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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1707-1712
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
From the Blood Research Institute of The Blood Center of Southeastern
Wisconsin, Milwaukee, WI.
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ABSTRACT |
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 Ca2+ 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 detergent-solubilized 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 detergent-dissociable inhibitor of this pathway.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
PLASMA 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 Mg2+- 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 Ca2+ 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 Ca2+-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 Ca2+-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 Ca2+-dependent,
bidirectional movement of PL between membrane leaflets, mimicking the
action of Ca2+ 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 Ca2+. 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 Ca2+.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 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
[Ca2+] 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 Ca2+-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
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
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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).
 -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 Ca2+.26
32P-labeling of cDNA probes.
cDNA of PL scramblase and  -actin were labeled with 1 mCi of
-32P-dCTP using random-primed DNA labeling kit. The
labeled probes were separated from free -32P-dCTP by
filtration through S-400 spin columns. The specific radioactivity of
the PL probes for PL scramblase and -actin was 4.6 × 108 and 8 × 108 dpm/µg, respectively.
Isolation of RNA and Northern blot.
Total cellular RNA was isolated from 4 × 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 -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
2-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 INVF' 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% -mercaptoethanol.
After SDS-polyacrylamide gel electrophoresis (PAGE) (lysate from 1 × 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).
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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 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. 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 dye-loaded 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.
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| 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
2-microglobulin (control gene), respectively. PCR
products were separated by agarose gel electrophoresis and visualized
by ethidium bromide staining. See Materials and Methods for details.
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| 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 + Ver)
or absence (3 hours) of 50 µmol/L verapamil. Aliquots were removed at
t = 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).
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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 Ca2+ at the
endofacial surface. Whereas intracellular Ca2+ 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 Ca2+, and these cells retain
their PL asymmetry despite entry of Ca2+ 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
Ca2+-induced transbilayer movement of the plasma membrane
PL. PL scramblase is an endofacially-oriented plasma membrane protein
that has been shown to mediate rapid transbilayer movement of all
plasma membrane PL upon binding Ca2+, mimicking the action
of Ca2+ at the internal surface of the plasma membrane. The
similarity of the function exhibited by PL scramblase to the observed
defect in 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 B-lymphoblast 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-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.
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| 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
-actin.
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| 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
2-microglobulin as control gene is also shown.
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| 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 µg/mL of rabbit anti-PL scramblase-E306-W318 raised against
the C-terminal peptide of PL scramblase. See Materials and Methods for
details.
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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 EBV-transformed 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 Ca2+-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 of the MDR family, it is also noteworthy that the
activity of these Pgps is ATP-, but not Ca2+-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 Pgp+ 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 Ca2+-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 Ca2+-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 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 Ca2+ 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).
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FOOTNOTES |
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 Affiliate (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: twiedmer{at}bcsew.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract
