©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Stimulation of Procoagulant Activity in Human Erythrocytes
ATP DEPENDENCE AND THE EFFECTS OF MODIFIERS OF STIMULATION AND RECOVERY (*)

Dwight W. Martin (§) , Jolyon Jesty

From the (1) Division of Hematology, Department of Medicine, State University of New York, Stony Brook, New York 11794-8151

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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] (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.

In both erythrocyte and platelets, elevation of cytoplasmic Ca 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.


EXPERIMENTAL PROCEDURES

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 NaHPO, 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.

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.

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 ( dA/ 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) .

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 (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 dA/ 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.


RESULTS

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 dA/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 mAmin.



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.

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.


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.

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.


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-induced Procoagulant Activity

Fig. 4 shows the Ca-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.

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 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.




DISCUSSION

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. 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.

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 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.

The direct or indirect relation of membrane PIP 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.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants HL-45955 and DK-19185. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 516-444-2059; Fax: 516-444-7530.

The abbreviations used are: Chromozym-TH, D-Phe-L-Pro-Arg- p-nitroaniline; D-FPRck, D-Phe-L-Pro-L-Arg-chloromethylketone; PS, phosphatidylserine; PC, phosphatidylcholine; BSA, bovine serum albumin; RBC, red blood cell(s); NEM, N-ethylmaleimide; N, N, N`, N`-tetraacetic acid; PIP, phosphatidylinositol 4,5-bisphosphate.


ACKNOWLEDGEMENTS

We thank Ann Lorenz and Cheryl Martin for excellent technical assistance.


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