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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 302-309
The Carboxy-Terminal Cell-Binding Domain of Thrombospondin Is
Essential for Sickle Red Blood Cell Adhesion
By
Cheryl A. Hillery,
J. Paul Scott, and
Ming C. Du
From the Department of Pediatrics, Medical College of Wisconsin,
Milwaukee; and the Blood Research Institute, The Blood Center of
Southeastern Wisconsin, Milwaukee.
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ABSTRACT |
Sickle red blood cells (SS-RBCs) have enhanced adhesion to the
plasma and subendothelial matrix protein thrombospondin-1 (TSP) under
conditions of flow in vitro. TSP has at least four domains that mediate
cell adhesion. The goal of this study was to map the site(s) on TSP
that binds SS-RBCs. Purified TSP proteolytic fragments containing
either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However,
a 140-kD thermolysin TSP fragment, containing the
carboxy-terminal cell-binding domain in addition to the type 1, 2, and
3 repeats fully supported the adhesion of SS-RBCs (126% ± 25%
adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK)
and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC
adhesion to TSP. In addition, monoclonal antibody C6.7, which blocks
platelet and melanoma cell adhesion to the cell-binding domain, did not
inhibit SS-RBC adhesion to TSP. These data suggest that a novel
adhesive site within the cell binding domain of TSP promotes the
adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind
SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to
immobilized TSP under conditions of flow, indicating that the adhesive
site on TSP that recognizes SS-RBCs is exposed only after TSP binds to
a matrix. We conclude that the intact carboxy-terminal cell-binding
domain of TSP is essential for the adhesion of sickle RBCs under flow
conditions. This study also provides evidence for a unique adhesive
site within the cell-binding domain that is exposed after TSP binds to
a matrix.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SICKLE CELL DISEASE is caused by a
genetic disorder of hemoglobin (hemoglobin  Glu6Val) that results in
hemolytic anemia.1-3 The major cause of morbidity and
mortality in sickle cell disease is tissue ischemia and infarction due
to vascular occlusion. The pathogenesis of vaso-occlusion remains
incompletely understood and likely involves many heterogeneous steps.
The sickle erythrocyte manifests numerous abnormalities that include
oxidative damage of membrane proteins and lipids,4 aberrant
cation homeostasis resulting in significant cellular
dehydration,5 abnormal clustering of surface
proteins,6 disruption of the membrane phospholipid asymmetry,7 and increased adhesive
properties.2,3 The increased adhesion of sickle red blood
cells (SS-RBCs) to vascular endothelium in vitro has been
described using both static adhesion assays8 and
endothelialized flow chambers.9 These observations have
been confirmed using live animal models by either infusing human
SS-RBCs into rats10,11or by studying transgenic sickle cell
mouse models.12 The enhanced adhesion of SS-RBCs to the
vascular endothelium and subendothelial matrix likely plays a
significant role in the pathogenesis of vascular occlusion in sickle
cell disease.
Thrombospondin-1 (TSP) is a 450-kD, homotrimeric glycoprotein present
in the subendothelial matrix, plasma, and platelet  storage granules
that can be released in high local concentrations by activated
platelets.13,14 TSP mediates cell attachment and spreading,
stabilizes platelet aggregation, regulates cell growth, and plays a
role in angiogenesis, wound healing, cell migration, and phenotypic
differentiation. TSP has at least four domains that mediate cell
adhesion, including the NH2-terminal heparin-binding domain
that also binds sulfated glycolipids and heparan sulfate proteoglycans,15,16 sequences within the type 1 repeats
that associate with CD36,17 the RGD integrin-binding site
within the last type 3 repeat of the calcium binding
domain,18 and the carboxy-terminal cell binding domain that
binds to platelets and transformed cells.19 Chondroitin
sulfate A also binds to TSP, likely through regions within the type 1 repeats or the carboxy-terminal cell binding domain.20
Similar to other adhesion molecules,21 the binding
characteristics of TSP can be affected by conformational changes.22
Soluble TSP has been shown to enhance the adhesion of SS-RBCs to
cultured endothelial cells.23,24 SS-RBCs also bind avidly to purified, immobilized TSP under conditions of flow in
vitro.25,26 The adhesion of sickle RBCs to TSP may
contribute to vaso-occlusive crises in sickle cell disease. Although
the sickle reticulocyte adhesive receptor CD36 is postulated to mediate
the adhesion of SS-RBCs to endothelial cells,23,24 the
adhesion of SS-RBCs to immobilized TSP is probably not mediated by
CD36.25,26 The specific molecular mechanisms by which TSP
binds sickle RBCs is not known. Therefore, in this study we sought to
determine the site on TSP that binds the sickle RBC under conditions of
flow in vitro. We found that the 20-kD carboxy-terminus of TSP that contains the cell-binding domain was required for SS-RBC adhesion. Additionally, we determined that site on TSP that binds SS-RBCs is
exposed after binding to a matrix and likely involves a unique adhesive
region(s) within the cell-binding domain.
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MATERIALS AND METHODS |
RBC and G361 cell preparation and flow adhesion assay.
After obtaining informed consent, blood samples were collected from
patients with hemoglobin SS disease in 3.8% sodium citrate (1:9). The
RBCs were washed three times and resuspended at a 2% hematocrit in
M199 serum-free cell culture medium (Sigma, St Louis, MO) containing
0.2% bovine serum albumin (BSA, buffer A) as previously described.25 G361 melanoma cells were obtained from
American Type Culture Collection (ATCC; Rockville, MD)
(CRL 1424) and cultured in McCoy's 5A medium supplemented with 10%
fetal calf serum at 5% CO2. Cells were detached using
trypsin-EDTA, washed twice with medium, and resuspended at 1 × 106/mL in buffer A. SS-RBC or G361 cell adhesion was
studied using a parallel plate perfusion chamber as previously
described.25,27 In brief, intact or purified TSP fragments
were coated on 35-mm2 tissue culture plates (2 µg/cm2, 60 minutes, room temperature) which served as the
adhesive surface of the flow chamber. An initial rinse period of 3 to 5 minutes with buffer A containing BSA served to block the treated
plates.25 Washed cells suspended in buffer A (2.5 to 3 mL)
were perfused through the flow chamber at a wall shear stress of 1 dyne/cm2, a force similar to that found in the
postcapillary venule. After a 5- to 10-minute rinse period, the number
of adherent cells per unit surface area were counted by direct
microscopic visualization of the adhesive surface in a previously
calibrated grid of known area in 4 to 6 random areas near the center of
the flow surface. For inhibition experiments, the reagent being tested
was incubated with either the cell suspension or the adhesive surface
for 30 minutes at 37°C before the initiation of the flow adhesion
assay. All flow adhesion experiments were performed at 37°C using
an air curtain incubator.
Purification and proteolytic digestion of TSP.
TSP was purified from washed, activated (thrombin receptor activation
peptide SFLL, 5 µmol/L and thromboxane A2 analogue U46619, 0.5 µmol/L) platelet releasate by gel filtration on Sepharose 4B
(Pharmacia, Piscataway, NJ) followed by affinity chromatography on
heparin-Sepharose as previously described.14 The 25-kD
NH2-terminal heparin-binding domain and the 140-kD
carboxy-terminal proteolytic TSP fragments were prepared by thermolysin
digestion (1:100, wt:wt) in the presence of 1 mmol/L CaCl2
in Tris-buffered saline (TBS, 20 mmol/L Tris-HCl, pH 7.6, 150 mmol/L
NaCl) for 60 minutes at room temperature. The digestion was terminated
by the addition of threefold excess (wt/wt) phosphoramidone and the
fragments purified by heparin-Sepharose FPLC affinity chromatography
(Pharmacia) as previously described.16 The 70-kD
proteolytic TSP fragment was generated by chymotrypsin digestion
(1:100, wt:wt) in the presence of 10 mmol/L EDTA in TBS, pH 7.4, for 30 minutes at room temperature followed by purification using gel
filtration chromatography (Superdex HR 75 FPLC; Pharmacia) as
previously described.28,29 To generate the 120-kD
proteolytic TSP fragment that contains the type 1, 2, and 3 repeats
(Ca2+ binding domain), TSP was treated with chymotrypsin
(1:100, wt:wt) in the presence of 1 mmol/L CaCl2 in TBS for
5 to 60 minutes at room temperature. All chymotrypsin digestions were
terminated by the addition of threefold excess (wt/wt)
phenylmethylsulfonyl fluoride (PMSF). The TSP digests and purified TSP
fragments were resolved by 4% to 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with or without
reduction using 2-mercaptoethanol followed by Coomassie blue staining.
Because the intact 20-kD TSP cell-binding domain remains disulfide
linked, we were not able to obtain this proteolytic fragment.
TSP peptides and monoclonal antibodies (MoAbs).
Mouse IgG1 MoAb C6.7, which recognizes the cell-binding
domain of TSP and functionally blocks the binding of platelets and melanoma cell to TSP,30,31 was provided by William A. Frazier (Washington University, St Louis, MO). Mouse IgM MoAb A4.1 is directed against the type 1 repeats of TSP29 and was
purchased from GIBCO-BRL (Gaithersburg, MD). The control
IgG1 MoAb MBC 35.5 is directed against protein C. The
control IgM MoAb MBC 45.7 is directed against protein S. The TSP
cell-binding domain adhesive peptides KRFYVVMWKK (4N1K) and FIRVVMYEGKK
(7N3), scrambled control peptide VKMKWKYVRF, and peptides GRGDW and
GRGEW were synthesized on a MilliGen 9050 PepSynthesizer (Millipore)
and purified by reverse-phase high-performance liquid chromatography
(HPLC). For studies requiring immobilized peptide, the 4N1K, 7N3, and
scramble control resin bound peptides were conjugated to BSA by
incubating with 1,3 diisopropylcarbodiimide and hydroxybenzotriazole in
dimethylacetamide overnight at room temperature. The BSA-coupled
peptides were cleaved from the resin and side chains groups removed
followed by precipitation in ether and desalting.
Flow cytometry experiments.
Washed SS-RBCs were resuspended to 10% hematocrit in TBS containing 1 mmol/L Ca2+ and 0.2% BSA (TBS-BSA/Ca2+) and
incubated with purified TSP (1 to 1.4 mg/mL) for 60 minutes at
37°C. After two washes in TBS-BSA/Ca+2, treated SS-RBCs
were incubated with mouse MoAb A4.1, isotype-specific negative control
MoAb MBC 45.7 (IgM, ascites, diluted 1:100 to 1:10,000), or positive
control MoAb E5 that recognizes glycophorin C (Sigma; IgG1,
ascites, diluted 1:600) for 60 minutes at room temperature. After two
washes, RBCs were incubated with fluorescein isothiocyanate
(FITC)-conjugated anti-mouse IgG or IgM (Jackson ImmunoResearch
Laboratories, West Grove, PA) for 30 minutes at room temperature and
bound antibody detected by flow cytometry.
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RESULTS |
To define the region of TSP that binds to sickle RBCs, several
strategies were used to generate and purify proteolytic fragments of
TSP. Thermolysin digestion of purified TSP in the presence of
Ca2+ yielded a 25-kD fragment that contains the
NH2-terminal heparin-binding domain, and a 140-kD
carboxy-terminal proteolytic fragment
(Fig 1).16
Chymotrypsin digestion of TSP in the presence of EDTA yielded a 70-kD
TSP fragment that contained the type 1 and type 2 repeats.
Alternatively, chymotrypsin digestion of TSP in the presence of
Ca2+, to protect the Ca2+-binding domain from
proteolytic cleavage, resulted in a 120-kDa carboxy-terminal
proteolytic fragment that is similar to the 140-kD fragment except for
cleavage of the terminal 18 to 20 kD of the carboxy-terminus that
contains the cell-binding domain.28 Similar to previous
reports,16,29,32 resolution of the TSP fragments by
SDS-PAGE under nonreducing conditions showed that the 25-kD fragment
was a monomer, while the 70-kD, 120-kD, and 140-kD fragments remained
trimers (data not shown). Additionally, the 18- to 20-kD C-terminal
fragment that is cleaved from the 140-kD fragment to form the 120-kD
fragment remains linked by disulfide bonds.31
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| Fig 1.
SS-RBCs bind to the 140-kD
carboxy-terminal proteolytic fragment of TSP. (A) Schematic
illustration of intact TSP with selected domains as described in the
text (HBD, heparin-binding domain; type 1, type 1 repeats; type 2, type
2 repeats; Ca++ Binding, Ca2+-binding
domains or type 3 repeats; CBD, cell-binding domain). The approximate
locations of the 25-kD, 140-kD, 120-kD, and 70-kD proteolytic fragments
as identified by amino-terminal sequencing and size16,29,32
are indicated by arrows. The 25-kD fragment is a monomer,16
the 70-kD, 120-kD, and 140-kD fragments are trimers,32 and
the 18- to 20-kD C-terminal fragment remains disulfide linked to the
120-kD fragment.31 (B) Proteolytic digestion and
purification of TSP fragments. TSP purified from platelet releasate was
treated with thermolysin and purified by heparin-Sepharose affinity
chromatography, or chymotrypsin in the presence of Ca2+
or EDTA and purified by gel-filtration FPLC as described in Materials
and Methods. Intact TSP (TSP), proteolyzed TSP (Thermolysin, Chtpn), or
purified proteolytic fragments (140 kDa, 25 kDa, 70 kDa, 120 kDa) were
resolved by 4% to 12% SDS-PAGE and stained with Coomassie Blue. (C)
Washed SS-RBCs were perfused through parallel plate flow chambers
coated (2 µg/cm2) with intact TSP (TSP, N = 12) or
purified 25-kD (25 kDa, N = 4), 140-kD (140 kDa, N = 4),
70-kD (70 kDa, N = 4), or 120-kD (120 kDa, N = 4) TSP fragments at
a wall shear stress of 1 dyne/cm2. After rinsing, adherent
RBCs per unit area were counted by direct microscopic visualization.
The results are shown as the mean ± SE of SS-RBC adhesion, normalized
to SS-RBC adhesion to intact TSP.
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When the purified 25-kD N-terminal heparin-binding domain of TSP was
immobilized on the surface of the flow adhesion chamber, it supported
only 9% of the binding activity of SS-RBCs to intact TSP under flow
conditions (Fig 1C). The 70-kD and 120-kD chymotryptic TSP fragments
that contain the procollagen-like segment, and type 1, 2, and 3 repeats
also did not support SS-RBC adhesion. However, the 140-kD TSP fragment,
containing the carboxy-terminal cell-binding domain in addition to the
type 1, 2, and 3 repeats of TSP, fully supported the adhesion of
SS-RBCs. As shown by the sequential chymotrypsin digestion of TSP in
Fig 2, the binding activity of TSP
dissipated coincident with the progressive cleavage of TSP fragments
larger than 120 kD containing the carboxy-terminal cell binding domain.
These data indicate that the 20-kD carboxy-terminal segment of TSP,
which contains the cell-binding domain, is required for SS-RBC adhesion
to immobilized TSP under flow conditions.
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| Fig 2.
Time course of TSP chymotrypsin digestion and its effect
on SS-RBC adhesion. (A) TSP was incubated in the presence of
CaCl2 (1 mmol/L) with control buffer containing
PMSF-inactivated chymotrypsin (time = 0 minutes) or active
chymotrypsin (1:100, wt:wt) for 5, 10, 30, or 60 minutes before
stopping the reaction with PMSF. Treated TSP was coated (2 µg/cm2) on flow-chamber wells followed by perfusion of
washed SS-RBCs as described in the legend to Fig 1. The results are
shown as the mean ± SE of SS-RBC adhesion, normalized to SS-RBC
adhesion to control-buffer-treated TSP (N = 3 to 9 for each time
point). (B) TSP treated with control buffer (PMSF-inactivated
chymotrypsin, 0) or chymotrypsin (5, 10, 30 or 60 minutes) was resolved
by 4% to 20% SDS-PAGE and stained with Coomassie Blue. Note that the
heparin-binding domain (25 kD) is cleaved during the incubation with
the control buffer, leaving the fully active 140-kD TSP fragment.
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In studies to further map potential adhesive sites within the
cell-binding domain of TSP that bind SS-RBCs, the anti-TSP MoAb C6.7,
which blocks the binding of platelets and transformed cells to the TSP
cell-binding domain,30 was tested for its effect on SS-RBC
adhesion. As shown in Fig 3A, anti-TSP MoAb
C6.7 did not significantly inhibit SS-RBC adhesion to TSP compared with isotype-specific control MoAb. The peptides 4N1K (KRFYVVMWKK) and 7N3
(FIRVVMYEGKK) are two well-characterized cell-binding domain adhesive
sequences that both inhibit as well as support TSP-mediated adhesion of
platelets and transformed cells.19 When the peptides 4N1K
or 7N3 were incubated with the SS-RBCs during the flow adhesion assay,
both peptides, either alone or in combination, failed to inhibit SS-RBC
adhesion to intact TSP (Fig 3A). In contrast, G361 melanoma cells,
which are reported to attach to TSP via the cell-binding
domain,19,33 bound to immobilized TSP under the same
conditions of low shear flow as the SS-RBC adhesion (Fig 3B). Similar
to previous reports under static conditions,19,33 the
adhesion of G361 cells to TSP was significantly inhibited by peptides
7N3 and 4N1K as well as MoAb C6.7 under flow conditions (Fig 3B,
P < .05). Additionally, when the albumin-conjugated peptides
4N1K and/or 7N3 were immobilized to the surface of the flow adhesion
assay (2 µg/cm2), neither peptide supported SS-RBC
adhesion (<5% adhesion, N = 3 to 9, data not shown). These data
suggest that these known adhesive epitopes within the TSP cell-binding
domain are not involved in the adhesion of sickle RBCs to immobilized
TSP under flow conditions.
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| Fig 3.
Effect of TSP cell-binding domain inhibitory antibodies
and peptides on SS-RBC adhesion to TSP. (A) Washed SS-RBCs were
incubated with control buffer (None), 7N3 peptide FIRVVMYEGKK (7N3, 200 µmol/L, N = 2), 4N1K peptide KRFYVVMWKK (4N1K, 200 µmol/L, N = 2), or both 4N1K and 7N3 peptides (4N+7N, 200 µmol/L, N = 2) for
60 minutes at 37°C before the flow adhesion assay. Treated RBCs
were perfused through flow chambers coated with immobilized TSP as
described in the legend to Fig 1. Immobilized TSP was incubated with 5 µg/mL purified anti-TSP MoAb 6.7 (C6.7) or isotype-specific control
MoAb MBC 35.5 (Control) for 30 minutes at 37°C before performing
the flow adhesion assay as described above (N = 3). Similar
results were found incubating SS-RBCs with MoAbs in ascites fluid (C6.7
and MBC 35.5, diluted 1:1,000) before the flow adhesion assay (N = 9). (B) G361 cells were incubated with control buffer (None, N = 4),
scramble control peptide (Scramble, 200 µmol/L, N = 8) 7N3 peptide
(7N3, 200 µmol/L, N = 4), 4N1K peptide (4N1K, 200 µmol/L, N = 4), or immobilized TSP was incubated with anti-TSP MoAb 6.7 (C6.7, N
= 4) for 30 to 90 minutes at 37°C before the flow adhesion assay.
G361 cells were then perfused through the flow chambers coated with
immobilized TSP as described in Fig 1.
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Because multiple adhesive epitopes may participate in the binding of
sickle RBCs to TSP, other potential adhesive domains of TSP were
studied. The last type 3 repeat within the TSP Ca2+-binding
domain contains the adhesive tripeptide RGD motif that participates in
the binding of TSP to integrin receptors.18 As shown in
Fig 4, the peptide RGDW, which blocks the
binding of cells to TSP via integrins, failed to significantly inhibit SS-RBC binding to TSP compared with control RGEW peptide. Although there was some decrease in SS-RBC adhesion in the presence of the RGDW,
the levels were similar to the inactive RGE control peptide, suggesting
a nonspecific peptide effect. Additionally, there was no dose-response
effect, with similar results obtained using concentrations of RGD
peptide ranging from 200 µmol/L to 1 mmol/L (data not shown).
Therefore, it is unlikely that the adhesive RGD motif contributes to
SS-RBC binding to immobilized TSP under low shear flow conditions. When
immobilized TSP was incubated with EDTA, known to affect the
conformation of both the Ca2+-binding domain as well as the
cell-binding domain, SS-RBC adhesion was inhibited by 66% (Fig 4).
When TSP was incubated with EDTA (5 mmol/L) followed by replacement of
Ca2+, SS-RBC adhesion to TSP was fully restored,
demonstrating that the EDTA effect on TSP was reversible (Fig 4).
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| Fig 4.
The role of calcium and RGD in SS-RBC adhesion to TSP.
Washed SS-RBCs were incubated with control buffer (None, N = 19),
GRGDW (RGD, 200 µmol/L, N = 12), or control peptide GRGEW (RGE, 200 µmol/L, N = 12) for 30 minutes at 37°C before perfusing through
flow chambers precoated with TSP. Immobilized TSP was incubated with
EDTA (EDTA, 5 mmol/L, 30 minutes, N = 5) or EDTA (5 mmol/L, 30 minutes) followed by 10 mmol/L CaCl2
(EDTA/Ca2+ N = 3) before performing the flow adhesion
assay as described above.
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To further study other potential adhesive regions on TSP, we developed
a murine MoAb (TSP-N1) that binds to the N-terminal 25-kD
heparin-binding domain of TSP as determined by immunoblot of intact and
thermolysin digested TSP (data not shown). However, this MoAb did not
significantly inhibit SS-RBC adhesion to immobilized TSP (104% ± 13% adhesion, N = 6). Furthermore, incubation of immobilized TSP with
anti-TSP MoAb 4.1 that is directed against the type 1 repeats16,19 failed to inhibit SS-RBC adhesion to TSP (data not shown). These data are in agreement with the above proteolytic TSP
fragment studies where neither the 25-kD N-terminal TSP fragment containing the heparin-binding domain nor the 70-kD fragment containing the type 1 repeats supported SS-RBC adhesion.
The binding characteristics of many of adhesive ligands can vary
depending on the shear force (eg, von Willebrand factor)34 and whether the ligand is in solution phase versus the solid phase (eg,
fibrinogen).21 Although immobilized TSP avidly binds
SS-RBCs under low shear conditions, soluble TSP did not bind SS-RBCs as detected by flow cytometry (Fig 5A). While
this study cannot rule out weak TSP binding to SS-RBCs that did not
tolerate the preparative washes, the tenacious adhesion of SS-RBCs
observed in the flow adhesion chambers was clearly not present.
Additionally, soluble TSP did not inhibit SS-RBC adhesion to
immobilized TSP (Fig 5B). This suggests that immobilization of TSP on a
matrix is required to expose the adhesive epitope within TSP that
recognizes SS-RBCs. Because proteins binding to plastic may not
accurately mimic in vivo conditions, TSP immobilized on a fibrinogen
matrix was also tested. TSP-immobilized on fibrinogen bound SS-RBCs
(mean 189 RBCs/mm2, N = 2), while the fibrinogen matrix
alone promoted minimal SS-RBC adhesion (mean 6 RBCs/mm2,
N = 2).
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| Fig 5.
Soluble TSP does not bind SS-RBCs. Washed SS-RBCs were
incubated with purified TSP (1.4 mg/mL) in the presence of 1 mmol/L
Ca2+ for 30 minutes, 37°C. Treated RBCs were incubated
with the nonblocking anti-TSP MoAb A4.1 (SS1 + TSP, SS2 + TSP), isotype-specific control MoAb MBC 45.7 (Control), or a positive
control MoAb directed against glycophorin C (glycophorin C), followed
by FITC-conjugated anti-mouse MoAb, and bound TSP detected by flow
cytometry. The above data are representative of six separate
experiments. (B) Washed SS-RBCs were incubated with purified soluble
TSP (100 µg/mL) for 30 minutes at 37°C before performing the flow
adhesion assay over immobilized TSP as described above (N = 3).
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DISCUSSION |
Using controlled proteolysis of TSP, we have shown that maintaining the
integrity of the 20-kD carboxy-terminal segment of TSP is essential for
binding to sickle erythrocytes. This region of TSP contains the
cell-binding domain that recognizes platelets, human melanoma cells,
and many other transformed cell lines.19 Using overlapping
serial peptide sequences throughout the cell-binding domain33 followed by subsequencing the active
sites,19 Kosfeld and Frazier have identified two adhesive
peptide sequences, termed 4N1K and 7N3, that share the VVM amino acid
motif. Although these adhesive sites are important for binding to
platelets and multiple transformed cell lines, we found that neither of
these adhesive peptides contributed to SS-RBC adhesion. In agreement,
the TSP MoAb C6.7, which binds the cell-binding domain and inhibits the binding of platelets and transformed cells to these adhesive sites, had
no blocking activity for SS-RBC adhesion. These data argue that SS-RBCs
recognize a novel adhesive site within the 20-kD carboxy-terminal TSP
cell-binding domain that is destroyed by proteolytic cleavage.
Alternatively, because the C-terminal 20-kD fragment remains disulfide
linked to the 120-kD fragment, the proteolytic cleavage may directly
destroy the adhesive epitope or essential interactions between the
20-kD and 120-kD TSP fragments. In this way, the cleavage of the
cell-binding domain abolishes a critical conformational structure of
the 20-kD fragment, the 120-kD fragment or a more distant region of
TSP. Thus, SS-RBCs could be binding to a site other than the TSP
cell-binding domain. However, the presence of the intact, uncleaved
cell binding domain must be required for the proper conformational
presentation of the alternative region of TSP. Additionally, because
MoAbs directed against the heparin-binding domain and the type 1 repeats also failed to affect SS-RBC adhesion, no alternative sites on
TSP that bind SS-RBCs were identified in this study.
The RGD peptide, which inhibits the binding of integrin receptors to
TSP, also did not significantly inhibit SS-RBC adhesion compared with
the control inactive peptide in this study. This is in contrast to
other studies where the RGD peptide has been observed to inhibit SS-RBC
adhesion to cytokine-treated35 or TSP-treated23
endothelial cells. The differences between these results
are likely due to distinct adhesive interactions occurring between
SS-RBCs and either activated endothelial cells, TSP bound to
endothelial cells, or immobilized TSP. Additionally, we25 and others26 have previously reported that both OKM-5, a
murine anti-CD36 MoAb, which blocks TSP binding to CD36, and the TSP type 1 repeat peptide, CSVTCG, which blocks the interaction of TSP with
CD36, failed to inhibit the binding of SS-RBCs to immobilized TSP.
These data would argue against other known adhesive TSP regions participating in SS-RBC adhesion to immobilized TSP.
The binding of TSP to SS-RBCs appears to be sensitive to conformational
changes. This was evident by both the effect of divalent cation
chelators on SS-RBC adhesion to TSP and the differences in binding
activity between soluble and immobilized TSP. The conformation of both
the Ca2+-binding domain and the cell-binding domain are
affected by divalent cations.22,36 When Ca2+ is
removed, the globular cell-binding domain unfolds and both the
cell-binding domain and the Ca2+-binding domain are more
sensitive to proteolytic cleavage.36,37 Because the removal
of Ca2+ can destabilize disulfide bonds and result in
extensive thiol-disulfide exchange, especially in the
Ca2+-binding domain, some conformational changes are
irreversible.38 Therefore, it is interesting that the
EDTA-induced inhibition of SS-RBC adhesion to TSP was fully reversed
after repletion of Ca2+.
Our observations that soluble TSP neither bound SS-RBCs nor inhibited
the adhesion of SS-RBCs to immobilized TSP lend further support for a
conformation-dependent epitope that is only expressed on TSP after
binding to a matrix. Similar findings have been reported for other
adhesive molecules such as fibrinogen, which is not recognized by the
integrin IIbIII on resting platelets
unless it binds to a matrix with an associated conformational
change.22 Our observation that TSP immobilized on wells
coated with fibrinogen also supported SS-RBC adhesion suggests that TSP
within a more natural extracellular matrix will likely bind SS-RBCs.
Alternatively, the high density of adhesive epitopes, resulting from
coating high concentrations of purified TSP onto a surface, may
optimize its affinity for SS-RBCs. Additionally, the finding by
Barabino et al,39 that von Willebrand factor can bind to
TSP and block SS-RBC adhesion, provides evidence that the adhesive
epitope on immobilized TSP that recognizes sickle RBCs is complex and
can be modulated by plasma and neighboring extracellular matrix proteins.
Two groups have previously reported that SS-RBCs bind to endothelial
cells after the addition of soluble TSP.23,24 In these studies, the adhesion of the sickle RBCs to the endothelial cells was
proposed to involve CD36 on the surface of sickle cells binding to
CSVTG within the type 1 repeats of TSP that was attached to an adhesive
receptor on the endothelial cell. Our observation that soluble TSP does
not bind sickle RBCs suggests that the attachment of the TSP to the
endothelial cells may induce a conformational change in TSP that
permits it to bind to SS-RBCs. However, the binding of sickle RBCs to
TSP bound to endothelial cells likely acts through different mechanisms
compared with SS-RBC adhesion to immobilized TSP. Neither CD36 nor the
type 1 repeats of TSP appear to be involved in the adhesion of SS-RBCs
to immobilized TSP.25,26 Additionally, while the
reports of sickle cell adhesion to endothelial cells via TSP show
a reticulocyte predominant subpopulation of RBCs
binding,23,24 we find that the SS-RBCs that bind to immobilized TSP under low shear flow conditions are not enriched for
reticulocytes (unpublished observations, November 1996). Thus, it is
likely that SS-RBC adhesion to intact endothelial cells versus
extracellular matrix involves diverse adhesive/ligand
interactions and conformational requirements.
In studying myoblast adhesion to immobilized TSP, Adams and
Lawler20 reported that myoblasts bound to the 140-kD TSP
fragment, but not to TSP fragments containing the heparin-binding
domain, or the type 1 and 2 repeats, and that this adhesion was not
affected by MoAb C6.7. These results are identical to
those found in this study for SS-RBC adhesion to surface-bound TSP.
Additionally, they found that either high-molecular-weight dextran
sulfate or chondroitin sulfate A inhibited the adhesion of myoblasts to
TSP and proposed that chondroitin sulfate proteoglycans on the myoblast surface were likely contributing to the adhesive
phenotype.20 We25 and others26 have
previously reported that the binding of SS-RBCs to immobilized TSP is
similarly inhibited by either high-molecular-weight dextran sulfate or
chondroitin sulfate A, and we25 proposed that sulfated
glycolipids may contribute to the SS-RBC adhesion. These data suggest
that analogous mechanisms may be contributing to the adhesion of either
myoblasts or sickle RBCs to immobilized TSP. It would be interesting to
postulate that an aberrant sulfated moiety in the sickle RBC membrane
may be binding to TSP through a mechanism that evolved for the inherent control of myoblast adhesion to matrix-bound TSP.
It is not known whether the adhesion of SS-RBCs to TSP contributes to
vaso-occlusive crises in sickle cell disease in vivo. However,
identification of the characteristics of SS-RBC adhesion will likely
improve our understanding of vaso-occlusive events in sickle cell
disease and may potentially result in improved therapy for this
disorder. The data in this study provide evidence for a unique
conformation- or valence-dependent adhesive site within the 20-kD
carboxy-terminal region of TSP required for the adhesion of SS-RBCs to
the subendothelial matrix protein TSP under conditions of flow in
vitro. Further characterization of this region within TSP that binds
the SS-RBCs and conditions that will inhibit this interaction will
further our understanding of RBC-adhesive ligand interactions and may
provide insight into the pathophysiology of vaso-occlusion in sickle
cell disease.
| |
ACKNOWLEDGMENT |
We thank William A. Frazier (Washington University, St Louis, MO) for
the gifts of MoAbs and peptides that were used in this study, as well
as for careful review and useful suggestions regarding the manuscript.
We also thank Evelyn Brown and Gwendolyn Lea for their assistance in
obtaining blood samples for this study.
| |
FOOTNOTES |
Submitted October 1, 1998; accepted February 22, 1999.
Supported by Public Health Services Grants No. K08-HL02858 (C.A.H.) and
Clinical Research Center Grant No. RR00058 from the National Institutes
of Health.
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 reprint requests to Cheryl A. Hillery, MD, Blood Research
Institute, The Blood Center of Southeastern Wisconsin, PO Box 2178, Milwaukee, WI 53201-2178; e-mail: chillery{at}bcsew.edu.
| |
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