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Sandor et al 2016 British Journal of Haematology .pdf

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Title: Effects of Poloxamer 188 on red blood cell membrane properties in sickle cell anaemia

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short report

Effects of Poloxamer 188 on red blood cell membrane
properties in sickle cell anaemia

Barbara Sandor,1,* Micka€el Marin,2,3,4,*
Claudine Lapoumeroulie,2,3,4 Miklos
Raba€ı,1 Sophie D. Lefevre,2,3,4,5 Nathalie
Lemonne,6 Wassim El Nemer,2,3,4 Ana€ıs
Mozar,4,7 Olivier Francßais,8 Bruno Le
Pioufle,8 Philippe Connes4,7,8,9,10,* and
Caroline Le Van Kim2,3,4,5,*

University of Pecs Medical School, Pecs, Hun-

gary, 2Inserm UMR_S1134, 3Institut National de
la Transfusion Sanguine, 4Laboratoire d’Excellence GR-Ex, 5Universit e Paris Diderot, Sorbonne
Paris Cit e, Paris, France, 6CHU de Pointe- aPitre, 7Inserm, Universit e des Antilles et de la

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia
(SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of
Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer
(P188) on red blood cell (RBC) properties by investigating haemorheology,
mechanical and adhesion functions using ektacytometry, microfluidics and dynamic
adhesion approaches, respectively. We show that poloxamer significantly reduces
blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the
beneficial use of this molecule in SS therapy.

Keywords: sickle cell anaemia, poloxamer, red blood cell.

Guyane, 97159, Pointe- a-Pitre, Guadeloupe,

Ecole Normale Sup erieure de Cachan, CNRS,

BIOMIS-SATIE, UMR 8029, Cachan,

Laboratoire CRIS EA647, Section “Vascular

Biology and Red Blood Cell”, Universit e Claude
Bernard Lyon 1, Lyon, and 10Institut Universitaire de France, Paris, France
Received 30 September 2015; accepted for
publication 4 December 2015
Correspondence: Caroline Le Van Kim,
Laboratoire d’excellence GR-Ex- UMR_S1134/
Institut National de la Transfusion Sanguine, 6
rue Alexandre Cabanel, 75015 Paris, France.
E-mail: caroline.le-van-kim@inserm.fr
*Equivalent position.

Sickle cell anaemia (SS) is a severe monogenic hereditary
haemoglobin disorder characterized by chronic haemolytic
anaemia and the occurrence of frequent painful vaso-occlusive crisis (VOCs). SS classical physiological scheme involves
haemoglobin S (HbS) polymerization under hypoxic conditions, which triggers the sickling of red blood cells (RBCs)
and the loss of their deformability. Recent studies demonstrated that the degree of haemorheological alterations, such
as blood hyper-viscosity, determines the risk for VOCs
(Nebor et al, 2011). Moreover, sickle RBCs (SS-RBCs)
abnormally adhere to vascular endothelium, triggering
microvascular occlusions (Hebbel et al, 1980). The generation of VOC results from a complex and partially known
combination of factors, such as the interaction of different
ª 2016 John Wiley & Sons Ltd
British Journal of Haematology, 2016, 173, 145–149

cell types including RBCs, activated endothelial cells, leucocytes and plasma factors.
Despite extensive molecular and genetic investigations and
an exquisitely precise molecular model of the primary pathophysiological event, very few drugs are available to efficiently
treat VOCs. Fluorocarbon emulsions have been studied in SS
since 1975 (Padilla et al, 1975). Poloxamer188 (P188)
belongs to the polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) family, which is listed as
pharmaceutical excipient in the U.S. and British Pharmacopoeia and has been used extensively in a variety of pharmaceutical formulations. The first clinical trial tested the
efficacy of P188 in a large sickle cell disease (SCD) cohort of
adults and children (Orringer et al, 2001). This study

First published online 5 February 2016
doi: 10.1111/bjh.13937

Short Report
demonstrated some reduction of pain duration in the subgroup of children treated with P188. Recently, a phase III
multi-centre trial was started to test the efficacy of a derivative product, velopoloxamer, during acute VOC in children
(Humphries et al, 2015). Although P188 has been reported
as a non-ionic block copolymer surfactant with antithrombotic and anti-inflammatory activities (Toth et al, 2000;
Armstrong et al, 2001; Hunter et al, 2010), its effects on
blood viscosity, RBC aggregation and adhesion under
haemodynamic conditions in the context of SS have not yet
been investigated. We conducted in vitro experiments using
the commercial formulation Kolliphor P188 to test the effects
of this molecule on blood rheology and RBC mechanical
properties using a new microfluidic device, and adhesive
function in SS patients.

Materials and methods
Venous blood samples (EDTA) from SS patients and healthy
individuals were collected with their approval and all measurements were performed within 6 h of collection. This
study is a part of the ‘sickle cell haemorheology’ study’
approved by the Ethics Committee (registration number:

P188 Treatment of RBCs
RBCs were washed in phosphate-buffered saline (PBS) and
incubated at 37°C for 30 min with 5 mg/ml of Kolliphor
P188 (Sigma-Aldrich, St Louis, USA), equivalent to the physiological concentration taken by patients (Orringer et al,
2001). P188 contain 80 ethylene oxide monomer units in the
PEO chains and 27 propylene oxide monomer units in the
central molecule. The haematocrit and buffer were chosen
according to the experimental procedure.

Haemorheological experiments
Haematocrit was adjusted to 40% in autologous plasma or in
3% dextran 70 (Sigma-Aldrich) with P188-treated and
untreated RBCs. Blood viscosity, RBC aggregation and
deformability were determined as previously described (Connes et al, 2014). Experiments were performed for 10 SS
patients and 6 controls.

RBC preparation and microfluidic trapping experiment
The microfluidic device was designed in order to sense RBC
rigidity through 8 parallel filter units. Microfabrication steps
were previously described (Picot et al, 2015).
P188-treated and untreated RBCs from 3 SS patients were
differentially stained and mixed as described (Picot et al,
2015). P188-treated RBCs were washed before mixing with

untreated RBCs. The RBC suspension was perfused at constant pressure (250 mBar, MFCSTM-EZ-1C pump, Fluigent
S.A., France). Occlusion of the filtering units was followed
over time using an inverted AxioObserver Z1 microscope
platform (Carl Zeiss, Le Pecq, France).

RBCs sickling in hypoxic conditions
P188-treated and untreated SS-RBCs were incubated in CellStab (Bio-Rad, Richmond, CA, USA). Slides were then submitted to stepwise deoxygenation by lowering O2 content
with nitrogen within the containment cabinet of the microscope platform (20, 10, 5, 3 and 0% O2, 15 min/step).

Endothelial cell culture in biochips and flow adhesion
Human microvascular endothelial cell line 1 (HMEC-1) cells
were seeded at 108 cells/ml in Vena8 Endothelial+ Biochips
(Cellix Ltd, Dublin, Ireland) previously coated with 40 ll of
0 2% gelatin in PBS. Cells were then incubated for 2 h at
37°C, permitting cell attachment and cultured for 48 h using
the Kima pump (Cellix Ltd.).
P188-treated and untreated RBCs were suspended at 1%
haematocrit in Hanks buffer with Ca2+ and Mg2+ supplemented with 0 4% bovine serum albumin and 1 mM
HEPES. Adhesion assay was initiated by perfusion of the
RBCs at a shear stress of 0 2 dyn/cm² for 10 min. This was
followed by 5-min of washes at 0 5, 1, 1 5, 2 and 3 dyn/
cm2.. The whole process was controlled using the Exigo
pumps and software (Cellix). For the hypoxic conditions,
HMEC-1 cells were incubated as above and then placed for
5 h in a 7% O2 incubator prior to the adhesion assay. Data
collection and analysis were done as previously described
(Chaar et al, 2014).

Statistical analysis
Results are presented as mean standard deviation (SD).
Non-parametric tests (paired Wilcoxon test or unpaired
Mann–Whitney test) were used to compare the groups and
to test the effect of P188 in each group. The significance level
was defined as P < 0 05. Analyses were conducted using SPSS
(v. 20, IBM SPSS Statistics, Chicago, IL, USA).

Results and discussion
P188 treatment decreases RBC aggregation and blood
viscosity in SS
In accordance with the literature (Vent-Schmidt et al,
2015), blood viscosity measured at an adjusted haematocrit
was higher in SS than in control (AA) samples at all shear
rates (Fig 1A). While P188 treatment did not significantly
affect blood viscosity in AA, it decreased blood viscosity at
ª 2016 John Wiley & Sons Ltd
British Journal of Haematology, 2016, 173, 145–149

Short Report

Blood viscosity (cP)

(A) 19

AA + P188
SS + P188


Untreated RBC














20 μm



RBC aggregation in
plasma (%)

Shear rate (/s)


With P188

RBC treated with P188





With P188








20 μm


Fig 1. P188 treatment decreases blood viscosity and SS-RBC aggregation. (A) Blood viscosity. RBC aggregation in plasma (B) and in dextran (C). *P < 0 05. P188, poloxamer; SS, sickle cell anaemia; AA,
controls; RBC, red blood cell.

the three lowest shear rates in SS. When measured in
plasma, RBC aggregation decreased with P188 in SS
patients but not in controls (Fig 1B). When RBC aggregation was promoted using dextran, P188 decreased RBC
aggregation in the two groups (Fig 1C). Indeed, the lowering of blood viscosity in SS is attributed to the inhibitory
effects of P188 on RBC aggregation. Given that Sharma
et al (1996) demonstrated that P188 adsorbs into the lipid
bilayers of cell membrane, it could be plausible that this
adsorption on damaged SS-RBC membranes decreases the
ability of adjacent RBCs to interact between them. All
these effects are of particular interest in the context of
ª 2016 John Wiley & Sons Ltd
British Journal of Haematology, 2016, 173, 145–149

Adherent cells (/mm²)


RBC aggregation in
dextran (%)



With P188






P5 Mean

Fig 2. Adhesion of non-treated SS-RBCs (A) or P188-treated SSRBCs (B) to the HMEC-1 cell line. (C) Graph representing adherent
RBCs per mm2 at a flow rate of 1 dyne/cm2. The mean of the 5
patients is expressed as the average number of adherent RBCs/
mm2 standard deviation. **P < 0 01. P188, poloxamer; SS, sickle
cell anaemia; AA, controls; RBC, red blood cell.

SCD, because both increased blood viscosity and RBCs
aggregation has been associated with a high risk of frequent VOC. It remains to be determined how much P188
is needed to adsorb on the RBC membrane to reduce cellcell interactions as an significant amount could also enter
into the cells (Gigout et al, 2008), thus limiting its antiaggregating effects.

Short Report

P188 treatment does not affect RBC deformability
RBC deformability, assessed by both ektacytometry (Figure S1A) and microfluidics (Figure S1B and C), was reduced
in SS compared to AA but was not affected by P188. Hypoxia
caused RBC sickling but the percentage of sickling remained
the same for the P188-treated and untreated RBCs (n = 5)
(Figure S2A), confirming the conclusion that P188 does not
act on the mechanical properties of RBCs. These results are in
agreement with the mode of action of P188 proposed by
Hunter et al (2010), who suggested that P188 might bind to
hydrophobic surfaces and lower surface tension without any
changes in the organisation of the cytoskeleton.

Decreased flow adhesion of SS-RBCs on endothelial cells
by P188 treatment
Next, we performed a functional study by examining the
effect of P188 treatment on SS-RBC adhesion to a monolayer
of the endothelial HMEC-1 cell line. We observed a mean
adhesion of 40 RBCs/mm2 for the untreated SS-RBCs versus
20 RBCs/mm2 in the case of the P188-treated RBCs, i.e. a
50% decrease upon P188 treatment (Fig 2A–C). Four AA
controls were analysed under similar conditions and no
adhesion was observed for the untreated or treated AA RBCs
(2–4 RBCs/mm2) (data not shown).
As VOCs occur in venules, we performed flow adhesion
experiments with deoxygenated RBCs and endothelial
HMEC-1 cultivated in hypoxic conditions (n = 5) (Figure S2B). We observed a dramatic decrease (76 3%) of RBC
adhesion on endothelial cells in hypoxic conditions upon
P188 treatment with a more pronounced effect than in normoxic conditions.
Together with RBC aggregation experiments, our findings
indicate that the binding of P188 to the SS-RBC membrane
highly reduces the interaction with endothelial cells and circulating cells. This is of particular importance in the context

Armstrong, J.K., Meiselman, H.J., Wenby, R.B. &
Fisher, T.C. (2001) Modulation of red blood cell
aggregation and blood viscosity by the covalent
attachment of Pluronic copolymers. Biorheology,
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Chaar, V., Laurance, S., Lapoumeroulie, C., Cochet,
S., De Grandis, M., Colin, Y., Elion, J., Le Van
Kim, C. & El Nemer, W. (2014) Hydroxycarbamide decreases sickle reticulocyte adhesion to
resting endothelium by inhibiting endothelial
lutheran/basal cell adhesion molecule (Lu/BCAM)
through phosphodiesterase 4A activation. Journal
of Biological Chemistry, 289, 11512–11521.
Connes, P., Lamarre, Y., Waltz, X., Ballas, S.K.,
Lemonne, N., Etienne-Julan, M., Hue, O.,
Hardy-Dessources, M.D. & Romana, M. (2014)
Haemolysis and abnormal haemorheology in


of SCA as increased RBC adhesiveness has been demonstrated to trigger VOC (Kaul et al, 1996).
In summary, in parallel with the on-going phase III study
focusing on the acute effects of P188 during VOCs in SS
patients (NCT01737814), our results bring new insights
regarding its mode of action on RBCs. We show that P188
directly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells in normoxic as well as in hypoxic
conditions, supporting the beneficial use of this safe drug to
treat SCD patients during acute events.

Funding sources: Travel grants of BS and MR were funded
by the Campus Hungary Short Term Study Program. Post
doc position of AM was funded by the Region Guadeloupe.
The authors thank Dr JA Ribeil (Necker hospital) for helpful

Author contributions
BS, MM, MR and PC designed the research; BS, MM, CL,
MR, SDL, AM, OF, BLP and PC performed the research; NL
provided the blood samples; BS, MM, CL, MR, SDL and PC
analysed the data; CL, SDL, PC and CLVK wrote the paper;
WEN reviewed the manuscript with critical reading and very
helpful comments.

Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig S1. P188 treatment does not change SS-RBC deformability.
Fig S2. P188 treatment on RBCs sickling and RBCs adhesion in hypoxic conditions.

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Short Report
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ª 2016 John Wiley & Sons Ltd
British Journal of Haematology, 2016, 173, 145–149

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