Journal of Thrombosis and Haemostasis, 9: 1049–1055
ORIGINAL ARTICLE
Red blood cell phosphatidylserine exposure is responsible
for increased erythrocyte adhesion to endothelium in central
retinal vein occlusion
M . - P . W A U T I E R , * à 1 E . H É R O N , § 1 J . P I C O T , * à Y . C O L I N , * à O . H E R M I N E – and J . - L . W A U T I E R * * *
*INSERM, UMRS665, Paris; Université Paris Diderot-Paris7, Paris; àInstitut National de la Transfusion Sanguine, Paris; §Centre Hospitalier
National dÕOphtalmologie des Quinze-Vingts, Paris; –Hôpital Necker, Paris; and **Groupe Hospitalier Lariboisière, Paris, France
To cite this article: Wautier MP, Héron E, Picot J, Colin Y, Hermine O, Wautier JL. Red blood cell phosphatidylserine exposure is responsible for
increased erythrocyte adhesion to endothelium in central retinal vein occlusion. J Thromb Haemost 2011; 9: 1049–55.
Summary. Background: Retinal vein occlusion (RVO) is a
common cause of permanent loss of vision. The pathophysiol-
ogy is uncertain, although enhanced erythrocyte aggregation
and blood hyperviscosity have been observed. Increased red
blood cell (RBC) adhesion has been associated with vascular
complications in several diseases, such as sickle cell anemia,
diabetes mellitus or polycythemia vera. Objectives: To measure
RBC adhesion to endothelial cells in RVO and to explore the
molecular basis of the adhesion process. Patients and methods:
We assessed RBC adhesion to endothelial cells and adhesion
molecule expression among 32 patients with RVO. Patients
with disease known to alter RBC adhesion were excluded
(n = 8), and further investigation was conducted in 20 patients
with central retinal vein occlusion (CRVO) and four patients
with retinal artery occlusion (RAO), compared with 25 normal
subjects. Results: Under static conditions, adhesion of CRVO
RBC was increased (135 ± 7 · 102 mm)2) compared with
RAO RBC (63 ± 5 · 102 mm)2) (P < 0.01) and normal
control RBC (37 ± 3 · 102 mm)2) (P < 0.001). Under flow
conditions, CRVO RBC adhered in greater numbers than
normal RBC (P < 0.001). Phosphatidylserine (PS) expression
on CRVO RBC was 2.4-fold higher than controls and
correlated with RBC adhesion (P = 0.001). In static condi-
tions, specific antibodies against PS receptor and annexin V
inhibited RBC adhesion. In flow conditions, the inhibitory
effect was in the same range with antibodies but was 2-fold
higher with annexin V. Conclusion: Increased CRVO RBC
Correspondence: Jean-Luc Wautier, INTS 6 rue Alexandre Cabanel
75015, Paris, France.
Tel.: +33 1 44 49 30 01; fax: +33 1 43 06 50 19.
E-mail: jlwautier@ints.fr
Received 20 October 2010, accepted 19 February 2011
1
These authors contributed equally to this work.
 2011 International Society on Thrombosis and Haemostasis
DOI: 10.1111/j.1538-7836.2011.04251.
adhesion is mediated by PS RBC and endothelial PS receptor.
This phenomenon may be one of the factors responsible for
CRVO.
Keywords: adhesion molecules, endothelial cell, phosphatidyl-
serine, red blood cell, retinal vessels.
Introduction
Retinal vein occlusion (RVO) is a common cause of
permanent visual loss and is the fifth cause of unilateral
blindness in an Australian population-based study [1]. The
most frequent and less severe type, branch retinal vein
occlusion (BRVO) [2], is possibly driven by a mechanical
factor because it generally occurs at an arteriovenous crossing
[3]. The most rare and sight-threatening form, central retinal
vein occlusion (CRVO) [2], remains of unknown pathophys-
iology [4,5]. Aging, arterial hypertension and glaucoma are
the only well-established risk factors for RVO [6–8]. Despite a
number of studies, thrombophilic risk factors have not been
strongly associated with RVO, which suggests a very limited
role of coagulation or anticoagulation factors in the patho-
physiology of the disease [5,9]. In contrast, substantial data
exist suggesting that blood hyperviscosity is an important risk
factor. Several blood viscosity parameters have been shown to
be increased in RVO patients compared with normal subjects,
including a higher hematocrit, higher whole blood viscosity,
reduced red cell deformability, and enhanced index of
erythrocyte aggregation [7,10–14]. We recently reported that
about 27% of patients with CRVO exhibit spontaneous in
vitro growth of erythroid precursors in the absence of any
detectable myeloproliferative disorder [15]. Red blood cells
(RBCs) from patients with polycythemia vera have an
abnormal CD239 (Lu/BCAM) phosphorylation, which is
responsible for abnormal interactions with endothelial cells
[16]. It was previously demonstrated that abnormal erythro-
cyte adhesion correlated with retinopathy in diabetes mellitus
1050 M.-P. Wautier et al

[17] and with vascular occlusion in sickle cell anemia [18]. Table 1 Clinical characteristics of the study population
These results prompted us to study RBC adhesion to Normal
endothelial cells in CRVO. Patients subjects
Variable (n = 20) (n = 25)

Patients and methods Men/Women 10/10 14/11

Age at the time of CRVO, 54 [21–75] 47 [32–63]
median [range], years
Study population
Body mass index, median [range], 25 [18–29] 20 [18–24]
The study was approved by the Internal Review Boards from kg m)2
participating institutions and signed informed consent was Arterial hypertension, n (%) 9 (45) 0
Dyslipidemia, n (%) 4 (20) 0
obtained in accordance with the Declaration of Helsinki. Diabetes 0 0
Between March 2007 and January 2009, 32 patients with a Current smokers, n (%) 2 (10) 0
history of decreased vision due to retinal vascular occlusion Bilateral CRVO, n (%) 5 (25)
were enrolled in the study. The general characteristics and a Retinal neovascular 5 (25)
detailed medical history of the patients were taken at enroll- complications, n (%)
Visual acuity* of involved
ment. Ophthalmological data, including a retinal angiography eye, n (%)
for each patient, were reviewed by an ophthalmologist. Retinal £ 1/10 10 (50)
vein occlusion was diagnosed by the presence of retinal 1/10 to £ 3/10 6 (30)
hemorrhages and venous dilation/tortuosity with or without > 3/10 4 (20)
retinal/disc swelling in a defined venous retinal territory: diffuse Chronic open angle glaucoma 0
for CRVO, and located within a hemiretina for hemiretinal *Best corrected visual acuity in case of bilateral CRVO.
vein occlusion (HRVO) or within a retinal quadrant or less for
branch retinal vein occlusion (BRVO). Central retinal artery
Table 2 Laboratory parameters of CRVO patients
occlusion (CRAO) was diagnosed in patients with a history of
sudden vision loss, presence of diffuse retinal pallor with Patients (n = 20)
delayed arterial filling during angiography, and a cherry red
Hematocrit, median % [range] 43.5 [37–50.1]
foveola. We undertook the current blind study to measure Reticulocytes, median, 71.6 [19.5–105.3]
RBC adhesion among RVO patients without knowing the 103 mm)3 [range]
ophthalmological diagnosis. We measured RBC adhesion and Blood platelet count, 260 [184–444]
adhesion molecule expression on RBC. After determining median, 103 mm)3 [range]
White blood cell count, 6.9 [4.6–11.2]
adhesion of RBC and expression of RBC receptor, patient
median, 103 mm)3 [range]
phenotypes were assessed. Two CRVO patients with sickle cell Protein C, protein S, or 1 (5)
anemia and three with diabetes mellitus, which are known antithrombin deficiency, n (%)
causes of increased RBC adhesion, were excluded. Two Antiphospholipid antibody*, n (%) 3 (15)
patients with BRVO and one with HRVO were also excluded Hyperhomocysteinemia 5 (25)
(> 15 lmol L)1), n (%)
to preserve the homogeneity of the study group. The study
Activated protein C resistance. 0
group comprised 20 patients with CRVO (Table 1). Their
laboratory parameters are presented in Table 2. Four patients *Anticardiolipin IgG antibody titer of 12, 19 and 44 UGPL (negative
< 10, undetermined 10–15 UGPL). None had a positive screening for
with CRAO, three women aged 47, 79 and 84 years and a man
anti-b2GP1 antibody or lupus anticoagulant.
aged 95, served as ophthalmological controls. None of the
patients had markers of myeloproliferative syndromes. The
time between diagnosis and blood sampling varied from interassay coefficient of variation was 13% ± 2%) all remain-
1 month to 3 years. Six patients have been tested at different ing patients were tested after storage.
times after occlusion. No patient was taking an anticoagulant,
nine took aspirin and five took troxerutin. The normal healthy Static conditions RBC adhesion was measured under static
controls (n = 25) were recruited for a previous study and are conditions using a radiometric technique as previously
members of the medical or laboratory staff of the different described [20]. Human microvascular endothelial cells
institutions (Table 1). (HMEC-1 Biological Branch Products; CDC, Atlanta, GA,
USA) were cultured in MCDB-131 (Invitrogen, Grand Island,
NY, USA) supplemented with 15% fetal calf serum (D.
Erythrocyte adhesion measurement
Dutscher, Brumath, France), endothelial growth factor
Blood samples were tested after storage at )196 C as (10 ng mL)1) and hydrocortisone (1 lg mL)1) (Invitrogen).
previously described [19]. For the first 10 patients, RBC Washed RBCs were resuspended to hematocrit 25% in Hanks
adhesion (on three occasions, n = 30) was measured just after Balanced Salt Solution (HBSS) plus 0.5% human serum
blood sampling and after storage (see Supporting Informa- albumin labeled with 51chromium, and incubated with HMEC
tion). As the results were similar before and after storage (the for 30 min at 37 C. Non-adhering RBCs were removed by six

 2011 International Society on Thrombosis and Haemostasis


successive washes with HBSS and the remaining adherent
RBCs were lysed with distilled water. Radioactivity in the
lysate was measured in a gamma counter, and adhesion was
then calculated as RBCs per square millimetre. In experiments
performed to identify the molecular basis of adhesion, a subset
of 10 patients, selected as representative of the whole group
according to their levels of adhesion, was tested. Inhibition of
RBC adhesion was assessed using HMEC preincubated with
specific antibodies directed against PS receptor (polyclonal
anti-PS receptor antibody, Sigma, St Louis, MO, USA;
polyclonal anti-human stabilin-2 antibody, R&D systems,
Minneapolis, MN, USA) or against human CD36
(monoclonal anti-CD36/SR-B3; R&D systems) or anti-
human Brain-specific angiogenesis inhibitor-1 (BAI-1; Santa
Cruz Biotechnology, Santa Cruz, CA, USA). Isotype-matched
rabbit IgG, sheep IgG or mouse IgG2b were used as controls as
appropriate. In other experiments, a blocking peptide against
the PS receptor (PSR (L-20) P; Santa Cruz Biotechnology) was
added to HMEC for 1 h at 37 C and included during the
adhesion assay. In some experiments, prior to the adhesion
measurement, RBCs were incubated with recombinant,
purified annexin V (BD Biosciences, San Jose, CA, USA).
Flow conditions Adhesion of RBCs to HMEC under flow
conditions was determined according to a previously published
technique [16]. Briefly, HMEC were cultured in gelatin (2%)
coated glass capillaries (microslides; Camlab Ltd, Cambridge,
UK) for 24 h. Microslides containing confluent HMEC were
mounted on the stage of a video-microscope. The RBC
suspension (hematocrit 0.5%) was perfused through the
microslide at a flow rate equivalent to a wall shear stress of
0.03 Pa for 10 min, followed by washout of non-adherent cells
for 10 min. The wall shear stress was then increased stepwise
(from 0.03 to 0.3 Pa) every 5 min. At each step, adherent RBCs
were video-recorded in 10 consecutive fields (one field measures
0.1 mm2), then were counted using a computerized image
analysis system (Optimas Media Cybernetics, Bethesda, MD,
USA; R & D Vision, Paris, France) and expressed as number
of RBCs per mm2. Inhibition of RBC adhesion was measured
in the presence of the antibodies used in static conditions or
after RBC incubation with annexin V.
Measurement of adhesion molecules and phosphatidylserine
(PS) on RBCs
The expression of the molecules CD31,CD36, CD47, CD49d,
CD71,CD99 (clone TÜ12) and CD239 was assessed using
corresponding specific monoclonal antibodies (Becton Dickin-
son, San Jose, CA, USA) and flow cytometry. Acquisition and
analysis were performed with the BD FACS Diva software
(v6.1.2) on a FACS Canto II flow cytometer (BD Biosciences).
Binding of annexin V-phycoerythrin (PE) (BD Biosciences)
to RBC was measured by flow cytometry to determine PS
exposure. One million washed RBCs were incubated with
annexin V-PE in the presence of either 2.5 mM CaCL2 or
2.5 mM EDTA [21]. The results were expressed as number of
 2011 International Society on Thrombosis and Haemostasis
Central retinal vein occlusion and RBC adhesion 1051
positive RBCs and mean fluorescence intensity (MFI). Blood
samples (five patients and five controls tested on two occasions,
n = 10) were tested the day after collection and also after
storage. The results of PS-positive cells were similar before and
after storage and are detailed in the Supporting Information
(the interassay coefficient of variation was 0.1%). We decided
to test all the patients after storage.
Statistical analysis
Results are presented either as the mean ± standard error of
the mean (SEM), or percentiles (10–90%, 25–75%, and
external values). Statistical significance was determined using
a one-way analysis of variance (ANOVA) followed by the
parametric DunnettÕs test. Correlation coefficient was calcu-
lated using the parametric PearsonÕs test.
Results
RBC adhesion to endothelium
Adhesion of RBC was enhanced in patients with RVO (32
patients, P < 0.001). Patients with CRVO (25 patients)
had higher RBC adhesion than patients with RAO (four
patients) or BRVO (two patients). One patient with hemiretinal
vein occlusion had RBC adhesion similar to patients with
CRVO.
Among the 20 CRVO patients selected for further analysis,
six have been tested at different time intervals: two patients
have been tested 1 and 2 years after the first investigation, one
after 2 years and three after 1 or 6 months. There was no
significant difference between the samples examined at different
times.
Under static conditions, adhesion of RBCs from patients
with CRVO (n = 20) to HMEC was significantly increased
compared with adhesion of normal RBCs (n = 25) and of
RBCs from patients with RAO (n = 4). The mean number of
adhering CRVO RBCs was 135 ± 7 · 102 mm)2 (mean ±
SEM) compared with the adhesion of RAO RBCs (63 ±
5 · 102 mm)2) and normal RBCs (37 ± 3 · 102 mm)2)
(Fig. 1A). A flow-based adhesion assay was used to test the
strength of RBC attachment to HMEC by allowing RBCs to
adhere at low wall shear stress (0.03 Pa) and then analyzing
washout with increasing stress (0.03–0.3 Pa). RBCs from
patients with CRVO adhered in greater numbers and were
more resistant to washout than control RBCs: at 0.07 Pa,
190 ± 8 and 30 ± 4 mm)2 RBCs respectively (P < 0.001).
The number of RBCs remaining adherent after washout at the
highest shear stress (0.3 Pa) was 20 ± 8 mm–2 for the patients
with CRVO and <1 mm–2 for the control group (Fig. 1B).
Adhesion of RBCs measured in previous studies [16,22] in Type
2 diabetic patients (n = 60) and in patients with polycythemia
vera (PV) (n = 38) is also presented in Fig. 1(B). The most
adherent were RBCs from patients with diabetes. PV RBCs
adhere more at low shear stress but similarly at 0.2 and 0.3 Pa
compared with CRVO RBCs.
1052 M.-P. Wautier et al

A Static conditions A
250 ***
***
4
200
RBC (×102)/mm2

PS positive RBC (%)

150 3

100
2
50

0 1
Controls RAO CRVO

B Flow conditions
0
Controls RAO CRVO
800 Diabetic patients B
PV patients 4

PS positive RBC (%)

CRVO
600 Controls 3
RBC/mm2

2
400

200
0
0.03 0.07 0.1 0.2
Shear stress (Pa) washout
Fig. 1. (A) Red blood cells (RBCs) from patients with central retinal vein
occlusion (CRVO) (n = 20) adhere in greater numbers on human
microvascular endothelial cells (HMEC) than do RBCs from patients with
retinal artery occlusion (n = 4) or RBCs from healthy volunteers (con-
trols, n = 25) (***P < 0.001) under static conditions. The unbroken line
within the box represents the median; the dotted line represents the mean.
The vertical lines extending beyond the boxes indicate the 25% and 75%
percentiles, while the horizontal bars outside the boxes represent the 10%
and 90% percentiles. The open circles indicate the values outside this
range. (B) RBCs from patients with CRVO (n = 20) adhere more effi-
ciently to HMEC than RBCs from healthy volunteers (n = 25) under flow
conditions and are resistant to washout by increasing shear stress
(P < 0.001). The more adherent were RBCs from patients with Type 2
diabetes (n = 60); adhesion of RBCs from patients with polycythemia
vera (PV, n = 38) was higher at low shear stress but was similar to that of
patients with CRVO at 0.2 and 0.3 Pa.
Expression of adhesion molecules and phosphatidylserine (PS)
expression on RBC
The expression of several adhesion molecules was tested by
flow cytometry in CRVO, RAO and control RBCs. CD36,
CD47, CD49d, CD99 and CD239 were similarly expressed on
normal and CRVO or RAO RBCs (no significant difference).
CRVO patients had a higher number of RBCs expressing
membrane phosphatidylserine (1.8% ± 0.1% positive RBCs)
compared with RBCs from normal subjects (0.7% ± 0.1%
positive RBCs, P < 0.001) or RAO patients (1.1% ± 0.1%
positive RBCs) (Fig. 2A). The mean fluorescence intensity
(MFI) was 3891 ± 208 and 413 ± 21 arbitrary units of
1
0
0 50 100 150 200
RBC×102 mm–2
0.3
Fig. 2. (A) Detection of phosphatidylserine (PS) exposure on red blood
cells (RBCs) by flow cytometry with annexin V-phycoerythrin. PS
expression was higher in patients with central retinal vein occlusion
(***P < 0.001). The percentage of PS-positive RBCs from retinal artery
occlusion patients is not statistically different from that of normal subjects.
The unbroken line within the box represents the median; the dotted line
represents the mean. The vertical lines extending beyond the boxes indicate
the 25% and 75% percentiles, while the horizontal bars outside the boxes
represent the 10% and 90% percentiles. The open circles indicate the
values outside this range. (B) A significant correlation between PS
expression and RBC adhesion was observed (Pearson correlation coeffi-
cient, r = 0.772, P = 0.001).
fluorescence (AUF) in CRVO patients and normal subjects,
respectively. Six patients have been tested on several occasions
from 3 months to 2 years after the first investigation. The
coefficient of variation was 0.1% ± 0.03%.
To exclude that microparticles may stick to RBCs and could
be responsible for increased PS, we have used a double labeling
technique using annexin V (annexin V-PE) and anti-PECAM
(CD31, anti-CD31-FITC). None of the RBCs was PECAM
positive, excluding that microparticles either from endothelial
cells or platelet origin were responsible for PS presence on
RBCs (Supporting Information). There was no correlation
between reticulocyte count and PS expression. Excluding that a
high reticulocyte count may be the cause of augmented
percentage of PS-positive red blood cells, double labeling with
anti-CD71 (anti-CD71-APC-A) and annexin V (annexin
V-PE) did not show that CD71-positive RBCs had a higher
PS expression (Supporting Information).
PS expression and RBC adhesion were measured at the same
time in the study group of patients with idiopathic CRVO

 2011 International Society on Thrombosis and Haemostasis

 Central retinal vein occlusion and RBC adhesion 1053

(n = 20). A significant correlation between PS expression and
RBC adhesion was observed (Pearson correlation coefficient,
r = 0.772, P = 0.001) and is presented in Fig. 2(B).
Molecular basis of the adhesion process
Several molecules have been described as potential receptors or
ligands for PS: PS receptor 40 KDa protein, Stabilin-2, BAI-1,
and a ubiquitous receptor, CD36 [23]. We tested the effect of
antibody in static conditions and flow conditions. Anti-PS
receptor, anti-stabilin-2 antibodies or peptide PS receptor
inhibited RBC adhesion (Fig. 3A,B). Anti-CD36 was used as a
positive control; anti-BAI-1 did not alter the adhesion of
CRVO RBCs. Proper isotype control antibodies had no effect.
Only rabbit IgG slightly enhanced RBC adhesion (Fig. 3A).
Except for annexin V, inhibition was similar in static and flow
conditions and anti-PSR was the most efficient (in static
conditions 55% of inhibition, in flow conditions between 60%
and 62% according to the shear stress). PSR peptide inhibited
RBC adhesion similarly in flow conditions at 0.1 and 0.2 Pa
(70% ± 2%), and to about the same extent in flow conditions
at 0.3 Pa and in static conditions (45% and 41%, respectively).
Annexin V is known to bind to PS and when preincubated with
RBC from CRVO it reduced the adhesion measured in static
conditions (35%, Fig. 3A), while it inhibited RBC adhesion
when determined in flow conditions between 60% and 72%
according to the shear stress (Fig. 3B). From these results it
appears that PS expressed on CRVO RBCs binds to endothe-
lial PS receptors and is responsible for the increased RBC
adhesion (Fig. 4).

Discussion
A Static conditions
Medium In this study we demonstrated that RBC adhesion to
endothelial cells was significantly increased in CRVO patients
Rabbit lgG
compared with normal subjects. The percentage of PS-positive
a.stabilin RBCs was significantly higher in CRVO patients and was
**

a.CD36 correlated with the extent of RBC adhesion. The anti-PS

**

receptor antibodies inhibited 55% of RBC adhesion in static

***

a.PSR
conditions and about 60% in flow conditions. Annexin V,
PSR peptide
**

which binds to PS, was the most efficient RBC adhesion

Annexin V blocker in flow conditions (68–74%). These results reinforce
*

0 50 100 150 200 the hypothesis that PS exposure on CRVO RBCs is an

RBC (×102)/mm2 important parameter for the increased RBC adhesion. No
significant difference was observed either in RBC adhesion or
B
Flow conditons PS expression in six patients tested at time of diagnosis and
200
after 3 months to 2 years. These results, concerning only a
limited number of patients, do not allow a firm conclusion but
160 Medium
a.stabilin suggest that the RBC abnormalities are permanent and not
PSR peptide only observed close to the ocular event. In different diseases,
RBC/mm2

120 a.PSR RBC adhesion was shown to be a factor responsible for

Annexin V
vascular occlusion. Increased exposure of PS was observed in
80 patients with sickle cell disease (SCD) [24] to a similar extent to
that found by us in CRVO (2.86% ± 2% vs. 1.8% ± 0.1%)
40 and vascular occlusion was only observed in certain circum-
stances. The percentage of CRVO RBCs that adheres is small
0
0.07 0.1 0.2 0.3
Shear stress (Pa) washout

Fig. 3. (A) Adhesion to human microvascular endothelial cells of red

blood cells (RBCs) from patients with central retinal vein occlusion
(n = 10) was measured under static conditions in the presence or absence
of antibodies or peptides. Anti-phosphatidylserine (PS) receptor antibody
blocked adhesion up to 50%, while anti-stabilin-2, anti-CD36 and peptide
PS receptor (PSR) ligand blocked adhesion up to 35%. Annexin V
(n = 16) inhibited adhesion by 35%. Bars denote SEM. ***P < 0.001,
**P < 0.01, *P < 0.05 vs. medium. (B) In flow conditions the different
antibodies inhibited adhesion. Anti-PSR and PSR peptide exhibited a high
inhibitory effect at 0.1 Pa (62% ± 8% and 70% ± 4%, respectively); at
0.3 Pa anti-stabilin and PSR peptide were in the same range of inhibition
(43% ± 3 and 45% ± 8%, respectively). Annexin V was the strongest
adhesion blocker at 0.1 and 0.3 Pa (75% ± 6% and 74% ± 8%,
respectively).
PS expression
Annexin V
BAI-1 Stab. PSR CD36
PSR
Fig. 4. Schematic representation of adhesion of red blood cells (RBCs)
from patients with central retinal vein occlusion (CRVO) to endothelium.
Blockade of phosphatidylserine (PS) RBCs and endothelial PS receptor by
annexin V or anti-PS receptor indicated that the couple PS–PS receptor is
responsible for increased adhesion of RBCs from patients with CRVO.

 2011 International Society on Thrombosis and Haemostasis

1054 M.-P. Wautier et al
and corresponds to 76 000 RBC mm)3, which may modify
vascular functions. In a rare disease, hereditary hydrocytosis, it
was reported that two patients had increased RBC adhesion to
endothelial cells as well as membrane phospholipid asymmetry
and the authors suggested that the increased adhesion may be
related to PS exposure [25]. Phospholipid asymmetry is
regulated by an ATP-dependent aminophospholipid translo-
case (APLT) [26,27]. Asymmetry may be lost by activation of
the phospholipid scramblase (PLSCR), which causes non-
specific bidirectional transport of phospholipids across the
RBC membrane [28]. It has been postulated that both
inhibition of APLT and activation of PLSCR results in PS
externalization. Externalization of erythrocyte PS occurs in
SCD [29] and may be important in the pathophysiology of
vascular dysfunction in SCD and also in CRVO. The
observation that RBC PS exposure is increased in patients
with CRVO may represent a risk factor that can be easily
explored in patients with retinal vascular occlusion. More
recently, it was demonstrated that ionophore-treated RBCs or
RBCs from patients with SCD bind to endothelium via a
specific receptor for PS [30]. As annexin V binds to PS exposed
by RBCs, we might expect that PS could also affect annexin V
anticoagulant activity.
Increased RBC adhesion to endothelium is also observed in
patients with vascular complications in diseases such as SCD,
diabetes mellitus and polycythemia vera. Different RBC
molecules and different endothelial cell receptors have been
identified as being responsible for the abnormal RBC-endo-
thelium interactions. In diabetes mellitus, RBC Band 3 protein
is glycated and binds to the receptor for advanced glycation end
products (RAGE) [22]. In PV the mutation of the kinase JAK2
results in phosphorylation of LuBCAM (CD239), which is a
ligand for the endothelial laminin alpha5 chain [16]. The same
RBC molecule, CD239, is implicated in RBC adhesion in SCD
[31]. These different examples indicated that RBC adhesion to
endothelium may participate in the development of vascular
complications in several diseases through different adhesion
mechanisms. Our results showed that it may also be a factor
that is associated with CRVO.
In SCD and PV a relationship between abnormal erythro-
poiesis and abnormal RBC behaviour has been proposed
[16,31]. Seven patients who participated in a previous study of
in vitro bone marrow culture in CRVO patients [15] have been
studied for RBC adhesion and PS expression. Five of them
showed endogenous erythroid progenitor growth; their results
in the present study did not differ from those of the other
patients. Further studies are needed to investigate the link
between bone marrow progenitor cells and mature red cell
abnormalities in CRVO disease.
In SCD, treatment by hydroxyurea reduced RBC adhesion
[32]. Hydroxyurea is also the first line treatment of PV patients,
reducing the risk of thrombotic events [16]. We can expect that
in various diseases with increased RBC adhesion, treatment
correcting the RBC abnormalities or modifying adhesion
molecule expression may improve the risk of vascular compli-
cations.
Modulation of APLT and PLSCR, which regulate phos-
pholipid asymmetry, is a possible candidate for the treatment
of patients with CRVO, as is the potential use of annexin V or
annexin peptides for masking PS at achievable concentrations.
Addendum
M.-P. Wautier conducted the study and wrote the manuscript.
E. Héron made the diagnoses, collected the patient data and
wrote the paper. J. Picot performed cytometry analysis and
analyzed the data. Y. Colin and O. Hermine discussed the
results, gave advice and commented on the manuscript. J.-L.
Wautier managed the project, designed the experiments and
wrote the manuscript.
Acknowledgements
We are grateful to M. Paques (Hôpital des Quinze-Vingts
Paris) for confirming the ophthalmological diagnosis, N. Sedira
(Hôpital des Quinze-Vingts Paris) for gathering patient files
and E. Vera (Centre National de Reference des Groupes
Sanguins Paris) for blood sample management. This work was
supported by Institut National de la Transfusion Sanguine,
Institut National de la Santé et de la Recherche Médicale,
Université Denis Diderot Paris 7, la Société Nationale Franç-
aise de Médecine Interne and Programme Hospitalier de
Recherche Clinique no AOR 09023/2009-A00983-54.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
File S1 Phosphatidylserine expression on Red Blood Cell
and RBC adhesion to endothelium were tested just after
sampling or after storage at minus 196C.
File S2 Flow cytometry analysis of RBC from normal
subjects or from CRVO patients labeled with anti-CD31
(PECAM) and annexin V or anti-CD71 (transferrin receptor)
and annexin V.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
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