Clinical Science (1991)80, 205-21 1
205
Alterations of platelet membrane microviscosity in essential
hypertension
KIM-HANH LE QUAN SANG, TI-&&SE MONTENAY-GARESTIER* AND MARIE-AUDE
DEVYNCK
Department of Pharmacology,CNRS SDI 6 1670, Faculty of Medecine Necker, Paris and *INSERM U 201, Museum National
&HistoireNaturelle, Paris, France
(Received 9 April/l2 September 1990; accepted 24 September 1990)
SUMMARY
INTRODUCTION
1. The metabolism of blood platelets, taken as an
accessible model of excitable cells, has been reported to
be altered in hypertension. Most of the identified alterations concern the functions of various plasma membrane
constituents.
2. A possible modification of membrane microviscosity was investigated by 1,6-diphenyl-l,3,5-hexatriene
and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene fluorescence depolarization. In order to determine
whether or not the membrane structures probed by these
indicators were related to platelet physiological functions,
the cytosolic free Ca2+concentration was determined in
parallel.
3. At physiological temperature, the fluorescence
anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyl1,3,5-hexatriene was decreased in untreated hypertensive
patients (0.276 k 0.002 versus 0.288 k 0.002, n = 23 and
22, P<O.OOl), indicating a lowered microviscosity at the
lipid-water interface of cell membrane. It correlated
inversely with blood pressure ( P < 0.001) and cytosolic
free CaZ+concentration ( P < 0.030). On the contrary, 1,6diphenyl-1,3,5-hexatriene fluorescence anisotropy was
observed to vary with sex but not with blood pressure.
4. These results suggest that structural membrane
modifications may participate in the various functional
abnormalities observed in platelets from hypertensive
patients.
Essential hypertension has been proposed by several
authors to be associated with (or to result from) hyperplasia and hypertrophy of vascular smooth muscle cells
[ 13 and altered cell membrane metabolism including
changes in ion transport and signal transduction [2-51. An
alteration of membrane structure is one of the various
mechanisms possibly involved. Changes in membrane
fluidity have indeed been shown to interfere with various
cell functions such as cell differentiation and proliferation,
accessibility of receptors, and modulation of membrane
transport and enzyme activities [6, 71. The abnormal
functions of membrane proteins in hypertension could
thus result in part from changes in their lipid environment
[8]. Such a hypothesis is supported by the high association
between lipid abnormalities and hypertension [ 91, the
hypotensive effect of dietary unsaturated fatty acids [lo],
and the effects of in vivo or in vitro changes in membrane
lipids on cell Ca2+handling [ 111 or various Na+ transport
systems [12,13].
Various methodologies have been developed to study
membrane fluidity: the most frequently used are e.s.r. and
fluorescence depolarization of probes embedded in the
membrane. Up to now, alterations in membrane microviscosity in hypertension have been investigated in only a
few studies: two of them concern the erythrocyte membrane. The rate of lateral diffusion of pyrene in the
erythrocyte membrane of patients with essential hypertension was reduced both in the lipid bilayer and in the
region of annular lipid compared with that of normotensive patients [ 141. The characteristics of 5-nitroxystearate, a spin-label agent, were also altered, indicating
that in the membrane region probed by this compound,
the fluidity of erythrocytes was lower in essential hypertension [ 151. In these two studies, interestingly, these
modifications were not observed in patients with secondary hypertension. As far as we know, there is only one
study concerning another type of cells: Naftilan et al. [ 161,
in 1986, reported in a preliminary observation on a small
Key words: cytosolic Ca2+,essential hypertension, fluorescence depolarization, membrane fluidity, platelets,
thrombin.
Abbreviations: DPH, 1,6-diphenyl-1,3,5-hexatriene;
PW,
platelet-rich plasma; TMA-DPH, 1-[4-(trimethy1amino)phenyll-6-phenyl-1,3,5-hexatriene.
Correspondence:Dr Kim-Hanh Le Quan Sang, Department
of Pharmacology, Faculte de Medecine Necker, 156 Rue de
Vaugirard,F-75015 Pans, France.
206
K.-H. Le Quan Sang et al.
number of patients that platelet membrane fluidity
analysed with 1,6-diphenyl-l,3,5-hexatriene(DPH)fluorescence depolarization was also decreased in hypertension.
The aim of the present study was to investigate whether
or not essential hypertension is associated with an altered
membrane fluidity. Membrane characteristics of platelets
unstimulated ex vivo were analysed with the use of two
fluorescent probes, DPH and 1-[4-(trimethy1amino)phenyll-6-phenyl-1,3,5-hexatriene(TMA-DPH),localized
respectively in the lipid membrane core and at the
lipid-water interface. Cytosolic free Ca2+concentration,
taken as an index of cell function, was measured in
parallel.
MATERIALS AND METHODS
Patients and subjects
Forty-five subjects were included in this study. Their
characteristics are given in Table 1. Twenty-three patients
had mild to moderate hypertension diagnosed on supine
diastolic blood pressure (Korotkoff phase V ) and World
Health Organization Criteria. Hypertension was considered to be essential on the basis of the classic biological
tests and timed intravenous pyelography. None had other
known associated diseases. All of them were normolipidaemic, without dietary restriction. Sixteen hypertensive patients had their antihypertensive treatment
interrupted for at least 4 weeks before blood sampling
and seven patients had never been treated. The 22
healthy normotensive subjects were free of any medication.
Twenty millilitres of venous blood were collected
between 09.00 and 11.00 hours in tubes containing
2.73% (w/v) citric acid, 4.48% (w/v) trisodium citrate and
2% (w/v) glucose as anticoagulant ( 1/ 10 v/v). The
platelet-rich plasma (PRP)was obtained by centrifugation
at 530 g, for 5 min at 20°C. Platelet sizes in the whole
blood and PRP averaged 8.5 kO.l fl and 8.6-tO.3 fl
( n= 4), respectively, indicating that no loss of platelets of
small sizes had occurred during PRP preparation. PRP
contamination by leucocytes averaged 1.3 k 0.3 x 1O4
cells/ml ( n= 5, compared with 4.5 k 0.6 X lo* platelets/
ml). All measurements were performed within 3 h of
blood sampling.
Measurement of membrane fluidity
For membrane fluidity studies, platelets from half of
each PRP sample were diluted five times with a medium
containing (in mmol/l): NaCl 145, KCl 5, Ca(NO,), 1,
MgSO,, 1, Na,HPO, 0.5, glucose 5 and 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid 10, pH 7.4 at
37°C (medium A). They were then centrifuged at 270 g
for 15 min at 20°C and resuspended in the same medium.
Their density was adjusted by Rayleigh scattering to a
value corresponding to a density of 2.5 X lo7cells/ml and
a turbidity of less than 0.2 absorbance units at 430 nm.
Under these conditions and with the microcuvettes used
(5 mm x 5 mm), the depolarization due to light scattering
was minimized. When platelets were lysed by sonication,
light scattering decreased further but DPH anisotropy did
not significantly change ( - 0.003 f 0.004, n = 3).
Stock solutions of TMA-DPH and DPH (Molecular
Probes) were prepared in dimethylformamide at a concentration of 1O-, mol/l. Dilutions were performed
extemporaneously. For labelling, TMA-DPH and DPH
were directly added in washed platelet suspensions at a
final concentration of 5 x
mol/l. Under these conditions, aggregation induced by ADP and thrombin were
observed not to be significantly altered by these probes.
In some experiments, to examine the role of extracellular Ca2+or of cell activation, platelets were suspended in
medium A without addition of Ca2+.
The fluorescence measurements were performed in a
Perkin-Elmer LS-5B spectrofluorimeter equipped with
polarizers. The excitation and emission monochromators
were positioned at fixed wavelengths, 350 and 430 nm,
respectively with a 5 nm bandwidth in excitation and in
emission. The temperature of the platelet suspension was
fixed at 37.0"C and accurately measured with a digital
thermometer. The platelet suspension was not stirred
during dye labelling and fluorescence measurements.
Before each experiment, signals from unlabelled cells
were first recorded to determine the blank values. Under
these conditions of incorporation, the output signals from
platelets labelled with TMA-DPH and DPH were 40fold greater than those obtained with unlabelled cells.
Under vertical excitation light (subscript V), the
fluorescence anisotropy is defined as:
R=
(&-
G ( I ,) v
(4)"+ 2 (311 1"
with a correcting factor
G=- ( I l l )ti
(I1)II
where the subscript H refers to horizontally polarized
excitation light. The subscripts II and 1 refer to components of the emitted light parallel and perpendicular to the
direction of polarization of the excitation beam.
The kinetics of TMA-DPH and DPH incorporation in
platelets membranes were studied first. For Th4A-DPH,
the fluorescence intensity increased immediately, then
decreased slightly to reach equilibrium and remained
stable for at least 30 min. In contrast, DPH fluorescence
intensity increased progressively in platelet membranes to
reach a stable value at 15 min. Polarization of fluorescence measurements was thus carried out at a fixed time
after marker addition: 10 min for TMA-DPH and 15 min
for DPH at 37°C.
The measurements of membrane fluidity were
made on each blood sample in duplicate.
The inter-assay variabilities (changes in fluorescence
anisotropy measured twice on platelets from the same
subject at intervals ranging from 2 weeks to 3 months)
were 1% and 0.5% for TMA-DPH and DPH, respectively
(n=4).
Membrane microviscosity and hypertension
The intra-assay variability (changes in fluorescence
anisotropy of the same platelet sample measured twice in
the same experiment) was 0.5% and 0.3% for TMA-DPH
and DPH, respectively ( n = 26).
Measurement of platelet cytosolic Ca2 concentration
+
In order to approach as nearly as possible the cytosolic
free Ca2 concentrations in vivo, platelets were loaded in
plasma with the fluorescent indicator Quin-2. A characteristic of Quin-2 is the requirement of higher intracellular concentrations than more recent indicators such as
Fura-2 and the introduction of a strong Ca2+buffer inside
the cell. Thereby it reduces and slows down Ca2+
transients. In this study, where only basal concentrations
were measured, this feature constitutes an advantage as it
stabilizes the initial Ca2+ values. Quin-2 also offers the
advantages of a low cell leakage rate (less than lO%/h)
and of the absence of known fluorescent Ca2+-insensitive
intermediate forms. Quin-2 was incorporated under conditions where the intracellular dye concentration did not
exceed 0.5 mmol/l [ 171, thereby minimizing the accumulation of unwanted hydrolysis compounds which, at
higher concentrations, may alter cell functions [18,19].
The inter-assay variability (platelet cytosolic free Ca2
concentration measured twice in the same subject at intervals ranging from 1 week to 2 months) averaged 4%
( n = 12).
The intra-assay variability (the same platelet measured
twice in the same experiment) was 5% ( n = 7).
Cytosolic free Ca2+ measurements were performed in
parallel on the remaining half of each PFW sample and in
duplicate as previously described [ 171.
+
+
Effects of thrombin on platelet membrane fluidity and
cytosolic CaZ+concentration
The effect of thrombin (Hoffmann-La Roche, Basel,
Switzerland)was studied in parallel on platelet membrane
fluidity and cytosolic free Ca2+ concentration. In the
fluidity studies, thrombin (0.01 unit/ml) was added to
platelet samples before TMA-DPH or DPH labelling. For
Ca2+ measurements, the same dose of thrombin was
added in washed platelets and fluorescence intensity was
recorded instantaneously.
RESULTS
TMA-DPH and
membranes
*
Results are expressed as means SEM. Differences
between the normotensive and hypertensive groups and
between the values in the presence and absence of
thrombin were calculated by using Mann-Whitney Utests and paired Wilcoxon tests, respectively. Relationships between the two variables were assessed by
Spearman correlations.
in
platelet
When compared with that of age-matched normotensive subjects, the fluorescence anisotropy of TMADPH (R,,,,,,)
was significantly decreased in the platelets of hypertensive patients, indicating that hypertension
was associated with an increased membrane fluidity in the
TMA-DPH local microenvironment (Fig. 1). This difference remained significant when male and female subjects were considered separately (Table 1).
No difference in TMA-DPH fluorescence anisotropy
was found between hypertensive patients who had never
been treated and patients who had interrupted their antihypertensive therapy for at least 4 weeks (0.273 0.004
versus 0.278 k 0.002, n = 7 and 16, respectively).
Fluorescence anisotropy of TMA-DPH was inversely
correlated with blood pressure ( r =0.543, P < 0.001, with
a slope of - 3.5 0.8 x
mmHg-', significantly different from zero) (Fig. 2). This correlation remained valid
when male and female groups were analysed separately
( r = -0.536, n=24, P=0.009 and r = -0.443, n=21,
*
*
0.31 (1 ( a )
o.20(1
x
a
2
2
..
I
A
B
Y
I
8
0.27b
Statistical analysis
incorporation
Hypertension and TMA-DPH anisotropy in platelets
'2 0.29 -
Responses of platelets to thrombin were recorded on
an aggregometer (Dual Aggro meter, Chronolog Corporation, Margency, France).
DPH
As TMA-DPH and DPH are fluorescent only when
incorporated into cell membranes and not in the medium,
their incorporation into platelets from normotensive subjects and hypertensive patients was evaluated by the total
fluorescence intensity in a given number of platelets. No
significant difference in the amount of dye incorporated
per platelet was found between the two blood pressure
groups (19.6k1.8 versus 16.2* 1.7, n = 7 and 9 for
TMA-DPH and 22.8 k 1.8 versus 22.2 k 2.7, n = 5 and 9
for DPH, in platelets from normotensive and hypertensive subjects, respectively).This indicates that each of
these probes, considered separately, had access to the
same membrane areas in platelets from the two blood
pressure groups.
c)
Platelet shape change and aggregation
207
0.25
fi
A
x
a
2
.g
0.18
'
c)
z
8 0.16
0.14
C
EHT
( n= 2 2 ) ( n= 2 3 )
(b)
a
C
EHT
(n=17) (n=17)
Fig. 1. Individual values of TMA-DPH ( a ) and DPH ( 6 )
fluorescence anisotropies in platelets of normotensive
subjects (C) and hypertensive patients (EHT).The mean
and SEM values are given in Table 1.( a )P< 0.001.
208
K.-H. Le Quan Sang et al.
Table 1. Biological parameters and platelet characteristics of normotensive subjects and hypertensive patients
Results are expressed as means fSEM. The number of hypertensive patients studied was reduced to 17 (eight males, nine
females) and that of normotensive subjects to 17 (seven males, 10 females)for DPH anisotropy measurements. Statistical
significance: * P < 0.05, **P< 0.01 and ***P< 0.001 compared with values obtained in normotensive subjects; t P < 0.05
and ttP< 0.01 compared with values obtained in male subjects of the same blood pressure group.
Normotensive subjects
Hypertensive patients
n
Age (years)
Mean blood pressure
(mmHg)
TMA-DPH fluorescence
anisotropy in platelets
DPH fluorescence
Females
Males and
females
Males
23
42f3
119 4***
14
42+3
118 3***
9
41 + 6
120 16***
Males and
females
Males
Females
22
38f2
93+2
10
36 + 2
94f4
12
39+4
91 f 2
+
0.276 + 0.002***
+
0.274 + 0.002**
0.279 k 0.003*
0.288 f0.002 0.288 f 0.003
0.288 f 0.003
0.170 k 0.002
0.166 f 0.002
0.173 k0.002tt 0.174f0.002 0.167 k0.003
0.177kO.002t
on average in females than in their male counterparts. The
same difference was observed between female and male
normotensive or hypertensive subjects (Table 1).
0.321
0 0
Relationship between platelet membrane fluidity and
cytosolic free Ca2
0
+
0
8
0.24
60
80
100
120
140
0
160
Mean blood pressure (mmHg)
Fig. 2. Correlation between individual values of TMADPH steady-state fluorescence anisotropy in blood platelets (37°C)and mean blood pressure in 22 normotensive
subjects ( 0 )and 23 untreated hypertensive patients ( 0 ) .
P < 0.001.
P = 0.045), but was not significant when only normotensive subjects were considered ( r = - 0.193).
When the hypertensive patients were restricted to
those who had never been treated, the correlation
between TMA-DPH fluorescence anisotropy and blood
pressure remained significant ( r = - 0.430, n = 29,
P = 0.01 9).
Fluorescence anisotropy of TMA-DPH did not correlate with plasma cholesterol content ( r = - 0.172).
Hypertension and DPH anisotropy in platelets
In contrast, DPH fluorescence anisotropy (R,,,) did
not differ between platelets from the two blood pressure
groups (Fig. 1), but was observed to be significantly higher
To investigate whether or not the hypertensionassociated structural features revealed by TMA-DPH and
DPH anisotropies were accompanied by alterations of
plasma membrane functions, the cytosolic free Ca” concentration which, in ‘unstimulated’ platelets, depends largely
on plasma membrane transport systems, was measured
concomitantly. The values measured by the fluorescent
chelator Quin-2, although higher than those obtained with
the Fura-2 chelator, were similar to those observed with
Indo-1 [20]. In agreement with previous results [17, 211,
platelet cytsolic free Ca2+ concentration was oberved to
be significantly higher in hypertensive patients than in
normotensive subjects (201f 7 versus 17 1 3z 6 nmol/l,
P< 0.01). Individual values of platelet cytosolic free Ca2+
concentration were inversely correlated with the corresponding values of TMA-DPH fluorescence anisotropy
[r= -0.319, P<O.O30, with a slope of - 1.23f
0.46 x
(nmol/l)-’, significantly different from zero]
(Fig. 3), but the significance of correlation disappeared
when calculated at constant blood pressure.
The relationship between DPH anisotropies and
cytosolic free Ca2+ concentrations did not reach significance(r= -0.258, n=37, P=0.118).
Influence of extracellular and intracellular Ca2
concentrations on TMA-DPH fluorescence anisotropy
+
To examine whether the hypertension-associated decrease in TMA-DPH fluorescence anisotropy could be
directly or indirectly due to the rise in platelet cytosolic
free Ca2 concentration, TMA-DPH fluorescence anisotropy was measured under two conditions where platelet
cytosolic free Ca2 concentrations were respectively
+
+
Membrane microviscosity and hypertension
I
0.30 -
0.32
2
*
8
8
s:
8
'5
3 o.28 -
8
8
8'
P
8
4
?
8
8
8
8
0.26
0.24l. I ' ' . ' . ' ' I . ' . ' . ' .
75 100 125 150 175 200 225 250 275 300
Platelet [Caz+](nmol/l)
Fig. 3. Relationship between individual values of TMADPH fluorescence anisotropy (37°C) and cytosolic free
Ca2+ concentration in 'unstimulated' platelets in 22
normotensive subjects and 23 untreated hypertensive
patients. P< 0.030.
decreased and increased. In the virtual absence of extracellular Ca2+ (about
mol/l, corresponding to the
value obtained without addition of Ca2+or EGTA which
may modify the membrane properties [22]),platelet cytosolic free Ca2+ concentration was found, as previously
reported [23], to be lower than in the presence of a
physiological extracellular concentration of Ca2+ (90 f 8
versus 189 f 10 nmol/l, n = 8, P < 0.001). The TMA-DPH
fluorescence anisotropies of the same platelet samples
distributed in two media with or without addition of 1
mmol/l Ca2+ were similar (0.283 f 0.004 versus
0.28 1 f0.002, in the presence and virtual absence of
extracellular Ca2 respectively).
Another means of modulating platelet cytosolic free
Ca2+concentration was to treat platelets with low concentrations of thrombin (external Ca2+ 1 mmol/l). In the
presence of thrombin (0.01 unit/ml), neither aggregation
nor shape change was observed but the cytsolic free Ca2+
concentration rose from 1 6 8 f 17 to 3 0 6 f 3 0 nmol/l
( n =6, P = 0.01). In agreement with previous reports [24],
this low dose of thrombin was found not to modify the
fluorescence anisotropy of either Th4A-DPH or DPH
(0.274 f0.003 versus 0.275 k 0.003, n = 6, for TMADPH and 0.174 k 0.002 versus 0.172 k 0.002, n = 5, for
DPH, in the presence and absence of thrombin, respectively).
+
DISCUSSION
The present study establishes that (i)blood platelets from
hypertensive patients are characterized by a modified
structure associated with a decreased fluorescence anisotropy of TMA-DPH, (ii) this membrane defect is present
only in the external leaflet of plasma membrane and not in
all cell membranes, and (iii)this structural characteristic is
associated with high blood pressure and elevated cytosolic free Ca2 concentration.
+
209
The analysis of membrane structure has been performed by using two fluorescent dyes with different
properties. Both DPH and its derivative TMA-DPH are
lipophilic probes with enhanced fluorescence properties
when inserted into biological membranes. Their incorporation is governed by a rapid partition equilibrium between
the medium (where they are not fluorescent) and the cell
membrane. TMA-DPH is assumed to be anchored to the
polar heads of the phospholipids by its positive trimethylamino group and to label essentially the glycerol backbone region and the fatty acyl chain region probably as far
down as C,-C,, [25]. In contrast, DPH is preferentially
located in the hydrocarbon core of the membrane and
progressively diffuses into all hydrophobic regions of the
cell. When excited by a polarized light, their fluorescence depolarization depends on their mobilizy in the
membrane and thereby reflects the rate and the angle of
rotation [26]. The measured data predominantly reflect
the structural order of membrane lipids, a high structural
order representing a high degree of packing or a high
mutual affinity for the lipid chains.
Previously reported values for DPH steady-state
anisotropy in 'unstimulated' platelets from normotensive
subjects range between 0.128 and 0.198 at 37°C [16,
27-31]. This variability is likely to be due to a large part
to the turbidity of platelet suspensions [32]. In the present
study, the density of platelets was carefully controlled to
minimize this depolarizing effect and to allow valid
comparison between groups. As far as we know, TMADPH fluorescence depolarization in human platelets has
been studied by only two groups and the anisotropy
values reported were close to those observed in the
present study [24,30].
Platelets from hypertensive patients had a significantly
decreased TMA-DPH fluorescence anisotropy, indicating
a lowered structural order at the external part of the
plasma membrane. This decrease, proportional to the rise
in blood pressure, was independent of sex or age. In
contrast, DPH fluorescence anisotropy was not modified
with hypertension but was observed to be significantly
higher in female than in male subjects. As all of these
patients were normolipidaemic, these differences could
not be explained by pathological alterations in the major
classes of blood lipids.
It is not possible from these data to determine whether
the decrease in TMA-DPH fluorescence anisotropy in
hypertensive patients is causally linked to high blood
pressure OJ is a consequence of hypertension. Studies are
in progress to determine whether it is modified after antihypertensive therapy. However, it does not result from
previous treatment, since similar values were observed in
patients who had never been treated and in those whose
treatment had been interrupted for at least 4 weeks. As
platelet activation and shape change have been reported
to induce a rise in TMA-DPH fluorescence anisotropy
[241, the lowered values observed in hypertensive patients
are not likely to result from such an activation in vivo or
ex vivo.
A physiological relevance of this alteration is, however,
supported by the observation that the higher the cytosolic
210
K.-H. Le Quan Sang et al.
free Ca2+concentration the more marked was the alteration in TMA-DPH fluorescence anisotropy. Membrane
fluidity is indeed known to modulate several membrane
functions including Ca2+ transport [33-361 and, conversely, cell Ca2+may modulate enzymic activities such as
that of calpain, a neutral proteinase which specifically
degrades membrane proteins (membrane-bound cytoskeletal proteins, protein kinase C, adrenergic receptors,
actin-binding proteins) [37]. In order to investigate the
mutual relationship between platelet Ca2+ handling and
TMA-DPH fluorescence anisotropy, the cytosolic free
CaZ+ concentration was modified in two ways: by a
change in the external Ca2+ concentration and after a
moderate stimulation by a low dose of thrombin. The lack
of change in TMA-DPH fluorescence anisotropy under
these conditions indicates that the environment of the
probe was not modified after short-term variations in
cytosolic free Ca2+ concentration. This, however, does
not imply that a chronic rise in cytosolic free Ca2+
concentration in vivo could not be involved in the
apparition of this structural membrane abnormality.
The absence of changes in DPH fluorescence anisotropy in platelets from hypertensive patients contrasts
with the previous observations in genetically hypertensive
rats indicating that, in erythrocyte ghosts, platelets, heart
plasma membrane and synaptosomes, the membrane
structures probed by DPH had an enhanced microviscosity [38-401. This discrepancy might be related to the
well-known species difference in membrane composition
or to the heterogeneity of diseases regrouped under the
name of hypertension. The present findings also disagree
with the preliminary observation by Naftilan et al. [16]
that DPH fluorescence polarization was enhanced in
platelets from hypertensive patients. The fact that, in their
study, the DPH fluorescence anisotropy values measured
in platelets from normotensive subjects were much lower
than those measured by other authors may account for this
difference [27, 29-31]. Another possible explanation
might be related to the sex-dependent difference in DPH
fluorescence depolarization. DPH fluorescence anisotropy values measured in females were indeed significantly higher than those measured in males,
independently of their age and blood pressure status.
Similar results have been previously reported in male and
female rats [40]. Sex differences have also been observed
in the aggregation responses of human platelets to ADP
and adrenaline [41,42] without a significant participation
of oestrogens [41,42] and in membrane functions such as
those of various Na+-transport systems [43, 441. The
present observation also implies that sex has to be taken
into account when comparing membrane properties by
using DPH fluorescence depolarization.
Although proportional to blood pressure, the
structural alteration of the platelet membranes revealed,
by TMA-DPH fluorescence anisotropy was not present in
all of the hypertensive patients studied. This probably
illustrates the heterogeneity of the disease. The exact
nature and the functional consequences of these
membrane abnormalities remain to be further defined.
ACKNOWLEDGMENTS
We gratefully acknowledge the help of Dr J. L. Elghozi
and Professor P. Meyer (Necker Hospital, Paris) and Dr J.
Levenson and Professor A. Simon (Broussais Hospital,
Paris), who selected the hypertensive patients.
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