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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2133-2138
Impaired Ca2+-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
By
David W.C. Dekkers,
Paul Comfurius,
Wim M.J. Vuist,
Jeffrey T. Billheimer,
Ira Dicker,
Harvey J. Weiss,
Robert F. A. Zwaal, and
Edouard M. Bevers
From the Department of Biochemistry, Cardiovascular Research
Institute Maastricht, Maastricht University, Maastricht, The
Netherlands; Dupont Merck Research Laboratory, Wilmington, DE; and the
Division of Hematology-Oncology, St. Luke's-Roosevelt Hospital Center,
New York, NY.
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ABSTRACT |
Scott syndrome is an hereditary bleeding disorder characterized by a
deficiency in platelet procoagulant activity. Unlike normal blood
cells, Scott platelets, as well as erythrocytes and lymphocytes, are
strongly impaired in their ability to scramble their membrane
phospholipids when challenged with Ca2+. In normal cells
this collapse of membrane asymmetry leads to surface exposure of
phosphatidylserine. Here we report that Scott erythrocytes show an
apparent defect in tyrosine phosphorylation on treatment with
Ca2+-ionophore. Diminished tyrosine phosphorylation was
also apparent in activated Scott platelets, but much less pronounced
than observed in red blood cells. On the other hand,
tyrosine phosphorylation profiles observed in Scott red blood cell
ghosts after sealing in the presence of adenosine triphosphate
(ATP) were indistinguishable from those obtained from
normal ghosts. Several observations argue in favor of a mechanism in
which tyrosine phosphorylation in red blood cells is facilitated by,
rather than required for scrambling of membrane lipids. Staurosporin
blocks tyrosine phosphorylation in normal red blood cells, but does not
inhibit the lipid scrambling process. White ghosts from normal
erythrocytes, resealed in the absence of ATP, exhibit
Ca2+-induced lipid scrambling without tyrosine
phosphorylation. A selective inhibitor of Ca2+-induced
lipid scrambling also showed an apparent inhibition of tyrosine
phosphorylation in ionophore-treated normal red blood cells, similar to
that observed in Scott erythrocytes. While this inhibitor also
suppressed Ca2+-induced lipid scrambling in ghosts that
were sealed in the presence of ATP, it did not inhibit tyrosine kinase
activity. We conclude that the apparent deficiency in tyrosine
phosphorylation in Scott cells is an epiphenomenon, possibly associated
with a defect in phospholipid scrambling, but not causal to this
defect.
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INTRODUCTION |
MEMBRANE PHOSPHOLIPID asymmetry is a
seemingly ubiquitous phenomenon that is generated by an adenosine
triphosphate (ATP)-dependent lipid pump, which specifically shuttles
aminophospholipids to the cytoplasmic membrane leaflet. Influx of
calcium, however, inhibits this aminophospholipid translocase activity
and activates a lipid scramblase that produces a progressive loss of
lipid asymmetry. This process exposes phosphatidylserine at the outer
surface, which can promote assembly on the membrane of the tenase and
prothrombinase complex of the blood coagulation cascade. This assembly
dramatically enhances the rate of thrombin formation responsible for
the procoagulant activity of activated platelets.1-3
A deficiency in platelet procoagulant activity associated with a
bleeding disorder was first described by Weiss et al4,5 and
is presently referred to as Scott syndrome. On activation, Scott
platelets show a strongly decreased lipid scrambling with little
surface exposure of phosphatidylserine.6 Moreover, these platelets express a considerably decreased number of factor
Va7 and factor VIIIa8 binding sites and are
markedly deficient in their ability to promote both tenase and
prothrombinase activity in response to agonists.6 They also
exhibit an impaired capacity to shed membrane-derived lipid-symmetric
microvesicles, a process often associated with lipid
scrambling.9 Recent studies on a newly discovered family
suggest that Scott syndrome is an inherited bleeding disorder
transmitted as an autosomal recessive trait,10 and this may
also be the case in the family of the originally described patient with
this disorder.11 The defect is not restricted to platelets,
but can also be demonstrated in the patients' erythrocytes and
lymphocytes.12,13 The observation that, unlike normal red blood cell ghosts, resealed ghosts from Scott erythrocytes do not show
Ca2+-induced scrambling of membrane phospholipids implies
that the lesion resides in the plasma membrane or in the tightly
associated cytoskeleton.12 Indeed, proteins fractionated
from platelet or red blood cell membranes have been reconstituted in
artificial lipid vesicles, which exhibited Ca2+-induced
lipid scrambling activity.14,15 Recently, it was shown that
proteoliposomes reconstituted from red blood cells from Scott syndrome
resulted in similar Ca2+-inducible lipid scrambling, as
observed for reconstitutes from normal red blood cells.16
Moreover, acidification caused Ca2+-independent lipid
scrambling in both proteoliposomes and inside-out vesicles derived from
normal and Scott erythrocytes, suggesting that the defect in Scott
syndrome is caused by an impaired regulatory mechanism rather than the
absence of a membrane protein responsible for lipid scrambling.
While hydrolyzable ATP does not seem to be required for lipid
scrambling, it has been reported that prolonged ATP depletion of
erythrocytes results in markedly diminished Ca2+-induced
randomization of lipids.17,18 Because this may suggest a
possible involvement of phosphorylated protein(s), it was intriguing to
observe a severely impaired tyrosine phosphorylation in Scott erythrocytes after treatment with Ca2+-ionophore. The
present study shows that the aberrant tyrosine phosphorylation is a
consequence rather than a cause of a defective lipid scrambling
process.
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MATERIALS AND METHODS |
Materials.
Ionomycin and bovine serum albumin (BSA; globulin and fatty acid free)
were obtained from Sigma Chemical Co (St Louis, MO). Coagulation
factors thrombin, prothrombin, factor Xa, and factor Va were purified
from bovine blood as described elsewhere.19 Thrombin-specific chromogenic substrate, S2238, was obtained from AB
Kabi Diagnostica (Stockholm, Sweden). Sodium orthovanadate was from
Janssen Chimica (Geel, Belgium). NBD-PS:
2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-oleoyl-sn-glycero-3-phosphoserine was obtained from Avanti Polar Lipids (Alabaster, AL). R5421, ethanimidothioic acid
N-[{N-butylthio-N-methylamino]-carbonyloxy}-methyl ester (see
insert Fig 3 for formula) was from Dupont-Merck (Wilmington, DE).
Monoclonal antiphosphotyrosine antibody (4G10) was obtained from UBI
(Lake Placid, NY). Enhanced chemiluminescence (ECL) materials were from
Amersham PLC (Aylesbury, UK). All other reagents were of the highest
grade commercially available.
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| Fig 3.
Upper panel: inhibition of
Ca2+-ionomycin-induced prothrombinase activity in
control erythrocytes by compound R5421. Cells were incubated with
increasing concentrations of R5421 for 10 minutes, followed by
ionomycin (5 µmol/L) in the presence of Ca2+ (1 mmol/L)
for 60 minutes. Subsequently, samples were analyzed for prothrombinase
activity as described in Materials and Methods. Insert: structural
formula of compound R5421. Lower panel: tyrosine phosphorylation in
control erythrocytes incubated for 15 minutes with
Ca2+-ionomycin in the absence (lane 1) and presence (lane
2) of R5421 (0.1 mmol/L). Lanes 3 to 6 represent phosphorylation
patterns of white ghosts incubated with four different concentrations
of ATP (0.05 mmol/L ATP, lane 3; 0.5 mmol/L ATP, lane 4; 1.0 mmol/L ATP, lane 5; and 2.0 mmol/L ATP, lane 6), all in the presence of R5421
(0.1 mmol/L). Contrary to the incubations with intact cells, the
experiments with ghosts were performed in the absence of
Ca2+ to ensure sufficient levels of phosphorylation (see
text).
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Isolation of erythrocytes and preparation of resealed ghosts.
Washed human red blood cells were obtained by differential
centrifugation of whole blood collected on citrate (3.8%) from healthy
volunteers. Blood from patient MS and appropriate control samples were
shipped at 4°C by air express to Maastricht and processed immediately on arrival. After washing in TEMS8 buffer (50 mmol/L TRIS,
120 mmol/L NaCl, 2 mmol/L MgCl2, 0.2 mmol/L EGTA, adjusted at pH 8.0), erythrocytes were finally resuspended at 10% hematocrit and stored on ice. Cells were used within 1 day. Ghosts were prepared by hypotonic shock in ice-cold lysis buffer (5 mmol/L TRIS, 12 mmol/L
NaCl, 2 mmol/L MgCl2, 0.2 mmol/L EGTA). After 15 minutes on
ice, tonicity was restored by adding one tenth volume 9% NaCl, followed by incubation at 37°C for 15 minutes. In some experiments, ATP (0.05 to 2.0 mmol/L) and/or R5421 (0.1 mmol/L) was added
before resealing. Resealed ghosts were collected by centrifugation at 2,250g for 20 minutes and washed three times in TEMS8 buffer. Ghost concentration was adjusted at 1 × 109/mL.
Erythrocytes and resealed ghosts were incubated at a concentration of 1 × 109/mL with ionomycin (final concentration 5 µmol/L)
in the presence of 1 mmol/L CaCl2 at 37°C.
Preparation of washed platelets.
Platelets were isolated from patient MS and an appropriate control by
differential centrifugation of whole blood, according to Bevers et
al.20 Because shipment of the blood samples took approximately 48 hours, the blood was collected in citrate to avoid a
prolonged period of acidity. Before centrifugation, acid citrate
dextrose (ACD: 0.18 mol/L glucose, 0.08 mol/L trisodium citrate, 0.052 mol/L citric acid) was added to the blood in a ratio 1:5 (vol/vol).
Washed platelets were finally resuspended in a HEPES buffer (136 mmol/L
NaCl, 2.7 mmol/L KCl, 2 mmol/L MgCl2, 10 mmol/L HEPES, 0.1 mmol/L EGTA, 5 mmol/L glucose, and 0.5 mg/mL human serum albumin, pH
7.5), and diluted to a final concentration of 5 × 108
platelets/mL.
Measurement of prothrombinase activity.
The rate of conversion of prothrombin to thrombin by the enzyme complex
factor Xa-factor Va has been shown to be a convenient, rapid, and
sensitive method to monitor in a semiquantitative way the extent of
exposure of phosphatidylserine at the outer cell surface.20
Briefly, 1 × 107/mL erythrocytes (or resealed ghosts)
were incubated at 37°C with factor Xa and factor Va at a final
concentration of 3 and 6 nmol/L, respectively, for 1 minute in the
presence of 3 mmol/L CaCl2. The reaction was started by
addition of prothrombin (final concentration 4 µmol/L); 1 minute
after addition of prothrombin, a sample from the incubation mixture was
transferred to a cuvette containing 1 mL buffer composed of 50 mmol/L
TRIS, 120 mmol/L NaCl, and 2 mmol/L EDTA (pH 8.0). The amount of
thrombin formed was determined by measuring the change in absorbance at
405 nm caused by the conversion of a thrombin-specific substrate, S2238
(0.2 mmol/L).
Measurement of scramblase activity in platelets.
A continuous assay for scrambling of platelet lipids was based on
reduction of a fluorescent lipid probe (NBD-PS) by
dithionite as previously described by Williamson et al.21
Washed platelets resuspended in HEPES buffer in the absence of albumin
were preincubated with 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF)
before labeling with 1 µmol/L NBD-PS. Addition of PMSF was required
to prevent degradation of the probe once it had been internalized by
the action of the aminophospholipid translocase. Platelets (2 × 108/mL) were loaded with NBD-PS for 45 minutes at 37°C,
followed by an additional incubation period of 45 minutes in the
presence or absence of R5421. A total of 50 to 100 µL of
NBD-PS-labeled platelets were diluted in 2 mL of HEPES buffer and
placed in the fluorimeter at 37°C under continuous stirring and
fluorescence was monitored at 534 nm ( ex 472 nm). At 60 seconds, dithionite was added to a final concentration of
5 mmol/L to quench NBD-PS present in the membrane outer leaflet. At
t=90 seconds, ionomycin was added (1 µmol/L final
concentration), and the scrambling process was started by the addition
of 1 mmol/L CaCl2 at t= 120 seconds. Fluorescence intensity was monitored for 7.5 minutes, after which Triton X100 was added to a final concentration of 1% to make all of
the NBD-PS available to dithionite. To determine IC50 and
half-time of R5421 inhibition, the rate of scrambling activity was
analyzed as the initial slope of the fluorescence decay after addition of Ca2+.
Tyrosine phosphorylation.
Protein-tyrosine phosphorylation was detected after sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blotting. Briefly, after the ionomycin treatment of intact erythrocytes, 0.2 mmol/L PMSF was added and cells were lysed by the
addition of 20 vol ice-cold lysis buffer. Ghosts were collected and
washed once by centrifugation (30,000g for 15 minutes). After resuspension, one fifth volume of electrophoresis sample buffer (312.5 mmol/L TRIS, 50% (vol/vol) glycerol, 15% (wt/vol) SDS, 0.17 mg/mL
Bromphenol blue, 50 mmol/L dithiothreitol, pH 6.8) was added. Samples
for electrophoresis from incubations with resealed ghosts (1 × 109/mL) or platelets (5 × 108/mL) were
prepared by addition of one fifth volume of electrophoresis buffer to
the incubation mixture. Proteins were separated by SDS-PAGE on 7.5%
gels and transferred to nitrocellulose. After blocking with 3% BSA for
1 hour, blots were incubated with the antiphosphotyrosine antibody,
4G10 (0.04 µg/mL) and after extensive washing, incubated with a
biotinylated goat antimouse antibody. Immunoreactive proteins were
detected by streptavidine horseradish peroxidase, visualized by ECL
(Amersham) according to the manufacturer's procedure.
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RESULTS AND DISCUSSION |
Incubation of human erythrocytes with Ca2+-ionophore
results in a rapid phosphorylation of tyrosine residues in certain
membrane proteins, of which band 3 appears to be the most
prominent.22,23 We have compared the phosphorylation
patterns of erythrocytes from healthy individuals with those from a
patient with Scott syndrome by Western blots using a monoclonal
antibody specific for phosphotyrosine residues.
Figure 1, upper panel, shows that tyrosine
phosphorylation is severely impaired in the patients' red blood cells
after treatment with Ca2+-ionophore (lanes 1 to 4). This
raises the question whether this impaired phosphorylation is related to
the lack of Ca2+-induced lipid scrambling in the patients'
blood cells, as shown by the lack of phosphatidylserine exposure
monitored by prothrombinase (Fig 1, lower panel).
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| Fig 1.
Tyrosine phosphorylation of membrane proteins and
development of prothrombinase activity in normal erythrocytes and
erythrocytes from Scott syndrome incubated with
Ca2+-ionomycin. Washed cells were incubated with
ionomycin in the presence of Ca2+ as described in
Materials and Methods, and samples were taken at 15 minutes for
analysis of tyrosine phosphorylation and at various time intervals for
measuring prothrombinase activity as a measure for surface exposed
phosphatidylserine. Phosphorylation patterns in red blood cells did not
appreciably change on varying the incubation time from 5 to 30 minutes.
Upper panel: normal erythrocytes in the absence (lane 1) and presence
of Ca2+-ionomycin (lane 2) and Scott erythrocytes in the
absence (lane 3) and presence of Ca2+-ionomycin (lane 4).
Lower panel: time course of appearance of prothrombinase activity in
normal ( ) and Scott erythrocytes ( ).
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To investigate if the impaired tyrosine phosphorylation observed in
Scott erythrocytes is caused by a deficiency in one or more of the
tyrosine kinases, we tested tyrosine phosphorylation in hemoglobin-free
(`white') erythrocyte ghosts. The major tyrosine kinase activity in
erythrocytes appears membrane-associated, and direct phosphorylation
can be observed in nonsealed ghost preparations after addition of
Mg-ATP.24 For this purpose, we used Ca2+-free
ghosts as a substrate because tyrosine phosphorylation in Ca2+-loaded ghosts is too low to detect a possible
deficiency of tyrosine kinase activity in Scott syndrome. As shown in
Fig 2, similar phosphorylation patterns
were found for both Scott and control ghosts when incubated with
increasing amounts of ATP. Because red blood cell ghosts still contain
protein tyrosine phosphatase activity associated with band
3,24 the level of protein phosphotyrosine is determined by
the activity of protein tyrosine kinases and the opposing activity of
protein tyrosine phosphatases. As expected, addition of
Mn2+ and VO43-, known inhibitors of
tyrosine phosphatase,23,24 causes higher levels of
phosphorylation, but again, no difference was found between control and
Scott ghosts. While these data suggest that neither enzyme is defective
in the patients' red blood cell ghosts, it can at present not be
excluded that diminished phosphorylation in Scott erythrocytes reflects
a difference in either a cytosolic kinase or phosphatase (or regulatory
component) that is washed away from intact erythrocytes on preparing
ghosts. However, the data from both intact cells and ghosts strongly
suggest that tyrosine phosphorylation is not required for lipid
scrambling. This is supported by two additional observations. First,
incubation of normal erythrocytes with staurosporin at a concentration
of 5 µmol/L, which completely blocks tyrosine kinase activity, does not prevent the generation of prothrombinase activity induced by
Ca2+-ionophore. Second, white ghosts from normal
erythrocytes resealed in the absence of ATP retain the ability to
scramble their phospholipids on Ca2+-influx,12
while no tyrosine phosphorylation can occur under these conditions.
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| Fig 2.
Western blots showing tyrosine phosphorylation in control
(lanes 1 to 4) and Scott erythrocyte ghosts (lanes 5 to 8) after a
15-minute incubation period with increasing concentrations of ATP: 0.05 mmol/L (lanes 1 and 5); 0.5 mmol/L (lanes 2 and 6); 1.0 mmol/L (lanes 3 and 7) and 2.0 mmol/L (lanes 4 and 8).
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It should be realized, however, that the identity of the (membrane)
protein(s) involved in the collapse of membrane lipid asymmetry is
still obscure, and these proteins may escape detection by the
phosphotyrosine Western blots. Although our observations rule out that
lipid scrambling requires stimulus-response coupled tyrosine
phosphorylation, it cannot be excluded that the putative protein(s)
involved in scramblase activity are already phosphorylated before cell
activation. Such involvement of constitutively phosphorylated proteins
may explain why red blood cells gradually lose their ability to undergo
Ca2+-induced lipid scrambling on prolonged ATP
deprivation.17,18
While impaired tyrosine phosphorylation does not underlie defective
Ca2+-induced lipid scrambling in erythrocytes from Scott
syndrome, it cannot be ruled out that the impaired tyrosine
phosphorylation results from defective scrambling. To address this
question, experiments were performed using R5421, which was found to
inhibit Ca2+-induced lipid scrambling in erythrocytes and
platelets. R5421 was found after screening a variety of organic
compounds using both the prothrombinase assay and the assay for
scramblase activity described in Materials and Methods. Its
pharmacology and mechanism of action is as yet unknown.
Figure 3 (upper panel) shows the effect of
increasing concentrations of R5421 on the
Ca2+-ionophore-induced prothrombinase activity in intact
normal erythrocytes. An IC50 of 35 µmol/L was found,
while complete inhibition was observed at a concentration of 100 µmol/L. Similar results were obtained when intact erythrocytes were
replaced by resealed ghosts. R5421 at concentrations up to 200 µmol/L
did not inhibit the prothrombinase assay in the presence of artificial
lipid vesicles. As shown in Fig 3 (lower panel), R5421 causes a
dramatic decrease in tyrosine phosphorylation on
Ca2+-influx into normal erythrocytes (lanes 1 and 2). To
investigate whether R5421 is a direct inhibitor of the major tyrosine
kinase in erythrocytes (or activator of tyrosine phosphatase), we
measured the effect of R5421 on tyrosine phosphorylation in `white'
ghosts incubated with ATP. As shown in Fig 3 (lower panel, lanes 3 to 6), no appreciable difference relative to ghosts without R5421 (Fig 2)
was found. Again, these findings do not rule out a possible effect of
R5421 on cytosolic kinase or phosphatase activities.
The present data may indicate that Ca2+-induced scrambling
of lipids facilitates protein tyrosine phosphorylation in intact erythrocytes. Because lipid scrambling is associated with membrane unpacking,25 it can be speculated that this increases
susceptibility of tyrosine residues for kinases. However, it can at
present not be excluded that erythrocytes from Scott syndrome (or
normal cells treated with R5421) are affected at an early step that
leads to both the collapse of lipid asymmetry and tyrosine
phosphorylation.
Originally, impaired lipid scrambling in Scott syndrome was discovered
in the patients' platelets.4 These platelets do not
develop appreciable procoagulant activity on activation with Ca2+-ionophore and have a strongly diminished procoagulant
activity in response to the combined action of collagen and
thrombin.6 As shown in Fig 4,
Scott platelets in response to various agonists consistently show a
lower degree of phosphorylation in comparison to healthy controls. This
effect is much less pronounced than observed for the patients'
erythrocytes, but this may be due to the presence of a wide variety of
different tyrosine kinases in platelets,26,27 which may
obscure a decreased activity of a single kinase. Also, platelets show a
significant extent of constitutive tyrosine phosphorylation.
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| Fig 4.
Tyrosine phosphorylation in normal (lanes 1 to 4) and
Scott syndrome platelets (lanes 5 to 8). Lanes 1 and 5 represent
unstimulated platelets; lanes 2 and 6, platelets stimulated with 4 nmol/L thrombin; lanes 3 and 7, platelets treated with 70 nmol/L
thapsigargin and subsequently activated with 4 nmol/L thrombin; lanes 4 and 8, platelets activated with 10 µg/mL collagen.
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Consistent with the observations in intact erythrocytes, R5421 also
inhibits prothrombinase activity in platelets stimulated with several
agonists including thrombin and Ca2+-ionophore
(IC50, 23 µmol/L). To further assess the effect of R5421
on scramblase activity in platelets, we used a continuous assay
recently described by Williamson et al,21 which allows monitoring real time movement of a fluorescent lipid analog, NBD-PS, from the inner to the outer leaflet of the plasma membrane. As shown in
Fig 5, preincubation of NBD-PS-loaded
platelets with 50 µmol/L R5421 inhibited
Ca2+-ionophore-induced transbilayer movement of PS by more
than 90% (curves a and b). For comparison, no scrambling of NBD-PS was found when Scott platelets were stimulated with
Ca2+-ionophore (Fig 5, curve c). Subsequent experiments
showed that the extent of inhibition by R5421 was time-dependent, with
50% inhibition obtained at 20 µmol/L for 60 minutes (Fig 5, insert). The inhibitory effect of R5421 could not be reversed by washing the
platelets, suggesting that R5421 is an irreversible inhibitor of the
scramblase.
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| Fig 5.
Effect of R5421 on Ca2+-dependent
scrambling of NBD-PS in human platelets stimulated with
Ca2+-ionomycin. Platelets were preincubated with 50 µmol/L R5421 for 60 minutes. At arrow 1, dithionite (5 mmol/L) was
added; at arrow 2, ionomycin (1 µmol/L); at arrow 3, CaCl2 (1 mmol/L); and at arrow 4, Triton X100 (1%). Curve
a: control platelets in the absence of R5421, curve b in the presence
of R5421. Curve c represents Scott platelets stimulated with
Ca2+-ionophore. Insert: effect of preincubation time with
R5421 (20 µmol/L) on rate of scrambling.
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Unlike the slightly diminished phosphorylation patterns observed with
Scott platelets, incubation of normal platelets with R5421 (100 µmol/L) does not cause appreciable changes in tyrosine phosphorylation patterns obtained on activation with a variety of
agonists. However, inhibition of scramblase activity by these high
levels of R5421 is not complete (compare curves b and c in Fig 5). If
this would be accompanied by a lower extent of inhibition of (a
single?) kinase, the effect of this compound may no longer be visible
on Western blots.
Of note, as in erythrocytes, aminophospholipid translocase activity in
platelets was not affected by R5421 up to a concentration of 100 µmol/L.
In summary, we found that the impaired Ca2+-induced lipid
scrambling in Scott syndrome is accompanied by a diminished tyrosine phosphorylation, an effect that was most pronounced in the patients' erythrocytes. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott syndrome cells is an epiphenomenon, possibly associated with defective lipid scrambling, but is not causal to this
defect. The data do not exclude the possibility that deficient lipid
scrambling in Scott syndrome directly affects tyrosine phosphorylation or that a step required for collapse of lipid asymmetry and tyrosine phosphorylation is aberrant.
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FOOTNOTES |
Submitted July 14, 1997;
accepted October 28, 1997.
Supported in part by Grant No. NHS 93.166 from The Netherlands Heart
Foundation (The Hague) (to W.M.J.V.).
The authors wish to dedicate this report to the memory of Mary
Ann Scott.
Address reprint requests to Edouard M. Bevers, PhD, Department of
Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht
University, PO Box 616, 6200 MD Maastricht, The Netherlands.
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.
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Abstract
Introduction
Abstract