From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Immuno-Electron
Human
Microscopical
Blood
Platelets
Jan J. Sixma,
By
Immunocytochemistry
cathepsin
D
human
lets
bone
from
applied
marrow
gold
blood.
(5 and
D was
somes
in
The
lysosomes
and
fixative
8) particles
platelets
2
were
used
and
in
in megakaryocytes.
of blood
Protein
Al
as second
and
label.
(ATP)
, adenosine
and pyrophosphates’;
and lysosomes.3
Lysosomes
were
similar
to a-granules,4
but the
lysosomal
enzymes
is different5;
of acid
ence
phosphatase
because
confirm
istry
were
of
using
megakaryocytes
somewhat
braneous
lighter
with often
halo. The secondary
Fixation:
Fifteen
normal
of
individuals
buffer
centrifuged
sion. The
(ten
platelets
into
at
minutes,
were
After
resuspended
10,000
for
five
panafonmaldehyde
treated
minutes
Blood,
hour
blood
Tynode.
platelets
The
at 37 #{176}C.
Human
Vol 65.
No
from
collected
diagnostic
into
of
buffer
at pH
fragments
were
collected
fixative.
The
further
platelets
and
according
alone
was
of
7.4
2%
incubated
as described
for
above.
to Geuze
0.1%
by
was
as
and
Slot.9
because
with
in three
the
The
pipetting
excess
for
blood
in paraformal-
fixation
glutaraldehyde
tissue
the
above
Fixation
mol/L
small
off
described
usual
patients
in 0.1
temperature.
a pellet
procedure
selected,
punctions
panafonmaldehyde
at room
into
of cathepsin
did
mixture
not
of 2%
preserve
the
performed
as
D.
microscopic
immunocytochemistry
previously.
labeled
with
We
cathepsin
D. The
has
the
embedded
in
8-nm
affinity
antibody
and
immunolabeling,
purified
or
used
of the enzyme
Ultrathin
10.1
5-nm
proteins.
colloidal
gold
rabbit
recognizes
extensively
sections
were
particles
indirectly
conjugated
anti-human
stained
and
with
to
amylase
was
native
forms
Following
uranyl
acetate
Tokuyasu.’4
used
to
placental
characterized.’2’3
according
pancreas
were
the precursor
been
anti-rat
was
cnyosections
purified
methylcellulose
rabbit
Bone
three
was
in 0. 1 mol/L
(20
was
fixed
and
Affinity-
as contnol.
RESULTS
lyso-
each
suspension
sternum
4 mL
phosphate
Marrow
A positive
temperature
was
of
5 (May),
gradually
fixation,
mol/L
and
#{176}C)
and
raised
platelet
phosphate
with
weeks.
were
the
pelleted
of gelatin
for up to two
platelets,
were
reaction
megakaryocytes,
cytes,
endothelial
from
The
subsequently
Megakaryocytes
reaction
ambient
on 0.1
minutes,
g, 20 #{176}C).Blocks
calcium-free
at
concentration
another
in 10% gelatin
equilibrated
as
are
or
METHODS
collected
Inc.
of 1 .0 U/mL.
a characteristic
submemlysosomes
are larger
and
was
stain-
tubules.
at 37 #{176}C
and
Electron
have
primary
reaction
cathepsin-D
was
found
localized
lysosomes.
for
cathepsin
macrophages,
cells,
and
in macrophages
predominantly
This
will
D was
found
monocytes,
fat cells.
The
where
in primary
be published
in
granulo-
strongest
cathepsin
and
D
secondary
elsewhere.’6
I 50 g, 20 #{176}C)
to obtain
a platelet
suspenpelleted
( 1 5 minutes,
I ,500 g, 20 #{176}C),
and
panaformaldehyde
hour.
7.4
minutes
a release
showed
& Stratton,
concentration
three
described
as well
35 mL of 2% paraformaldehyde
pH
a final
an
Immunocytochemistry
Platelets
blood
by Grune
1985
lacking
of a transparent
undergoing
surface-connecting
antigenicity
We
primary
lysosomes
and of equal
density
AND
Blood
milliliters
phosphate
the
primary
in the
the
in platelets
smaller,
presence
thrombin
on
lysosomes
lysosomes
being
by the
Platelets
with
localization
secondary
The
by
and
halo.
paraformaldehyde
in vesi-
D. We
core,
stimulation
their
and
inclusions.
dense
dehyde
The precise
be described,
inclusions.
Blood platelets
contain
similar
to those in megakaryocytes.
MATERIALS
one
sulphatase
contain
lysosomes.
The
than
the a-granules
Tissue
a-
enzymes,6
contain
electronmithe pres-
anti-cathepsin
secondary
smaller
show
somes
aryl
to be
for
covered
with reaction
product.
these data with immunocytochem-
on cryosections
that
and
of
electron
Fragments
lacking
of lysosomal
a-granules,
Recently,
demonstrated
smaller
than a-granules.8
the vesicles
could
not
they were
and extend
found
thought
pattern
patients
granules
have normal
contents
and
cell
fractions
containing
almost
no lysosomal
enzymes.7
croscopic
histocytochemistry
des, which
morphology
diphosphate
peroxisomes,2
initially
secretion
presence
by
complex
a-granules
S
UMAN
BLOOD
PLATELETS
contain
several
types of granules:
a-granules,
containing
various
proteins; dense granules, containing serotonin, adeno-
Golgi
from
ing
in mega-
identified
of the
D
and Hans J. Geuze
differed
after
H
sine
triphosphate
(ADP)
calcium,
were
trans-side
in
Anti-Cathepsin
Kurt von Figura,
submembrane
lyso-
secondary
lysosomes
Hasilik,
by the
parafor-
secondary
primary
Primary
of
plate-
was
8%).
-‘
in primary
and
sections
used
Using
karyocytes
anti-human
frozen
gradient
localized
blood
van den Berg, Andrej
ultrathin
of Lysosomes
Megakaryocytes
affinity-purified
to
(concentration
Cathepsin
Agnes
megakaryocytes
peripheral
maldehyde
colloidal
with
was
and
Demonstration
In order
first
buffer,
again
tissue
was
thrombin
(Sigma,
St Louis)
pp
1287-1291
From
Microscopy,
7.4,
Department
the
thrombinin
for
ten
Sept
Address
was
added
to
Utrecht,
© I 985
of
/3,
reprint
of
and
Utrecht,
Chemistry,
/983;
Center
The
for
Electron
Netherlands,
M#{252}nster, West
and
Germany.
accepted
Nov
27,
to
J.J.
Sixma,
Department
P0
16250.
requests
University
The
Hematology
University
ofPhysiological
Hematology,
warmed
Department
State
Submitted
in 8%
by gelfiltration
suspension
in
was
minutes,
stoned
to obtain
washed
pH
(five
were
platelet
1985:
to 8%
pellet
Hospital
Dr
Utrecht,
1984.
Box
3500
of
CG
Netherlands.
by Grune
& Stratton,
Inc.
0006-4971/85/6505-0036$03.00/0
1287
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1288
SIXMA
ET
AL
Fig 1 .
Overview
of Golgi region
of a megakaryocyte.
Primary
lysosomes
are indicated
by single arrows.
A secondary
lysosome
is
indicated
by a double arrow.
a. Typical
a-granules;
N, nucleus.
Areas (A) and (B) are shown at higher magnification
in the insets. The bars
in the overview
and inset (A) correspond
to 0.50
sm; in inset (B) to 0.25 m.
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MEGAKARYOCYTE
AND
PLATELET
LYSOSOMES
1289
Fig 3.
Detail of a platelet
containing
labeled
lysosomes
(1) and
several
a-granules
(a). The lower
lysosome
may represent
a
secondary
one in view
of its size
and the heterogeneity
of its
content.
The bar corresponds
to 0.25 m.
on one single
granule
Label
was also found
connected
tubules,
This was confirmed
D was localized
release
Fig 2.
Localization
of cathepsin
D in a blood
platelet.
lysosome
(L) is heavily labeled.
a, Representative
a-granules;
surface-connected
tubular
system.
The bar corresponds
zm.
In megakaryocytes,
lysosomes,
which
were
diameter
neous
than
size
a-granules
translucent
tion with the
where
primary
Secondary
and the
label
was
characterized
halo
with
and
trans-side
lysosomes
found
in
by
often
a
The
SCS,
to 0.5
Blood
sections
showed
primary
smaller
acid
a submembrafrequent
associa-
complex
(Fig
I),
characterized
by their larger
of the contents,
were also
with
at all.
anti-rat
in general
granules
reaction
density,
was
which
hydrolases
requires
occupancy
lysosomal
with
thrombin
anti-rat
(Fig
4). Con-
pancreas
(Figs
2, 3). These
label was found only
a stronger
Recent
Likewise,
amylase
granules
Secretion
stimulus
in
of
as well
receptor
in
both the contents
are
diminished
unchanged.’8
studies
histochemical
aryl sulphatase
megakaryocytes
platelet
syndrome6
of a
and
as secretory
documented.
of membrane
content.6
storage
disease,
and
a-granules
enzymes
remain
and
and
observed
on granules
were somewhat
lighter
smaller
than a-granules
were scarce.
In general,
induced
as
for throm-
bin in comparison
to the secretion
of the contents
of
a-granules
and dense
bodies.’7
Patients
with the gray
platelet
syndrome
lack a-granules
and have a normal
pancreas
Platelets
A positive
homogenous
been
presence
of lysosomes
blood platelets
is well
constant
by their
incubated
no reaction
profiles.
surface-
presumably
as a result of secretion.
in experiments
in which
cathepsin
in surface-connected
tubules
after
trol sections
incubated
with
were completely
negative.
The
human
labeled
(Fig I , inset A). No label was observed
in the
Golgi complex
or in the rough
endoplasmic
reticulum.
Control
amylase
platelet
dilated
DISCUSSION
of the Golgi
develop.
lysosomes,
heterogeneity
has
in every
three
in occasional
localization
and
the
demonstrated
the
combined
of acid
phosphatase
in normal
platelets
in
the presence
platelets8
the gray
of reac-
tion product
in vesicles
in blood
platelets
and
karyocytes
and also of acid phosphatase
in some
nae of the
observations.
Golgi
complex.
Our
a-5
of the dense granules
but
the
lysosomal
studies
extend
megacisterthese
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1290
SIXMA
sensitivity
complex.
to
fixation
of
cathepsin
Our
immunocytochemistry
cathepsin
D also visualized
cytes,
which
were evidently
D
in
ET AL
the
Golgi
studies
with
antigranules
in megakaryosecondary
lysosomes.
As
no evidence
of active
phagocytosis
has been found
in
blood platelets,’9
the presence
of secondary
lysosomes
was somewhat
surprising.
The most likely explanation
is
that
secondary
lysosomes
are
caused
by autophag-
ing. This may indicate
the presence
of a rapid
down of cellular
material
in megakaryocytes.
break-
The presence
of cathepsin
D in surface-connected
tubules
is in accordance
with the idea that secretion
lysosomes
occurs
via the surface-connected
tubules,
has previously
a-granules
such
Prolonged
retention
connected
tubules
slow and
A similar
factor
The
before
fixation.
of a platelet
treated
Cathepsin
D
(5
localized
in the surface
connected
0.25 pm. SCS. surface-connected
that
We found
cathepsin
were smaller
than
slightly
lower
complex.
This
present-day
was seen
concept
over the
findings
strated
Golgi
In
the
granules
with the
localization
of Bentfeld-Barker
gold
particles.
of thrombin
arrows)
is
The bar corresponds
system.
to
localized
and had
vicinity
in granules
the same or
of
the
and
Golgi
were found,
some of
trans-side
of the Golgi
is in agreement
of lysosome
Golgi
complex
acid phosphatase
complex.
These
1 .0 units
system.
tubular
D to be
a-granules
density.
complex,
still-smaller
which
were associated
nm
with
with
our
formation.
No label
in contrast
to the
Bainton,8
who
but not aryl sulphatase
data are also in contrast
demonin the
to the
findings
in macrophages
where
some label was found
over the Golgi stack.
It may well be that the amount
of
cathepsin
D in the Golgi
cistemnae
is below the detection level for immunoelectronmicroscopy
personal
communication).
This absence
be due to the low local
concentration
(H.J. Geuze,
of label
may
or increased
for proteins
stored
in
and platelet
factor
4#{149}20.21
hydrolases
in the
be responsible
observed
has been
surface-
for the relatively
for acid
suggested
hydrolases.22
for platelet
423
granules
been
Detail
of acid
may
partial
release
mechanism
necessarily
Two types
Fig 4.
been
reported
as fibrinogen
of
as
labeled
with
anti-cathepsin
representative
for all
of acid hydrolase-containing
recognized
with
the
D are
not
lysosomal
granules.
granules
have
digitonin
lysis
technique24
and in patients
with
lysosomal
heterogeneity
fication
of lysosomes.
storage
pool deficiency.25
This
may interfere
with the identiCathepsin
D may be present
in
one type of lysosomes
ance of the granules
and
not in another.
that
are
labeled
The appearwith
anti-
cathepsin
from that
D in platelets
of a-granules
is not sufficiently
different
to exclude
the possibility
that
cathepsin
D is present
in a subclass
Theoretically,
double-label
of
experiments
a-granules.
should
offer
direct
evidence.
We have previously
used two different
gold labels to demonstrate
the contribution
of albumin
and f3-thromboglobulin
in ct-granules.26
We have also
used double-immunofluorescent
labeling
to demonstrate
the
globulin,
we tried
level
sons.
with
The
resolution
codistribution
of
fibrinogen,
fl-thrombo-
and platelet
factor
427
Experiments
in which
double
labeling
at the electron
microscopical
two gold labels
immunofluorescent
in order
to
failed
ascertain
for
studies
that
unexplained
reahad insufficient
the
occasional
granules
stained
with anti-cathepsin
D were different
from
the many
a-granules
stained
with anti-fibrinogen.
The presence
of cathepsin
D in a subclass
of
a-granules
or conversely
the presence
of a-granule
proteins
because
granule
of the
patients
in lysosomes
seems
less
likely,
however,
of the different
secretion
patterns
for aproteins
and lysosomal
enzymes,
and because
normal
presence
of lysosomal
enzymes
in
with the gray platelet
syndrome.6
Moreover,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MEGAKARYOCYTE
AND
PLATELET
in megakaryocytes
cathepsin
D differ
LYSOSOMES
the granules
morphologically
1291
I 4.
stained
with
antifrom a-granules.
Tokuyasu
sections.
KT:
I 5. Geuze
Griffith
ACKNOWLEDGMENT
thank
Roy
Annemieke
the
Geenaths
Beyen
and
for
Maeyken
preparing
the
Hoeneveld
for
photographs
the
and
preparation
I
Holmsen
.
Rev
2.
H,
Med
Bneton
Weiss
Hi:
30:1 19,
1979
Gonius
megakanyocytes
method.
and
3.
Bentfeld
somal
enzyme
4.
ME,
as revealed
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rat
pools
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23:197,
Bainton
in
storage
in platelets.
lJ,
types
by the
ofgnanules
diamino
in
DF:
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and
May
B, Hasslen
R: Electron
in blood
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H,
MC:
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Weiss
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storage
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patients
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storage
HJ,
hydro-
pool
in
NP,
and
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Gray
Platelet
Cohen
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mitochondnia
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Hi,
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human
Dangelmeien
to
Galjaard
and
cells.
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their
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HR:
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iG:
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JG:
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receptor
occu-
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in
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latex
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in I 8
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1985 65: 1287-1291
Immuno-electron microscopical demonstration of lysosomes in human
blood platelets and megakaryocytes using anti-cathepsin D
JJ Sixma, A van den Berg, A Hasilik, K von Figura and HJ Geuze
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