From the
The human erythrocyte membrane is generally considered to have
no procoagulant activity. The normal membrane is characterized as
having an asymmetric distribution of phospholipid species such that
negatively charged and aminophospholipids are predominantly located on
the inner leaflet of the membrane bilayer. Elevation of cytoplasmic
Ca
The rate of conversion of prothrombin to thrombin in the Factor
V- and Factor Xa-dependent step of the coagulation cascade is greatly
enhanced by a negatively charged phospholipid membrane surface.
Normally this surface is provided by damaged endothelial cells or
activated platelets. Erythrocytes are usually not procoagulant since
the negatively charged phospholipids (predominantly phosphatidylserine)
reside exclusively on the inner leaflet of the membrane bilayer,
creating an asymmetric phospholipid distribution between the outer and
inner leaflets
(1, 2, 3, 4, 5) .
A similar asymmetric distribution of phospholipid exists in
nonactivated platelets
(6) . Earlier studies, which focused on
the distribution of endogenous phospholipid, suggested that the
phospholipid asymmetry was largely due to phospholipid-cytoskeletal
interactions
(7, 8) . Initial studies of lipid asymmetry
were limited by cumbersome protocols requiring side-specific enzymatic
degradation or specific chemical modification of membrane
phospholipids, often with reagents that were not totally membrane
impermeable. Because of the extended time required for phospholipid
hydrolysis or labeling, these methods largely focused on static states
of phospholipid distribution. More recently, studies using
membrane-incorporated exogenous spin labeled, fluorescently labeled,
and radioactively labeled phospholipid analogues have indicated that
phospholipid asymmetry appears to be actively maintained under normal
conditions by an ATP-dependent process
(1, 9, 10, 11, 12) . The
apparent phospholipid specificity
(11, 13, 14) and the sensitivity of this process to sulfhydryl reagents
(13, 14, 15, 16, 17) , vanadate,
vanadyl, cytoplasmic [Ca
In both erythrocyte and
platelets, elevation of cytoplasmic Ca
Experiments concerning the
temperature dependence of the assay were done on a Hewlett-Packard
8452A spectrophotometer equipped with a thermostatted cell holder of
1-cm pathlength.
In
practice, ideal parabolic data sets are rarely seen. In particular,
initial rate conditions do not hold for the cleavage of the chromogenic
substrate by thrombin; when high concentrations of Factor Xa or
phospholipid are assayed, a significant proportion of the thrombin
substrate is consumed even over relatively short time intervals.
Although it is possible to raise the Chromozym-TH concentration, this
results in significant inhibition of Factor Xa, reducing the
sensitivity of the assay. Rather then restrict the usable range of the
assay, nonideal data are managed as follows.
First, absorbance data
that exceed the zero time absorbance by more than 0.8 absorbance units
are discarded. The relatively high value of this limit extends the
upper limit of the assay. If it is set lower, e.g. to 0.2
absorbance units, then the number of available data points in samples
of high activity is correspondingly reduced.
Second, the data
analysis program fits the data to a cubic function in addition to a
quadratic, and accepts the function that produces the better fit, as
measured by
Fig. 2A shows a standard curve generated
using varying concentrations of 50:50 PS:PC vesicles and demonstrates
the sensitivity of the assay for procoagulant phospholipid and its wide
usable range. Fig. 2 B shows a standard curve generated
from phospholipid vesicles (10 µM) of varied PS:PC.
Fig. 3
shows a standard curve
using a sonicated preparation of red blood cells, expressed as
nanoliters of sonicated packed cells. The assay of red blood cells,
particularly intact cells of very low procoagulant activity, is a
severe test of a chromogenic assay because hemoglobin, which is present
in the cells at about 4 mM, absorbs strongly at the absorption
maximum of p-nitroaniline. A partial solution to this problem
is to operate the microplate reader or spectrophotometer in
dual-wavelength mode, with a reference wavelength in the 450-490
nm region. The major part of the solution, however, lies in the
sensitivity of the assay. For example, the procoagulant activity of
freshly prepared RBC, which is of the order of 0.5% of that attainable
by sonication, is quite measurable using 10 nl of cells in the assay.
Inspection of the above figures
revealed that even in the presence of high concentrations of
translocase inhibitors there is still a partial recovery from the
maximal levels of procoagulant activity after adding EGTA.
Fig. 9
compares the relative recoveries from activation of
procoagulant activity in systems treated with the three translocase
inhibitors. We find that there appears to be two components to the
recovery: 1) an initial rapid phase that represents about 40% of the
recovery and is unaffected by the translocase inhibitors (increasing
inhibitor concentration does not influence this result) and 2) a second
component that represents more than half of the recovery, which is
inhibited by the translocase inhibitors and may be ascribed to the
translocase. It is useful to keep in mind that each of these inhibitory
reagents affects the Ca
Taken together, our data suggest the following conclusions.
1) The phospholipid translocase is not responsible for the loss of
asymmetry due to elevated cytoplasmic Ca
Prior studies
(22) indicated that the outward movement of
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled PC, PE, and PS
in the absence of elevated cytoplasmic Ca
The direct or indirect relation of membrane
PIP
We thank Ann Lorenz and Cheryl Martin for excellent
technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in erythrocytes produces an assortment of
biochemical and structural responses that include diminished
phospholipid asymmetry and an elevation in procoagulant activity.
Maintenance of the normal asymmetric distribution of phospholipid
species is believed to be largely mediated by a phospholipid
translocase mechanism. We have utilized a recently developed
single-step kinetic assay of procoagulant activity to investigate the
mechanisms of Ca
stimulation of procoagulant activity
and recovery from the procoagulant state upon removal of
Ca
. This study demonstrated that stimulation of
procoagulant activity by elevated cytoplasmic Ca
is
greatly diminished in ATP-depleted erythrocytes. Phospholipid
translocase inhibitors failed to fully inhibit recovery from the
procoagulant state after removal of Ca
. The data
indicate that recovery of endogenous lipid from a procoagulant
configuration may not be entirely mediated by the phospholipid
translocase. Additionally, the data are inconsistent with the
phospholipid translocase mediating the Ca
-induced
elevation of procoagulant activity, although the involvement of other
protein(s) is indicated.
]
(18) , and
temperature
(14, 19) have lead to the conclusion that
this process involves a functionally specific protein or protein
complex termed the aminophospholipid translocase (flippase)
(20) . It has been shown that this process has the affinity and
velocity to rapidly transport aminophospholipids from the external
leaflet to the internal leaflet. However, it has also been shown that
the process displays a different transport velocity depending on the
type of phospholipid or phospholipid analog
(21, 22) ,
and therefore the capacity and velocity of the process with regards to
endogenous phospholipids remain uncertain.
results in a
loss of phospholipid asymmetry and a concomitant rise in procoagulant
activity. The mechanism by which cytoplasmic Ca
alters membrane phospholipid asymmetry remains unknown. Elevated
erythrocyte cytoplasmic Ca
has been associated with
numerous phenomena including the release of microvesicles
(17, 23, 24) , KCl efflux and cell shrinkage
(25, 26, 27) , breakdown of
polyphosphoinositides with associated enzymatic activations
(28, 29, 30) , transamidase activation and
protein aggregation
(31, 32) , elevated proteolytic
activity (calpain), and the cleavage of cytoskeletal anchoring proteins
2.1 and 4.1
(23, 24, 31, 33, 34, 35) .
To date, the evidence suggests that neither destruction of
phospholipid-cytoskeleton interactions nor the
Ca
-induced inhibition of the phospholipid translocase
could be responsible for most of the Ca
-induced
destruction of phospholipid asymmetry prompting the suggestion for the
existence of a third mechanism or flippase process, which responds to
cytoplasmic Ca
(20, 36, 37) .
Using a recently developed single-step kinetic assay of procoagulant
activity, we have initiated investigations of the response of the
endogenous phospholipid to elevated cytoplasmic Ca
.
Our studies demonstrate that Ca
-induced stimulation
of procoagulant activity in human red blood cells is greatly diminished
in ATP-depleted cells and suggest that a significant fraction of the
recovery of endogenous phospholipid back to the asymmetric state upon
removal of elevated Ca
may not be mediated by the
normally characterized aminophospholipid translocase.Our data also
argue that a protein moiety may be involved in
Ca
-induced procoagulant activity.
Materials
D-Phe-L-Pro-Arg- p-nitroaniline
(Chromozym-TH)(
)
is a product of Boehringer
Mannheim.
D-Phe-L-Pro-L-Arg-chloromethylketone
(D-FPRck) was obtained from Calibiochem (La Jolla, CA).
Phosphatidylserine (PS), phosphatidylcholine (PC), bovine serum albumin
(BSA, grade V, fatty acid-free), N-ethylmaleimide (NEM),
penicillin G, streptomycin, and A23187 were obtained from Sigma. A23187
was stored in isopropyl alcohol prior to use. Vanadyl sulfate was
purchased from Aldrich. All other purchased reagents were research
grade. Factor Xa was prepared from purified human Factor X
(38) by the method of Jesty and Nemerson
(39) .
Prothrombin was prepared as a byproduct of the Factor X preparation
after separation from Factors IX and X on dextran sulfate-agarose
(38) .
Preparation of Factor Va
Factor Va, which is
required as a cofactor in the activation of prothrombin by Factor Xa
was prepared from 1500 ml of bovine plasma (Pel-Freez, Rogers, Ark) by
a modification of the method of Nesheim et al.(40) .
Three changes were made in their procedure. 1) DEAE-Sephadex A50 was
used instead of DEAE-cellulose. 2) After absorbing the plasma proteins,
the DEAE-Sephadex was packed in a column (5 15 cm) and eluted
at 150 ml/h with a linear 2500-ml gradient, from 0.1 to 0.35 M
NaCl, in 20 mM imidazole-HCl, pH 7.5, 5 mM
CaCl
, 5 mM benzamidine. 3) The improved
purification provided by gradient elution of the DEAE-Sephadex meant
that only the octyl-Sepharose chromatography was necessary to obtain
Factor V of >90% purity by gel electrophoresis; chromatography on
phenyl-Sepharose was not performed. The pooled active fractions from
the octyl-Sepharose column ( ca. 36 mg of Factor V in 20 ml of
elution buffer) were treated immediately with 60 nM human
thrombin for 3 min at 37 °C. The thrombin inhibitor
D-FPRck was then added to a concentration of 1
µM. After a 10-min incubation at 37 °C, the resulting
Factor Va was dialyzed at 4 °C once against 1 liter of
Tris-buffered saline, 1 mM CaCl
, and then twice
against 500 ml of 50% glycerol in Tris-buffered saline, 1 mM
CaCl
. The product, which is free of single-chain Factor V
by gel electrophoresis, was stored at -22 °C. Its
concentration was determined from the absorbance at 280 nM(40) . The total yield was 32 mg. This material contains no
detectable thrombin activity, nor does it contain residual
D-FPRck, as judged by its ability to inhibit thrombin (0.1
nM) in a chromogenic assay.
Red Blood Cells (RBC)
Red blood cells were
prepared fresh from the blood of normal volunteer donors. Blood (10 ml)
was collected into 0.1 ml of 1.36 M sodium citrate and
centrifuged at 2500 g for 15 min. The plasma and buffy
coat were removed by aspiration. The resulting RBC were washed twice by
gently suspending in 10 ml of 0.1% BSA in 120 mM NaCl, 2.7
mM KCl, 0.5 mM NaH
PO
, 1.0
mM MgCl
, 0.1 mM EGTA, 5 mM
glucose, 50 units/ml penicillin G, 50 µg/ml streptomycin, and 50
mM Hepes, pH 7.4 (termed HTT buffer). With each wash, the
cells were recentrifuged as above, and the supernatant and pellet
surface (
2 mm) were aspirated. The packed cells were resuspended to
give a 10% hematocrit in HTT buffer, unless otherwise indicated, and
stored on ice until used. Many of the experiments were also conducted
using buffer with high concentrations of KCl to reduce
Ca
-induced microvesiculation
(23) . This
buffer was termed HHTHK and had the same composition as HHT except that
KCL = 100 mM and NaCl = 22.7 mM.
ATP-depleted Red Blood Cells
RBC were prepared as
described above, except they were washed and resuspended in HTT without
glucose. A 10% hematocrit was incubated at 37 °C overnight to
metabolically exhaust the cellular ATP. Alternatively, the ATP in the
red cells was depleted by incubating the cells for 3 h at 37 °C in
HTT without glucose but containing 10 mM NaN and
50 mM deoxyglucose.
NEM-treated RBC
RBC were prepared and washed as
described above using HTT buffer with glucose. The cells were
resuspended to a 10% hematocrit in HTT buffer containing 5 mM
NEM. The suspension was incubated 30 min at 37 °C and washed twice
with HTT buffer without NEM. The cells were stored on ice until used.
Determination of Intracellular ATP
At varied
points throughout the experimental protocols, the ATP content of the
RBC was determined using the luciferase-luciferin method
(41) .
Coupled Assay of Factor Xa
PS:PC vesicles were
used as the lipid source to develop the assay. All assays except those
on temperature dependence were done at room temperature (20-25
°C) on a VMax microplate reader (Molecular Devices, Menlo Park,
CA). Each microplate well contained the following final concentrations:
0.28 µM prothrombin, 25 µM PS:PC, 0.4
mM Chromozym-TH, and the Factor Xa sample (20 µl, varying
concentration). The well buffer was 100 mM NaCl, 1 mM
EDTA, 0.1% BSA, 50 mM Tris, pH 7.5 (termed Tris-buffered
saline buffer). To start the assay, 50 µl of a mixture containing 1
µg/ml Factor Va and 20 mM CaCl was added,
making a total volume in each well of 200 µl. The absorbance,
A
, was read at intervals over 5-15 min,
depending on the Factor Xa activity of the sample. The plate-reading
frequency, which can be as high as eight readings/min on this
instrument, was adjusted to obtain 30-60 absorbance readings over
the course of an assay. In general, the larger the number of readings,
the more accurate the results.
Prothrombin Activation Assay of Red Blood
Cells
The single-step coupled chromogenic assay described above
was used to measure the procoagulant activity of the RBC preparations.
Each microplate well contained the following final concentrations: 0.28
µM prothrombin; 0.3 mM Chromozym-TH; and 0.625
ng/ml Factor Xa. At the indicated timed intervals, a 10-µl sample
of RBC suspension (usually 10% hematocrit) was added to 490 µl of
Tris-buffered saline buffer. This 1:50 dilution of the blood suspension
was immediately vortexed, and 20 µl of the dilution were
immediately added to an assay well. The assay well immediately received
a 50-µl addition of a mixture of Factor Va (1 µg/ml) and
Ca (20 mM) (final concentrations, Factor Va
= 0.3 µg/ml, Ca
= 6.25
mM). The contents of the well were mixed, and the absorbance
at 405 nm was read at 20-s intervals for 15 min. The assay was
performed with the microplate reader operating in dual-wavelength mode,
with a reference wavelength of 450 nm to minimize light scattering and
hemoglobin interferences. When performed by a practiced technician,
less than 1 min elapses between aliquoting the 10-µl sample of RBC
suspension and obtaining the first absorbance reading. The
computer-controlled multireading capacity of the apparatus and software
permits new data samples to be initiated as prior assays are
continually read. Thereby, the kinetics of generation and loss of
procoagulant activity can be followed.
Data Analysis
If initial-rate conditions are met,
then thrombin is generated linearly with time in these assays, the rate
of its generation being a function of Factor Xa or phospholipid
concentration. At the same time as it is generated, thrombin cleaves
Chromozym-TH, the rate of cleavage being proportional to thrombin
concentration. Under perfect conditions, where thrombin generation is
linear with time, the generation of p-nitroaniline is
parabolic with time. The absorbance data are fitted to a quadratic,
A = A+ Bt + Ct
, where the value of C,
which equals
( d
A/ dt
),
will be proportional to the Factor Xa or phospholipid concentration.
B is the initial rate of color generation and should be very
close to 0. Under near ideal conditions where B = 0, it
may not be necessary to fit the data to a polynomial, and C can be obtained by plotting A against
t
, as described by Carson
(42) .
(sum of squares of residuals divided by
degrees of freedom). In this case, where A =
A
+ Bt +
Ct
+ Dt
, it is
the initial value of d
A/ dt
at t = 0 that is obtained from the value
of C. Only negative cubic coefficients are allowed in fitting
to the cubic function, representing a decay of the second derivative
with time.
Data Analysis Software
This report does not
concern reader-control software, since that is instrument-specific. The
data analysis program is available on request (contact J. Jesty) for
IBM personal computers or compatible machines (running Microsoft or IBM
DOS, versions 2 and above). The program also permits editing of raw
absorbance data. This is occasionally necessary when a bubble is
introduced into a microplate well on sample addition and later burst,
causing aberrant early absorbance values. If the user's
spectrophotometer or microplate reader can write absorbance-time data
sets to an ASCII file, then all that is required to use the program is
a short translation program to put the data into the format that the
data analysis requires. The full format specifications are available
along with an example of code (QuickBasic) for writing such data to a
compatible file.
Sensitivity
It should be noted for all assays that
20-µl samples (Factor Xa, phospholipid, and sonicated RBC) have
been used throughout. Although more expensive in terms of experimental
volumes, the assay of low concentrations can be substantially improved
by using 100-µl or even larger samples. In addition, the
sensitivity can be significantly increased by simply raising the Factor
Xa concentration in the well. For example, raising it to 100
pM increases the sensitivity approximately 4-fold. In addition
to these methods, increasing temperature increases the sensitivity, the
value of C increasing 4-fold over a 10 °C rise in
temperature. The raw data ( i.e.dA/ dt
) are
generally given and based on calibration curves that are strictly
linear; these can be converted to units of nmol of thrombin/min when
multiplied by the factor 0.0004 (nmol of
thrombin-min
A
). To avoid rate
differences due to variations in cell concentration between different
suspensions, in some instances the rates were normalized to the maximal
rate obtained with a sonicated sample of the RBC suspension of
interest.
The Assay of Procoagulant Activity
Fig. 1
illustrates the range of Fig. 1assay response curve,
which may be analyzed by our protocol. The three curves show typical
raw absorbance data from the assay of Factor Xa at concentrations
ranging over 3 orders of magnitude (4.4, 0.44, and 0.044 fmol). (For
illustrative purposes Factor Xa was varied, as opposed to phospholipid,
to permit overlapping absorbance origins; however, qualitatively
similar curves can be obtained with varied phospholipid concentration.)
The lines show the best-fit quadratic or cubic function, from
which a value of the parameter C at time = 0 is derived
(see ``Experimental Procedures''). Occasional gaps in the
absorbance data, as seen in linesA and B,
occur when new samples are added to a microplate that is already being
read. The simultaneous assay of several samples is a key strength to
the use of the microplate reader. The fit of the data below A = 0.8 ( dashedline) is excellent, and it
is reflected in the low error estimates (Fig. 1,
legend). For Factor Xa concentrations in the range of
5-100 pM, the error in the value of C, which is
estimated in the fitting routine is typically less than 5%. Below 5
pM, the error in C is typically < 0.1
mA/min
.
Figure 1:
Analysis of nonlinear
p-nitroaniline generation. Twenty-microliter samples of Factor
Xa at 220 ( A), 22 ( B), and 2.2 ( C)
pM were assayed as described under ``Experimental
Procedures'' using a microplate reader at room temperature.
Absorbance readings less than ( A +
0.8), shown by the dashedline, were fitted to
quadratic and cubic equations, the better fit being shown by the
lines, to determine the initial value
d
A/dt
at t = 0.
Values of the square-term coefficients, 1000
C (see
``Experimental Procedures''), and estimates of the standard
error were, respectively, 69.2 ± 3.2, 6.61 ± 0.11, and
1.17 ± 0.004
mA
min
.
The Assay of Procoagulant Phospholipid
Negatively
charged phospholipids support the activation of prothrombin by factors
Xa and Va, and can thus be measured using this method. We have
successfully measured the generation of procoagulant activity in both
platelets and red blood cells upon stimulation by various means. To
measure procoagulant phospholipid, the assay is run with a fixed
concentration of Factor Xa (22 pM in the microplate well), so
that the phospholipid becomes limiting in thrombin generation.
Standardization can be done with a preparation of negatively charged
vesicles such as sonicated vesicles of PS and PC. Alternatively, when
one is studying the procoagulant activity of cells, a suitable standard
is a sonicated or frozen-thawed preparation of the cells in question.
Intact unstimulated cells generally have low procoagulant activity
because they have little or no negative phospholipid in the outer
leaflet of the membrane. Freshly prepared red blood cells, for example
typically show procoagulant activity of about 0.5% of their sonicated
maximum.
Figure 2:
The assay of procoagulant lipid.
A, duplicate 20-µl samples of phospholipid vesicles, 50:50
in PS:PC, containing 0.25-2500 nM phospholipid (PS
+ PC), were assayed as described under ``Experimental
Procedures.'' The line is a linear regression of the data
in the range of 0.63-125 nM. B, preparations of
PS:PC vesicles were made at constant phospholipid concentration and
varying PS:PC ratio from a PS fraction of 0-0.5 (50%). The
concentration of total phospholipid in each assay was 12.5
µM; conditions otherwise were as described under
``Experimental Procedures.''
Three observation in Fig. 2 B should be noted. First,
the plot of activity against PS content has a positive x intercept, indicating that below a small but definite PS content
vesicles show no detectable procoagulant activity. Second, the activity
at 100% PC confirms the lack of significant contamination of the PC
with acidic phospholipids. Third, it is clear that at low PS fraction,
the procoagulant activity of the vesicles is not the same as that
produced by an equivalent concentration of PS in 50:50 PS:PC vesicles;
in fact the line in Fig. 2 B is significantly steeper
than that of Fig. 2 A. We note that in most experimental
studies of cells, the total phospholipid concentration remains
constant, and it is the fraction of active acidic phospholipids that is
measured, i.e. analogous to Fig. 2 B. If we
assume that the PS content of an average cell membrane is of the order
of 15% of total phospholipid, the data show that the assay easily
supports measurement of just 1% exposure of PS on the cell surface
( i.e. PS:PC = 0.02, assuming random distribution of
PS). Obviously biological membranes are more complex than PS:PC
vesicles, and the incremental sensitivity of the assay to PS exposure
at the cell surface may be different (though not necessarily less) than
that observed with vesicles.
Figure 3:
The assay of RBC. Fresh RBC at a
hematocrit of 10% were sonicated and diluted to give the equivalent
packed-cell volumes (per 20-µl sample) over the range of 0.004 to
10 nl. Duplicate assays of procoagulant phospholipid were done as
described under ``Experimental Procedures.'' The inset shows a linearly scaled subrange of total data set. Lines are by linear regression. The log-scaled line was derived
from data between 0.005 and 0.5 nl of RBC.
Ca
Fig. 4
shows the
Ca-induced Procoagulant
Activity
-induced generation of procoagulant activity under
varied incubation conditions. The data demonstrate that in intact red
blood cells, Ca
-induced activity is dependent upon
incubation time, Ca
concentration, and the
concentration of the Ca
ionophore A23187. Increasing
either A23187 or extracellular Ca
enhances the rate
of production of procoagulant activity. Elevation of procoagulant
activity has been observed in cells incubated with as low as
10
µM Ca
. Incubations of up to 60 min in
the presence of Ca
and A23187 produced a continual
rise in procoagulant activity, longer times were not studied. At given
conditions of [Ca
], [A23187], and
time, the level of procoagulant activity varied between different RBC
preparations. No correlation could be made between the level of
activity and specific individual blood donors. The lone addition of
either Ca
or A23187 to the extracellular media did
not stimulate procoagulant activity. The data are consistent with a
rise in cytoplasmic [Ca
] leading to the
generation of procoagulant activity.
Figure 4:
The generation of procoagulant activity
was dependent on time, [Ca], and
[A23187]. Red blood cells were prepared as described under
``Experimental Procedures'' except that the cells were
finally resuspended in HHT buffer, which did not contain glucose or
BSA. The cells were incubated in the presence of Ca
and A23187 at the indicated concentrations at 37 °C, and the
procoagulant activity was assayed at the indicated times as described
under ``Experimental Procedures.'' Each data point represents
the mean of duplicate determinations ± S.D. indicated by the
errorbars.
Exposure of
Ca-stimulated red cells to EGTA resulted in a loss of
procoagulant activity (Fig. 5). Activity decreased with a t of about 3-5 min, which is consistent with reported values
for the phospholipid translocase-mediated flop of PS from the outer
leaflet to the inner leaflet of the cell membrane
(43) . The
rate of activity loss could be slowed somewhat by exposing
Ca
-loaded cells to BSA, which removes
membrane-incorporated A23187. Under those conditions, reducing
cytoplasmic [Ca
] relied more substantially
upon Ca
pump activity. Qualitatively, stimulation and
recovery from procoagulant activity could be linked to the appearance
and disappearance of 2,4,6-trinitrobenzenesulfonic acid-reactive
aminophospholipid on the cell surface (data not shown).
Ca
tracer studies showed that the extent
of procoagulant activity and recovery could be qualitatively correlated
to the influx and efflux of Ca
and time of exposure
of the cytoplasm to elevated Ca
. However, the time
course of removal of cytoplasmic Ca
was much more
rapid than that of recovery from procoagulant activation.
Ca
is reported to be an inhibitor of the translocase
(18) , and the data are consistent with a requirement that
cytoplasmic Ca
be either removed or sequestered in
order for recovery to occur. The Fig. 5 inset shows that
after a 30-min exposure to EGTA, about 20% of the stimulated activity
remains. A significant fraction of this residual activity appears to be
due to procoagulant activity released into the supernatant, since it
cannot be removed by centrifugation (200
g, 5 min).
The level of this residual activity varied between experiments and most
likely represents microvesicles, which are known to be released upon
incubating red cells with Ca
(17, 23) . Conducting experiments in HHTHK buffer
([K
] = 100 mM) resulted in
an
50% reduction in the residual activity. There were no others
significant changes in the stimulation and recovery curves obtained in
HHTHK buffer.
Figure 5:
EGTA induced recovery from
Ca-stimulated procoagulant state. Red blood cells
were prepared as described under ``Experimental Procedures.''
The cells were incubated 30 min at 37 °C in the presence of 10
µM A23187 and Ca
at 10 µM (
) and 100 µM (
). At the end of the
incubation ( t = 0 in the above figure) 1 mM
EGTA and 5 mg/ml BSA were added and procoagulant activity was assayed
at the indicated times as described under ``Experimental
Procedures.'' Inset, replot of data expressed as activity
at time = t divided activity at time =
0.
The Effects of Phospholipid Translocase
Inhibitors
Addition of vanadate to the cell suspension either
before or after exposure of the cells to Ca and
ionophore did not substantially affect the level of procoagulant
activity induced by Ca
(Fig. 6). However,
vanadate did at least partially inhibit the EGTA-induced recovery from
the procoagulant state. This is consistent with reports that vanadate
inhibited the aminophospholipid translocase
(11, 18) .
Procoagulant activity was not stimulated either by the addition of
vanadate in the absence of A23187 and Ca
or by the
addition of A23187 in the absence of Ca
(data not
shown). Since exposure to vanadate neither inhibited nor stimulated the
procoagulant response of the cells to Ca
, the data
indicate that inhibition of the translocase does not affect
Ca
induction of procoagulant activity and suggest
that the translocase does not mediate the generation of the
procoagulant state.
Figure 6:
Vanadate
inhibits the EGTA-induced recovery from the procoagulant state. Red
blood cells were prepared as described under ``Experimental
Procedures'' and treated as follows: () the cells were
incubated 30 min at 37 °C in the presence of 10 µM
A23187 and 100 µM free Ca
, at the end of
the incubation ( t = 0) 1 mM EGTA and 5 mg/ml
BSA were added and the procoagulant activity was assayed at the
indicated times as described under ``Experimental
Procedures.'' (
), the same as (
) except that, after a
15-min incubation in presence of A23187 and Ca
, 200
µM sodium vanadate was added and the incubation continued
an additional 15 min followed by EGTA and BSA addition as in (
).
(
) same as (
) except that the cells were first incubated
with 200 µM sodium vanadate 15 min, 37 °C prior to
adding A23187 and Ca
. A 30-min incubation in the
presence of A23187 and Ca
followed by EGTA and BSA
were subsequently performed as in (
).
Vanadyl (VO), has also been
reported to be a potent inhibitor of phospholipid translocase activity
(18) . We found that vanadyl is also a potent inhibitor of
Ca
-stimulated procoagulant activity (Fig. 7).
Vanadyl also inhibited the rate of recovery from the procoagulant state
after adding EGTA. This was more apparent when the activity data were
expressed as a fraction of the maximal activity obtained prior to
addition of EGTA as discussed below (see Fig. 9).
Figure 7:
Vanadyl inhibits
Ca-stimulation of procoagulant activity and EGTA
induced recovery. Red cells were prepared as described under
``Experimental Procedures'' and treated as follows: (
)
the cells were incubated 30 min at 37 °C in the presence of 10
µM A23187 and 100 µM free
Ca
, at the end of the incubation ( t =
0) 1 mM EGTA and 5 mg/ml BSA were added and the procoagulant
activity was assayed at the indicated times as described under
``Experimental Procedures.'' (
) same as (
) except
the incubation media also contained 500 µM vanadyl sulfate
in addition to Ca
and A23187. The initial
procoagulant activity of the red blood cells prior to the 30-min
incubation is indicated at t =
-30.
Figure 9:
The effects of NEM, vanadate, and vanadyl
on the EGTA induced recovery from Ca-stimulated
procoagulant activity. Red blood cells were washed and incubated
± 5 mM NEM as described in the legend to Fig. 5.
(
), (
), and (
), cells not treated with NEM;
(
), cells treated with NEM. For (
) and (
), the cells
were incubated 30 min at 37 °C in the presence of 10
µM A23187 and 100 µM free
Ca
, at the end of the incubation ( t =
0), 1 mM EGTA and 5 mg/ml BSA were added, and the procoagulant
activity was assayed at the indicated times as described under
``Experimental Procedures.'' (
), same as (
)
except the cells were incubated with 200 µM sodium
vanadate in addition to Ca
and A23187. (
), same
as (
) except the cells were incubated with 500 µM
vanadyl sulfate in addition to Ca
and A23187. The
initial procoagulant activity of the red blood cells prior to the
30-min incubation is indicated at t =
-30.
NEM is
another reagent that has been reported to substantially inhibit
translocase activity
(9) . Cells pretreated with NEM showed a
substantially greater response to Ca stimulation of
procoagulant activity (Fig. 8).
Figure 8:
NEM enhances Ca
induction of procoagulant activity. Red blood cells were washed as
described under ``Experimental Procedures'' and resuspended
at 10% hematocrit in HHT ± 5 mM NEM. The cells were
incubated 30 min at 37 °C, washed twice in HHT (no NEM) and
resuspended at 10% hematocrit in HHT. (
), cells not treated with
NEM. (
) and (
), cells treated with NEM. For (
) and
(
), the cells were incubated 30 min at 37 °C in the presence
of 10 µM A23187 and 100 µM free
Ca
, at the end of the incubation ( t =
0), 1 mM EGTA and 5 mg/ml BSA were added and the procoagulant
activity was assayed at the indicated times as described under
``Experimental Procedures.'' For (
), the cells were
incubated as above but without Ca
and A23187. The
initial procoagulant activity of the red cells prior to the 30-min
incubation is indicated at t =
-30.
Typically, NEM-treated cells
developed 3-5-fold as much procoagulant activity in response to
Ca stimulation as did that of non-NEM-treated
controls. In the absence of Ca
, NEM did not affect
the procoagulant state of the cells. A similar augmentation of the
Ca
effect was not observed when cells were treated
with vanadate (Fig. 6), indicating that the NEM effect was not
simply the result of translocase inhibition and probably reflected the
involvement of other proteins. Taken together, the above data
demonstrate that Ca
induction of procoagulant
activity is more complex than merely the inhibition of the
aminophospholipid translocase.
stimulation curves
differently (see Figs. 6-8), yet the recovery curves are
strikingly similar when the data are expressed as a fraction of the
maximal activity suggesting separate, uncoupled mechanisms of
stimulation and recovery.
The Effects of ATP Depletion
The phospholipid
translocase is reported to be an ATP-dependent system
(10, 11) . Fig. 10compares
Ca-stimulated procoagulant activity and recovery in
ATP-replete and ATP-depleted cells. We find that
Ca
-induced procoagulant activity is greatly reduced
in cells that have been ATP depleted. This difference was not due to a
reduced capacity for procoagulant activity in the ATP-depleted cells
since sonicated samples of ATP-replete and ATP-depleted cell
suspensions had similar procoagulant activity. The maximal activity at
time 0 for the ATP-replete cells was about 20% of that obtained with
sonicated ATP-replete cells, whereas the maximal activity for
ATP-depleted cells was about 3% of that obtained with sonicated
ATP-depleted cells. The same results were obtained whether
ATP-depletion was accomplished by overnight incubation in glucose-free
media as in the data of Fig. 10or by a 3-h incubation in
deoxyglucose media (data not shown). If recovery in the ATP-depleted
system is analyzed relative to the maximal stimulation, we observe
recovery curves that are similar to those seen in Fig. 9for NEM,
vanadate, and vanadyl. This is consistent with a portion of the
recovery being due to the phospholipid translocase, which is inhibited
in the ATP-depleted state.
Figure 10:
The effects of ATP-depletion on
Ca-stimulated procoagulant activity and EGTA-induced
recovery. Red blood cells were washed and resuspended at 10% hematocrit
in HHT buffer ± 5 mM glucose. The cells were incubated
overnight (
16 h) at 37 °C in vessels sealed with Parafilm®.
(
), cells incubated in HHT with glucose; (
), cells incubated
in HHT without glucose. Subsequently, the cells were incubated 30 min
at 37 °C in the presence of 10 µM A23187 and 100
µM free Ca
, at the end of the incubation
( t = 0), 1 mM EGTA was added, and the
procoagulant activity was assayed at the indicated times as described
under ``Experimental Procedures.'' The initial procoagulant
activity of the red cells prior to the 30-min incubation is indicated
at t = -30.
. Vanadate
and NEM at concentrations that should have been more than sufficient to
inhibit the translocase did not inhibit Ca
stimulation of procoagulant activity, and NEM actually enhanced
the stimulation. 2) Stimulation and recovery from the procoagulant
state involve different mechanisms, both of which have a requirement
for ATP. 3) Recovery from the procoagulant state is not entirely
mediated by the phospholipid translocase, since a substantial degree of
recovery can occur even in the presence of translocase inhibitors.
were
mediated by an ATP-dependent, sulfhydryl reagent-sensitive process
consistent with the involvement of a protein-mediated mechanism. The
apparent ATP requirement for stimulation of
Ca
-induced procoagulant activity may in fact reflect
a requirement for a phosphorylated moiety and not a metabolic
requirement. Recently, Sulpice et al.(44) reported
that Ca
-induced redistribution of spin-labeled
phospholipids was enhanced by the presence of PIP
in
studies involving red blood cells and red blood cell inside-out
vesicles. In preliminary studies (data not shown), we have observed
that Ca
-induced procoagulant activity is enhanced in
ATP-depleted red blood cells if the phosphatase inhibitors, vanadate or
okadaic acid, are present during ATP depletion. The inclusion of
vanadate during ATP depletion resulted in roughly a doubling of the
Ca
-induced procoagulant activity, whereas inclusion
of okadaic acid produced about a 50% increase in
Ca
-induced procoagulant activity. Our studies argue
for the participation of protein(s) in Ca
-induced
procoagulant activity since NEM-enhanced Ca
-induced
activity in ATP-replete red blood cells (see Fig. 8) and okadaic
acid, which is a specific inhibitor of protein phosphatases
(45) , enhanced Ca
-induced activity in
ATP-depleted red cells.
to Ca
-stimulated procoagulant
activity is unclear. PIP
is metabolized slowly by the red
blood cell
(46, 47) . Sulpice et al.(44) report that after 40 h of glucose starvation at 37 °C,
about 90% of the red cell PIP
had been depleted. In
Fig. 10
, we demonstrate a substantial reduction in
Ca
-induced procoagulant activity after a 16-h
incubation in glucose-free media, after which substantial PEP
would have still been present in the cells. Furthermore, we see
identical results when ATP is depleted by a 3-h incubation in the
presence of deoxyglucose. Only a minor amount of PIP
would
have been metabolized during this short incubation, yet about 90% of
the Ca
-induced procoagulant activity was suppressed.
These data suggest the involvement of a phosphorylated species that
undergoes a more rapid rate of dephosphorylation than that of
PIP
. Obviously more investigations are needed to resolve
the molecular details of these processes.
, phosphatidylinositol 4,5-bisphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.