From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Effect
of Cell
Shape
By
Although
it has
shape
effect
study,
rat
been
causes
of
uptake
in the
shape
alteration,
only
5%-i
after
cells)
there
prior
agents
HE
variety
and
cause
was
OF
not
an
they
binding
whereas
of
with
if cells
erythrocytes
Erythrocyte
shape changes
vivo
hemolysis
anemias.6
In
the morphology
hereditary
of the
surface
organs7
hemolysis
of aged
ciated
with
signposts
a
possibly
their
or
treatment
effect
rat
of shape
for example,
progressively
Male
Charles
or irreversibly
sickled
either
dysfunction
in the
hemolysis.8
Furthermore,
erythrocytes
membrane
is believed
loss
and
to be asso-
sphering,9
and
the spherical
morphology
can result
from severe crenation or cupping.’#{176} All of these
findings
and others
support
the hypothesis
that shape
alteration
per se can
cause extravascular
hemolysis.
These
arguments,
however,
are
indirect
and
no
Sprague-Dawley
River
Breeding
(D
3260),
and
tetracaine
Mo.
2,4
The
source
demonstrated
selectively
that
were
that
“fixed”
crenated
erythrocyte
with
the
diamide
remained
washed
away, whereas
with
diamide
returned
by
Blood,
Vol. 59. No. 2 (February),
coworkers”
membranes
could
be
oxidant,
diamide.
Cells
by a crenating
agent
and
“fixed”
crenated
when
the agent
was
induced
echinocytes
not treated
to the disc
form.
Further,
1982
the
lgG.
clearance
of
membrane
factors.
did
not
result
in
their
the circulation.
In
employed
to measure
on erythrocyte
England
Nuclear,
Rat
for
this
the
clearance
in a
10 mM
times.
The
20%.
as
sodium
by cardiac
and
NaH2PO4,
Chromium5’
pH
was
(“Cr)
I 5 mm.
to remove
Co.
St.
chromate
into
blood
Louis,
was
buffy
coat
7.4).
This
resuspended
was added
Following
unbound
were
New
procedure
in PBS
the
(I 100 g
and
(PBS;
was
the
140 mM
repeated
3
to a hematocrit
and the mixture
then
contain-
aspirated,
saline
incubation,
51Cr,
a syringe
was centrifuged
in phosphate-buffered
pellet
(C8138),
Sigma
puncture
The
supernatant
final
at 37#{176}C
for
times
from
of Erythrocytes
was resuspended
NaCI,
purchased
from
Mass.
Diamide
Mass.
was drawn
the
were
chlorpromazine
7004),
as an anticoagulant.
10 mm),
METHODS
purchased
chromium-51
Labeling
blood
pellet
(D
Boston,
51Chromium
ing heparin
AND
were
of
cells
resuspended
of
was incubated
were
in
washed
PBS
to
3
20%
hematocrit.
Shape-Change-Inducing
Health
and
discs
dinitrophenol
plasm.
Haest
of
deformability
altered
rats (200-400
g)
Labs,
Wilmington,
(T7508)
2,4-Dinitrophenol
ments
were achieved
however,
that
their
of
clearance
binding
the
mechanical
from
was
alteration
direct
causal
relationship
between
the alteration
of
shape
and
in vivo clearance
has been
found.
The
problem
has been that most agents
that
preserve
or
“fix” erythrocyte
shape also fix the erythrocyte
cytoRecently,
of
MATERIALS
spherocytosis,
cells
becomes
severe
suggest
than
in vivo
the
It is
alteration
model.
Materials
of
the
altered
from
rather
immediate
clearance
study
diamide
fixation
to contribute
spherocytic
as in acanthocytosis
are
we
controls,
observed.
increase
alone
results
properties
a
serum
shape-
over
were
stomatocytes,
discs.
cells
diamide
the
more spherocytic
with longer
in vitro incubations.
The
absence
of spherocytes
in freshly
drawn
blood, except
in severe
cases,
is taken
as an indication
that
the
spherocytes
are cleared
from
the circulation.
When
abnormally
shaped
erythrocytes
are found
in penipheral blood,
cells,
they
clearance
the
3-8-fold
binding
will
treatment
of
to
if
clearance,
cells,
disc
of
the
by
in C3
diamide-flxed
case
diamide
are believed
the hereditary
in
the
shape-altered
the
occurs
by
opsonization,’
and
in
determine
were
from
oxidation,2’3
in
in
shape
in vitro
in autologous
plasma.
but not severe
echinocytes.
the
increased
a biconcave
properties
of the
was
that
Because
washed
cells
Further.
lgG
without
of their
To
involved
no differences
from
and,
echi(cupped
be
incubation
conversion
clearance.
might
of
shape
spleen
through
then
were
incubated
stomatocytes
concluded
Whereas
depletion
6 hr.
reduced
altered
cells
With
severe
of
effect
ATP
i 2-i
components
51Cr
were
by extravascular
hemolysis
mechanisms
involving
IgG
membrane
form
by
of severe
treatment
clearance.
discoid
extent
stomatocytes
since
In this
to fix
addi-
by the
by
for
of cell shape.45
It is the question
of the role
shape
in clearance
that
we will
address.
alteration
of cell
in
This
agents.
not
C3 opsonization,2
to
severe
liver.
injection.
cleared
clearance
and
CLEARANCE
blood
of
were
cupping.
Hemolysis
P. Sheetz
crenated
in membrane
the
Michael
glucose
the
measured
with
or
50%-80%
cupping
did
as
crenation
cells)
to
alone
correlated
by the
or
away
liver
was
primarily
crenating
and
cells
by
changes
in vivo
of discoid
(crenated
by
cells
and
the
of
has been
made.
was
employed
of
either
0%
1 hr.
nocytes
spleen
measure
produced
or
Cosgrove
of erythrocyte
direct
shapes
amphipaths
Clearance
Patricia
alteration
no
in altered
different
structure.
T
that
clearance.
of varying
shape
on clearance
the protein
oxidant,
diamide,
erythrocytes
tion
inferred
cell
on Extravascular
From
the
Department,
Farmington,
Supported
in part
Submitted
June
reprint
ogy
Department.
Farmington.
Conn.
© I 982
(DNP),
chlorpromazine,
by mixing
equal
volumes
Physiology
Center,
Address
Treatments
by Grune
by NIH
treatcells
University
Grant
HL
accepted
requests
to Michael
& Stratton,
tetracaine
of 5tCr-labeled
of
Connecticut
Conn.
15. 1Q81;
University
06032.
and
of
23359-03.
October
19. 1981.
P. Sheezz.
Connecticut
Ph.D.,
Health
PhysiolCenter,
Inc.
0006-4971/82/5902-0031$1.00/0
421
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
COSGROVE
422
(hematocrit
20%)
10% and
a final
and
chemical
stocks
concentration
of the
giving
a final
treating
agents
Erythrocytes
were
in PBS
12-22
for
mg/mi
the cells
these
in
protein’3
for
remove
unbound
Because
ATP-depleted
from echinocytes
Diamide
An
to become
10 mM
extensive
and
with
of
suspension
mM
diamide.
times
with
The
chromium
prepared
after incubation.
treated
of 100
mM
erythrocytes
The cells
were
diamide
to give
incubated
PBS, and resuspended
was
a final
added
to a 10%
concentration
at 37#{176}C
for 45 mm,
to a 50% hematocrit
gel
of Crenating
A crenating
degree
index
of shape
cytes
were
or cupping
change
fixed
The
gradations,5
and each
severely
2%
of 1
washed
3
category
crenated.
by the value
for that
index.
category.
cupping
index
was
cell
given
a value of two
was
calculated
in
Analysis
the
samples
drawn
were
microliters
jugular
of
5’Cr-labeled,
of an ether
by retroocular
puncture
vein
to determine
rate ofclearance
of labeled
stream.
Tests were done
with a Coulter
the hematocrits
spleen,
location
were uniform.
liver,
kidneys,
of cleared
were
After
and
the
to treated
serum
for 30 mm
cells.
Reduced
paper
lose
rabbit
systematic
blood
of
samples
7000
remaining
in
the
of sample
the
The
blood
51Cr
organs
(The
determined
counts
injected
found
in
and
on
a
erythroratio
of
of injection.
organ
was
of
effect
cells
of treated
erythrocytes
1:100
sion
volume
g for
a fluffy
of fresh,
cells
buffer
10 mm)
were
on
membrane
membrane
proteins
Membrane
a 50%
phosphate,
at 4#{176}C.
This
ghosts
erythrocyte
pH
procedure
suspen-
7.4)
and
was
repeated
twice
with
equal
centrifug-
IgG
were
heparinized
in the serum
3 times
and
incubated
plasma
to bind
resuspended
or PBS
to the
in PBS.
proteins
was
incubated
then
was incubated
A protein
was
As determined
well,
“51-Staph-A
Fc portion
dried,
dilutions
conditions
were
nitrocellucontaining
with
to the
these
The
in a buffer
rinsed
by analyzing
under
erythrocytes
proteins
paper.
bound
as a Shape-Stabilizing
of the
and
exposed
of whole
serum,
was
approximately
were
tested
Agent
of diamide
concentrations
to stabilize
echinocytes.
Maintenance
was not observed
at 0. 1 mM or 0.5 mM,
5.0, and 10.0
no significant
an
for
30 mm
treated
cells.
Aliquots
to test
order
over
time,
bated
in
mM were
difference
control
PBS
the
of
whereas
all found
to be effective
in the degree
of shape
effect
of diamide
10 mM
of shapediamide
at
cell shape
on erythrocytes
and diamide-treated
with
for
of cell
cells
glucose
for
were
up
incu-
to 4 hr.
Control
cells remained
as discs throughout
the incubation period.
Diamide-treated
cells
became
progressively
more crenated,
with the most dramatic
change
occurring
between
1 hr, when 66% of the diamidetreated
cells
were
discs,
and 2 hr, when
22% were
discs. Therefore,
although
diamide
could
be expected
to maintain
cell shape
for a period,
it is evident
that
necessary
It was
to
“fixed”
with
being
injected
into
hundred
injected
removed
the
percentage
these
scopic
basis
that
ascertain
stomatocytes
after
Binding
50%)
ghost
nitrocellulose
these
separated
C3, was rinsed,
of sensitivity
Diamide
of
pellet.
homologous
washed
gels.’2
by diluting
(5 mM
(hematocrit
the
the
polyacrylamide
prepared
white
treatments
by separating
oflgG
cells
at 37#{176}C
to allow
The
on SDS
were
Determination
Treated
determined
in a lysis
ing (35,000
to obtain
shape-change-inducing
was
The
to nitrocellulose
this lock on shape
is not permanent.
problem
of changing
cell shape,
a 1 -hr
tion period was chosen.
Gel Electrophoresis
The
gel
gels.
the
treated
to the
RESULTS
In
directly.
composition
polyacrylamide
from
maintenance.
the
an
membranes)
of
3 (C3)
homologous
to bind
points
counted
by
in serum
C3
fresh
time
in organ
at time
to
(hematocrit
cells
of
stabilization.
Incubation
of a 10% suspension
altered
erythrocytes
in PBS with 1 mM
37#{176}Cfor 45 mm resulted
in reproducible
of labeled
counts/mi
Treated
volume
from
The
film.
limit
the
electrophoresis
an equal
(10’
“51-Staph
IgG.)
1.0,
with
to determine
were
and
bound
component
were
Blood
differences
percentage
was
to calculated
of
percentage
calculated
and
counter.
counts
for complement
membranes.
with
ghosts
with
anti-rat
Several
Each
1 hr. the rat was sacrificed
No
to
PBS,
polyacrylamide
at 37#{176}C
to allow
on SDS
run
ability
shape
from the blood
to determine
that
removed
of
cells
rat.
treated
anesthetized
were
erythrocyte
incubated
electrophoresed
products
manner.
at various
in
was
spherostomatocyte
erythrocytes
counter
heart
erythrocytes.
gamma
counts/mi
The
each
4 being
noted.
Aliquots
Beckman
cytes
a similar
and
3 times
resuspended
as a ratio
to assay
the
category
of these
Staph-A
washed
cells.
for
method
four
of Clearance
One-hundred
injected
into
sizes
sum
expressed
iodonated
were
10 ngofC3.
a value of four.
given
the
1-4,
in each
The
under
to Weed’s
a value
of cells
the
erythro-
observed
according
with
cells
protein,
PBS-treated
was used
were
to x-ray
to represent
of treated
and
scored
number
the crenating
cupped
Portions
was assigned
The
represents
was
cells.
were
Index
transfer
50%)
protein.
in PBS.
was designed
glutaraldehyde
echinocytes
multiplied
The
index
in treated
in
microscope.
most
or Cupping
incubated
37#{176}C.The
of C3 Binding
et al.’4
rabbit
Determination
were
versus
bound
were
amount
Results
Towbin
Treatment
appropriate
at
“51-Staph-A
plasma-treated
1
washing
stomatocytes
were
mm
Determination
and
16 hr. then
30
by
5 mM
cytidine,
incubation
cells
counted.
cell
at 37#{176}C
containing
at 37#{176}C
for
were 5’Cr-Iabeled
a 20%
stomatocytes
buffer
myo-inositol,
and streptomycin
lysis,
incubating
and streptomycin
in a PBS
10 mM
in PBS.
cell
causes
by
induced
suspension
glucose,
penicillin
ATP
penicillin
were
a 10% cell
10 mM
of
1 mg/ml
hr. Erythrocytes
incubating
DNP,
depleted
and
suspensions
of
the text.
suspension
cell
hematocrit
as indicated
AND SHEETZ
diamide
the
To avoid
the
in vivo incubaechinocytes
remained
blood
stream.
or
as such
Five-
microliters
of 5tCr-labeled
treated
cells were
into a rat. After
5 mm, aliquots
of blood were
by retroocular
puncture
and were fixed. On
of cpm/ml,
it was possible
to predict
the
of
treated
cells
that
should
be found
in
samples.
With
4 mM DNP-treated
cells, microobservation
revealed
that 0.4%
± 0.3%
(count-
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EFFECT
OF CELL
SHAPE
IN HEMOLYSIS
423
Table
1 . Time
Dependence
of Clearan
cc from
Blood
Diamide#{176}
Untreated
DNPt
Control
Chlorpromazine
Time
C
10-20mm
102±
16(16)
72±
12(20)
69±5(7)
lhr
121
±
11(16)
61
±
13(18)
50±
4 hr
94
±
6 (2)
43
±
1 2 (2)
3 1
24hr
79±9(2)
mM
diamide.
mM
DNP.
O.4
mM
error)
of
ing predicted
mM
of total
injected
cpm remaining
the cells were crenated,
that 1 .4% ± 0.4% were
Tetracaine-treated
cells,
cells
in the blood
while ‘Cr
crenated.
1 .9%
±
0.4%
.
because
of clearance,
measurements
were
observations
did
the statistical
diminished.
reveal
that
stream
cupped
were observed,
while
1 I % ± 0.4% cupped
predicted.
After
I 5 mm, increased
numbers
shape-altered
cells were
found
in the bloodstream,
cells
of
but
significance
of the
Thus,
microscopic
erythrocytes
shapes were in the bloodstream
and
observed
were
within
experimental
with
altered
the proportions
error
of the
predicted values.
In Vivo
Clearance
Studies
were then done to determine
the
diamide
treatment
on erythrocyte
longevity
blood
stream
(Table
1). The initial
cpm/ml
blood stream
was calculated
by determining
effect
of
in the
in the
the total
blood
volume
from the body weight
(4.95
ml blood/
100 g body weight)’5
and dividing
total counts
injected
by this
volume.
The cpm/ml
of blood
injected
is
than
the calculated
cpm/ml
with the untreated
indicating
a possible
problem
of incomof injected
cells
with
the total
blood
data do show a more
rapid
clearing
of
cells
than
untreated
cells
from
the
erythrocytes,
plete
mixing
volume.
The
diamide-treated
blood
stream,
particularly
changes
might
cupping
or crenating
more
4 (2)
11
2 (2)
±
20±1(2)
countWith 4
were
greater
±
45±8(4)
-
chlorpromazine.
§Values are percentage
ing
17±2(4)
53±9(4)
11(7)
rapid
after
be expected
agent
clearance
Clearance
of
reflected
in the
from
1 hr when
to occur.
to the
of
the
SD. values
kidney,
in parentheses
and
heart
mately
the
diamide-treated
same
represent
stream.
from
the
blood
is
total
5’Cr injected
that was found
in the organs
(Table
2). The liver and
spleen
are
thought
to be the major organs
of clearance.
clearance
clearance
of
animal.
To
diamide
check
levels
cells
for
in
remain
other
possible
approxiof the
loss of
regions
clearance
of
of
the
diamide
by the first capillary
bed encountered,
the lungs
excised
and
counted,
but no clearance
was
cells
were
observed.
Rather,
it seems
more likely that the difference represents
a change
in blood pool size and to an
extent
a statistical
variation
in the blood
counts.
Because
of the reproducibility
and the direct
interpretation
of
counts,
the
the
organs
clearance
as determined
percentage
of counts
primarily
was
used
Effect ofShape-Inducing
of Erythrocytes
The effects
phenol
Figure
the
of two
by
found
Treatments
crenating
Table
and
2.
treatments,
of Altered
Untreated
Spleen
(12)’
Diamide(12)
2,4-dinitro-
were
studied.
representative
of
erythrocytes.
Cells
Percent
Agent
of clearance.
of ATP-depleted
Clearance
organ
various
on Clearance
(DNP)
and
ATP
depletion,
1 illustrates
cell populations
DNP-crenated
the
in the
as a measure
Liver
by Organ
Clearance
±
Kidney
1±.4
8±1
1±3
7±2
6±2
2±.9
SD
Heart
O.8±.3
1±.3
DNP,
6 mM (3)
14±5
30±1
3±1
O.9±.2
DNP,
4 mM
(4)
11±1
22±2
2±7
0.8±.3
DNP,
2 mM (2)
8±3
ATP
depleted
(5)
9±1
2±.3
O.6±.2
23±8
70±20
1±.5
O.4±.2
74±7
2±1
23±13
1±.4
Tetracaine,
6 mM
(5)
12±8
Tetracaine,
4 mM
(4)
9±3
Tetracaine,
2 mM (4)
the liver and
or by labeling
the
Washed
6%
in
increase
of experiments.
counts
from the blood (Table
1) without
their appearance in the spleen
or liver (Table
2). This difference
could
represent
an increase
in the blood
pool size or
Kidneys
clear lysed cells and the heart is included
as a
control
organ.
There
are very few labeled
untreated
cells in the spleen
(1%),
kidney
(1%),
or heart
(1%),
and slightly
more in the liver (8%).
These
liver cells
presumably
represent
the volume
of blood
residing
in
filtering
of cells damaged
by handling
with 51Cr. Diamide
treatment
causes
a
cells trapped
in the spleen,
while liver,
number
as the control.
In the case
cells,
there
is an apparent
shape
The addition
of a
cells causes
an even
the blood
erythrocytes
percentage
the
±
Chlorpromazine,
0.4 mM
mM
(cupped)
‘Number
1±.1
4±2
9±2
2±.2
33±6
33±15
2±.1
0.6±
9±1
9±.1
2±.1
0.7±.1
1±.3
0.5±.1
(2)
Chlorpromazine.
0.2
2±.7
O.6±.2
(2)
incubated
(2)
of rats.
24±6
50±4
1
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
COSGROVE
424
AND
SHEETZ
‘C
a,
V
C
a1
0
C
0
C)
0
C
a,
U
DNP
Fig.
treated
1.
Scanning
with
4 mM
electron
micrographs
of (A) erythrocytes
DNP
+
1 mM
diamide,
(B) ATP-depleted
+ 1 mM
diamide.
(C) 4 mM tetracaine
+ 1 mM
and
(D) untreated
erythrocytes
+
1 mM
diamide
erythrocytes
diamide,
(x2000).
An
increase
in
the
in DNP
of
number
concentration
causes
echinocytes
and
to
more
severely
crenated
cells (as defined
by Weed).5
The echinocytes
formed by treatment
with DNP or
by ATP depletion
and held in shape by diamide
were
cleared
from
the blood
by the organs
more
rapidly
than
diamide-treated
erythrocytes
after
1 hr (Table
1 ). Examination
of individual
organs
reveals
an
increase
in splenic
clearance
of l%-7%
by all concentrations
of DNP
erythrocytes.
cally
increased.
64% more
mM
DNP
tested
Liver
and
of
clearance,
16%
cells
clearance
in the liver
and diamide
a 24%
and diamide
with
2 mM
was
plus
of DNP
concentrations
of clearance
in liver ().
in spleen
diamide
(1 mM)
(A-A).
Cells
were
for 45 mm at 37’C
tion of clearance
Chlorpromazine
on
crenation
index
and percent
of
incubated
and then
with
DNP
and
washed.
by the spleen
as well
at 0.2 mM did not change
as the liver.
the level of
clearance
from that of diamide.
Again,
the association
between
the severity
of shape change as represented
by
CI and the amount
of clearance
by the organs
suggests
that changed
shape
causes
perhaps
also by the spleen
zine
(Figs.
3 and 4).
Drug
dramati-
diamide
had
than diamide
alone; 6
increase;
4 mM DNP
a 1 6% increase,
and
DNP
and
diamide.
clearance
Effect
percent
clearance
by the liver and
in the case of chlorproma-
by ATP-depleted
however,
ATP-depleted
2.
(mM)
concentration
of tetracaine
(2 mM)
caused
no change
in clearance.
Both
0.4
mM
chlorpromazine
and
washed
incubated
cells resulted
in a substantial
eleva-
an increase
a shift
Fig.
(O-O),
Concentration
little change
noted
The
association
Effects
Several
possibility
cytes
and
Membrane
experiments
Changes
were
that clearance
is due to a direct
dependent
change
in
done
to
of echinocytes
drug
effect
the
eliminate
the
or stomatoor to a drug-
erythrocyte
membrane.
between
CI (crenation
index)
and
liver
clearance
suggests
that the echinocyte
is being cleared
from the
blood by the liver specifically
as a consequence
of the
changed
shape
(Fig. 2).
Stomatocytes
were induced
promazine,
and by washing
by tetracaine,
cells that had
by chlorbeen incu-
0
m
‘C
.
bated
overnight
10 mM cytidine,
echinocytes,
larger
increasing
percentage
a greater
population
is shown
increase
the blood
diamide
in
the
drug
concentrations
cells
to be cupped
of the
severity
in
representative
in Fig.
Stomatocytes
from
with
with
5 mM
DNP,
10 mM
and 10 mM myo-inositol.
the
degree
of 0.4
of
mM
glucose,
As with
caused
a
and caused
.
a
0)
.C
C)
a.
a
a.
0
cupping.
A cell
chlorpromazine
1.
held in shape by diamide
were
cleared
stream
more rapidly
than discs treated
(Table
1). There
is a corresponding
percentage
organs
(Table 2). With
there
1 7%,
were increases
in
respectively,
over
of
6 mM
counts
found
and 4 mM
liver
clearance
diamide
levels.
in
tetracaine
of
Tetracoine
Concentration
(mM)
the
68% and
The
lower
Fig.
(O-O),
3
Effect
percent
clearance
in spleen
and diamide
(1 mM)
of tetracaine
of clearance
concentrations
in liver
(-*),
on
cupping
index
and percent
of
(A-A).
Cells were incubated
with tetracaine
for 45 mm at 37’C and then washed.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
EFFECT
OF CELL
SHAPE
IN HEMOLYSIS
425
Table
3.
Effect
of Sh ape Recovery
on Clearance
Percent
Clearance
Liver
Spleen
ATP depleted
+
diamide
()t
34
ATP depleted
+
10 mM
ads-
nosine
caine
C)
a,
0
+ 2 mM tetra-
+ diamide
±
3
4/97/G
7
±
2
±
2
98/0/2
3H-
+ diamide
(2)
+ diamide
(2)
represent
or
of
than
labeled
remained
Protein
2% of the 3H-chlorpromazine
Further
evidence
for noninvolvement
inducing
agent
itself in clearance
comes
that altered
shape needed
to be preserved
to occur.
Erythrocytes
were
treated
concentrations
30
maintain
of DNP
and
mm
at 37#{176}C,which
the crenated
shape.
for
discocytes
after
percentage
heart
were
of counts
the same
the
DNP
membrane
that
Reversibility
Not
results
ofShape
with
was
not
The cells
was
found
for all
diamide
and for diamide
DNP
itself does not cause
reversible,
1 mM
sufficient
to
then became
away.
The
in the spleen,
liver,
DNP
concentrations
and
plus
that
in the
and
is seen
is a corresponding
reduction
and
with
liver.
If
the
tetracaine
and
in the
in these
not
alterations
triggers
in membrane
the
clearing
mecha-
gel
analysis
electrophoretic
of
ghosts
crosslinking
Electrophoresis
cytes treated
of membrane
proteins
as in Palek et al.’6
of membrane
proteins
from erythrowith shape-change-inducing
agents
and
revealed
that
diamide
resulted
in the same
that
of
ethanol
diamide
of ghosts
alone.
from
resulted
ghosts
in
from
protein
Echinocytes
ing as control,
times
control
cupping
extent
All
pattern
Reduction
diamide-treated
separation
untreated
diamide
caused
S-S
of separation
with
as
/3-mercaptoerythrocytes
identical
to
that
of
erythrocytes.
and C3 Binding
IgG
is
in
/
erythrocyte
in
were incuof the cells
stomatocytes
Crosslinking
and
stomatocytes
with
the
approximately
DNP
percent
was also a decrease
3). Shape
reversal
change,
treated
showed
with
reversed
processes,
Change
3 with ATP depletion.
Erythrocytes
were incufor 12 hr at 37#{176}C
in PBS with added
penicillin
There
shape
in IgG and
(see Materials
shape-inducing
morphology
I
spleen
the
there
(Table
increase
binding
as was seen above
to discs.
treatment,
ofclearance
in clearance.
and streptomycin.
When these echinocytes
bated with 10 mM adenosine
for I hr, 59%
reverted
diamide
alone.
This suggests
a chemical
alteration
by
was
Polyacrylamide
of the shapefrom the fact
for clearance
with
several
washed
only are there
no effects
of the
itself,
but
also
the changed
agent
Table
bated
then
cells
shape
that
nism.
to the stomatocytes.
discocytes
of rats.
metabolic
for 45 mm at 37#{176}C.
The
PBS and counted.
Less
to be bound
percent
6
experiments
was not uniform,
and a range of shapes
from cupped
to crenated
cells was observed
in each
sample.
There
was,
however,
an increase
in the
percentage
of cupped
cells and a decrease
in clearance
with
increasing
tetracaine
concentration.
This
sug-
plus diamide
washed
with
echi-
3 mM
echinocytes.
chlorpromazine
cells were then
seemed
0/48/52
32
on cupping
and percent
of
with chlorproand then were
the
14
0.2
(mM)
with
±
±
gests
DNP
59
5
caine
diamide
amount
Approximately
10
3/74/23
echinocytic
1% of the
±
10
clearance
nocytes.
2 1
(2)
±
+
tNumber
or
59/34/7
50
‘Values
3H-DNP
5
.8
percent
with
±
±
U
incubated
16/0/84
16
9
Control
were
1
tetra-
C
Erythrocytes
±
caine + diamide
(2)
ATP depleted
+ 4 mM tetra-
a
Fig. 4.
Effect
of chlorpromazine
concentrations
index (0-O),
percent
clearance
in liver (I),
clearance
in spleen (A-A).
Cells were incubated
mazine
and diamide
(1 mM)
for 45 mm at 37’C
washed.
13
ATP depleted
a
Concentration
18
Morphology’
±
0
0
C)
Chiorpromazine
46
a
3
C
11
(3)
ATP depleted
‘C
±
SD
±
control,
levels
agents,
highest
the
4 mM
of the
treatments
tetracaine-treated
there
was
and the
revealed
tested.
assayed
for
an
component
3 (C3)
for assays).
Cells
concentrations
same amount
and 0.4 mM
of IgG binding
of clearance
transfer
analyses
were
complement
and Methods
chlorpromazine
(see Table
a correlation
of
of lgG
cells
4).
almost
4
10 times
With
the
between
amount
of IgG
no C3 binding
DNP
bind-
the
binding.
with any
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
COSGROVE
426
Table
4.
lgG
and
C3
B inding
to
Altered
Earlier
Erthrocytes
studies
of
erythrocytes
1 mM diamide
-
1.0
phenylhydrazine,
IgG, or heat
with
mild
damage,
and with
6mMDNP
1.0
1.0
hepatic
clearance.’7”8
4mMDNP
1.1
1.0
4mMtetracaine
3.0
1.0
tion or
increase
cupping
index,
there
in hepatic
clearance.
2mMtetracaine
1.2
1.0
0.4
mM
chlorpromazine
8.8
-
0.2
mM chlorpromazine
0.9
-
lgG
Binding’
Serums
‘Values
represent
tValues
densometric
ratio
each in three
of treated
versus
Binding
diamide
counts/cell
of
baseline
peaks
from
with
mechanism
chlorpromazine
of clearance
ance
be
the
result
ng of C3 applied.
analogous
studies
we have
concentrated
on clearance
early
times,
since
significant
time-dependent
changes
in cell shape occur in the diamide-treated
cells
ity
of
An
in
important
time
early time point also reduces
the possibilmodification
of damaged
cells, which
is
vivo
identifying
the state of the cell at the
clearance.
The counts
found
in the organs
a reliable
and well-defined
measure
of the
of labeled
erythrocytes
from
the blood
in
of
provided
clearance
necessary,
but
not
desirable,
to use
diamide
altered
cell shape
maintenance.
However,
the
observed
changes
in clearance
did not correlate
with
an effect
of diamide.
Treatment
with higher
concentrations
of diamide
produced
splenic clearance
and not
the hepatic
clearance
that
was observed
with
the
for
altered
shapes
(Thimmappaya
et al.,
results).
In addition,
protein
crosslinking
was not altered
when shape was altered.
unpublished
by diamide
Several
methods
of altering
shape
were
control
for amphipath
or system-dependent
employed
effects.
particular
with either
cells
or
interest
washing
the
are the
of the
addition
of
a
reversibility
drug
from
cupping
to
Of
experiments
DNP-treated
agent
to
ATP-
depleted
cells. ATP-depletion
and crenation
has been
extensively
studied9
and has been shown
to produce
hepatic
clearance.’8
Reversal
of the cell shape
change
with tetracaine
is sufficient
to decrease
the extent
of
clearance.
Tetracaine
would
be expected
to reverse
only the shape change
and not the other effects
of ATP
depletion.
clearance
decreases
it alters
respectively.
of
Further,
normal
erythrocyte
the shape
This
4 mM
tetracaine
cells. Thus, tetracaine
increases
increases
the
or
clearance
depending
on whether
from or towards
a biconcave
disc,
and
explained
by a direct
longevity.
other
observations
effect
of shape
different
from
greater
cells.
the rat’s
crena-
the
an
other
alternative
cells or may
binding
IgG
erythroclastic
human.
The
to
system
histologic
is
struc-
can
on
best
erythrocyte
humans.’7’8
Although
the spleen is thought
to be the
major
organ
for clearance
of aged
cells
in both
animals,
in neither
case is there
a change
in erythrocyte lifespan
ence between
after
splenectomy.23’24
The major
differhuman
and rat erythrocytes
is that rat
erythrocytes
are
undergo
much
Numerous
more
fragile
mechanisms
be
and
sphering
in vitro
and
can
lysis.2
spontaneous
been
have
clearance
of damaged
erythrocytes.
shape
changes
are believed
to
stream.
It was
in the
only a correlative
The
exception
was
shown
to displace
calmodulin
unexpected
pattern
of clear-
much
of the
with
clearance
damage,
of the spleen
and its relative
size is similar.2#{176}Both
IgG-opsonized
and
heat-denatured
erythrocytes
are
primarily
cleared
by the spleen
in rat52122 and in
at
in vitro.
increase
was
may reflect
for shape-altered
the
to that
splenic
severe
ture
DISCUSSION
In these
of
chlorpromazine-treated
In many respects
SHEETZ
damaged
noted
more
an
was
in
from
(total
which
that it has been
membranes.’9
The
agents
experiments).
represent
ratio of areas of treated versus
scans of the gel transfer autoradiographs.
With
chlorpromazine,
2.0
-
six samples
5O
C3
AND
decreased
proposed
for
the
In one case, the
lead
to erythrocyte
deformability,
which
results
primarily
presented
mability
changes
alternative
in splenic
trapping.
The
observations
here do not support
that theory,
since deforis already
decreased
by diamide,”
and shape
result
in hepatic,
not splenic, clearance.
An
model
to the mechanical
mechanism
of
clearance
the
IgG
is the alteration
of surface
receptors
such as
receptor.
Studies
by Kay25
indicate
that
surface
These
IgG binding
increases
data show a correlation
an increase
in IgG
ing
IgG
ance’
with
agents.
binding
binding
with erythrocyte
between
clearance
for cupping
per se leads
and would
explain
why
chlorpromazine
cupped
most strongly.
The correlations
ance
and the crenation
and
explained
by IgG binding.
there
is splenic
cells,
which
is logical
The
to
suggest
properties,
exact
surface
not
crenatclear-
clearance
binds
IgG
between
hepatic
clearcupping
indexes
are not
Because
the liver is presumed
cytes on the basis of their altered
surface
but
to splenic
aging.
and
to sequester
erythrosurface
properties,
it
that
the shape
change
alters
which
in turn
produce
clearance.
property
that
is correlated
with
clearance
is not known.
Maintenance
of the biconcave
disc
morphology
appears
from
these
studies
to be
important
not only
for 02 exchange,
but
also
for
erythrocyte
survival.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
EFFECT
OF CELL
SHAPE
IN HEMOLYSIS
427
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1982 59: 421-427
Effect of cell shape on extravascular hemolysis
P Cosgrove and MP Sheetz
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