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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1422-1429
By
From the Department of Chemical Engineering, Northeastern University,
Boston, MA; the Department of Medicine, Albert Einstein College of
Medicine, Bronx, NY; and the Hematology Division, Brigham & Women's
Hospital, Boston, MA.
The abnormal adherence of sickle red blood cells (SS RBC) to
vascular endothelium may play an important role in vasoocclusion in
sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance
adhesion of SS RBC. Also, these endothelial proteins bind to sulfated
glycolipids and this binding is inhibited by anionic polysaccharides.
Reversible sickling may expose normally cryptic membrane sulfatides
that could mediate this adhesive interaction. In this study, we have
investigated the effect of anionic polysaccharides, in the presence or
absence of TSP, on SS RBC adhesion to the endothelium, using cultured
human umbilical vein endothelial cells (HUVEC) (for the adhesion assay)
and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The
baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most
effectively inhibited by high molecular weight dextran sulfate (HDS),
whereas low molecular weight dextran sulfate (LDS) and the
glycosaminoglycan chondroitin sulfate A (CSA) also had significant
inhibitory effects. Heparin was mildly effective whereas other
glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and
fucoidan) were ineffective. Similarly, HDS and CSA resulted in an
improved hemodynamic behavior of SS RBC. Soluble TSP caused significant
increases in SS RBC adhesion and in the peripheral resistance. Both HDS
and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities.
Thus, anionic polysaccharides can inhibit SS RBC-endothelium
interaction in the presence or absence of soluble TSP. These agents may
interact with RBC membrane component(s) and prevent TSP-mediated
adhesion of SS RBC to the endothelium.
THE PATHOPHYSIOLOGY OF SICKLE CELL anemia
is due to a single amino acid substitution at the sixth position of the
A number of mechanisms have been proposed to account for SS
RBC-endothelium interactions. Factors such as RBC age and density, RBC
shape, RBC membrane alterations, endothelial abnormalities, and plasma
factors may influence these mechanisms. Although all SS RBC are
potentially adherent,11,12 specific receptor-ligand interactions involve only a subset of sickle reticulocytes, ie, stress
reticulocytes. In both static and flow systems, SS RBC adhesion to the endothelium is
enhanced by soluble TSP.16,17 Recent studies have shown
that SS RBC also adhere avidly to immobilized TSP and that this
interaction is not inhibited by antibodies to CD36, a TSP receptor,
suggesting yet another mechanism by which SS RBC may bind to
TSP.25,26 In one study,25 soluble TSP did not
inhibit SS RBC adhesion to immobilized TSP, suggesting that epitopes
responsible for mediating adhesion might be different for soluble
versus immobilized TSP, and quite possibly for TSP expressed on the
endothelium as well.
TSP-mediated adhesion may involve some of the same molecules that bind
vWF. For example, both vWF and TSP can bind to
The present studies were designed to resolve the following issues. Do
anionic polysaccharides inhibit SS RBC adhesion to the vascular
endothelium under shear-flow conditions and what is the hemodynamic
impact of this inhibition? What are the hemodynamic consequences of
TSP-enhanced adhesion? Can TSP-enhanced adhesion be inhibited by
anionic polysaccharides? To resolve these issues, we have used two
complementary dynamic assay systems. In one assay, SS RBC were infused
into a parallel-plate flow chamber lined with human umbilical vein
endothelial cells (HUVEC), and in the second assay, we evaluated their
hemodynamic behavior in the ex vivo mesocecum vasculature of the rat.
Reagents.
TSP purified from human platelets was generously provided by Dr J. Lawler (Harvard University, Cambridge, MA). All other reagents and
anionic polysaccharides used in these studies were purchased from Sigma
(St Louis, MO).
Preparation of cells.
Blood was drawn into heparinized tubes from normal adults (n = 3) and
from sickle cell anemia patients (n = 9) who were not in crisis and had
not received blood transfusion in the preceding 4 months. Blood samples
from four sickle cell anemia patients were obtained at the Boston
Comprehensive Sickle Cell Center and five patients were drawn at the
Heredity Clinic of the Albert Einstein College of Medicine, Bronx, NY.
Blood samples from three patients were shared between the two laboratories.
Endothelial cells.
HUVEC were harvested from two to five umbilical cord veins, pooled, and
grown in primary culture as previously described.34 Cultures were serially passaged (1:3 split ratios) with M199-20% fetal
calf serum supplemented with 50 to 100 µg/mL endothelial cell growth
factor and 100 µg/mL porcine intestinal heparin in Costar tissue
culture flasks (Cambridge, MA) coated with purified gelatin.
Experiments were performed with second-passage HUVEC that were grown to
confluence on fibronectin-coated LabTek slides (LabTek, Naperville, IL).
Erythrocyte adherence assay.
A parallel-plate flow chamber with an endothelial cell-coated slide as
its base was used to assess the adhesion of RBC under defined fluid
dynamic conditions. The flow chamber and the glass slide were held
together by a vacuum maintained at the periphery of the slide forming a
channel of parallel-plate geometry. The height of the flow channel, as
determined by a silastic gasket, was 100 microns. After assembling the
endothelial-coated slide in the flow chamber, RBC suspensions (Hct 1%)
were drawn into the chamber from a reservoir maintained at 37°C in
a water bath using a syringe pump (Model 956, Harvard Apparatus, South
Natick, MA) at a controlled-flow rate to give a venular wall shear
stress of 1 dyne/cm2. The flow chamber was mounted on an
inverted-phase contrast microscope (DIAPHOT-TMD, Nikon, Garden City,
NY) equipped with a CCD video camera (Model 72, Dage-MTI, Michigan City, IN). The microscope stage was
maintained at 37°C by a thermostatic air bath (Model ASI-400,
Nicholson Precision Instruments, Gaithersburg, MD). All of the
experiments were recorded in real time on a 0.5 inch video cassette
recorder (Model BV-1000, Mitsubishi, Cypress, CA) and displayed on a
high-resolution monitor (Model PM-127, Ikegami, Maywood, NJ). A
PC-based image processing system (Optimas, Bioscan, Edmunds, WA) was
used for digitization of video recordings and for further image
processing and analysis. For each adherence assay, the endothelial cell
monolayer was rinsed for 2 minutes with HBSS followed by a 10-minute
perfusion with the RBC suspension that had been untreated or incubated
with a given anionic polysaccharide at 200 µg/mL for 30 minutes. In
experiments involving the inhibition of TSP-enhanced adhesion, RBC
suspensions were incubated sequentially with 200 µg/mL of a specific
anionic polysaccharide for 30 minutes followed by a 30-minute
incubation with 1 µg/mL of TSP. The treated suspensions (without any
further manipulation) were then used for flow adhesion assay. The
number of adherent erythrocytes remaining after a 10-minute rinse
period were counted in a minimum of 24 fields and reported as the
number of adherent RBC per mm2.
Hemodynamic studies using the ex vivo mesocecum preparation.
Hemodynamic studies were performed in the isolated, acutely denervated,
artificially perfused rat mesocecum vasculature according to the method
of Baez et al35 as modified for the study of
erythrocytes.36 Arterial perfusion pressure (Pa) was
rendered pulsatile with a peristaltic pump. Venous outflow pressure
(Pv) was kept at 3.8 mm Hg, and the venous outflow rate (Fv) was
monitored with a photoelectric drop counter and expressed as
milliliters per minute. All perfusion experiments were performed at
ambient air and at 37°C as described.6 During perfusion
with Ringer's at Pa of 60 mm Hg, a bolus of 0.3 mL red-cell
suspensions (Hct 30%) was infused. Washed SS RBC suspensions were
either untreated or incubated with a given polysaccharide (250 µg/mL)
for 30 minutes. In experiments involving the inhibition of TSP-enhanced
adhesion, SS RBC suspensions were incubated sequentially with 250 µg/mL of a specific anionic polysaccharide for 30 minutes followed by
a 30-minute incubation with 2.5 µg/mL of TSP. The treated samples
were then used for bolus infusion. Peripheral resistance units (PRU)
were determined as described37 and expressed in mm
Hg/mL/min/g. PRU = Statistical analysis.
Paired t-test or unpaired Student's t-test was applied
to analyze data. The tests for hypothesis were performed using a Type-I error of 0.05 and were two-tailed. The statistical analyses were performed using Statgraphics Plus (version 3.1 ) program for Windows (Manugistics, Inc, Rockville, MD) and an IBM-compatible computer.
We evaluated the effect of anionic polysaccharides on SS RBC adhesion
to the vascular endothelium and its hemodynamic consequences in the
presence or absence of TSP, using two complementary dynamic assays. A
parallel-plate flow chamber lined with HUVEC was used for the adhesion
assay, whereas the hemodynamic behavior was evaluated in the ex vivo
mesocecum vasculature.
Inhibition of SS RBC adhesion to HUVEC by anionic polysaccharides.
Anionic polysaccharides inhibit sickle erythrocyte binding to
immobilized TSP.25,26 To determine if these compounds would also be effective inhibitors of sickle RBC adhesion to HUVEC, we
screened a number of anionic polysaccharides in our adhesion assay. RBC
suspensions in HBSS/BSA (hematocrit, 1%) were incubated with a given
anionic polysaccharide at 200 µg/mL for 30 minutes before being
perfused over HUVEC monolayers. The high molecular weight form of
dextran sulfate (HDS, MW 500,000) (n = 9) was found to be the most
effective inhibitor, blocking 78% of SS RBC adhesion to HUVEC
(P < .0001) whereas the low molecular weight form of dextran
sulfate (LDS, MW 5,000) (n = 7) inhibited 52% of SS RBC adhesion
(Fig 1). The sulfated glycosaminoglycan
chondroitin sulfate A (CSA) (n = 6) inhibited 40% of SS RBC adhesion
to HUVEC (P < .001), whereas chondroitin sulfate B (CSB or
dermatan sulfate) (n = 6) and chondroitin sulfate C (CSC) (n = 4) had
no significant effect on adhesion (Fig 1). CSB and CSC differ from CSA
in the substitution of iduronic acid for glucuronic acid, and in the position of sulfation of N-acetyl-
The effect of anionic polysaccharides on the hemodynamic behavior of
SS RBC.
Next, we evaluated the hemodynamic behavior of SS RBC treated with
selected anionic polysaccharides. In a series of experiments, washed SS RBC (Hct 30) were incubated with a given AP (250 µg/mL) and
their hemodynamic behavior was compared with untreated SS RBC obtained
from the same patient. A bolus of SS RBC (treated or untreated) was
infused into the isolated mesocecum vasculature during perfusion with
Ringer-albumin solution at an arterial pressure (Pa) of 60 mm Hg. In
experiments (n = 13) in which hemodynamic behavior of human normal (AA)
RBC was compared with SS RBC, the resulting PRU for SS RBC
was almost twofold greater than that for AA RBC (%PRU: AA RBC, 20.0 ± 4.3; SS RBC, 38.7 ± 10.1; P< .0001, paired
t-test) (Fig 1). This increase in PRU for SS RBC was
accompanied by a significantly prolonged pressure-flow recovery time
(Tpf, sec; AA RBC, 40.4 ± 15.3; SS RBC, 81.5 ± 17.5, P<.0001, paired t-test). Both higher PRU and a
delayed Tpf reflect abnormal rheological and adhesive properties of
oxygenated SS cells as described earlier.5
Thrombospondin-enhanced adhesion of SS RBC to HUVEC and its
prevention by anionic polysaccharides.
We next investigated whether TSP-enhanced adhesion of SS RBC could be
prevented by anionic polysaccharides. In these experiments, SS RBC were
treated with TSP (1 µg/mL) alone or incubated sequentially with a
given anionic polysaccharide (CSA, HS, heparin, or HDS) at 200 µg/mL
for 30 minutes and then with TSP (1 µg/mL) for 30 minutes. TSP alone
enhanced endothelial cell adhesion of SS RBC by ~50% (P < .01) (Fig 4). The prior addition of either
CSA or HDS caused a significant inhibition of adhesion of SS RBC
(P < .03 to .05). The decrease (ie, ~87% decrease relative
to the untreated control) in adhesion observed with HDS was comparable
to that observed with HDS in the absence of TSP. The presence of CSA
was also inhibitory. Adhesion showed 28% decrease compared with
untreated cells (Fig 4) whereas CSA in the absence of TSP resulted in a greater inhibition (ie, ~40%) of adhesion (Fig 2). Thus, sequential treatment of SS RBC with CSA and TSP resulted in less-effective inhibition of adhesion. Sequential treatment of SS RBC with heparin and
TSP (n = 4) caused a significant reduction in adhesion compared with
TSP-enhanced adhesion (P < .05), but the resulting adhesion was still somewhat higher as compared with untreated SS RBC (Fig 4).
Pretreatment of SS RBC with HS (n = 3) or fucoidan (n = 5) had no
effect on TSP-enhanced adhesion.
TSP-induced hemodynamic abnormalities are prevented by CSA and HDS.
Because TSP caused a significant increase in adhesion of SS RBC to
HUVEC that was abrogated by the presence of HDS and CSA, we evaluated
the effect of a similar sequential treatment on the hemodynamic
behavior of these cells in the mesocecum vasculature. Figure 5 depicts the results of five sets
of experiments designed to evaluate the effect of TSP (2.5 µg/mL)
alone and of sequential treatment with a given anionic polysaccharide
(CSA, HS, heparin, HDS, and fucoidan, each 250 µg/mL) and TSP (2.5 µg/mL). In each group, incubation of SS RBC with TSP invariably
caused a significant increase in PRU (P < .05 to .02) (Fig
5A-E), as well as in Tpf (P < .05)
(Table 2) compared with untreated cells.
These changes reflected an increase in SS RBC adhesion accompanied by
modest postcapillary blockage. Adhesion was confined to venules and was characterized by frequent cluster formation of adherent SS RBC in the
affected venules (Fig 6). The presence of
CSA caused significant decreases in both PRU (P < .01) and
Tpf (P < .05) (Table 2, Fig 5) compared with
TSP-treated SS RBC; the resulting PRU was not significantly different
from that observed with untreated cells. The presence of HS (n = 4) or
fucoidan (n = 3) had no significant effect on PRU or Tpf as compared
with TSP-treated SS RBC (Table 2, Fig 5). Sequential treatment with
heparin and TSP (n = 5) caused a moderate decrease in PRU and Tpf
(P < .5) as compared with that for TSP-treated SS RBC (Table
2, Fig 5), although the resulting PRU was still higher than that for
untreated cells. The presence of HDS resulted in maximal decreases in
both PRU and Tpf as compared with TSP-treated SS RBC (P < .01) (Table 2, Fig 5).
The present study shows for the first time the efficacy of anionic
polysaccharides to inhibit SS RBC interaction with vascular endothelium
under dynamic flow conditions, both in the presence and absence of
exogenous soluble TSP. In particular, the results show that the
baseline adhesion of washed SS RBC to HUVEC under venular flow
conditions is substantially inhibited in the presence of HDS, LDS, and
CSA, whereas heparin is mildly effective and other sulfated
polysaccharides (ie, CSB, CSC, and fucoidan) are ineffective.
Similarly, when SS RBC are pretreated with HDS, CSA, and heparin and
infused into the ex vivo mesocecum rat vasculature, a significant
improvement in their hemodynamic behavior is observed as indicated by a
decrease in the peripheral resistance. Furthermore, TSP-enhanced SS RBC
adhesion to HUVEC, as well as attendant hemodynamic abnormalites in the
mesocecum are most effectively ameliorated by pretreatment of SS RBC
with HDS or CSA.
Submitted January 1, 1998; accepted October 5, 1998.
Supported by NIH Grant No. HL45931 (D.K.K.) and by NIH
Comprehensive Sickle Cell Centers at Boston, MA (HL15157; G.A.B. and B.M.E.) and Bronx, NY (HL 38655).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to G.A. Barabino, PhD, Department of Chemical
Engineering, Northeastern University, 342 Snell Engineering Center,
Boston, MA 02115.
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Top
Abstract
Introduction
chain ( 
val) of the hemoglobin (Hb) S
molecule, which results in polymerization of HbS and sickling of red
blood cells (RBC) under deoxygenated conditions. Although HbS
polymerization is central to the pathophysiology of sickle cell
disease, multiple factors (both primary and secondary) may be involved
in the initiation of a vasoocclusive event. Among these factors, the
abnormal adherence of sickle red blood cells (SS RBC) to the vascular
endothelium may play an important role in vasoocclusion.
4
P/Q, in which 
