[CANCER RESEARCH 38, 2950-2955, September 1978]
0008-5472/78/0038-0000$02.00
Aggregation of Platelets and Cell Membrane Vesiculation by Rat Cells
Transformed in Vitro by Rous Sarcoma Virus1
Gabriel J. Gasic,2 David Boettiger,3 James L. Catalfamo, Tatiana B. Gasic, and Gwendolyn J. Stewart
Departments of Pathology fG. J. G., J. L. C., T. B. G.Jand Microbiology (D. B.J, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104, and Specialized Center for Thrombosis Research, Temple University, Philadelphia, Pennsylvania 19140 (0. J. S.J
ABSTRACT
Primary rat embryo cells and established normal rat
kidney cells transformedIn vitro by Rous sarcoma virus
induced the aggregation of rat platelets In vitro. The
aggregating activity was shown to be specific for the
transformedcells and was absent in the normal parent
cells. The aggregation reaction is accompaniedby the
release of serotonin from the platelets. Further analysis
and purificationof this activityfromthe transformedcells
demonstrated that the activity is shed from the cells
growing in culture and is associated with membrane
vesicles of heterogenous size. The normal cells also
producedvesiclesin culture;however,the level of vesicle
productionwas less than that fromtransformedcells, and
the platelet aggregationand serotoninrelease activities
were greatly reducedor absentin these vesicles.
INTRODUCTION
parameter in the characterization of the transformed cell
phenotype.
MATERIALS AND METHODS
Cell and Cell Cuftures.NRKcellsare an establishedcell
line of normal cells originally from the kidney of an Os
borne-Mendel rat (6). These cells havebeen transformed by
the B77 strain of Rous sarcoma virus (2). LR cells are early
passagecells obtained from individual 16-day-old Lewis rat
embryos. These cells were transformed by the Schmidt
Ruppin strain of Rous sarcoma virus, as described previ
ously (30). All cultures were maintained in Dulbecco's
modified Eagle's medium supplemented with 5% fetal calf
serum. Roller cultures were grown in disposable glass
bottles (growth area, 790 sq cm) rotated at 6 to 12 revolu
lions/hr. For shedding experiments cells were incubated
overnight in 50 ml medium or for 1 hr in 15 ml of serum-free
medium.
Platelet Aggregation and Serotonin Release Assays.
Platelet-rich plasma (5 x 108/ml) and platelet-poor plasma
were prepared as described by Gasic et al. (15). Sodium
heparin (The Upjohn Co., Kalamazoo, Mich.) was used as
indicating
thattumor cellsmay aggregateplatelets
invivo anticoagulant (10 units/mI). Platelet aggregation was mea
as well (5, 10, 16, 18, 22, 32) and that this activity conlrib
sured by a lurbidimetric method (4), with the use of a
ules to metastasis(10). For example, thrombocytopenia, or Paytondual-channell Aggregometer attached to a dual-pen
impaired platelet function, interferes with the development recorder (Payton Inc., Buffalo, N. V.). The aggregometer
of metastases(10, 12, 14). Recently, it has been suggested was standardized to a 0 and 100%transmission with the use
that the release of a growth factor from platelets may be a of platelet-rich plasma and platelet-poor plasma, respec
factor in the establishment of metastatic cell foci (25).
lively. For the assay,0.05 ml of a cell suspensionwas added
If platelets do play an important role in tumorigenesis in to 0.45 ml of rat platelet-rich plasma, or 0.1 ml of the cell
vivo, then there would be a positive selective pressure on
fraction was added to 0.40 ml of platelet-rich plasma, and
tumor cells to produce platelet-aggregating material. A the mixture was stirred at 850 rpm at 37°;aggregation was
similar selective advantage appearsto occur in the produc
recorded as a function of time.
lion of plasminogen activator by tumor cells (23). For exam
The labeling of platelets and measurement of serotonin
inahionof the acquisition of platelet-aggregating activity in releasewere performed according to the method of Jerush
vitro , in the absence of obvious selection for this property, almy and Zucker (21), as modified by Gasic et a!. (15).
a series of independently isolated Rous sarcoma virus
Briefly, platelets were labeled by incubation of heparinized
transformed cells were examined. In each case these in platelet-rich plasma with [14Clseroloninat 37°for 15 mm.
vitro transformed cell lines induced platelet aggregation,
For calculation of the uptake of [‘4C]serolonin
by platelets,
whereas normal cells, either from an established “normal―
the free radioactivity in the supernalanl of labeled platelet
cell line or from primary culture, did not.
rich plasma was subtracted from the total radioactivity
The characteristic of platelet-aggregating activity from releasedby sonic disruption. For assayof serotonin release,
the Rous sarcoma virus-transformed cell lines and partial the labeled platelet-rich plasmawas exposed to cells or cell
purification of this activity may be used as an additional fractions for 5 mm as in the platelet aggregation test. The
mixture was then centrifuged in an Eppendorf centrifuge at
Previouswork has demonstrated that several established
transplantable tumor lines can induce the aggregation of
homologous platelets in vitro (10, 15). There is evidence
12,000
I
Supported
by
the
National
Cancer
Institute
(Grants
CA-15278
and
CA
16502) and the National Heart and Lung Institute (Grants HL 18827 and HL
14217).
2 To
whom
requests
for
reprints
should
be
addressed.
3 Leukemia Society of America Scholar.
ReceivedMarch9, 1978;acceptedJune9, 1978.
2950
rpm
for
1
mm,
and
0.1
ml
of
supernatanl
was
counted by liquid scintillation. The release of serotonin was
calculated as a percentage of the labeled serolonin taken
up by the platelet suspension that was released into the
supernalant.
CANCERRESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Aggregation of Platelets by Transformed Cells
Table 1
RESULTS
Two systems of Rous sarcoma virus-transformed cells
were used as model systemsfor this study. The NRKsystem
represents an established cell line, whereas the LR system
is a primary culture system with an inbred rat strain. The
Iransformed colonies that were developed into the Irans
Aggregation of platelets by normal and Roussarcoma virus
transformed rat cells
AggregationRelease
Sero
toninCell
formed lineswere selectedfrom a cultureof infected
cells (%)Normal
lineNo.ofcells
testedaLag
@
on the basis of morphology. All transformed cell lines were
morphologically distinct from the parent lines, produced
colonies in soft agar with high efficiency, and grew to a
high saturation density producing multilayered cultures in
vitro (Ref. 2; D. Boettiger, J. Brooks, and K. Sleimer,
unpublished observations). The transformed cell lines were
testedfor lumorigenicityin inbred Lewis rats.The synge
neic lines LR 3/1 , LA 3/2, LA 3/3, and LA 5/1 all induced
tumors in50% of the recipients
following
s.c.injection
of
10@to 10@cells; Ihe exact dose differed for each line. The
transformed NAK cells were not tested in theseexperiments
becauseof the lack of a suitable syngeneic system in which
to assay tumorigenicity. All Rous sarcoma virus-Irans
formed lines were negative for the release of infectious
transforming virus, but Rous sarcoma virus could be res
cued from each of these lines by fusion with appropriate
chicken cells (30). In each case the rescue efficiency was
high, which
@
@
has been correlated
formed Cells. The kinetics of the aggregation of platelets
by transformed cells and by ADP is shown in Chart 1: The
aggregation induced by transformed cells is irreversible
and is preceded by a lag period. No lag is observedfor ADP
abbreviation
used
is:
PBS,
phosphate-buffered
saline
(0.5
m@i
MgCI,,I .5 mMKH@PO4,
2.7 mi.i KCI,8.1 [email protected]
Na@HPO4,
and 137mMNaCI).
SEPTEMBER
3/25
(3)NTLA
x [email protected]
3/35
x 1O@1
(1)NTLR5/15x1052.290
(min)'@Plateletag
gregation(%)release
(4)(@
±04d0
±1.032
±0.485
6
0
(7)
±11
(2)
76 (2)
(4)2
±10
.695
(1)NT
a Number of cells in 50 @I
used for test.
b Lag between
the addition
of platelets
and
the
initiation
of
aggregation.
Numbersin parentheses,
numberof separateexperiments.
d Mean ±S.E.
eNT,nottested.
a
with high levels of virus
specific ANA in the cells (3) and high levels of virus-coded
proteins (31).
Platelet Aggregationby TransformedCells. In prelimi
nary experiments and surveyexperiments, whole cells were
used to induce platelet aggregation, as described previ
ously (10, 15). Confluent cultures of normal or transformed
cells were washed twice with PBS,4dissociated mechani
cally, and resuspendedin PBSplus 0.9 mMCaCI2(complete
PBS). The cell suspension was dialyzed overnight at 4°
against complete PBSto removeADP,which may leak from
the damaged cells and induce platelet aggregation itself.
Direct determination of ADP in the supernalant of the
dialyzed cell suspension according to the method of HoIm
sen et a!. (19) revealed less than 0.1 @M
ADP, which is
insufficient to induce the aggregation of rat platelets. Sixty
to 80% of the cells still exclude Irypan blue stain following
treatment.
No aggregation was observed when 10@NRK or 2 x 10@
rat embryo cells/assay were used, whereas 5 x 10@Irans
formed cells induced 85 to 95% aggregation following a
prolonged lag period (Table 1). The transformed NRKcells
were also tested for induction of serotonin release. These
cells induced 76% release, whereas no significant release
of serotonin was induced by the normal NRK parent cell
line (Table 1). Aggregation of the dialyzed whole-cell sam
pie suggests that the activity may be associated with the
cell surface.
Characteristicsof the Platelet Aggregation by Trans
4 The
NRK
NRK/B77TI
10°
NTeLA LA10° 2 x 10.2.6
Normal
(3)NTLA
3/15
x [email protected]
z
I
0
TIME
Chart 1. Plateletaggregationinduced by 10 @tM
ADP(A)or by 1.2 x 10@
LR 5/1 cells (B). The addition of cells (arrow) is followed by a lag period and
then by complete, irreversibleaggregation.The addition of ADP (arrow)
causedimmediatereversibleaggregation.
induced aggregation, and the aggregation is reversible.
The lag period representsthe delay between mixing of cells
with platelet-rich plasma and the initiation of the turbidity
change due to aggregation.
Shedding of Platelet-aggregatingMaterial by Trans
formedCells. In additionto the aggregatingmaterialasso
cialed with intact cells, platelet-aggregating material was
found in the supernatant medium in which cells had been
incubated. Since the activity present in the media from cells
incubated in culture dishes was less active than that from
the intact cells, large-scaleroller bottle cultures were estab
lished to collect sufficient material for further study of its
properties. Roller bottles containing 1 to 5 x 10@cells were
incubated either overnight in 50 ml of culture medium or
for 1 hr in 15 ml serum-free medium (serum-freeshedding).
The shed material from several bottles was pooled; free
cells and gross cell debris ‘were
remOvedby 2 successive
centrifugation steps (270 x g for 10 mm and 1000 g for 10
mm).
To determine whether the active material was particulate
or soluble, we subjected the cell-free 1000 x g supernalant
to successive rounds of centrifugation at increasing gravi
tational forces. Table 2 demonstrates that activity was
recovered from the 4,000 x g, the 50,000 x g, and the
100,000 x g pellet but not from the 100,000 x g superna
tant, even after concentration by ultrafiltration. These re
1978
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
2951
G. J. Gasic et al.
Table 2
cellsA Particulate
nature of platelet-aggregating
material
shed from LA 3/2
for60pool of shed material was subjected to successiverounds of centrifugation, each
wereresuspended
mm, and pellet and supernatant fractions were collected. Pellet fractions
@
theserotonin
in 5 ml complete
PBS, and 100
of each fraction
were
used in
release
assay.Serotonin
releasePlatelet
(%)4,000xgpellet
Fraction
DistributionTotal
agprogregation
Units/mi
tein (mg)
(%)a
protein
protein
1.4
1.6
2.3
97
95
95
36
31
22
50.4
49.6
50.6
NTC
0
3350,000xgpellet
33100,000
34100,000xgsuperx g pellet
<2natant
a The platelet aggregating
Units/total of activity
activity of a 4,000 x g pellet, 50,000 x g pellet, and 100,000
x g pellet, with serotonin release of 50 ±10%, was tested by adding 28, 32, and 46 @.&g
protein suspension, respectively,to 0.4 ml platelet-rich plasma.The same activity of the
100,000 x g supernatant
was assayed by the addition
of 100 pi of the 50-fold concentrate
to 0.4 ml of platelet-rich plasma.
b Unit of serotonin release. One unit is defined as the amount of activity that causes
the releaseof 50 ±10%of the serolonin from platelets in the standard assay.
C NT, not tested.
Material
was concentrated
50-fold
by means
of ultrafiltration
with the
use of UM1OAmicon membranes. Protein concentration was not measured becauseof
the presenceof serum proteins.
@
suIts indicate that the activity is contained in particulate
material that is probably of diverse sizes.
For quantitation of the platelet aggregation for this shed
material, the lag period, percentage of platelet aggregation,
and serotonin release were measured with the use of serial
dilutions of the 50,000 x g pellet resuspended in complete
PBS (hereafter called crude fraction). Chart 2 shows the
relationship among the parameters associated with the
platelet aggregation induced by the crude fraction from LA
3/2 cells. The percentage of platelet aggregation appears to
be a threshold phenomenon, whereas the lag period shows
a dose-dependent, inverse relationship with the concentra
lion of platelet-aggregating material. The relationship be
tween amount of platelet-aggregating material and percent
age of serotonin release was linear in the range of 10 to
60% serotonin
release. These parameters
can be used to
compare the relative release of platelet-aggregating mate
rial by the 4 transformed cell lines. For LA 3/1 , LA 3/2, and
LA 3/3, the aggregation response dilutes out around 5 to 10
/Lg of protein, whereas LA 5/1 dilutes out around 40 @g
of
protein . Both LA 3/1 and LA 5/1 tended to be less active in
releasing platelet-aggregating material. A comparison of
the transformed LA cell lines indicated that the intact cells
of each transformed line exhibit similar amounts of platelet
aggregating activity on a per cell basis, but the material
shed from LA 5/1 was about one-fifth as active in the
platelet aggregation assay (data not shown). If the cell
bound and shed material are compared on a per cell basis,
the amount of platelet-aggregating activity released during
1 hr of shedding represents 1 to 5% of the total cell-bound
activity. In 1 representative experiment with LA 3/2 cells
and cell sheddings, a dilution end point of 2.5 x 10@intact
cells induced platelet aggregation, while shed material from
10@cells was needed to produce the same effect. A similar
observation has been made with mouse ascites 15091A
tumor cells, in which neither mechanical dissociation nor
2952
—
LR
S@oIonin
Ralsose
@@---•
PlateletAg@sqató@
..—..
Log
l0
8
zw
6
IC)
3
4
2
0
PROTEINTESTED (j@g/0.4 PRP)
Chart 2. The relationship between protein concentration of crude pellet
materialfrom LR3/2 cells and 3 parametersassociatedwith aggregationof
platelets in heparinized platelet-rich plasma (PAP). 0- - - 0, Percentage of
maximum aggregation; 0-'-.- 0, length of the lag period preceding aggre
gation; 0—0,
percentage of release of [@C]serotonin from radiolabeled
platelets. See text for additional information. Vertical bars, S.E. for each
determination.Resultsfrom 5 independentexperimentsare included.
dialysis was required to measure the platelet aggregation
induced by intact cells (11).
Purificationof Platelet-aggregatingMaterial Shed by LR
3/2 Cells. Further purification was carried out with the use
of isopyknic, sucrose density gradient centrifugation. For
this purpose EDTA was added to the cell-free 1,000 x g
supernatant to a final concentration of 0.001 M and centri
fuged at 50,000 x g for 60 mm. The resulting pellet was
resuspended in PBS containing 0.002 M EDTA, mixed with
CANCER RESEARCHVOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Aggregation of Platelets by Transformed Cells
an equal volume of sucrose density (1.17 g/sq cm), and
layered out on a discontinuous sucrose density gradient for
isolation of plasma membrane vesicles, essentially as de
scribed by Aossomando and Cutler (28). The gradient
contained sucrose densities of 1.17, 1.18, and 1.19 g/sq
cm, which were previously cleaned by Millipore filtration.
The gradient was centrifuged at 100,000 x g for 18 hr in a
Beckman SW-27rotor. Eighteen 2.0-mI fractions were col
lected, diluted 1.5 with PBS, and pelleted at 50,000 x g for
1 hr. Each fraction was resuspended in complete PBS and
tested for the induction of platelet aggregation and sero
toninrelease.
Activity was found in 2 peaks: (a) a low-density peak (1.11
g/sq cm) consisting of the first 6 ml of the gradient with an
isopyknic density that is characteristic of plasmamembrane
vesicles (hereafter called the vesicular fraction); and (b) a
high-density peak (1.19 g/sq cm) present in the pellet
(hereafter called the granular fraction). Table 3 shows the
activity of crude pellet and granular and vesicular fractions
of sheddings taken from normal and transformed Lewis rat
embryo cells. Material shed from transformed cells present
in the crude fraction induced both platelet aggregation and
sion electron microscopy. The material from each sample
was pelleled, placed in cold 2.5% glutaraldehyde in 0.1 M
cacodylale buffer (pH 7.4), and held in the cold overnight.
The samples were then fixed in cold with 1% osmium
tetroxide in the same buffer for 1 hr, dehydrated with
ethanol, and embedded in Epon 812. Sections were stained
with uranyl acetate followed by lead citrate. Fig. 1 shows
that the active low-density fraction contains mostly vesicles
of varying sizes up to 400 nm. The thickness of the vesicle
membrane is about 100 A at the narrowest point. No
contamination of this fraction with mitochondria, ribo
somes, viruses, or other identifiable subcellular compo
nents is evident. The purified fraction from normal cells was
virtually indistinguishable from the transformed cell vesic
ular fraction with respect to the types of structures present.
The granular fraction contained numerous ribosome-like
bodies and probably represents debris from dead or dis
rupted cells. Transformed cells produced larger granular
fractions, as expected from the increased amount of cell
debris in the media from transformed as compared to
normal cells.
serotoninrelease(Table3, Lines 1 and 2). Following DISCUSSION
purification by sucrose density gradient, both granular and
vesicular fractions caused platelet aggregation, but only
the vesicular fractions induced significant serotonin release
(Table 3, Lines 3 and 4). Material shed from normal cells,
when used at a concentration similar to that of the trans
formed cell material, did not produce significant serotonin
release, but some platelet aggregating activity was present
in the purified fractions (Table 3, Lines 6 and 7). The
increased activity of the purified fractions from normal cells
may result from either separation of inhibitors of aggrega
lion or concentration of active material. A quantitative com
parison of the relative levels of shed material from normal
and transformed cells is difficult becauseof the presenceof
serum proteins in most experiments, which tends to mini
mize the difference in total shed material when compared
on a mg protein basis for each fraction.
Vesicular Nature of the Serotonin-releasingFraction.
For further characterization of the vesicular fraction, mate
rial from the sucrose gradient was examined by lransmis
The data presented above describe 2 cell properties that
are modified following in vitro transformation of cells by
Rous sarcoma virus: (a) increased shedding of vesicles,
probably from the plasma membrane; and (b) the ability of
both the transformed cells and the vesicles shed from
transferred cells to induce aggregation of homologous
platelets. In contrast, the normal parents of the transformed
cells, either the NAK cell line or the LA embryo cells,
releasedfewer vesicles, and these vesicles failed to induce
significant platelet aggregation; no serolonin release was
observed, even when the vesicles were concentrated or
purified by sucrose density centrifugation. One question
that arises is whether the observed difference in platelet
aggregating activity between normal and transformed cells
is related to variations in the susceptibility of the cell to
damage during preparation and the subsequent leakage of
internal cell components. This appears not to be the case,
since normal cells attach more lightly to the glass substrate
Table 3
linesCrude
Platelet response
to crude and purified
fractions
from normal and virally transformed
embryo cell
volumewere
fractions were 100-fold concentrated; purified fractions resuspended in one-fifth of the crude fraction
appliedto thedensitygradient.Protein
tested(@g/0.4ml
SerotoninCell
(%)LR
line
@
platelet-rich
Status
3/2
Fraction
b Mean
C Numbers
SEPTEMBER
EDTA
Platelet
ag-
Lag (mm)
gregation
(%)
release
(10)cTransformed
Transformed
Cruder'
52 ± 5b
.o ±o.i
se ± 2
62 ±9
(5)Transformed
(5)Transformed
Crud&'
Granular
Vesicular
10 ± 2
10 ± 0
10 ± 0
1.7 ±0.3
1.0 ±0.1
1.2 ±0.2
51 ± 9
70 ± 8
82 ± 5
5 ±2
5 ±2
29 ±2
(4)Normal
Normal
(4)Normal
Crudea
Granular
56 ±20
8 ± 1
0.8
0
47 ±16
0
2 ±1
(5)LA
a After
plasma)
treatment,
CaCI2
was
Vesicular
13 ± 3
added
ionic
to restore
calcium
1.6 ±0.1
concentration
41 ±22
to permit
platelet
2 ±1
(4)
aggregation.
±S.E.
in
parentheses,
number
of
separate
experiments.
1978
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
2953
@
@,
..
I.
G. J. Gasic et al.
@i.,,
@
‘
w*
‘,
,
@.:
•‘
(
$1
/@,?,
.--.
‘
@‘
@!1.t.
; ;@
@
@.‘ w
: â€ẫ€¢@
‘
‘@ ,@i/.
@
@
..@
.
@@1r
@
@
,..
;3@;
Fig. 1. Transmission electron micrograph of the low-den
sity fraction obtained by isopyknic sucrose density gradient
centrifugation of material shed by LR 3/2 cells. After fixation
with glutaraldehyde and osmium tetroxide, sections were
stained with uranyl acetate followed by lead citrate. See
‘Materials
and Methods―
for additionaldetails. x 27,000.
@). %,@&1i#..,
; ,:@?:::@a@
‘@
¶.\@ 4.;
. ( @‘.r'
.
./@e:
•c
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•
‘@
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,
.%@_@.•)
9@,
:‘@@
ae@@'•
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0,
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:
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@
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-
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and therefore are more likely to be disrupted by mechanical
dissociation than are transformed cells. Furthermore, sub
cellular fractions from TA3tumor cells, which do not induce
physiologically. This category of tumors forms a special
group within cancers in general.
Aggregation of platelets by conventional agents may be
aggregation,failto show platelet-aggregating
activity,immediate or may be preceded by a lag period of variable
whereas similar fractions from an aggregating tumor length. The pattern of aggregation produced by Irans
(15091A) were active (G. Gasic, J. Catalfamo, and T. Gasic,
formed cells falls within the latter category and is similar to
unpublished observations). Thus, the difference in aggre that produced by collagen (29) or immune complexes (20).
gating activity does not appear to be related to cellular However, these agents are not involved in platelet aggre
disruption.
gation by transformed cells. Collagen can be excluded on
Workers from several laboratories have examined Irans
several grounds: (a) the platelet-aggregating activity of
formed cells with the useof a scanning electron microscope transformedcellsisnot inhibited
by collagenase(11);(b)
and have observed that transformed cells have a ruffled transformation of collagen-producing cells by Aous sar
surface consisting of many microvilli. When temperature coma virus results in a suppression of collagen production
sensitivetransformation mutants of Roussarcoma virus are (24); and (C)the kinetics of platelet aggregation and secre
used as the transforming agent, the appearance of the lion by collagen, as studied in the Lumi-Aggregomeler
ruffles is rapid following a shift to permissive temperature (ChronoLog Corp., Philadelphia, Pa.), is distinctive from
(1). it @5probable
that
the observed
membrane
vesiculalion
could result from a pinching off of the microvilli. The
disruption of the cyloskeleton, which also accompaniescell
transformation (7), may play a role in allowing the enhanced
releaseof membranevesicles (26).
Some types of normal cells may induce platelet aggrega
lion. This has been observed
in tests on human cells from
an established line of fibroblasts and from a line of smooth
muscle cells after more than 8 in vitro passages (17). Al
though their aggregating activity may be attributed to the
secretion of collagen, it is also possible that these cells
have undergone some degree of cell transformation.
Although cells from many solid tumors, asciles tumors,
and cells transformed in vitro by oncogenic viruses induce
platelet aggregation, in certain cases well-established can
cers do not. This has been observed with cells of a mouse
mammary asciles tumor (11) and with several mouse plas
macylomas and human chronic leukemias (G. Gasic and T.
Gasic, unpublsihed observations). II is of great interest that
severalother exceptions belong to neoplasmsof the reticu
loendothelial system, the normal cells of which metastasize
2954
thatinducedby 15091Atumorvesicles.5
Immune complexes
from transformed cells can also be ruled out, since the cells
are grown in vitro under conditions that preclude the
formation of these complexes.
Although thrombin causes immediate platelet aggrega
lion, the possibility that the platelet-aggregating activity
displayed by tumor material may be mediated by lhrombin,
generated after some delay by the procoagulant activity of
the tumor material, cannot be ruled out. However, this
interpretation is unlikely because: (a) no correlations have
been observed
between the procoagulant
and the platelet
aggregating activity induced by tumor cells (15); (b) all tests
have been performed with heparinized platelet-rich plasma
(see “Materials
and Methods―);
and (c) hirudin, which is a
specific inhibitor of thrombin, used in doses as high as 10
antilhrombin units/mI, did not prevent platelet aggregation
by the tumor material (G. Gasic, J. Catalfamo,T. Gasic, and
J. B. Smith, unpublished observations). We are at present
SG. Gasic, J. Catalfamo, T. Gasic, and J. B. Smith, manuscript in
preparation.
CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Aggregation of Platelets by Transformed Cells
further investigating the nature of aggregating activity of
the vesicles and the potential mechanisms (for example,
thromboxane formation) by which they may induce aggre
PlateletAggregatingActivityas a Markerof CellTransformation.Feder
ation Proc.,36: 350a,1977.
14. Gasic, G. J., Gasic, T. B., and Stewart, C. C. AntimetastaticEffects
Associated with Platelet Reduction. Proc. NatI. Acad. Sci. U. S.. 61: 46-
gation.
Our preliminary communications on the platelel-aggre
gating activity of cells transformed in vitro by oncogenic
viruses (8, 9, 13)haverecently been confirmed by Pearlstein
and Karpatkin (27)using BHKcells transformed by polyoma
virus and BALB/3T3
13. Gasic, G. J., Gasic, T. B., Steimer, K., Boettiger, D., and Jimenez, S. D.
cells transformed
by SV4O virus.
ACKNOWLEDGMENTS
We thank S@Bobyock and R. A. Kuprionas for excellent technical assist
ance.
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2955
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Aggregation of Platelets and Cell Membrane Vesiculation by Rat
Cells Transformed in Vitro by Rous Sarcoma Virus
Gabriel J. Gasic, David Boettiger, James L. Catalfamo, et al.
Cancer Res 1978;38:2950-2955.
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