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Expression of Proteins Controlling Transbilayer Movement of Plasma Membrane
Phospholipids in the B Lymphocytes From a Patient With Scott Syndrome
By Quansheng Zhou, Peter J. Sims, and Therese Wiedmer
Scott syndrome is a rare inherited bleeding disorder in which
platelets and other blood cells fail to promote normal
assembly of the membrane-stabilized proteases of the plasma
coagulation system. The defect in Scott blood cells is known
to reflect inability to mobilize phosphatidylserine from inner
plasma membrane leaflet to the cell surface in response to
an elevation of Ca21 at the endofacial surface. To gain insight
into the molecular basis of this membrane defect, we examined the expression in Scott cells of plasma membrane
proteins that have been implicated to participate in the
accelerated transbilayer movement of plasma membrane PL.
By both reverse transcriptase-polymerase chain reaction
(RT-PCR) and functional assay, the level of expression of the
multidrug resistance (MDR)1 and MDR3 P-glycoproteins in
immortalized B-lymphoblast cell lines from the patient with
Scott syndrome were indistinguishable from matched cell
lines derived from normal controls. Whereas the plasma
membrane of Scott cells are insensitive to activation of the
plasma membrane PL scramblase pathway, it had been
shown that PL scramblase protein isolated from detergentsolubilized Scott erythrocytes exhibits normal function when
incorporated into proteoliposomes (Stout JG, Basse F, Luhm
RA, Weiss HJ, Wiedmer T, Sims PJ: J Clin Invest 99:2232,
1997). Consistent with this finding in Scott erythrocytes, we
found that Scott lymphoblasts expressed normal levels of PL
scramblase mRNA and protein, and that the deduced sequence of PL scramblase in Scott cells is identical to that of
normal controls. These data suggest that the defect in Scott
syndrome is related either to aberrant posttranslational
processing of the PL scramblase polypeptide or to a defect or
deficiency in an unknown cofactor that is required for normal
expression of plasma membrane PL scramblase function in
situ, or alternatively, reflects the presence of a detergentdissociable inhibitor of this pathway.
r 1998 by The American Society of Hematology.
P
required for normal plasma clotting.18-23 The plasma membranes of Scott cells contain normal amounts of PS and other
PL, and exhibit normal aminophospholipid translocase activity.19,24 Nevertheless, the plasma membrane of these cells are
unresponsive to elevations in [Ca21] at the endofacial surface,
which in normal cells evokes the accelerated movement of all
PL between inner and outer plasma membrane leaflets. Recent
evidence indicates that Scott syndrome is an autosomal recessive trait affecting all hematologic lineages that results in
abrogation of normal Ca21-stimulated transbilayer movement
of plasma membrane PL.23,25,26 However, the defective gene
giving rise to this disorder remains to be identified. It had been
observed that upon activation by thrombin plus collagen, Scott
syndrome platelets showed reduced protein tyrosine phosphorylation, implying a potential enzyme defect affecting one or more
intracellular protein tyrosine kinases.27 Nevertheless, a subsequent study demonstrated that the reduced protein phosphorylation observed in Scott syndrome cells is likely an epiphenomenon relating to the aberrant stability of the plasma membrane
PL distribution in these cells and is not causally related to the
LASMA MEMBRANE phospholipids (PL) are normally
asymmetrically distributed, with phosphatidylcholine (PC)
and sphingomyelin located primarily in the outer leaflet, and the
aminophospholipids, phosphatidylserine (PS), and phosphatidylethanolamine (PE) restricted to the cytoplasmic leaflet.1,2
This membrane lipid asymmetry is maintained at least in part
through the activity of a Mg21- and adenosine triphosphate
(ATP)-dependent aminophospholipid translocase that selectively transports PS and PE from outer to inner membrane
leaflet.3-5 Upon an increase in intracellular Ca21 due to either
cell activation, cell injury, or apoptosis, rapid bidirectional
movement of the plasma membrane PL between leaflets is
observed, resulting in exposure of PS and PE at the cell
surface.1,6-8 This exposure of plasma membrane amino PL has
been shown to promote assembly and activation of several key
enzymes of the coagulation and complement systems, and to
accelerate the clearance of injured or apoptotic cells by the
reticuloendothelial system, suggesting that Ca21-induced remodeling of plasma membrane PL is central to both vascular
hemostasis and cellular clearance.1,9-12 The molecular mechanism(s) underlying this intracellular Ca21-initiated remodeling
of the transbilayer distribution of plasma membrane PL remains
poorly understood. We recently identified and cloned an integral
plasma membrane protein, PL scramblase, that mediates Ca21dependent, bidirectional movement of PL between membrane
leaflets, mimicking the action of Ca21 at the endofacial surface
of the plasma membrane.13-17 PL scramblase was shown to be
expressed in erythrocytes, platelets, endothelium and a variety
of other cells and tissues that are known to expose plasma
membrane PS in response to elevated cytosolic Ca21. Furthermore, we demonstrated that the level of expression of PL
scramblase in the plasma membrane determines the extent to
which PS becomes exposed at the cell surface upon an increase
in cytosolic Ca21.17
Scott syndrome is a rare bleeding disorder in which platelets
and other blood cells show a diminished capacity to mobilize PS
to the cell surface, resulting in impaired assembly and activation
of the surface-catalyzed coagulation enzyme complexes that are
Blood, Vol 92, No 5 (September 1), 1998: pp 1707-1712
From the Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee, WI.
Submitted March 4, 1998; accepted April 29, 1998.
Supported in part by National Heart, Lung and Blood Institute Grant
No. HL36946 from the National Institutes of Health (to P.J.S. and T.W.)
and a Grant-In-Aid from the American Heart Association (Grant No.
95013720 to T.W.). Q.Z. is the recipient of a Research Fellowship Award
from the American Heart Association, Wisconsin Affıliate (Grant No.
96-F-Post-50).
Address reprint requests to Therese Wiedmer, PhD, Blood Research
Institute, The Blood Center of Southeastern Wisconsin, PO Box 2178,
Milwaukee, WI 53201-2178; e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9205-0012$3.00/0
1707
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1708
ZHOU, SIMS, AND WIEDMER
disorder.28 Recently, Toti et al29 reported that Epstein-Barr virus
(EBV)-transformed B lymphocytes from a patient with Scott
syndrome lacked expression of multidrug resistance (MDR)
genes MDR1 and MDR3. They proposed that deficiency in the
expression of the MDR proteins was responsible for the
aberrant properties of the plasma membrane of Scott cells, and
they proposed that the phenotype observed in Scott syndrome
resulted through mutation in an unlinked gene coding for a
regulatory protein that is required for normal MDR gene
expression.
In this study, we report that EBV-transformed B lymphocytes
from a patient with Scott syndrome exhibit normal expression
of both PL scramblase and MDR genes. Furthermore, cDNA
sequence encoding PL scramblase in Scott syndrome cells is
identical to that we previously reported for cells exhibiting
normal PL scramblase function.15
MATERIALS AND METHODS
Materials. Fetal bovine serum, RPMI 1640, rhodamine 123 (Rh123),
and verapamil were purchased from Sigma, (St Louis, MO). PCR2.1
vector and T-A Cloning kit were from Invitrogen (Carlsbad, CA). All
restriction enzymes were from New England BioLabs, Inc (Beverly,
MA). ExpressHyb, Klentaq Polymerase, and Advantage reverse transcriptase-polymerase chain reaction (RT-PCR) kits were from Clontech
Laboratories (Palo Alto, CA). a-32P-deoxycytidine triphosphate (dCTP)
was purchased from Dupont (Wilmington, DE). Random Primed DNA
Labeling Kit was from Boehringer Mannheim (Indianapolis, IN),
Hybond-N Nylon membrane from Amersham (Arlington Heights, IL),
and SuperSignal ULTRA Chemiluminescence Kit from Pierce Chemical Co (Rockford, IL).
B-cell lines. EBV-transformed B-lymphoblast cell lines were established from peripheral B lymphocytes of a single patient with Scott
syndrome (patient MS; now deceased) as previously described.26 The
clinical and cellular abnormalities identified in this patient have been
reviewed in detail by Weiss.18,19 Three independent transformations of
B lymphocytes were performed using venous blood obtained on
separate occasions from MS, and from three separate normal volunteers. The lymphoblasts were cultured in RPMI 1640 containing 10%
fetal bovine serum. All three cell lines derived from the patient with
Scott syndrome exhibited a marked defect in capacity to expose PS at
the cell surface upon ionophore-induced increase in intracellular
Ca21.26
32P-labeling of cDNA probes. cDNA of PL scramblase and b-actin
were labeled with 1 mCi of a-32P-dCTP using random-primed DNA
labeling kit. The labeled probes were separated from free a-32P-dCTP
by filtration through S-400 spin columns. The specific radioactivity of
the PL probes for PL scramblase and b-actin was 4.6 3 108 and 8 3 108
dpm/µg, respectively.
Isolation of RNA and Northern blot. Total cellular RNA was
isolated from 4 3 107 Scott or normal control B lymphoblasts by the
guanidinium thiocyanate cell lysis method using a Total RNA isolation
kit (Ambion, Austin, TX). A total of 10 µg of RNA from Scott or normal
B lymphoblasts were loaded to each lane of an agarose gel, separated by
electrophoresis, and transferred to a nylon membrane. The membrane
was prehybridized with ExpressHyb solution at 68°C for 30 minutes
and hybridized with ExpressHyb containing 5 ng/mL 32P-labeled PL
scramblase cDNA probe at 68°C for 1 hour, then washed, and exposed
to x-ray film. After development, the membranes were stripped and
hybridized with 32P-labeled b-actin cDNA probe using identical conditions.
RT-PCR. A total of 1 µg RNA was reverse transcribed using
Moloney murine leukemia virus transcriptase and oligo (dT) primers.
cDNA representing 50 ng RNA was then subjected to PCR for 35 cycles
in a final volume of 100 µL using 2.5 U of Klentaq polymerase to ensure
high-fidelity amplification. The primers to amplify PL scramblase,
MDR1, MDR3, and b2-microglobulin cDNA sequences were identical
to those previously described by us15 and by Toti et al,29 respectively.
After an initial denaturation of 2 minutes at 94°C, each cycle consisted
of 1 minute at 94°C, 1 minute at 55°C, and 2 minutes at 68°C. PCR
products were separated on a 2% agarose gel. Bands were visualized by
ethidium bromide staining and photographed.
cDNA cloning and sequencing. After RT-PCR and electrophoresis,
PL scramblase cDNA derived from Scott B lymphoblasts was cut from
the gel, purified with Wizard kit, and directly cloned into T-A cloning
vector pCR2.1. Escherichia coli strain INVaF’ was transformed, and
the presence of a 954-bp cDNA insert in the plasmid isolated from a
single colony was confirmed by digestion with EcoRI. The plasmid was
purified by Qiagen Kit. DNA was sequenced on an ABI DNA Sequencer
Model 373 Stretch (Applied Biosystems, Foster City, CA) using PRISM
Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin
Elmer-Cetus, Norwalk, CT). To avoid any PCR-mediated errors, two
batches of independent RT-PCR products were separately cloned and
each sequenced in duplicate.
Western blot. B lymphoblasts from the patient with Scott syndrome
or normal controls were lysed (60 minutes at 4°C) with 2% (vol/vol)
NP-40 in Hanks’ balanced salt solution (HBSS) containing 5 mmol/L
EDTA, 50 mmol/L benzamidine, 50 mmol/L N-ethyl maleimide, 1
mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L leupeptin. The
lysates were centrifuged (250,000g, 30 minutes, 4°C), and the supernatants denatured (100°C, 5 minutes) in 10% (wt/vol) sodium dodecyl
sulfate (SDS) sample buffer containing 2% b-mercaptoethanol. After
SDS-polyacrylamide gel electrophoresis (PAGE) (lysate from 1 3 106
cells per lane) and transfer to nitrocellulose, the blocked membrane was
incubated with 10 µg/mL of rabbit anti-PL scramblase-E306-W318, a
specific antibody raised against the C-terminus of PL scramblase.15 The
blots were incubated with horseradish peroxidase-conjugated goat
antirabbit IgG and developed by SuperSignal ULTRA chemiluminescence.
Dye efflux assay for MDR1 P-glycoprotein (Pgp) activity. B
lymphoblasts from a patient with Scott syndrome or normal controls
were suspended in RPMI 1640 complete medium at 106 cells/mL and
incubated in presence of 5 µg/mL Rh123 for 10 minutes at 37°C. After
washing, the rhodamine-loaded cells were suspended in RPMI 1640
complete medium and incubated in the presence or absence of the
MDR1 Pgp inhibitor verapamil (50 µmol/L final concentration) at 37°C,
5% CO2 for up to 3 hours. Aliquots of 0.5 mL were removed at times 0
hour and 3 hours, the cells were recovered by centrifugation (735g, 1
minute), and suspended in 0.5 mL HBSS. Single cell fluorescence was
quantified by flow cytometry monitoring cell-associated Rh123 fluorescence in the FL1 channel (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA).
RESULTS
Expression of MDR genes in Scott B lymphoblasts. Toti et
al29 recently reported that the genes MDR1 and MDR3 were not
expressed in EBV-transformed lymphoblasts from a patient
with Scott syndrome and inferred a role of this family of
proteins both in surface exposure of PS and in the plasma
membrane defect manifest in Scott syndrome. Therefore, it was
of interest to investigate MDR gene expression in the immortalized B cells obtained from the single other individual (MS) who
was well documented to exhibit the Scott syndrome.18-22
RT-PCR was performed on RNA isolated from each of three
Scott and three normal control EBV-transformed B-lymphoblast cell lines, using oligonucleotides specific for MDR1 and
MDR3, respectively. As shown in Fig 1, transcripts of both the
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PL SCRAMBLASE AND MDR GENES IN SCOTT SYNDROME
Fig 1. Expression of MDR genes in Scott and normal control
EBV-transformed B lymphocytes. RNA was extracted from three
EBV-transformed B-lymphoblast cell lines derived from a patient with
Scott syndrome (S0, S1, S2) and three normal volunteers (C1, C2,
W9), and RT-PCR performed with primers specific for MDR1, MDR3,
and b2-microglobulin (control gene), respectively. PCR products were
separated by agarose gel electrophoresis and visualized by ethidium
bromide staining. See Materials and Methods for details.
MDR1 and MDR3 genes were detected in all cell lines
analyzed. Although absolute amounts of each of these mRNAs
varied slightly among the individual cell lines, we observed no
consistent differences in the levels expressed by Scott versus
normal control cells. To confirm the presence of functional
MDR1 Pgp in these cells, B lymphoblasts were loaded with the
fluorescent dye Rh123, and efflux of Rh123 was quantified by
flow cytometry. As shown in Fig 2, Rh123 was efficiently
extruded from both Scott and normal control cells, as evidenced
by a time-dependent decrease in cell-associated fluorescence
upon incubation of the Rh123-loaded cells in dye-free medium.
Fig 2. Flow cytometric analysis of MDR1 Pgp activity in B
lymphoblasts. B lymphoblasts
from a patient with Scott syndrome and normal controls were
loaded with Rh123 (10 minutes,
37°C), washed and reincubated
in Rh123-free medium in the
presence (3 hours 1 Ver) or absence (3 hours) of 50 µmol/L
verapamil. Aliquots were removed at t 5 0 hour and 3 hours,
and analyzed by flow cytometry.
Dot plots are shown for one normal control (C2; lower panels)
and one Scott lymphoblast cell
line (S2; upper panels). An arbitrary gate is drawn to facilitate
comparison between samples.
All three Scott and three normal
control cell lines displayed MDR1
activity in the absence of verapamil, although differences in activity between cell lines were observed (not shown).
1709
Although the rate of dye efflux varied somewhat from cell line
to cell line, each of the three Scott B-lymphoblast cell lines
tested was capable of actively extruding Rh123, and no
significant difference in MDR1 Pgp activity was discerned
between the Scott and normal control cell lines (data not
shown). In all cases, the efflux of Rh123 from the B lymphoblasts (Scott or control) was completely inhibited when dyeloaded cells were incubated in the presence of the MDR1 Pgp
inhibitor, verapamil. These data confirm the presence of functional MDR1 Pgp in the B lymphoblasts obtained from this
patient with Scott syndrome and suggest that the abnormality in
transbilayer mobilization of plasma membrane PL that is
characteristic of these cells must relate to a defect or deficiency
in another plasma membrane PL transporting protein.
Expression of the PL scramblase gene in Scott B lymphoblasts. The functional abnormality in the blood cells of
patients with Scott syndrome has been shown to specifically
relate to a defective response of the plasma membrane to an
elevation of Ca21 at the endofacial surface. Whereas intracellular Ca21 normally initiates collapse of the plasma membrane PL
asymmetry by stimulating the rapid transbilayer movement of
all plasma membrane PL, in Scott syndrome cells, the plasma
membrane remains unresponsive to such elevation in intracellular Ca21, and these cells retain their PL asymmetry despite entry
of Ca21 into the cytosol in the circumstance of either plasma
membrane injury or cell activation. This implied that Scott cells
are either deficient or defective in the protein that is responsible
for mediating Ca21-induced transbilayer movement of the
plasma membrane PL. PL scramblase is an endofaciallyoriented plasma membrane protein that has been shown to
mediate rapid transbilayer movement of all plasma membrane
PL upon binding Ca21, mimicking the action of Ca21 at the
internal surface of the plasma membrane. The similarity of the
function exhibited by PL scramblase to the observed defect in
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1710
ZHOU, SIMS, AND WIEDMER
Fig 3. Northern blot analysis of PL scramblase in
Scott and normal B lymphoblasts. RNA was extracted
from Scott and normal control B lymphoblasts, and
Northern blot performed with 32P-labeled PL scramblase cDNA probe as described in Materials and
Methods. Results are shown for Scott S2 and normal
control W9 (upper panel). Comparable amounts of
transcripts were observed for all cell lines tested.
Lower panel shows Northern blot of b-actin.
Scott syndrome cells, suggested that this syndrome was likely
related to an abnormality specifically affecting this protein. To
gain further insight into the origin of the functional defect in the
Scott syndrome cells, we quantified the level of PL scramblase
expression and obtained the cDNA sequence of the PL scramblase that is expressed in the aberrant Scott cell lines for
comparison to that in normal cells (Figs 3 and 4). Expression of
PL scramblase RNA was examined in three different Scott and
three normal control B-lymphoblast cell lines. Northern blot
with PL scramblase cDNA probe showed two transcripts of
approximately 1.6 and 2.6 kb in both Scott (S2) and normal
control (W9) B lymphoblasts (Fig 3), in agreement with what
we previously observed in a number of other tissues and cell
lines examined.15,17 RT-PCR performed with the same Blymphoblast cell lines indicated the presence of PL scramblase
cDNA in Scott cells of the same size as that found in normal
control lymphoblasts (Fig 4). Furthermore, Western blotting
with antibody directed against the C-terminal peptide of PL
scramblase15 showed the presence of , 37 kD PL scramblase
protein at comparable amounts in both Scott and normal control
B lymphoblasts (Fig 5). Complete identity in the predicted
amino acid sequence of PL scramblase expressed in Scott
syndrome cells to that previously reported for wild-type human
PL scramblase (GenBank accession number AF008445) was
also confirmed by sequencing of cDNA obtained from duplicate
RT-PCR reactions performed for three different Scott B-
Fig 4. RT-PCR analysis of PL scramblase in Scott
and normal B lymphoblasts. RNA was extracted from
three Scott (S0, S1, S2) and three normal control (C1,
C2, W9) B-lymphoblast cell lines and RT-PCR performed with PL scramblase-specific primers. PCR
products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
See Materials and Methods for details. RT-PCR of
b2-microglobulin as control gene is also shown.
lymphoblast cell lines. Thus, as demonstrated for the MDR1
and MDR3 Pgps, the phenotype in Scott syndrome also cannot
be attributed to mutation in the PL scramblase gene.
DISCUSSION
The multidrug resistance Pgps MDR1 and MDR3 are members of the superfamily of ATP-binding cassette transporters.
Whereas MDR1 Pgp is thought to primarily function to extrude
cytotoxic agents from the cell, MDR3 Pgp has no drug-pumping
activity, but there is strong evidence that MDR3 Pgp is a
PC-specific flippase.30,31 Mice deficient in mdr2, the homologue
to human MDR3, display a defect in PL secretion into the bile.32
Evidence for MDR1 Pgp-mediated transport of PC and PE, but
not PS, from inner to outer plasma membrane leaflet has also
been presented.33,34 Toti et al29 recently reported the lack of
expression of both MDR1 and MDR3 genes in EBVtransformed B lymphocytes derived from a patient with Scott
syndrome and inferred a link between transbilayer transport of
PS and the MDR family of Pgp. In this study, we present
evidence that both MDR1 and MDR3 Pgp are normally
expressed in another well-documented case of Scott syndrome.
Our results suggest that neither MDR1 nor MDR3 plays a role
in Ca21-induced surface exposure of PS, the transbilayer PL
transport activity that is deficient in Scott syndrome. Although
the possibility exists that the patient with Scott syndrome
described by Toti et al is deficient in an as yet unknown member
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PL SCRAMBLASE AND MDR GENES IN SCOTT SYNDROME
1711
Scott syndrome cells express normal levels of PL scramblase
mRNA and protein, and that the deduced sequence of the
expressed polypeptide is identical to that of PL scramblase
found in normal cells.15 Thus, the molecular identity of the
defect leading to the aberrant phenotype of the Scott syndrome
cell remains elusive. Among the possibilities now being pursued are (1) the presence in Scott cells of an inhibitor of the PL
scramblase pathway that dissociates from the protein upon
solubilization in detergent; (2) a deficiency in a protein that
normally interacts with PL scramblase and is necessary for its
function in situ; (3) a defect in Scott cells in a posttranslational
modification of the PL scramblase polypeptide affecting either
its affinity for Ca21 or the expression of its PL mobilizing
function. In this context, we recently showed that the activity of
PL scramblase is dependent upon thioesterification of one or
more cytoplasmic cysteinyls with fatty acid.37 Nevertheless,
preliminary experiments in Scott syndrome lymphoblasts suggest that the PL scramblase expressed in these cells is normally
palmitoylated (data not shown).
REFERENCES
Fig 5. Western blot of PL scramblase in Scott and normal B
lymphoblasts. Lysates of Scott and normal B lymphoblasts were
subjected to SDS-PAGE, transferred to nitrocellulose, and the blot
was developed with 10 mg/mL of rabbit anti-PL scramblase-E306W318 raised against the C-terminal peptide of PL scramblase. See
Materials and Methods for details.
of the MDR family, it is also noteworthy that the activity of
these Pgps is ATP-, but not Ca21-dependent. It is also worth
considering that expression of MDR genes has been reported to
vary between individuals, with differentiation stage and age.
For instance, Pilarski et al35 reported that 50% to 80% of normal
blood T or B lymphocytes were positive for cell surface MDR1
Pgp, with the proportion of Pgp1 cells decreased in young
children and individuals over age 60. Furthermore, the possibility exists that MDR expression in the study by Toti et al was
decreased upon EBV-transformation of B lymphocytes and
does not reflect the level of MDR Pgp expression in the
circulating blood cells of this patient, as neither analysis by
Northern blot nor by MDR1 Pgp function apparently yielded
conclusive results.29,36
There is now strong evidence that cell surface exposure of PS
that occurs upon cell activation or injury is mediated by the
Ca21-activated PL scramblase, a plasma membrane protein that
mediates rapid transbilayer movement of all plasma membrane
PL. Although Scott cells are characterized by an apparent
functional defect in this Ca21-activated plasma membrane PL
scramblase pathway, we had previously shown that Scott
erythrocytes contain a protein that exhibits normal PL scramblase function, after it is extracted from the erythrocyte with
detergent and reconstituted into liposomal membranes.14 This
implied that the gene defect in Scott syndrome affecting the PL
scramblase pathway is only manifest in situ, and that the protein
functions normally after exposure to detergent and reconstitution with exogenous PL. Our current data also indicate that
1. Schroit AJ, Zwaal RFA: Transbilayer movement of phospholipids
in red cell and platelet membranes. Biochim Biophys Acta 1071:313,
1991
2. Devaux P: Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30:1163, 1991
3. Dolis D, Moreau C, Zachowski A, Devaux PF: Aminophospholipid translocase and proteins involved in transmembrane phospholipid
traffic. Biophys Chem 68:221, 1997
4. Tang XJ, Halleck MS, Schlegel RA, Williamson P: A subfamily of
P-type ATPases with aminophospholipid transporting activity. Science
272:1495, 1996
5. Zwaal RFA, Schroit AJ: Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121, 1997
6. Williamson P, Kulick A, Zachowski A, Schlegel RA, Devaux PF:
Ca21 induces transbilayer redistribution of all major phospholipids in
human erythrocytes. Biochemistry 31:6355, 1992
7. Chang C-P, Zhao J, Wiedmer T, Sims PJ: Contribution of platelet
microparticle formation and granule secretion to the transmembrane
migration of phosphatidylserine. J Biol Chem 268:7171, 1993
8. Smeets EF, Comfurius P, Bevers EM, Zwaal RFA: Calciuminduced transbilayer scrambling of fluorescent phospholipid analogs in
platelets and erythrocytes. Biochim Biophys Acta Bio-Membr 1195:
281, 1994
9. Bevers EM, Comfurius P, Zwaal RF: Platelet procoagulant
activity: Physiological significance and mechanisms of exposure. Blood
Rev 5:146, 1991
10. Sims PJ, Faioni EM, Wiedmer T, Shattil SJ: Complement
proteins C5b-9 cause release of membrane vesicles from the platelet
surface that are enriched in the membrane receptor for coagulation
factor Va and express prothrombinase activity. J Biol Chem 263:18205,
1988
11. Wang RH, Phillips G Jr, Medof ME, Mold C: Activation of the
alternative complement pathway by exposure of phosphatidylethanolamine and phosphatidylserine on erythrocytes from sickle cell disease
patients. J Clin Invest 92:1326, 1993
12. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL,
Henson PM: Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macrophages.
J Immunol 148:2207, 1992
13. Bassé F, Stout JG, Sims PJ, Wiedmer T: Isolation of an
erythrocyte membrane protein that mediates Ca21-dependent transbilayer movement of phospholipid. J Biol Chem 271:17205, 1996
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1712
14. Stout JG, Bassé F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ:
Scott syndrome erythrocytes contain a membrane protein capable of
mediating Ca21-dependent transbilayer migration of membrane phospholipids. J Clin Invest 99:2232, 1997
15. Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ:
Molecular cloning of human plasma membrane phospholipid scramblase — A protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem 272:18240, 1997
16. Zhou Q, Sims PJ, Wiedmer T: Identity of a conserved motif in
phospholipid scramblase that is required for Ca21-accelerated transbilayer movement of membrane phospholipids. Biochemistry 37:2356,
1998
17. Zhao J, Zhou Q, Wiedmer T, Sims PJ: Level of expression of
phospholipid scramblase regulates induced movement of phosphatidylserine to the cell surface. J Biol Chem 273:6603, 1998
18. Weiss HJ, Vicic WJ, Lages BA, Rogers J: Isolated deficiency of
platelet procoagulant activity. Am J Med 67:206, 1979
19. Weiss HJ: Scott Syndrome: A disorder of platelet coagulant
activity (PCA). Semin Hematol 31:312, 1994
20. Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ: Assembly
of the platelet prothrombinase complex is linked to vesiculation of the
platelet plasma membrane. Studies in Scott syndrome: An isolated
defect in platelet procoagulant activity. J Biol Chem 264:17049, 1989
21. Miletich JP, Kane WH, Hofmann SL, Stanford N, Majerus PW:
Deficiency of factor-Xa-factor Va binding sites on the platelets of a
patient with a bleeding disorder. Blood 54:1015, 1979
22. Ahmad SS, Rawala-Sheikh R, Ashby B, Walsh PN: Platelet
receptor-mediated factor X activation by factor IXa: High-affinity factor
IXa receptors induced by factor VIII are deficient on platelets in Scott
syndrome. J Clin Invest 84:824, 1989
23. Toti F, Satta N, Fressinaud E, Meyer D, Freyssinet J-M: Scott
syndrome, characterized by impaired transmembrane migration of
procoagulant phosphatidylserine and hemorrhagic complications, is an
inherited disorder. Blood 87:1409, 1996
24. Bevers EM, Wiedmer T, Comfurius P, Zhao J, Smeets EF,
Schlegel RA, Schroit AJ, Weiss HJ, Williamson P, Zwaal RFA, Sims PJ:
The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions
is not responsible for Ca21-induced loss of phospholipid asymmetry in
the human erythrocyte. A study in Scott Syndrome, a disorder of
calcium-induced phospholipid scrambling. Blood 86:1983, 1995
25. Bevers EM, Wiedmer T, Comfurius P, Shattil SJ, Weiss HJ,
Zwaal RF, Sims PJ: Defective Ca21-induced microvesiculation and
deficient expression of procoagulant activity in erythrocytes from a
patient with a bleeding disorder: A study of the red blood cells of Scott
syndrome. Blood 79:380, 1992
26. Kojima H, Newton-Nash D, Weiss HJ, Zhao J, Sims PJ, Wiedmer
ZHOU, SIMS, AND WIEDMER
T: Production and characterization of transformed B-lymphocytes
expressing the membrane defect of Scott syndrome. J Clin Invest
94:2237, 1994
27. Dachary-Prigent J, Pasquet JM, Fressinaud E, Toti F, Freyssinet
JM, Nurden AT: Aminophospholipid exposure, microvesiculation and
abnormal protein tyrosine phosphorylation in the platelets of a patient
with Scott syndrome: A study using physiologic agonists and local
anaesthetics. Br J Haematol 99:959, 1997
28. Dekkers DWC, Comfurius P, Vuist WMJ, Billheimer JT, Dicker
I, Weiss HJ, Zwaal RFA, Bevers EM: Impaired Ca21-induced tyrosine
phosphorylation and defective lipid scrambling in erythrocytes from a
patient with Scott syndrome: A study using an inhibitor for scramblase
that mimics the defect in Scott syndrome. Blood 91:2133, 1998
29. Toti F, Schindler V, Riou JF, Lombard-Platet G, Fressinaud E,
Meyer D, Uzan A, Le Pecq JB, Mandel JL, Freyssinet JM: Another-link
between phospholipid transmembrane migration and ABC transporter
gene family, inferred from a rare inherited disorder of phosphatidylserine externalization. Biochem Biophys Res Commun 241:548, 1997
30. Gottesman MM, Pastan I: Biochemistry of multidrug resistance
mediated by the multidrug transporter. Annu Rev Biochem 62:385,
1993
31. Elferink RPJO, Tytgat GNJ, Groen AK: The role of mdr2
P-glycoprotein in hepatobiliary lipid transport. FASEB J 11:19, 1997
32. Crawford AR, Smith AJ, Hatch VC, Elferink RPJO, Borst P,
Crawford JM: Hepatic secretion of phospholipid vesicles in the mouse
critically depends on mdr2 or MDR3 P-glycoprotein expression —
Visualization by electron microscopy. J Clin Invest 100:2562, 1997
33. Bosch I, Dunussi-Joannopoulos K, Wu RL, Furlong ST, Croop J:
Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. Biochemistry 36:5685,
1997
34. Van Helvoort A, Smith AJ, Sprong H, Fritzsche I, Schinkel AH,
Borst P, van Meer G: MDR1 P-glycoprotein is a lipid translocase of
broad specificity, while MDR3 P-glycoprotein specifically translocates
phosphatidylcholine. Cell 87:507, 1996
35. Pilarski LM, Paine D, McElhaney JE, Cass CE, Belch AR:
Multidrug transporter P-glycoprotein 170 as a differentiation antigen on
normal human lymphocytes and thymocytes: Modulation with differentiation stage and during aging. Am J Hematol 49:323, 1995
36. Grulois I, Fardel O, Drenou B, Lamy T, Le Prise PY, Fauchet R:
Multidrug resistance in B-cell chronic lymphocytic leukemia. Acta
Haematol 94:78, 1995
37. Zhao J, Zhou Q, Wiedmer T, Sims PJ: Palmitoylation of
phospholipid scramblase is required for normal function in promoting
Ca21-activated transbilayer movement of membrane phospholipids.
Biochemistry 37:6361, 1998
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1998 92: 1707-1712
Expression of Proteins Controlling Transbilayer Movement of Plasma
Membrane Phospholipids in the B Lymphocytes From a Patient With Scott
Syndrome
Quansheng Zhou, Peter J. Sims and Therese Wiedmer
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