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CONCISE
REPORT
Evidence
That
Platelet
Density
Content
By Bernard
The
relation
between
thromboglobulin
granule
)
(1
a
was
assessed
Platelets
discontinuous
Stractan
tions.
in density
ranging
(20’C).
The
with
the
(20%
/9-TG
(1 5%
by
platelets)
most-dense
1 .061
(mean
from
three
up to
65.6
platelets).
(2)
n
8.6
±
and
to
density
ance of cyclooxygenase
aspirin
in humans.23
activity
By contrast,
That
the lessfraction
was
the reappear-
after
a single
dose of
in rabbits
and subhu-
man primates,
it was found that the more-dense
platelets had significantly
longer
survival
times
than
the
less-dense
platelets,
suggesting
that dense platelets
are
mainly
young
cells.4
Conash
et al.6 published
the first reliable
method
of platelets
on iso-osmolan
Stractan
separation
for
gra-
dients,
which
yielded
intact
and functional
platelets.
They
noted
that the number
of granules,
apart
from
mitochondnia,
was increased
in association
with platelet density.67
In this article,
we have tested
the hypothesis that alpha-granules,
as measured
by intraplatelet
f3-thromboglobulin
(f3-TG),
are responsible
for the
heterogeneity
in platelet
density.
Adenosine
bitartrate)
The
diphosphate
were
from
prostaglandin
METHODS
and
(ADP)
obtained
Netherlands;
AND
SaIm
E, (PGE,)
and
(l-epinephnine
Kipp,
Breukelen,
and hirudin
from
Munich,
(I5
plasmin
Utrecht,
Department
of
Hematology,
University
Hospital
The Netherlands.
Supported
in
part
Blood
Grant
78.059
from
the
Dutch
Heart
Submitted
Address
1 9, 1983;
reprint
requests
accepted
to Dr.
Medicine.
Department
of
Columbia,
2146
Sciences
Canada.
(r)
1984
Health
theophylline
citrate
Bernard
Biochemistry.
Mall,
29. 1983.
A. van Oost,
University
Vancouver,
Faculty
B.C.
of
British
V6T
I W5
by Grune
& Stratton,
Inc.
than
the
from
U/mg)
from
collagen
from
Won-
Hormon
Organon-Iechnika,
from
UCB,
from
Oss,
Brussels,
AB
Belgium;
KABI,
Stockholm,
and f-TG
content
and
collected
processed
was
content
Platelet
discontinuous
Stractan
thrombin
Collagen,
(2
used,
of
(see
tion).
below)
plasma
mixture
After
was
mM);
plasmin
yields
to
f3-IG
measured
clotting9
by
added
ADP
U/mI),
(1,000
and
(final
(7
streptokinase
mixtures
platelets
(1,000
(7 zM),
theophylline
PGE,
U/mI),
within
CaCI2
PGE,
U/mI),
(10
(0.15
(3 mM),
designed
had
deag-
heparin
(2 U/mI),
were
tube
added,
plasmin
sM),
activation:
( I ,000
were
following
theophylline
hirudin
added,
the
aggregates
the
concentrations):
U/mI),
streptokinase
of deaggregated
platelet
PGE,
epinephnine
activation:
deaggregation
the
of platelets,
zg/ml),
observawere
tilting
agents
to a
deag-
interfering
(0. 1 ml)
deaggregation
until
platelet
without
gently
10 mm.
to PRP
unpublished
37#{176}Cwhile
the
37#{176}Cfor
added
facilitated
or epinephnine
at
(20
thrombin
(0.15
The
was
was
peptide
activation
were
(0. 15 CU/mI),
after
ADP,
incubated
streptokinase
after
warmed
secretion’#{176} (and
collagen
agents
mM);
platelets
on
the
Plasma
by preventing
5 mm,
collagenase
was
The
incubated
After
the
plasmin
ml)
I mM.
thrombin,
dispersed.
and
centrifuged
at 4#{176}C,
and
Gly-Pro-Arg-Pro
thrombin-induced
the
were
gradients
in Platelet-Rich
was
concentration
the
sodium
Fractions
suspensions
medium
plasma
Platelet-rich
with
platelet
vol of 0.1 1 M
as described.8
Activation
Before
of
mM
For in vitro
in 0.1
Density
or platelet
of the extracellular
EDTA/30
M
temperature.
plasma
radioimmunoassay,
vol of 0.27
collected
at room
ofPlatelet
multistep
distribution
in 0.1
at 4#{176}C
as described.t
blood
Platelet-rich
mal
Processing
processed
studies
Analysis
(13
of
was
and
activation
mM).
0006-4971/84/6301-0036$Ol.OO/O
482
September
density
more-dense
narrower
NJ;
hepanin
of the density
blood
CU/mI),
April
the
(395
collagenase
Freehold,
and
For determination
platelets,
U/mI),
Foundation.
by
treated
and
much
and streptokinase
Collection
gregation
by
isolation
Sweden.
and
the
MO;
Germany;
CU/mg)
after
platelets.
Gly-Pro-Arg-Pro
end-over-end.
Front
were
there
less-dense
separately
less-dense
and
Corp.,
when
The
degranulation.
originally
Louis,
Netherlands;
and
Sigma
St.
Chemie,
gregation
epinephrine
Dade,
Co.,
Biochemical
final
MATERIAL
complete
diphos-
a decreased
seen
(3)
were
of resting
thington
only
fractions.
identical
in
fl-TG.
platelet
distribution
Chemical
was
of
centrifugation.
the
were
density
The
This
After
of
adenosine
resulted
secretion
thrombin.
platelets
of platelets
thrombin.
epinephrine
more-dense
distribution
tg $-TG/109
J. Sixma
platelets.
gradient
with
cells
the
the
and
I RCU LATING
PLATELETS
are heterogeneous
in size and density.’
The question
of whether
the
heterogeneity
in density
is due to events
that occurred
before
or after
platelets
were
released
from
megabeen resolved.
is the younger
that followed
of
simultaneous
frac-
Activation
a-Granule
with
or
was
g/mI
Jan
plasma
collagen.
density
increased
and
platelet-rich
phate.
C
karyocytes
has not yet
dense
platelet
fraction
suggested
by experiments
in
five
/3-TG/109
1 5.5
Timmermans,
1 .091
1 7)
=
g
±
/3-
alpha-
iso-osmolar
into
g/mI
SD.
±
27.8
P. M.
independent
on
on the
in Platelets
Ans
platelet
gradients
from
Oost,
density
for
separated
density
density
less-dense
cells
marker
were
content
platelet
van
buoyant
(/3-TG),
content.
approaches.
platelet
A.
Depends
(3
(7 tiM),
theophylline
to achieve
I hr
after
(3
maxi-
secretion
induction.
Blood.
Vol.63, No. 2 (February),1984:
pp. 482-485
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PLATELET
DENSITY
DEPENDS
ON
Table
1 . Platelet
483
a-GRANULES
Density
Distri bution’
and lntraplat
elet fi-TG
Content’
Percent
13
4.7
Plateletfraction(%)
±
ND
(sg/1O9celIs)
are presented
The subjects
1 3% Stractan
45.0
were
was
8.3
±
2.2
Stractan
i6
i5
6.1
20.3
±
27.8
± 8.6
Plasma
20
33.3
±
2.6
26.2
± 4.2
15.5
± 4.2
42.4
± 6.7
55.9
± 9.5
65.6
±
content
(3-TG
‘Data
14
in Platelet-Rich
as means
taken
cells (mean
platelet
unfractionated
a healthy
not determined
jg/1O9
for 1 7 subjects.
± SD
from
young
because
n
± SD,
sample.
15.5
population
described
of the low platelet
17). The mean
=
The slightly
higher
in detail
concentration.
fl-TG
content
mean f3-TG content
by us
12
The intraplatelet
in the platelet
in the platelet
The fl-TG
fl-TG
fractions
fractions
content
content
was
104%
is probably
of platelets
with
in the unfractionated
±
caused
9 (n
=
17) of the /-TG
by neglecting
density
platelet
the
less than
sample
content
least-dense
was
of the
platelet
fraction.
Isolation
Fractions
and
Activation
Twenty-seven
was
separated
milliliters
2.5 ml
Stractan,
2.5
pooled
15%/l6%
gel-filtered
and
on Sepharose
recentrifuged
2.5
ml
15%
20%
Stractan.
The
Stractan
Stractan
platelets.
The
2B, treated
on Stractan
ml
Stractan,
ml
1.5
with
gradients
cells/z1)
106
of 2.5
platelets.
16%/20%
more-dense
14%
13%/14%
less-dense
(2 x
consisting
and
and
l0%/l3%
concentrate
2.5 ml
Stractan,
designated
Density
gradient
Stractan,
16%
on the
designated
and
13%
ml
and were
lets on the
and
ofplatelet
on a discontinuous
Stractan,
platelets
ofPlatelet
interface
Likewise,
platelet
50
50
25
25
were
the
interface
graded
10%
plate-
were
pooled
fractions
were
doses of thrombin,
C
0
U
a
.
S
as described.”
S
a
RESULTS
(3-TG
content
platelet
viduals
density
is shown
platelets
was
of
platelet
distribution
in Table
density
fractions.
in 17 young
I . The density
The
healthy
of 80%
mdiof the
0
L-#.,’
%Stractan
SOr
50
between
1.061
g/mI
(13%
Stractan)
and
1.074 g/ml
(16%
Stractan).
The intraplatelet
3-TG
content
rose steadily
and significantly
from 27.8
.og/
i09 cells in the less-dense
platelets
up to 65.6 tg/lO9
cells
(p
in
the
most-dense
platelet
fraction
0.0 1 , Wilcoxon
paired
rank test).
Platelet
secretion
and density
decrease
rich plasma.
Incubation
of platelet-rich
thrombin,
ADP,
collagen,
or epinephrine
25
<
in plateletplasma
with
resulted
in
0
platelet
secretion,
and
deaggregated
platelets
density
1E/-TG
which
decrease
secretion.
did not
the density
of the subsequently
was decreased
(Fig.
I ). This
was only observed
in conjunction
with
At
lower
agonist
concentrations,
provoke
secretion,
the density
did not
change.
For example,
with 1 .25 tM
ADP,
which
only primary
aggregation
without
secretion,
the
sity
was
unchanged
(data
not shown).
Thrombin
lions.
The
fractions
Stractan
treatment
less-dense
of platelet-rich
gradients
and
of platelet
density
and the more-dense
dense
density
platelet
decrease
content.
The
fraction
reacted,
and the secretion
more-dense
fracplatelet
plasma
were
separated
treated
with graded
doses
thrombin
after
removal
of Stractan
(Fig.
2A).
With
0.05 U/ml
thrombin,
platelet
by
gave
den-
on
of
gel-filtration
only the less-
as detected
by the
of 53% of its f3-TG
fraction
showed
no
-i:2i:
25
io
13
ii.
15
16
20
i1i
Fig. 1 .
Platelet
density
decrease
after
induction
of platelet
secretion
in platelet-rich
plasma.
Platelet-rich
plasma
was incubated
with
0.5 U/mI
thrombin
(A). 10 oM ADP
(B). 4 g/ml
collagen
(C). or 5 sM epinephrine
(D). After
3 mm. the platelet
aggregates
were
deaggregated
as described
in Materials
and
Methods.
The recovery
of the degranulated
platelets
was for
thrombin:
89%; ADP: 92%: collagen:
61 %; epinephrine:
61 %. The
extent
of /3-TG
secretion
was for thrombin:
70%;
ADP:
73%;
collagen:
100%;
adrenalin:
49%.
The data shown
are based on
separate
experiments
using different
donors.
All density
determinations
were done in duplicate
and repeated
several
times with
similar results
using different
platelet
suspensions.
The recovery
of platelets
from the Stractan
gradient
was 1 00% ± 7% ( mean ±
SD. n = 32) and the precision
ranged from 2% to 3%. as reported
before.’1
The abscissa
shows
the concentration
of the Stractan
solutions
used to construct
the gradient.
and the platelet
fraction
found at each Stractan
interface
is expressed
as percent
of the
platelets
applied
onto the gradient
(ordinate)
and is identical
in all
four panels. (#{149})
Control
platelet
suspension.
(0) activated
platelet
suspension.
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VAN
484
SOr
25
50
TIMMERMANS,
AND
SIXMA
have a significant
effect on platelet
density
and may be
largely
responsible
for the decrease
in density
following platelet
secretion
as observed
in these and previous
studies.”Platelets
of high density
contain
more 13-TG
B
C
0
U
S
;
OOST.
than less-dense
platelets,
which
confirms
the morphological
observations
by Corash
et al.6’7 and Caranobe
et
25
S
S
al.’6
a
demonstrated
uptake
increased
the same
trend
that
the
with
in our
platelet
gro”p
serotonin
content
density.
of normal
We observed
subjects
and
found
a significant
correlation
between
serotonin
uptake
and f3-TG content
in the platelet
density
fractions,
as well as in the total platelet
samples
(unpublished).
However,
it is very unlikely
that dense
granules are responsible
for the heterogeneity
in platelet
density.
Dense
granules
are I 00-fold
less numerous
S Stractsn
50
than
total
25
alpha-granules’7
platelet
volume.’4
and comprise
Moreover,
only
platelets
pool patients
with no dense
granules
density’5
(also
van Oost,
unpublished)
ii
13
20
15
density
of platelets
of a
deficiency
in alpha-granules
shifted
towards
lower
_
0
9
Fig. 2.
Density
decrease
of platelet
density
fractions
(I).
Platelets
from a platelet
concentrate
were
separated
in a lessdense platelet
fraction
(0) and a more-dense
platelet
fraction
(#{149})
as described
in the text. The fractions
were treated
with 0 U/mI
thrombin
(A). 0.05 U/mI
(B). 0.1 U/mI
(C). and 0.5 U/mI (D). The
less-dense
platelet
fraction
contained
27 zg $-TG/109
cells. and
the more-dense
platelet
fraction
48 .sg $-TG/1O’
cells. A typical
experiment
representative
for three
separate
experiments
is
shown.
For further
details
see legend to Fig. 1.
change
in density
and only
I 1% f./-TG
was secreted
The
less-dense
platelet
fraction
secreted
completely
in response
to 0.1 U/ml
thrombin,
as the
density
decrease
was maximal
and
100% fl-TG
was
secreted.
With
this concentration
of thrombin,
the
dense platelet
fraction
also decreased
in density,
while
secreting
73% 3-TG (Fig. 2C). With
0.5 U/mI
thrombin, alpha-granule
secretion
was complete,
and the
(Fig.
2B).
density
decrease
the degranulated
dense
ping
and
maximal.
platelets
more-dense
(Fig.
The density
originating
platelet
distributions
from
the
fractions
were
of
less-
overlap-
2D).
DISCUSSION
Platelets
contain
several
granule
types
in variable
amounts.
To study
the relationships
between
granule
constituents
and platelet
density,
we selected
/3-TG as
the granule
marker.
fl-TG
is exclusively
located
in the
alpha-granules,’3
which
comprise
10% of the platelet
volume.’4
The density
range
of alpha-granules
measured
with
iso-osmolar
Percoll
gradients
is I . I 21I .I
45 g/ml.’5
plasma,
gradients,
as
The
and
density
measured
is 1.061-1.091
of platelets
here
on
g/mI.
in platelet-rich
iso-osmolar
Stractan
Thus,
alpha-granules
Platelets
decreased
secrete
fl-TG.
This
for washed
human
Cieslar’9
for rabbit
by showing
ADP,
and
platelet
their
have a normal
whereas
the
with
dense
density
a
combined
granules
when
thrombin,
were
able
provided
that
is
induced
has been shown
previously
thrombin-treated
platelets
platelets.
We extend
these
that not only
epinephrine,
density,
patient
and
0.1% of the
of storage
to
by us”
and by
findings
but also collagen,
to decrease
the
the
strength
of
the
inducer
was
high
enough
to induce
secretion.
We
previously
found,
with thrombin
dose-response
curves,
that
platelet
secretion
and
the
density
decrease
occurred
in parallel.’1
Moreover,
we demonstrate
here
that secretion-associated
density
decrease
of platelets
also takes
place in the plasma
milieu,
which
supports
the hypothesis
that platelet
density
is decreased
after
secretion
in vivo.’2
Thrombin
treatment
made
it possible
ity of platelets.
of
to analyze
We found
platelet
density
fractions
the functional
heterogenethat the less-dense
platelets
had a lower threshold
thrombin
concentration
than the
more-dense
platelets.
In view of the lower
granule
content
of the less-dense
platelet
fraction,
it might
be
speculated
that
induce
secretion
less second
by thrombin
messenger
is required
than in the more-dense
to
fraction.
On the other
hand,
at saturating
thrombin
concentrations,
the more-dense
platelets
secreted
more
f3-TG and decreased
more
in density
than
the leastdense platelets.
It was also noted that the density
of the
degranulated
platelet
fractions
was unrelated
to their
original
density
and that
the density
distribution
of
degranulated
platelets
was much
narrower
than
the
density
distribution
of the parent
platelet
suspension.
This
leads
to
the
conclusion
that
the
alpha-granule
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PLATELET
DENSITY
DEPENDS
content
largely
is tightly
responsible
density.
The
conclusions
ON a-GRANULES
485
associated
with platelet
for the heterogeneity
derived
from
ments
described
in this article
for the interpretation
of in vivo
density
and is
in platelet
the
in vitro
been suggested
by some investigators320
increase
in density
in the circulation.
that platelets
By contrast,
our
data
are compatible
with either
the hypothesis
that
platelets
do not change
their density
in the circulation,
experi-
may have implications
platelet
kinetics.
It has
or that
tinuing
platelets
secretion
decrease
in density,
in the circulation.
caused
by con-
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I.
Penington
Platelets
DG:
Formation
in Biology
and
of
platelets,
2. Amsterdam,
Pathology
IL
in Gordon
Elsevier,
(ed):
platelets:
1981,
62:433,
p19
12.
2.
Boneu
dehyde
B. Sie
(MDA)
tions after
3.
man.
I Hematol
age
ML,
6.
buoyant
density,
Corash
7.
tionship
(eds):
platelets:
between
that
of
dense
‘Cr-
and
most
HR.
and
and
Shafer
“In-labeled
dense
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Biologic
Therapy.
TI,
in Greenwalt
Research,
New
York,
1977
R. Liss,
APM,
mans
human
tests
BA,
Oost
Sixma
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for
bind
Ii:
platelet
Proc
10.
NatI
and
Harfenist
use of the
El,
synthetic
prevent
Sci
ii:
The
vanOost
Blood
1978,
p
effect
of
MA,
peptide,
rabbit
thrombin
of
other
that
of fibnin
mono-
platelets.
MA,
Mustard
IF:
in the preparation
Blood
59:952,
density
distribution
of
1W,
Blood
and
acute
peripheral
1982
BN,
PA, Pepper
Bolhuis
of fibrinogen,
in thin
frozen
DS,
platelet
sections
of human
1983
H, Dame
normal
HC,
changes,
mellitus
48:289,
72:1277,
Houwelingen
platelet
localization
K, Themann
on
van
for
in diabetes
Bouma
Invest
GO:
W: Ultrastructural
human
blood
A method
platelets.
platelets.
for
Thromb
I 6.
Caranobe
C, Sie
human
platelet
density
17.
Nossel
age
the
morphomet-
Haemostasis
isolation
Res 20:669,
7:242,
of alpha-granules
1980
B: Serotonin
P. Boneu
subpopulations.
uptake
Br
Hi, Witte
LD, Kaplan
H, Goodman
DS, Baumgartner
pool
Blood
deficiency:
including
I
and storage
Haematol
in
52:253,
Blood
Wi,
of
Thromb
in 18
substances
platelet-derived
A,
in storplatelet
growth
factor.
Mustard
normal
platelets
that
density:
(in
IP,
a-granules
in
storage
pool
Packham
MA,
Kin-
are
a
press)
Rand
IF:
platelet
Studies
ML,
Separation
of thrombin-treated
by density-gradient
centrifugation.
1979
Boneu
age.
Haemostas
RL,
from
platelet
Chernoff
alpha-granules,
in
Evidence
platelet
P, Greenberg
53:867,
on
BA,
Heterogeneity
on granule-bound
deficient
and
HI:
Weiss
Cieslar
studies
Lages
HR:
1979
determinant
deficiency.
platelets
Studies
variants
4, fl-thromboglobulin,
54:1296,
1 8. Vicic
19.
KL,
Weiss
patients
20.
1982
IH,IimmermansAPM,Sixma
the
I Clin
lough-Rathbone
1978
Packham
lipids
beta-thromboglobulin
human
major
1982
derivatives
polymerization
gly-pro-arg-pro,
Hien-Hagg
on
47:239,
peptide
with
Tests
Haemostas
Slot
investigations
factor
Timmer-
distribution
comparison
Haemostas
Synthetic
the
Guccione
BA,van
and
75:3085,
USA
ofthrombin-degranulated
11.
RF:
BFE,
density
of the
aspects
Thromb
Doolittle
Acad
Veldhuyzen
Determination
activation.
AP,
to fibrinogen
mers.
IH,
Methodological
Laudano
9.
van Hien-Hagg
circulation.
I 982
GA
Jamieson
79
8. van
4, and
platelets.
of
Rela-
vol 28: The
Alan
Hi,
BFE,
II:
serum
Thromb
I 5. Gogstad
between
49:71,
in the
I 978
B: Heterogeneity
heterogeneity:
Veldhuyzen
lmmunocytochemical
blood
platelets
Relationship
ultrastructure.
age,
and
II:
factor
nc
platelets
Sixma
and
disease.
14. Stahl
of density
BA,
reactants
Sixma
1982
or
Oost
13. Sander
age in
to demonstrate
Survival
discussion-Platelet
in Clinical
Evidence
platelet
60: 166,
IF:
subpopulations.
volume,
in Transfusion
RH:
of activated
APM,
vascular
1980
from
H, Gralnick
density
Progress
Platelet
of least
1983
Invited
Blood
Use
survival
cell
LM:
phase
subpopula-
with
rabbits
Mustard
61:362,
blood platelet
whole
related.
rabbit
L, Tan
Corash
are
MA,
Blood
density
van
Timmermans
1981
B: Use of asplenic
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R: Malondial-
Res I 9:609,
P. Aster
Catalano
Packham
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platelet
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not size, correlates
I 1:61,
and
subpopulations
human
K-I.
but
L, Shafer
platelet
C, Bierme
in human
of aspirin.
density,
5. Rand
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intake
D, Hwang
buoyant
Am
C, Nouvel
reappearance
4. Corash
that
P. Caranobe
a single
Mezzano
platelet
The
Detection
1983
B, Vigoni
the
Am
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J Hematol
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between
1 3:239,
A,
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1982
C, Sic
buoyant
P: Further
density
and
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1984 63: 482-485
Evidence that platelet density depends on the alpha-granule content in
platelets
BA van Oost, AP Timmermans and JJ Sixma
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