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Presence of phosphatidylserine in the outer membrane bilayer of newborn human erythrocytes
Vol.
136,
May
14,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1986
Pages
PRESENCE
OF PHOSPHATIDYLSERINE IN THE OUTER BILAYER OF NEWBORN HUMAN ERYTHROCYTES Sushi.1
Department of State University Shreveport,
Louisiana
Received
February
K.
25,
914-920
MEMBRANE
Jain Pediatrics School of LA 71130
Medicine
1986
The phospholipid distribution across red cell membrane bilayer is asymmetrical. Sphingomyelin and phosphatidylcholine are predominantly present in the outer membrane bilayer, whereas only small amounts of phosphatidylethanolamine and no phosphatidylserine are present in the outer membrane bilayer. The present study, using specific phospholipase, chemical probe, and Russell's viper venom clotting time has found that in neonatal red cells a portion of PS is 0 1986 AcademicPress, Inc. also present in the outer membrane bilayer.
It
is
present
now
only
in
al.
(2)
Zwaal
et
inner
membrane
mechanism In
initiate
the
inner
have
in
outer
found
that
outer
surface, present
and
localization
in
of
Copyright All righfs
red
may
to inner
adult
human
side
of
in
the
(3)
h ave
suggest
membrane outer
that
914
that
red to
the
PS can
cells
with
microvascular
The
present
study
PS is
present
at
cells
where
bilayer. and
in
hemostasis. shown
adherence
red
(I).
PS only
inner
all
bilayer
has
the the
The
PS, phosphatidylserine; PE, viper venom; TNBS, trinitro: chromatography.
$1.50 Inc. reserved.
regulating
of
is
physiological
in
a portion
cells
of
important
to
(PSI
red
presence
occlusions.
cells
phospholipids
0 1986 by Academic Press, of reproduction in any ftirm
human
and
susceptible
Abbreviations: PC, phosphatidylcholine; phosphatidylethanolamine; RW, Russell's benzenesulfonilic acid; TLC, thin-layer 0006-291X/86
an
microvascular
the
adult the
which
are
contrast in
phosphatidylserine
Papahadjopoulos
contact,
neonatal
only
that
thrombosis
surface
in
of
represent
avoiding
causing
the
bilayer
may
cell-to-cell the
that
suggested
Wilschut
endothelium,
is
documented
bilayer
both
addition,
PS at
well
of
PS
Vol.
136,
No.
neonatal cells
to
BIOCHEMICAL
1986
and
phosphat in
3,
adult
human
specific
red
outer
membrane
present
outer coagulant
>,
bilayer, acid
in
surface
the of
activity
outer red
was
of
cells
using
MATERIALS
detected
by
degrades
PS,
phosphatidylcholine
and
by
using
(TNBS),
was
RESEARCH
and
membrane cells
BIOPHYSICAL
which (PE
trinitrobenzenesulfanilic PE
cells
phospholipase,
idylethanolamine
the
AND
also
chemical
indirectly
AND
these
with
the
PS
externalization assessed
viper
present
probe
reacts PS
Russell’s
exposing
(PC)
which
bilayer.
COMMUNICATIONS
venom
at by
and the
the
(RVV).
METHODS
Blood from the fresh placental cord of infants born at gestational age 38-41 weeks and from adult volunteers was collectedinto tubes containing ethylenediamine tetraacetate (EDTA, 10.5 mg/7 ml). Blood was then centrifuged at 2000 rpm for seven minutes in a refrigerated centrifuge. Plasma and buffy coat were discarded. The red cells were washed with cold 0.15 M sodium chloride solution three times after one to ten dilution. Complete blood count by electronic counter done on some washed red cell samples showed contamination of less than 0.15 white blood cells per 100 red blood cells. Treatment with phospholipase: In order to determine phospholipid organization, red cells were suspended in glycylglycine buffer at pH 7.4 containing (in mM) KC1 100, NaCl 50, MgC12 0.25, sucrose 44, glycylglycine 10 and CaC12 0.25. The red cell suspensions were exposed to nonpermeable Bee Venom phospholipase-A2 (Specific activity 2220 U/mg protein, Sigma Chemical Company, St. Louis, Missouri) for two hours in a shaking water bath at 37°C. The incubation medium contained 0.25 ml of packed cells + 5 ml glycylglycine buffer +‘50 Ill of phospholipase. Various incubations contained respective phospholipase-untreated red cells as controls. At the end of incubation time, phospholipase action was terminated by adding 5 mM EDTA to the incubation medium. A portion of the first supernatent was used to determine the extent of hemolysis during phospholipase treatment. Red cells were subsequently washed with cold 0.15 M sodium chloride two times to remove any lysed cell and enzyme. TNBS labeling: TNBS labeling of adult and neonatal red cells was done as described bv Marinetti and Crain (4). The react ion mixture contained 0.2 ml packed cells + 4 ml of 40 mM sodium bicarbonate - 120 mM sodium chloride buffer (pH 8.5) + 2 mM TNBS. The incubation was done at 25°C in a shaking water bath for one hour. At the end of incubation, cells were washed with 0.15 M sodium chloride three times to remove free TNBS. Lipid analyses: Lipid extraction of washed phospholipase-treated and TNBS labeled red cells was carried out using isopropanol and chloroform by the method of Rose and Oklander (5). Lipid extracts were dried with nitrogen and then redissolved in Phospholipid classes in the lipid extracts of 50 pl of chloroform. phospholipase treated cells were separated by two-dimensional thin-layer chromatography (TLC) on silica gel H plates (Brinkmann NY) using a solvent system consisting of Instruments, chloroform/methanol/acetic acid/water (50/25/8/4) (v/v) in the first directiou and chloroform/methanol/water (5/10/l) (v/v> in the second direction. Phospholipid classes in the lipid extracts of
915
Vol.
136,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TNBS-labeled cells were separated by the method of Schick et al. (6) on silica gel G plates (Brinkmann Instruments, NY) using solvent system chloroform/methanol/concentrated ammonia (70/30/5) (v/v). After developing in this solvent to two-third the height of the plate, the TLC plate was dried and then developed again in the same direction in chloroform only. This TLC separated TNBS-labeled PE from other phospholipid classes whereas TNBS labeled PS migrated along with PC. The amount of unlabeled PS or PE is divided by sphingomyelin (SM), and the percentage decrease in the ratio of PS or PE in TNBS-reacted red cells in comparison to control red cells determines the percentage of PS and PE in the outer half of membrane bilayer. Visualization of various lipid spots on TLC plate and phospholipid-phosphorus determination was carried out as described earlier (7). The extent of phospholipid degradation by phospholipase was assessed by calculating the decrease in the ratio of unhydrolyzed phospholipid to SM. Phospholipase used does not act on SM. Two-dimensional TLC used separates lysolipids from other lipids. Phospholipase or TNBS treatment of neonatal or adult red cells for up to three hours did not cause more than 2% hemolvsis. Coagulant activity: Coagulant activity of- red cells was determined by their ability to shorten the clotting time on recalcification of normal human platelet-poor plasma in the presence of RVV (Signa Chemical Company). RVV clotting time was determined as described by Zwaal et al. (2). For this assay, fresh titrated human plasma was obtained from healthy adult volunteers. Plasma was centrifuged at 5000 rpm in a Sorvall RC-5B superspeed centrifuge at 4°C for 15 minutes. One volume of clear supernatant plasma was diluted with two volumes of 0.15 M NaCl before use. 0.1 ml of diluted plasma was incubated in a fibrometer at 37’C for 30 seconds followed by an addition of 0.1 ml of washed with 10 pl of 12 ng RVV, unhemolyzed packed red cells (85% hematocrit and similar amount of phospholipid) and 0.1 ml of 25 mM calcium chloride. Clotting time of is0 tonic saline, instead of red cells, was about 100 seconds and was used as a control. Clotting time of adult and newborn red cells is expressed as a percent of the control run on the same day. In this assay system, clotting factor X is activated by RVV. Because the conversion of prothrombin to thrombin by prothrombinase is the last step in the coagulation cascade requiring phospholipid, this assay system is used to assess the exposure of procoagulant lipid PS in the red cell membrane. carried out using non-paired Student’s Statistical analyses were ‘t’ test. RESULTS Treatment hydrolysis that
of of
all
PS
hydrolyzed
ps,
present
by
with
and in
in
9% the
PE, and,
thus,
with
PS
side
inner
neonatal
but
phospholipase
PE,
red
less
PC,
are
membrane.
916
present
caused unaffected,
of
cells
This
phospholipase.
more
cell
cells
PC
contrast,
action red
red 63%
is
significantly
phospholipase neonatal
PS In
I).
was
nearly
the
(Table
adult
membrane 57%
suggests are
the
bilayer
PC,
16%
PE,
that
a
portion
available in
suggesting
and
8% of
for outer
bilayer
of
Vol.
136,
BIOCHEMICAL
No. 3, 1986
AND
BIOPHYSICAL
Table
COMMUNICATIONS
I
Phospholipid hydrolysis in and adult red cells treated Bee Venom phospholipase-A2
neonatal with
PS Red
RESEARCH
PE
PC
Cells % hydrolyzed
Adult Neonatal
0
9*1
63*1
a*2
16*2
57*2
P value
Table
II
cells,
took
a
fibrin
firm
Values neonatal of PS
are Mean l samples. by phospholipase
shows
that
nearly
22X
clot
SE of Note
washed less
when
eight adults significant in neonatal
neonatal
time
<0.05
red
than
recalcified
cells,
washed with
and ten hydrolysis red cells.
free
adult
of
red
titrated
cells
any
ghost
to
form
platelet-poor
plasma. Table PS
and
III
PE
cells.
This
of
PE
in
comparison
conditions,
are
by
shows
that
TNBS
in
also
suggests
present
in
there
neonatal
red that
the
outer
to
adult
red
TNBS
could
permeate
RVV
Red
was cells a
membrane
red
cell
Table
II
labeling
comparison
to
PS
increased
of bilayer
Under
adult
greater
in
portion
cells.
clotting and
significantly
and of
certain
neonatal
red amounts
experimental
membrane
and
label
Clotting time (X, of control*)
Adult
37*2
Neonatal
29*2
P value
Values are MEAN * SE of 11 adults and 11 neonatal blood samples. *Saline was used instead of cells a control, and its clotting time on a particular was taken as control for all calculations of red cells on that day.
917
adult
red
time of neonatal red cells
cells
of
as day
inner
cells
Vol.
136,
BIOCHEMICAL
No. 3, 1986
AND
Table TNBS
Red
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
III
labeling neonatal
of red
adult cells
cells
and
PS
PE % labeled
Adult
1*1
12*1
Neonatal
9*1
18+2
P value
(0.01
Values neonatal labeling
membrane
bilayer
study, cells
of
suggests
experimental
of
1
SE of eight adults Note significantly and PE in neonatal red
(8).
TNBS
that
labeling
TNBS
PS
illustrates
with
may
not
linear
externalization
simultaneously
on
negative
correlation
in
clotting
vitro
*
be
and ten greater cells.
However,
in
the
phospholipase
present
treatment
permeable
under
the
of present
conditions.
Figure extent
Mean samples. of PS
aminophospholipids
consistency
red
are
and
neonatal
red
(r time
regression
= of
the
in
cells.
-0.66)
analysis vitro There
between
neonatal
the
red
cells.
between
clotting was PS
a
time
done
significant
externalized Data
the
in
and Figure
1
the is
45 . r* -I&
f !I I P . *-a5 #
.
.
l
t
F
P
.
l
F % u’
.
25 “\
0
Figure
5
IO
15
1:Linear regression analysis between the extent of PS externalization and in vitro clotting time of neonatal red cells. externalieation was ztxed by exposing red cells to Bee venom phospholipase-A2 and in vitro clotting time using RVV as described Materials and Methods. Note significant correlation coefficient -0.66) between the PS externalization and the clotting time.
918
PS
(r
in =
Vol.
136,
from
BIOCHEMICAL
No. 3, 1986
red
suggests
cells
of
that
analytic
contribute
to
other
the
than
AND
the
BIOPHYSICAL
above
observation
newborn
of
physiological
RESEARCH
PS
COMMUNICATIONS
infants.
This
externalization
coagulation
also
may
function
in
newborn
infants.
DISCUSSION PS
is
a
important
role
cascade the
of
PS
study
(11)
does compared
the
have with
any
observed
PS
externalization
intravasc:ular
outer
cell
any
affect
red
(1.2,13).
neonatal
of
cells
bringing newborn The
the
red
is
membrane
spectrin
have of
cells
the
shown
to
the are
(14,16,17).
has
been
cells that
the
contribute
in
the
Disseminated during
the
the
outer may
altered
newborn
surface have
of
some
hemostasis
role
during
the shown
of of
side
known
of to
actual
same that
as
externalization
Haest
important
(11,151
The is
an
oxidation
outer
the
known.
bilayer
lipids
treatments
cells
inner
for
not
plays
that
membrane
inner
at
red
the
period.
neonatal
in
to
study
and
in
suggests
cells.
present
hypercoagulability
responsible
PE
PS
resulted
of
frequently of
A
reticulocytes
unlikely
quite
the
which
This
red
shown
cells.
organization
rats.
neonatal
in
mechanism
and
PS
is
presence
observed
about
the
has
red
as
very
coagulation
study
rats,
such
a
blood
neonatal of
unbled
occurs
Thus)
red
of
the
play
present
bleeding
precursors
coagulation
period
of
precursors
cells
cells
the
surface
on
can
in
cells,
that
red
and
cofactor red
shown
red
of
the
adult
of not
phospholipid
enhancing
has
presence
in
rate
at
enrichment
blood, when
a Unlike
presence
the
charged
as
(9,101.
previous in
negatively
et
can
cause
red
cell
movement membrane
spectrin
domains
found of
in
adult
receptor
919
of
human mobility
PE
from
All in
spectrin
peroxidation
and
bilayer.
in red
the
and
of red
neonatal cells
PS
studies
and
PS
that of
Previous (14),
in
shown
stablization
alterations of
PS
have
the
groups
concentration that
in
(14)
erythrocytes.
sulfhydryl
cause
al.
role adult
of
the these cells red
(181, endocytosis
but
it in
Vol.
the which
136,
BIOCHEMICAL
No. 3, 1986
membranes may
have
of
neonatal
a role
red in
the
AND
cells
BIOPHYSICAL
are
RESEARCH
deficient
externalization
in of
PS in
COMMUNICATIONS
spectrin
(191,
their
membranes. ACKNOWLEDGEMENTS This 30247 and author is assistance, for editing
study was supported in part by the NIH grant lRO1 HL a grant-in-aid from the Hoffmann-La Roche, Inc. The grateful to Ethelyn B. LaHaye for her technical to Kathy Jones for typing, and to Barbara MacRoberts this manuscript. REFERENCES
1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15.
16. 17.
18. 19.
van Deenen, L.L.M. (1981). FEBS Letters 123:3-15. Zwaal, R.F.A., Comfurius, P., and van Deenen, L.L.M. (1977). Nature (London) 268:358-360. and Papahadjopoulos, D. (1979). Nature (London) Wilschut, J., 281:690-693. Marinetti, G.V., and Crain, R.C. (1978). J Supramolecular Struct 8:191-213. J Lipid Res 6:528-531. Rose, H.G., and Oklander, M. (1965). Schick, P.K., Kurica, K.B., and Chacro, G.K. (1976). J Clin Invest 57:1221-1226. Jain, S.K., Subrahmanyam, D. (1978). Italian J Biochem 27:11-19. Ann Rev Biochem 48~47-71. Opdenkamp, J.A.F. (1979). Adv Lipid Res 4:1-37. Marcus, A.J. (1966). (1984). Brit Mannhalter, C., Schiffman, S., and Deutsch, E. Haematol 56:261-271. Jain, S.K. (1985). J Clin Invest 76:281-286. McDonald, M.M., Hathaway, W.E. (1983). Seminars in Perinatology 7~213-225. Corrigan, J.J. (1979). Amer J Pediatric Hematology/Oncology 1:245-249. Haest, C.W.M., Plasa, G., Kamp, D., and Deuticke, B. (1978). Biochim Biophys Acta 509:21-32. Jain, S.K. (1984). J Biol Chem 259:3391-3394. Jain, S.K., and Hochstein, P. (1980). Biochem Biophys Res Commun 92~247-254. Jain, S.K., and Hochstein, P. (1980). Arch Biochem Biophys 201:683-687. (1978). Shapiro, D.L., Pasqualini, P. Pediatric Res 12:176-178. J Cell Tokuyasu, K.T., Schekman, R., and Singer, S.J. (1979). Biology 80:481-486.
920
J
136,
May
14,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1986
Pages
PRESENCE
OF PHOSPHATIDYLSERINE IN THE OUTER BILAYER OF NEWBORN HUMAN ERYTHROCYTES Sushi.1
Department of State University Shreveport,
Louisiana
Received
February
K.
25,
914-920
MEMBRANE
Jain Pediatrics School of LA 71130
Medicine
1986
The phospholipid distribution across red cell membrane bilayer is asymmetrical. Sphingomyelin and phosphatidylcholine are predominantly present in the outer membrane bilayer, whereas only small amounts of phosphatidylethanolamine and no phosphatidylserine are present in the outer membrane bilayer. The present study, using specific phospholipase, chemical probe, and Russell's viper venom clotting time has found that in neonatal red cells a portion of PS is 0 1986 AcademicPress, Inc. also present in the outer membrane bilayer.
It
is
present
now
only
in
al.
(2)
Zwaal
et
inner
membrane
mechanism In
initiate
the
inner
have
in
outer
found
that
outer
surface, present
and
localization
in
of
Copyright All righfs
red
may
to inner
adult
human
side
of
in
the
(3)
h ave
suggest
membrane outer
that
914
that
red to
the
PS can
cells
with
microvascular
The
present
study
PS is
present
at
cells
where
bilayer. and
in
hemostasis. shown
adherence
red
(I).
PS only
inner
all
bilayer
has
the the
The
PS, phosphatidylserine; PE, viper venom; TNBS, trinitro: chromatography.
$1.50 Inc. reserved.
regulating
of
is
physiological
in
a portion
cells
of
important
to
(PSI
red
presence
occlusions.
cells
phospholipids
0 1986 by Academic Press, of reproduction in any ftirm
human
and
susceptible
Abbreviations: PC, phosphatidylcholine; phosphatidylethanolamine; RW, Russell's benzenesulfonilic acid; TLC, thin-layer 0006-291X/86
an
microvascular
the
adult the
which
are
contrast in
phosphatidylserine
Papahadjopoulos
contact,
neonatal
only
that
thrombosis
surface
in
of
represent
avoiding
causing
the
bilayer
may
cell-to-cell the
that
suggested
Wilschut
endothelium,
is
documented
bilayer
both
addition,
PS at
well
of
PS
Vol.
136,
No.
neonatal cells
to
BIOCHEMICAL
1986
and
phosphat in
3,
adult
human
specific
red
outer
membrane
present
outer coagulant
>,
bilayer, acid
in
surface
the of
activity
outer red
was
of
cells
using
MATERIALS
detected
by
degrades
PS,
phosphatidylcholine
and
by
using
(TNBS),
was
RESEARCH
and
membrane cells
BIOPHYSICAL
which (PE
trinitrobenzenesulfanilic PE
cells
phospholipase,
idylethanolamine
the
AND
also
chemical
indirectly
AND
these
with
the
PS
externalization assessed
viper
present
probe
reacts PS
Russell’s
exposing
(PC)
which
bilayer.
COMMUNICATIONS
venom
at by
and the
the
(RVV).
METHODS
Blood from the fresh placental cord of infants born at gestational age 38-41 weeks and from adult volunteers was collectedinto tubes containing ethylenediamine tetraacetate (EDTA, 10.5 mg/7 ml). Blood was then centrifuged at 2000 rpm for seven minutes in a refrigerated centrifuge. Plasma and buffy coat were discarded. The red cells were washed with cold 0.15 M sodium chloride solution three times after one to ten dilution. Complete blood count by electronic counter done on some washed red cell samples showed contamination of less than 0.15 white blood cells per 100 red blood cells. Treatment with phospholipase: In order to determine phospholipid organization, red cells were suspended in glycylglycine buffer at pH 7.4 containing (in mM) KC1 100, NaCl 50, MgC12 0.25, sucrose 44, glycylglycine 10 and CaC12 0.25. The red cell suspensions were exposed to nonpermeable Bee Venom phospholipase-A2 (Specific activity 2220 U/mg protein, Sigma Chemical Company, St. Louis, Missouri) for two hours in a shaking water bath at 37°C. The incubation medium contained 0.25 ml of packed cells + 5 ml glycylglycine buffer +‘50 Ill of phospholipase. Various incubations contained respective phospholipase-untreated red cells as controls. At the end of incubation time, phospholipase action was terminated by adding 5 mM EDTA to the incubation medium. A portion of the first supernatent was used to determine the extent of hemolysis during phospholipase treatment. Red cells were subsequently washed with cold 0.15 M sodium chloride two times to remove any lysed cell and enzyme. TNBS labeling: TNBS labeling of adult and neonatal red cells was done as described bv Marinetti and Crain (4). The react ion mixture contained 0.2 ml packed cells + 4 ml of 40 mM sodium bicarbonate - 120 mM sodium chloride buffer (pH 8.5) + 2 mM TNBS. The incubation was done at 25°C in a shaking water bath for one hour. At the end of incubation, cells were washed with 0.15 M sodium chloride three times to remove free TNBS. Lipid analyses: Lipid extraction of washed phospholipase-treated and TNBS labeled red cells was carried out using isopropanol and chloroform by the method of Rose and Oklander (5). Lipid extracts were dried with nitrogen and then redissolved in Phospholipid classes in the lipid extracts of 50 pl of chloroform. phospholipase treated cells were separated by two-dimensional thin-layer chromatography (TLC) on silica gel H plates (Brinkmann NY) using a solvent system consisting of Instruments, chloroform/methanol/acetic acid/water (50/25/8/4) (v/v) in the first directiou and chloroform/methanol/water (5/10/l) (v/v> in the second direction. Phospholipid classes in the lipid extracts of
915
Vol.
136,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TNBS-labeled cells were separated by the method of Schick et al. (6) on silica gel G plates (Brinkmann Instruments, NY) using solvent system chloroform/methanol/concentrated ammonia (70/30/5) (v/v). After developing in this solvent to two-third the height of the plate, the TLC plate was dried and then developed again in the same direction in chloroform only. This TLC separated TNBS-labeled PE from other phospholipid classes whereas TNBS labeled PS migrated along with PC. The amount of unlabeled PS or PE is divided by sphingomyelin (SM), and the percentage decrease in the ratio of PS or PE in TNBS-reacted red cells in comparison to control red cells determines the percentage of PS and PE in the outer half of membrane bilayer. Visualization of various lipid spots on TLC plate and phospholipid-phosphorus determination was carried out as described earlier (7). The extent of phospholipid degradation by phospholipase was assessed by calculating the decrease in the ratio of unhydrolyzed phospholipid to SM. Phospholipase used does not act on SM. Two-dimensional TLC used separates lysolipids from other lipids. Phospholipase or TNBS treatment of neonatal or adult red cells for up to three hours did not cause more than 2% hemolvsis. Coagulant activity: Coagulant activity of- red cells was determined by their ability to shorten the clotting time on recalcification of normal human platelet-poor plasma in the presence of RVV (Signa Chemical Company). RVV clotting time was determined as described by Zwaal et al. (2). For this assay, fresh titrated human plasma was obtained from healthy adult volunteers. Plasma was centrifuged at 5000 rpm in a Sorvall RC-5B superspeed centrifuge at 4°C for 15 minutes. One volume of clear supernatant plasma was diluted with two volumes of 0.15 M NaCl before use. 0.1 ml of diluted plasma was incubated in a fibrometer at 37’C for 30 seconds followed by an addition of 0.1 ml of washed with 10 pl of 12 ng RVV, unhemolyzed packed red cells (85% hematocrit and similar amount of phospholipid) and 0.1 ml of 25 mM calcium chloride. Clotting time of is0 tonic saline, instead of red cells, was about 100 seconds and was used as a control. Clotting time of adult and newborn red cells is expressed as a percent of the control run on the same day. In this assay system, clotting factor X is activated by RVV. Because the conversion of prothrombin to thrombin by prothrombinase is the last step in the coagulation cascade requiring phospholipid, this assay system is used to assess the exposure of procoagulant lipid PS in the red cell membrane. carried out using non-paired Student’s Statistical analyses were ‘t’ test. RESULTS Treatment hydrolysis that
of of
all
PS
hydrolyzed
ps,
present
by
with
and in
in
9% the
PE, and,
thus,
with
PS
side
inner
neonatal
but
phospholipase
PE,
red
less
PC,
are
membrane.
916
present
caused unaffected,
of
cells
This
phospholipase.
more
cell
cells
PC
contrast,
action red
red 63%
is
significantly
phospholipase neonatal
PS In
I).
was
nearly
the
(Table
adult
membrane 57%
suggests are
the
bilayer
PC,
16%
PE,
that
a
portion
available in
suggesting
and
8% of
for outer
bilayer
of
Vol.
136,
BIOCHEMICAL
No. 3, 1986
AND
BIOPHYSICAL
Table
COMMUNICATIONS
I
Phospholipid hydrolysis in and adult red cells treated Bee Venom phospholipase-A2
neonatal with
PS Red
RESEARCH
PE
PC
Cells % hydrolyzed
Adult Neonatal
0
9*1
63*1
a*2
16*2
57*2
P value
Table
II
cells,
took
a
fibrin
firm
Values neonatal of PS
are Mean l samples. by phospholipase
shows
that
nearly
22X
clot
SE of Note
washed less
when
eight adults significant in neonatal
neonatal
time
<0.05
red
than
recalcified
cells,
washed with
and ten hydrolysis red cells.
free
adult
of
red
titrated
cells
any
ghost
to
form
platelet-poor
plasma. Table PS
and
III
PE
cells.
This
of
PE
in
comparison
conditions,
are
by
shows
that
TNBS
in
also
suggests
present
in
there
neonatal
red that
the
outer
to
adult
red
TNBS
could
permeate
RVV
Red
was cells a
membrane
red
cell
Table
II
labeling
comparison
to
PS
increased
of bilayer
Under
adult
greater
in
portion
cells.
clotting and
significantly
and of
certain
neonatal
red amounts
experimental
membrane
and
label
Clotting time (X, of control*)
Adult
37*2
Neonatal
29*2
P value
Values are MEAN * SE of 11 adults and 11 neonatal blood samples. *Saline was used instead of cells a control, and its clotting time on a particular was taken as control for all calculations of red cells on that day.
917
adult
red
time of neonatal red cells
cells
of
as day
inner
cells
Vol.
136,
BIOCHEMICAL
No. 3, 1986
AND
Table TNBS
Red
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
III
labeling neonatal
of red
adult cells
cells
and
PS
PE % labeled
Adult
1*1
12*1
Neonatal
9*1
18+2
P value
(0.01
Values neonatal labeling
membrane
bilayer
study, cells
of
suggests
experimental
of
1
SE of eight adults Note significantly and PE in neonatal red
(8).
TNBS
that
labeling
TNBS
PS
illustrates
with
may
not
linear
externalization
simultaneously
on
negative
correlation
in
clotting
vitro
*
be
and ten greater cells.
However,
in
the
phospholipase
present
treatment
permeable
under
the
of present
conditions.
Figure extent
Mean samples. of PS
aminophospholipids
consistency
red
are
and
neonatal
red
(r time
regression
= of
the
in
cells.
-0.66)
analysis vitro There
between
neonatal
the
red
cells.
between
clotting was PS
a
time
done
significant
externalized Data
the
in
and Figure
1
the is
45 . r* -I&
f !I I P . *-a5 #
.
.
l
t
F
P
.
l
F % u’
.
25 “\
0
Figure
5
IO
15
1:Linear regression analysis between the extent of PS externalization and in vitro clotting time of neonatal red cells. externalieation was ztxed by exposing red cells to Bee venom phospholipase-A2 and in vitro clotting time using RVV as described Materials and Methods. Note significant correlation coefficient -0.66) between the PS externalization and the clotting time.
918
PS
(r
in =
Vol.
136,
from
BIOCHEMICAL
No. 3, 1986
red
suggests
cells
of
that
analytic
contribute
to
other
the
than
AND
the
BIOPHYSICAL
above
observation
newborn
of
physiological
RESEARCH
PS
COMMUNICATIONS
infants.
This
externalization
coagulation
also
may
function
in
newborn
infants.
DISCUSSION PS
is
a
important
role
cascade the
of
PS
study
(11)
does compared
the
have with
any
observed
PS
externalization
intravasc:ular
outer
cell
any
affect
red
(1.2,13).
neonatal
of
cells
bringing newborn The
the
red
is
membrane
spectrin
have of
cells
the
shown
to
the are
(14,16,17).
has
been
cells that
the
contribute
in
the
Disseminated during
the
the
outer may
altered
newborn
surface have
of
some
hemostasis
role
during
the shown
of of
side
known
of to
actual
same that
as
externalization
Haest
important
(11,151
The is
an
oxidation
outer
the
known.
bilayer
lipids
treatments
cells
inner
for
not
plays
that
membrane
inner
at
red
the
period.
neonatal
in
to
study
and
in
suggests
cells.
present
hypercoagulability
responsible
PE
PS
resulted
of
frequently of
A
reticulocytes
unlikely
quite
the
which
This
red
shown
cells.
organization
rats.
neonatal
in
mechanism
and
PS
is
presence
observed
about
the
has
red
as
very
coagulation
study
rats,
such
a
blood
neonatal of
unbled
occurs
Thus)
red
of
the
play
present
bleeding
precursors
coagulation
period
of
precursors
cells
cells
the
surface
on
can
in
cells,
that
red
and
cofactor red
shown
red
of
the
adult
of not
phospholipid
enhancing
has
presence
in
rate
at
enrichment
blood, when
a Unlike
presence
the
charged
as
(9,101.
previous in
negatively
et
can
cause
red
cell
movement membrane
spectrin
domains
found of
in
adult
receptor
919
of
human mobility
PE
from
All in
spectrin
peroxidation
and
bilayer.
in red
the
and
of red
neonatal cells
PS
studies
and
PS
that of
Previous (14),
in
shown
stablization
alterations of
PS
have
the
groups
concentration that
in
(14)
erythrocytes.
sulfhydryl
cause
al.
role adult
of
the these cells red
(181, endocytosis
but
it in
Vol.
the which
136,
BIOCHEMICAL
No. 3, 1986
membranes may
have
of
neonatal
a role
red in
the
AND
cells
BIOPHYSICAL
are
RESEARCH
deficient
externalization
in of
PS in
COMMUNICATIONS
spectrin
(191,
their
membranes. ACKNOWLEDGEMENTS This 30247 and author is assistance, for editing
study was supported in part by the NIH grant lRO1 HL a grant-in-aid from the Hoffmann-La Roche, Inc. The grateful to Ethelyn B. LaHaye for her technical to Kathy Jones for typing, and to Barbara MacRoberts this manuscript. REFERENCES
1. 2. 3. 4. 5. 6. 7.
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J