 8F11 and 20A11 to Various Clones of
Colon 26. The reactivity of mAbs 8F11 and 20A11 to the four
clones of colon 26 was determined by cell-binding radioimmunoassay. In preliminary experiments the binding of both
125I-labeled mAbs was saturated at 1 Mg/ml in these clones and
the complete inhibition of the binding of respective labeled
mAbs occurred at 1 mg/ml of unlabeled mAbs. Thus, the
binding of labeled mAb was measured at 1 Mg/ml of the mAb
and the amounts of mAb bound to these clones was calculated
by subtracting the amount of the bound mAb in the presence
of 1 mg/ml of unlabeled mAb from that of the bound mAb in
the presence of 1 mg/ml of normal rat Igs.
The amount of 8F11 bound to NL-17 was 1.5 fmol/1 x 10"
cells and was twofold higher than that bound to NL-14 (Fig.
4A). Other two clones, NL-4 and NL-44, which possessed
higher platelet-aggregating activity than NL-14 (Fig. 1), also
exhibited a two- or threefold higher levels of the binding of
8F11 than NL-14 (Fig. 4A). 20A11 reacted to the four clones
in a similar manner as 8F11 (Fig. 4B). The amount of 20A11
bound to NL-17 was 1.1 fmol/1 x IO4 cells at saturation, and
NL-4, NL-17, and NL-44 showed a two- to threefold higher
levels of the binding of 20A11 than NL-14. These findings
indicated that the reactivity of both mAbs to respective colon
26 correlated well with the platelet-aggregating activity of each
clone.
Effect of 8F11 and 20All on Platelet Aggregation Induced by
NL-17. Two mAbs inhibited platelet aggregation induced by
NL-17 cells (Fig. 5, A and B). 8F11 at 0.1 mg/ml prolonged
the lag time and at 1 mg/ml, completely inhibited the platelet
aggregation induced by NL-17. 20A11 showed weak inhibition
at 0.1-1 Mg/ml and markedly prolonged the lag time at 10 Mg/
ml. Complete inhibition occurred at 100 Mg/ml. The concentra
tion of mAbs required for the inhibition of platelet aggregation
by NL-17 cells were relatively high. However inhibition of
platelet aggregation did not occur with another mAb, 13G7, at
1 mg/ml, which was highly immunoreactive to NL-17 cells and
had the same immunological subclass (IgG2a) as 8F11 and
20A11. The inhibition of platelet aggregation by mAbs 8F11
and 20A11 is, therefore, a specific reaction mediated by specific
antigens, and is not an inhibitory effect mediated by the binding
to Fc receptors via the Fc portion of the molecules.
Determination of Antigen Recognized by mAbs 8F11 and
20A11. The antigen recognized by 8F11 or 20A11 was deter-
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IDENTIFICATION
OF A PLATELET-AGGREGATING
FACTOR
A
B
Mrx10
-3
- 200
- 97.4
- 68
- 43
1.0
Bound
2.0
mAb
( fmol/1x104
cells
)
- 25.7
Fig. 4. Reactivity of two mAbs to various clones of colon 26. The cells were
plated at a density of 1 x 10* cells/well in 96-well nitration plates, blocked with
3% bovine serum albumin and incubated at room temperature with l «¿g/ml
of
'"1-labeled 8F11 (A) or 20A11 (B) in the presence of 1 mg/ml of unlabeled mAbs
or normal rat Igs fraction. After several washes, the filters were removed and the
amounts of mAbs bound to cells was calculated as described in "Materials and
Methods." Values are means and SD (bar) of three independent determinations.
„¿80
- 18.4
•¿5
60
a
40
S
20
Fig. 6. Western blot analysis of antigens recognized by mAbs. Proteins of the
crude membrane fraction of NL-17 were separated by electrophoresis on sodium
dodecyl sulfate-10% polyacrylamide gel and then transferred electrophoretically
to nitrocellulose paper. The nitrocellulose was reacted with '"I-labeled 8FI1 (A)
or 20A11 (/>'l and an autoradiogram of the paper was obtained. Molecular weights
of marker proteins were indicated on the right.
c.d
0
12
6810
14
Time (min)
~
~
f
80
Õ
60
<D
O
E
O.
o
n
O
40
-
8
20
>-
02468
10
12
14
4
co
Time (min)
Fig. S. Inhibition of NL-17-induced platelet aggregation by mAbs.. I. NL-17
cells were incubated on ice for 20 min with 0.1 to 10 mg/ml of 8F11. Then the
platelet-aggregating activity of cells was examined, a, control; />.0.1 mg/ml; c, 1
mg/ml; d, 10 mg/ml. B, NL-17 cells were incubated with 0.1 to 100 ng/ml of
20A11 as described above, and the platelet-aggregating activity was examined, a,
control; ft, O.I fig/ml; c, 1 eg/ml; d, 10 pg/ml; e, 100 <ig/ml.
10
mined by Western blot analysis. Both mAbs reacted with the
same protein band having a molecular weight of 44,000 under
reducing conditions (Fig. 6). 20A11 recognized two other pro
teins having molecular weights of 26,000 and 23,000.
The reactivity of 8F11 to NL-17 cells decreased when the
60
30
Time
(min)
Fig. 7. Change in the binding of 8F11 to NL-17 cells after treatment of the
cell with trypsin. NL-17 cells (2 x 10*cells/ml) were incubated with trypsin (200
yV-a-benzoyl-L-arginine ethyl ester U/ml) at 37°Cfor various times. Reactions
were terminated by adding soybean trypsin inhibitor (10 *ig/ml). After three
washes, the amount of I25l-labeled 8F11 bound to the cells were determined.
Values are means and SD (bar) at three determinations.
6414
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IDENTIFICATION
OF A PLATELET-AGGREGATING
cells were treated with trypsin. Approximately 86% of the
binding was lost after 60 min (Fig. 7). These results indicate
that the antigen recognized by 8F11 is a trypsin-sensitive pro
tein.
DISCUSSION
In previous studies we have established several high or low
metastatic clones from a metastatic variant of murine colon
adenocarcinoma 26 (24) and demonstrated that platelet aggre
gation induced by tumor cells was an indispensable event for
both experimental and spontaneous metastasis of colon 26
clones (10, 25, 35). However, the mechanism(s) of platelet
aggregation induced by colon 26 clones is not fully understood.
In this report, we found that a high metastatic clone, NL-17,
induced platelet aggregation, and the aggregation was mediated
by a trypsin-sensitive cell surface protein (Fig. 2A). Neither the
generation of thrombin through a procoagulant mechanism
(Fig. 3) nor the cellular efflux of ADP is involved in platelet
aggregation induced by NL-17 cells. The mechanism of platelet
activation by NL-17 cells resembles that reported previously by
Lerner et al. (16).
Reportedly, one factor responsible for platelet aggregation
has been purified from murine 15091A mammary adenocarci
noma and rat Walker 256 adenocarcinoma and the factor was
named PAA/PCA protein (17, 18). This factor aggregated
platelets through a thrombin-generating process (17). Colon 26
cells induce platelet aggregation by a thrombin-independent
mechanism (Fig. 3). The factor involved in such a mechanism
has not been purified, although a partial characterization has
been reported for several tumor cells (13,14,16). We attempted
to purify the factor present on the membrane of NL-17 cells to
elucidate the molecular mechanism involved in platelet aggre
gation. In preliminary experiments we attempted to purify the
factor by standard chromatography methods after membrane
solubili/n iion of NL-17 cells by several detergents. We exam
ined various detergents, such as Triton X-100, Nonidet P-40,
/V-octyl-glucoside,
3-[(3-cholamidopropyl)dimethylamino]-1
propansulfonate, and deoxycholate. However, platelet-aggre
gating activity was lost after membrane solubilization. Similar
detergent inactivation of platelet-aggregating activity has been
reported in other tumor cells (14). We therefore decided to
generate mAbs against the platelet-aggregating factor(s) of NL17 cells by satisfying the following two criteria: (a) showing
higher reactivity to NL-17 than to NL-14 (the latter is a low
metastatic clone that possesses a lower platelet-aggregating
ability than the former as shown in Fig. 1); (b) possessing an
inhibitory activity of platelet aggregation induced by NL-17
cells. In the present study we produced two mAbs, designated
8F11 and 20A11, which satisfied the above criteria. Both anti
bodies recognized a membrane protein with a molecular weight
of 44,000 as antigen.
Platelet-aggregating activity was not detected when the cell
membrane was solubilized with detergents. The platelet-aggre
gating factor of colon 26 might be composed with a large
membrane complex. The M, 44,000 membrane protein recog
nized by these mAbs therefore might be a component of a large
membrane complex. However, the M, 44,000 protein seems to
be an important part of the active site of the platelet-aggregating
complex, because: (a) both mAbs showed a two- to threefold
higher binding to NL-17, NL-4, and NL-44 clones possessing
a high platelet-aggregating activity than to NL-14 possessing
low aggregating activity (Figs. 1,4A, and 4Ä);(b) the treatment
of NL-17 with these mAbs resulted in an inhibition of platelet
FACTOR
aggregation induced by NL-17 (Fig. 5, A and B)\ (c) treatment
of NL-17 cells with trypsin resulted in a decrease of the binding
of 8F11 (Fig. 7), indicating that the antigen recognized by the
mAb was a trypsin-sensitive protein.
The M, 44,000 protein identified in this study is apparently
distinct from the PAA/PCA protein mentioned above in both
molecular weight and platelet-aggregating ability. The PAA/
PCA protein has been reported to have a molecular weight of
51,000 (murine 15091A mammary adenocarcinoma) or 58,000
(rat Walker 256 adenocarcinoma) and induce platelet aggrega
tion via the generation of thrombin through direct activation of
Factor X (17, 18). In conclusion, our results indicate that the
M, 44,000 protein which is expressed on the membrane of
metastatic colon 26 clones plays an important role in platelet
aggregation induced by tumor cells. As the present mAbs are
reactive to intact tumor cells and inhibit the platelet aggregation
induced by the tumor cells, such mAbs might be useful for the
elucidation of the mechanism of tumor cell induced platelet
aggregation, as well as for the inhibition of tumor metastasis.
Experiments along these lines are now under progress in our
laboratory.
ACKNOWLEDGMENTS
We are grateful to Dr. H. Mamada for his helpful suggestion through
out the study. We thank N. Aihara for preparing the manuscript.
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6416
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Identification of a Platelet-aggregating Factor of Murine Colon
Adenocarcinoma 26: Mr 44,000 Membrane Protein as
Determined by Monoclonal Antibodies
Masahiko Watanabe, Etsuko Okochi, Yoshikazu Sugimoto, et al.
Cancer Res 1988;48:6411-6416.
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