From the Unité 353 INSERM, Institut
d'Hématologie, Université Paris VII, Hôpital St
Louis, Cedex 10, Paris, France, the § Department of
Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada, and
the
Department of Biochemistry, Wake Forest University
School of Medicine,
Winston-Salem, North Carolina 27157-1072
Received for publication, November 6, 2000, and in revised form, November 27, 2000
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ABSTRACT |
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Thrombospondin-1 (TSP) may, after secretion from
platelet Thrombospondin-1 (TSP)1
represents 20-30% of the glycoproteins stored in human platelet
The participation of TSP in platelet aggregation has been demonstrated
by a variety of studies reporting inhibition of platelet aggregation
and secretion, by anti-TSP antibodies (18-23) and synthetic or
recombinant peptides of TSP (6, 24). Leung (19) suggested that the
interaction of TSP with Fg on the surface of activated platelets
stabilizes the binding of Fg to its receptor, the activated integrin
GPIIbIIIa (GPIIbIIIa*), with only Fg participating in direct
cross-bridges. Recent studies propose that TSP also interacts with the
integrin-associated protein (IAP/CD47) (11) and functions as a
costimulator of platelet integrin GPIIbIIIa and GPIaIIa (25, 26). A
direct role for TSP as a cross-linker of platelets involving TSP/Fg
interactions was supported by studies performed with isolated platelet
membranes or activated fixed platelets (AFP) bearing Fg (27, 28).
However, in these models, the correlation between numbers of
ligands/receptors and kinetics/extent of aggregation was not addressed.
Moreover, studies were generally performed under nearly static
conditions (29) or in an aggregometer (27), i.e. not
representative of the physiological flow environment and shear stresses.
In the present study, using well defined in-flow experimental models,
free of plasma, red blood cells, platelet signaling, and secretion, we
isolated, measured and modelled (i) the capacity of TSP to form and
support inter-platelet cross-bridges through its interactions with Fg
or with other TSP molecules and (ii) the contribution of TSP-mediated
cross-bridges in the aggregation of platelets driven by Fg/GPIIbIIIa* interactions.
Reagents--
Human platelet TSP was purified as published (30)
and characterized by Western blot for the absence of fibrinogen, von
Willebrand factor and fibronectin. The recombinant protein TSP18,
corresponding to amino acid residues 1-174 of human platelet TSP, was
purified from inclusion bodies as previously described (6). Human Fg, depleted of von Willebrand factor and fibronectin, was from Enzyme Research Laboratories (South Bend, IN). Purified human platelet GPIIbIIIa receptor was isolated from human platelet membranes by lentil
lectin affinity chromatography followed by Sephacryl S-300 HR gel
filtration chromatography and eluted from the column with an HSC buffer
(5 mM HEPES, 150 mM NaCl, 3 mM
CaCl2, pH 7.4) containing 30 mM of
n-octyl- Preparation of Fg- or TSP-coated Beads--
Polystyrene latex
beads were washed three times at ~0.5% solids and incubated with
either 500 nM Fg in phosphate-buffered saline or 200 nM TSP in Tyrode buffer containing 2 mM
Ca2+, pH 7.4, for 30 min at room temperature and processed
as previously published for Fg (32). The beads were finally centrifuged
and resuspended in distilled and deionized water (Fg-beads), or in Tyrode containing 2 mM Ca2+ (TSP-beads) at a
concentration of 250,000 beads/µl and stored at 4 °C. Beads coated
only with BSA (BSA-beads) following the same procedure served as
controls in aggregation or protein binding studies. The number of Fg or
TSP molecules bound to the beads was measured with FITC-labeled protein
(diluted 1:10 with unlabeled protein) as previously reported (34), with
an average of 183,034 ± 11,740 Fg or 149,070 ± 26,942 TSP
molecules per bead (i.e. 2882 ± 185 and 2347 ± 424 molecules/µm2), respectively. The FITC/molecule ratio
was also used to determine the number of FITC-labeled molecules bound
per bead or platelet in equilibrium binding studies.
GRGDSP-activated GPIIbIIIa*-beads--
GRGDSP-activated
GPIIbIIIa beads (GPIIbIIIa*-beads) were prepared with aldehyde/sulfate
polystyrene latex beads as previously described (34) with 110 nM GPIIbIIIa and 1 mM GRGDSP at room temperature. Final beads were washed with BSA to block unoccupied sites, then washed, and stored at 4 °C in phosphate-buffered saline, 61 µM HEPES, 0.1% BSA, pH 7.4 (200,000 beads/µl). The
beads obtained had a total of 51,678 ± 4,935 GPIIbIIIa as
measured by FITC-Reopro binding and 38,747 ± 7,948 GPIIbIIIa*, as
measured by FITC-Fg binding at saturating concentrations
(i.e. 610 ± 125 GPIIbIIIa* molecules/µm2).
Washed Platelets--
Washed platelets were prepared from
platelet-rich plasma by the single centrifuging and dilution procedure
described by Goldsmith et al. (35). Briefly, blood was taken
from healthy volunteers not on any medication added into 3.8% sodium
citrate (1:9 v/v blood), followed by centrifugation (150 × g, 15 min). Plasma-rich platelet was removed and acidified
to pH 6.5 with 0.1% citric acid, and ZK 36 374 was added to 50 nM followed by centrifugation (800 × g for
15 min). The platelet pellet was gently redispersed and resuspended in
Ca2+-free modified Tyrode buffer containing 0.35% (w/v)
BSA, pH 7.4 (BAT buffer), and kept at 37 °C.
GPIIbIIIa-activated and Fixed Platelets--
GPIIbIIIa-AFP were
prepared according to Du et al. (36) with modifications as
previously reported (37). Briefly, plasma-rich platelet was diluted
10-fold with Ca2+-free BAT buffer, incubated at 37 °C
for 5 min with 10 nM ZK 36 374 and then for 5 min with
CaCl2 (1 mM), followed by 200 nM Ro 44-9883 to activate the GPIIbIIIa receptors for another 5 min (all at
37 °C). The platelets were then fixed with freshly prepared 0.5%
(w/v) paraformaldehyde, washed with BAT buffer, and stored at
4 °C.
Fg-GPIIbIIIa*-beads and Fg-AFP--
GPIIbIIIa*-beads
(10,000/µl in a total volume of 400 µl) were incubated with 150 nM Fg in BAT buffer, 1 mM CaCl2,
for 30 min at room temperature. Beads were then incubated with 1 µM Ro 44-9883, for 30 min at room temperature to
displace reversibly bound Fg from the beads and block free GPIIbIIIa*
to prevent aggregation caused by GPIIbIIIa*-Fg-GPIIbIIIa*
cross-bridges. In these conditions, 50% of bound Fg was irreversibly
bound to GPIIbIIIa* receptors, as measured using FITC-Fg. After
incubation, beads were pelleted (10,000 × g, 30 s), resuspended in 400 µl of BAT buffer, 1 mM CaCl2 containing 500 nM Ro 44-9883. Fg-AFP
(40,000/µl in a total volume of 400 µl) were prepared using the
same procedure, except that 500 nM Fg instead of 150 nM were used during the first incubation step. With AFP,
however, only ~ 5% of the surface bound Fg remained irreversibly attached after incubation of the platelets with Ro 44-9883. Fg-GPIIbIIIa*-beads and Fg-AFP were used for equilibrium binding and aggregation studies (see below).
Equilibrium Binding Studies--
All incubations were done at
room temperature in the dark. Fg- or TSP-beads (10,000/µl) were
incubated for 1 h with increasing concentrations of FITC-TSP or
FITC-TSP18, in Tyrode buffer, pH 7.4, supplemented with 1 mM CaCl2, 1 mM MgCl2,
1% (w/v) BSA, and 0.05% (v/v) Tween 20 (modified Tyrode buffer).
GPIIbIIIa*-beads or Fg-GPIIbIIIa*-beads (10,000/µl) were similarly
incubated for 1 h with increasing concentrations of FITC-TSP but
in modified Tyrode buffer. For effect of preincubation of TSP on
GPIIbIIIa*-beads, the latter (10,000/µl) were incubated for 1 h
in modified Tyrode buffer, with increasing concentrations of FITC-Fg
(0-100 nM) preincubated 30 min with buffer or 80 nM TSP. Binding studies were done in parallel on albumin
coated beads (BSA-beads) used as a control for the nonspecific binding
and for calculation of Kd and
Bmax.
Fg Occupancy of GPIIbIIIa* on AFP and Effects of TSP--
We
prepared AFP with Fg occupancy of GPIIbIIIa* at 3-6, 20, and 35%, by
incubating AFP in BAT buffer, 1 mM CaCl2, with
20 nM Fg for 2 min (3-6% occupancy) or 30 min (20%), or
with 50 nM Fg for 30 min (35%) at room temperature, prior
to shear. The occupancy was determined from the ratio of bound
fluorescence to the maximal fluorescence obtained at saturating Fg
concentration (33). To study the effect of TSP on Fg-mediated
aggregation of AFP, Fg and TSP were preincubated 15 min at room
temperature in a molar ratio of 1/4 (i.e. Fg/TSP = 20/80 or 50/200 nM), and AFP were incubated with this
Fg/TSP mixture in the same conditions as for Fg alone. We found no
changing in the specific FITC-Fg binding to AFP with TSP addition.
Effect of the Addition of TSP on Aggregation Efficiency of
GPIIbIIIa Beads Decorated by Fg at Very Low Receptor Occupancy
(0.5%)--
GPIIbIIIa*-beads (7000/µl) were incubated in BAT
buffer, 1 mM CaCl2, with 0.15 nM of
FITC-Fg for 30 min at room temperature to reach 0.5% receptor
occupancy. Beads were then incubated 30 min at room temperature with
buffer or 180 nM TSP and sheared for 0-60 s at 300 s Aggregation in Flow--
Kinetics of aggregations of AFP or
GPIIbIIIa*-beads and/or ligands (Fg, TSP) were determined in a
microcouette, as previously described (34, 37). Briefly, suspensions
(400 µl) were loaded in the gap between the two cylinders at a fixed
shear rate (G). Shear was stopped at selected times, and 20-µl
subsamples were taken, fixed with 0.8% (v/v) glutaraldehyde (5 volumes), and analyzed by flow cytometry. The fraction of particles
recruited into aggregates, PA (the percentage of
aggregation) was calculated by monitoring the decrease of single bead
particles/unit volume. Aggregation efficiencies (
Aggregation of AFP during incubation with soluble Fg or Fg plus TSP was
negligible, as confirmed by phase contrast microscopy (Zeiss, 500×
magnification). Moreover, we did not detect disappearance of platelets
or beads from the samples because of adhesion to the walls of the
microcouette during shear.
TSP-beads coaggregate during storage at 4 °C to partially form
doublets and triplets (with no significant higher multiplets): ~23
and 13%, respectively (mean of 15 measurements), of the total number
of particles at time 0 of the shear. We corrected for this effect on
calculated Data Analysis--
Data are expressed as the means ± S.E.
To fit the nonlinear equations to our data, we used a nonlinear
regression curve fitter software (Sigma Plot, Jandel Scientific
Software, San Rafael, CA), as previously described (37).
Interactions of TSP and TSP18 with Fg-beads--
Soluble FITC-TSP
(200 nM) bound to immobilized Fg (Fg-beads) with a
half-time of about 10 min and saturation by ~60 min of incubation at
room temperature (Fig. 1). Specificity of
the binding was demonstrated with a recombinant
NH2-terminal domain of TSP, TSP18, as shown previously with
Fg immobilized on microtiter wells (6), with up to 90% inhibition of
FITC-TSP binding to Fg-beads at 2 µM, and 50% inhibition
(IC50), of about 550 nM (Fig.
2). The adsorption isotherm curve of
FITC-TSP binding to Fg-beads was distinctly biphasic, as seen by
curves 1 and 2 in Fig.
3A (n = 3).
The Kd value for the initial phase ([FITC-TSP] < 150 nM (curve 1) was 52 ± 16 nM, corresponding to soluble TSP binding to immobilized Fg
with high affinity (38, 39), with a maximum binding
(Bmax) of 4,172 ± 473 molecules. The
second phase ([FITC-TSP] > 200 nM)
(Bmax of ~6,000 molecules) may correspond to a
low affinity TSP to TSP binding as described previously (40) and
explored further below. By comparison, FITC-TSP18 binding to Fg-beads
was fitted with a one-binding site model with a Kd of 369 ± 31 nM and a Bmax
of ~ 190,000 TSP18/bead (Fig. 3B). The difference of
Bmax obtained with the entire TSP molecule as
compared with the TSP18 fragment is explained by steric hindrances
caused by (i) the high density of Fg adsorbed on the beads (2882 Fg/µm2, i.e. maximal density as previously
published (34)) and (ii) the fact that Fg is horizontally elongated
when adsorbed on the beads (34). Under similar conditions, we did not
detect any binding of soluble FITC-Fg to TSP-beads (results not
shown).
Aggregation of Fg-beads by Soluble TSP--
In the absence of TSP,
Fg-beads did not aggregate during 120 s of shear at 300 s Effect of TSP on Fg Binding to GPIIbIIIa*-beads--
Soluble Fg
competed with Fg-beads for the binding of FITC-TSP with an
IC50 of ~240 nM (results not shown),
indicating that TSP interacted with both soluble and immobilized Fg, as
also shown previously (22). For this reason, we studied the influence
of the preincubation of FITC-Fg with TSP on the affinity
(Kd) and the Bmax of FITC-Fg
binding to GPIIbIIIa*-beads. The isotherm curve of FITC-Fg binding to
GPIIbIIIa*-beads was not modified when FITC-Fg was preincubated 30 min
with 80 nM of TSP (Fig. 5). In both conditions, FITC-Fg bound to GPIIbIIIa*-beads with a
Kd of 23 ± 2.4 nM and a
Bmax of ~39,000 ± 8000 FITC-Fg
molecules.
Binding of FITC-TSP to GPIIbIIIa*-beads and Fg-GPIIbIIIa*-beads or
Platelets--
Soluble TSP did not bind to GPIIbIIIa*-beads (Fig.
6). However, when GPIIbIIIa* receptors
were decorated by irreversibly bound Fg molecules
(Fg-GPIIbIIIa*-beads), soluble TSP bound to the beads in a saturable
manner. The corresponding isotherm binding curve, fitted to the data
using an equation for a one-binding site model, gave a
Kd of ~23 nM. However, the curve was
best fitted by a two-binding site equation model, which gave two
Kd values of ~5 and 163 nM,
corresponding to Bmax values of 2105 ± 178 and 4315 ± 806 molecules/bead, respectively. These observations were qualitatively confirmed for AFP and ADP (10 µM)-activated washed platelets, with no binding of
FITC-TSP (100 nM, 45 min), in absence of platelet secretion
verified by the lack of detectable surface expressed TSP. However,
preincubation for 5 min with 100 nM Fg led to binding of
FITC-TSP to the platelet surface (results not shown).
Aggregation of Fg-GPIIbIIIa*-beads by TSP--
We next determined
the capacity for soluble TSP to form cross-bridges between
receptor-bound Fg, in the absence of direct Fg cross-bridging to free
GPIIbIIIa* receptors. For this experiment, GPIIbIIIa*-beads were first
decorated by irreversibly bound Fg (Fg-GPIIbIIIa*-beads), and the free
receptors were blocked by the RGD mimetic Ro 44-9883 (Fig.
7, top panel). Thus, without TSP (Fig. 7A), the aggregation of Fg-GPIIbIIIa*-beads was
almost completely inhibited by Ro 44-9883 (PA = 13.6 ± 4.4%, after 120 s of shear rate at 300 s Effect of the Addition of TSP on Aggregation Efficiency of
Fg-GPIIbIIIa*-beads at Low Receptor Occupancy
(0.5%)--
Fg-GPIIbIIIa*-beads were prepared at low receptor
occupancy (0.5%) in a range where aggregation efficiency varies
rapidly with percent occupancy, previously reported to be between 0 and 20% for platelets (37) and 0 and 5% for our model beads (34). Incubation of these beads with 180 nM of TSP gave a partial
decoration of bound Fg by TSP (10-15% of the bound Fg). In the
presence of TSP, the kinetics of aggregation at 300 s Effect of the Addition of TSP on Aggregation Efficiency of AFP by
Fg--
We first varied the Fg receptor occupancy at a fixed shear
rate of 300 s
We next varied the shear rate at a fixed receptor occupancy of 20%.
Thus, AFP were incubated with Fg (20 nM) or Fg plus TSP (20/80 nM) for 30 min. In the absence of TSP, Interactions of TSP with TSP-beads--
Soluble FITC-TSP could
bind to TSP immobilized on polystyrene latex beads (TSP-beads) with a
Kd of 732 ± 118 nM (Fig. 9). Aggregate formation mediated by
TSP-TSP interaction(s) was investigated in flow by shearing TSP-beads
from 100 to 2000 s We studied the involvement of TSP in platelet aggregation through
its interaction with Fg bound to its receptor, GPIIbIIIa*. We used
model particles, either Fg-beads or Fg-GPIIbIIIa*-beads, or Fg-AFP.
These models were chosen to mimic the surface of activated platelets
but in the absence of any signaling or secretion process, thereby
isolating the cross-bridging functions of TSP while monitoring qualitative and quantitative surface expression of ligands.
Soluble TSP induced aggregation of Fg-beads
dose-dependently, with a maximal effect observed at 200 nM, and an aggregation efficiency ( Preincubation of FITC-Fg with TSP did not modify the affinity
(Kd) nor the maximum binding
(Bmax) of FITC-Fg to GPIIbIIIa* immobilized on
beads (Fig. 5). This is in accord with the study of Boukerche and
McGregor (23) who showed that a monoclonal antibody anti-TSP
(P8) that inhibits platelet aggregation by low doses of thrombin
(0.05-0.06 unit/ml) did not affect the dissociation constant of Fg
binding to platelets stimulated with ADP (10 µM) or
thrombin (0.4 unit/ml). However, Leung (19) postulated that TSP, by
interacting with Fg at a site different from its GPIIbIIIa*-binding site, increases the affinity of Fg for GPIIbIIIa*, thereby stabilizing the aggregates. This assumption, based on the observation that an
anti-TSP Fab decreased the affinity of Fg binding to thrombin activated
platelets, could rather reflect a steric hampering caused by the Fab
bound to TSP, that would disable the closely located Fg/GPIIbIIIa*
interactions described by several authors (9, 43, 44).
Surprisingly, our studies suggest that a receptor-bound Fg with
attached TSP is no longer capable of interacting with another GPIIbIIIa*, because incubation of Fg-GPIIbIIIa*-beads (0.5% of receptor occupancy) with TSP decreases the aggregation efficiency by
~30%. In this experiment, we expect that Fg-bound TSP (~10-15% of receptor-bound Fg contain a TSP molecule) cannot find a counterpart Fg molecule on an adjacent bead because of the low surface density of
Fg and TSP molecules (~200 Fg/bead and ~25 TSP/bead). This limitation is not encountered by the Fg-GPIIbIIIa* cross-bridging system because ~39,000 GPIIbIIIa* are available for each Fg on adjacent beads. Using a standard curve reporting the efficiency of
GPIIbIIIa*-beads aggregation for varying Fg receptor occupancies, as
previously published (34), we determined that the aggregation efficiency in the presence of TSP is equivalent to the aggregation efficiency that would be obtained with ~15% fewer functional Fg on
GPIIbIIIa*-beads, closely corresponding to the percentage of Fg
occupied by TSP. This hypothesis may explain the "anti-adhesive" property of TSP previously reported in the literature for platelet adhesion to immobilized Fg or fibronectin, after preincubation of these
supports with soluble TSP (45, 46). Our results support the idea that
the anti-adhesiveness of TSP is induced by the loss of the reactive
site on Fg or fibronectin for its platelet receptor, following its
occupancy by TSP.
Nevertheless, when testing the effect of addition of TSP on the
aggregation efficiency of AFP mediated by Fg and GPIIbIIIa*, we found
an increase of 30-110% at all Fg receptor occupancies (3-35%) and
all shear rates (300-2000 s granules, participate in platelet aggregation, but its
mode of action is poorly understood. We evaluated the capacity of TSP to form inter-platelet cross-bridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on
activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms. Aggregation at
physiological shear rates (100-2000 s
1) was studied in a
microcouette device and monitored by flow cytometry. Soluble TSP bound
to and induced aggregation of Fg-coated beads dose-dependently, which could be blocked by the
amino-terminal heparin-binding domain of TSP, TSP18. Soluble TSP
did not bind to GPIIbIIIa*-coated beads or AFP, unless they were
preincubated with Fg. The interaction of soluble TSP with
Fg-GPIIbIIIa*-coated beads or Fg-AFP resulted in the formation of
aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in
a system where direct cross-bridges mediated by GPIIbIIIa*-Fg on one
particle and free GPIIbIIIa* on a second particle were blocked
by the RGD mimetic Ro 44-9883. Soluble TSP increased the efficiency of
Fg-mediated aggregation of AFP by 30-110% over all shear rates and
GPIIbIIIa* occupancies evaluated. Surprisingly, TSP binding to Fg
already bound to its GPIIbIIIa* receptor appears to block the ability
of this occupied Fg to recognize another GPIIbIIIa* receptor, but this
TSP can indeed cross-bridge to another Fg molecule on a second
platelet. Finally, TSP-coated beads could directly coaggregate at shear rates from 100 to 2000 s
1. Our studies provide a model
for the contribution of secreted TSP in reinforcing inter-platelet
interactions in flowing blood, through direct Fg-TSP-Fg and
TSP-TSP cross-bridges.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-granules (1). Upon platelet activation and degranulation, TSP is
released, and an important fraction is found associated with the
platelet surface (2, 3). Several putative receptors and ligands for TSP
at the surface of activated platelets have been described, including
fibrinogen (Fg) (4-7), sulfatides (8) glycoprotein IV (GPIV, CD36) (9,
10), and integrin-associated protein (IAP/CD47) (11). TSP may also
interact with several integrins including
v
3 and
IIb
3
(GPIIbIIIa), through its cryptic RGDA sequences (12). However, the
interaction of TSP with GPIIbIIIa is controversial (13-17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (31). All
three proteins (TSP, Fg, and GPIIbIIIa) appeared undegraded, with
appropriate molecular weights, when analyzed in reduced/unreduced forms
by SDS-polyacrylamide gel electrophoresis. Peptide GRGDSP and
fluorescein isothiocyanate (FITC) celite were from Calbiochem (La
Jolla, CA). Ro 44-9883, a nonpeptide analogue of the RGD peptide but
1000 times more potent and selective for
IIb
3 (GPIIbIIIa) than for
v
3 (32), was kindly provided by Dr. T. Weller (F. Hoffmann-La Roche, Basel, Switzerland). Dr. T. Krais
(Schering Co., Berlin, Germany) generously provided ZK 36 374, a stable
prostacyclin analogue. Polystyrene latex beads (diameter, 4.5 µm)
were from Polyscience (Warrington, PA), and surfactant-free
aldehyde/sulfate polystyrene latex beads (diameter, 4.5 µm) were from
Interfacial Dynamics Corporation (Portland, OR). FITC-labeled TSP
(FITC-TSP), TSP18 (FITC-TSP18), or Fg (FITC-Fg) were prepared as
previously described by Xia et al. (33) for FITC labeling of
Fg. FITC-Reopro was a gift from Centocor (Malvern, PA).
1. These studies were compared with beads prepared with
5% Fg receptor occupancy that yield maximal kinetics of aggregation,
as previously reported (34).
), defined as the
fraction of all shear induced collisions that result in the formation
of doublets (34), was determined from experimentally measured initial
rate of beads or AFP removal into bead-bead or AFP-AFP doublet
formation, as previously described (34, 37).
by modifying our equations as previously described
(34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Kinetics of FITC-TSP binding to
Fg-beads. Fg-beads ( ) or BSA-beads (
) (control)
(10,000/µl) were incubated with FITC-TSP (200 nM) in
modified Tyrode buffer for increasing time up to 3 h at room
temperature in the dark. The number of FITC-TSP molecules associated
with the beads was calculated from the fluorescence bound to the beads
measured by flow cytometry. Results from one experiment representative
of three separate assays.
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Fig. 2.
Inhibition of FITC-TSP binding to Fg-beads by
TSP18. Fg-beads or BSA-beads were incubated with FITC-TSP (80 nM) in the presence of increasing concentrations of TSP18
(0-2000 nM), for 1 h at room temperature in the dark.
The fluorescence associated with the beads was measured by flow
cytometry. The results are presented as the percentages of FITC-TSP
specifically bound to Fg-beads (fluorescence measured on BSA-beads was
subtracted from the fluorescence measured on the Fg-beads). Means ± S.E. of three experiments are shown.
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Fig. 3.
Binding isotherms of FITC-TSP or FITC-TSP18
to Fg-beads. Fg-beads ( ) or BSA-beads (
) were incubated with
increasing concentrations of FITC-TSP (0-400 nM)
(A) or FITC-TSP18 (0-2000 nM) (B)
for 1 h. The fluorescence associated with the beads was measured
by flow cytometry. The results are expressed as the number of FITC-TSP
or FITC-TSP18 molecules bound to the beads. The binding of FITC-TSP to
BSA-beads, considered as nonspecific, amounted to about 30% of binding
to Fg-beads. The binding of FITC-TSP to the Fg-beads (mean ± S.E.
of three experiments) is best fitted by the nonlinear regression
curves 1 and 2 constructed from [TSP] < 150 nM and [TSP] > 200 nM data,
respectively.
1. However, Fg-beads preincubated for 20 min with
50-200 nM TSP before shear, aggregated
dose-dependently. Aggregation efficiencies (
) measured
from initial rates of aggregation increased from 5.3 ± 0.1% to
14.0 ± 2%, and the extent of aggregation at 120 s increased
from 63 ± 2% to 85 ± 2%. For 200 nM of TSP,
about 1-2% of all Fg at the surface of the beads was occupied by TSP (measured with FITC-TSP), corresponding to a surface density of ~29-58 TSP molecules/µm2 (n = 3).
TSP18 (4 µM), added before starting the shear, inhibited TSP (200 nM)-induced Fg-beads aggregation (
decreased
from 14.0 ± 2% to 4.0 ± 0.4%). However, TSP18 added only
after 120 s of aggregation of Fg-beads by TSP (200 nM)
could not dissociate the formed aggregates (Fig.
4).
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Fig. 4.
Aggregation of Fg-beads by soluble TSP.
Fg-beads (10,000/µl) were incubated for 20 min at room temperature
with increasing concentrations of TSP and then sheared at 300 s 1. After 120 s of shear buffer or 4 µM of TSP18 (black or white crossed
squares, respectively) was added to the Fg-beads aggregated by 200 nM TSP, and samples were sheared for additional 120 s.
Means ± S.E. of three experiments are shown.
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Fig. 5.
Binding isotherm of FITC-Fg to
GPIIbIIIa*-beads; influence of preincubation of FITC-Fg with TSP.
GPIIbIIIa*-beads were incubated for 1 h in modified Tyrode buffer,
with increasing concentrations of FITC-Fg (0-100 nM)
preincubated 30 min with buffer ( ) or 80 nM TSP (
).
BSA-beads were used instead of GPIIbIIIa*-beads, as a control for
nonspecific binding. The results are expressed as the number of FITC-Fg
molecules specifically bound to GPIIbIIIa*-beads. Results from one
experiment representative of three separate assays. Inset,
schematic representation describing the assay. Black
bar, Fg; shaded square, TSP; white oval with
indentation, GPIIbIIIa*.
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Fig. 6.
Binding isotherm of FITC-TSP to
GPIIbIIIa*-beads or Fg-GPIIbIIIa*-beads. GPIIbIIIa*-beads ( ) or
Fg-GPIIbIIIa*-beads (
) were incubated with increasing concentration
of FITC-TSP as described under "Experimental Procedures." Results
are expressed as the number of FITC-TSP molecules bound per bead. The
data from one typical experiment are shown. The curves
constructed from the data correspond to the best fit equations for
either a one-binding site (dotted line) or a two-binding
site association model (solid line). Inset,
schematic representation describing the assay. Black
bar, Fg; shaded square, TSP; white oval with
indentation, GPIIbIIIa*.
1). However, in presence of 200 nM TSP,
aggregation rapidly reached 70.1 ± 2.5% by 120 s with an
efficiency of 18.1 ± 4.8%. In such conditions, about 10-15% of
GPIIbIIIa*-bound Fg was occupied by TSP (calculated from Fig. 6),
corresponding to ~31-44 TSP molecules/µm2
(n = 3), very similar to the density of TSP on Fg-beads
mentioned above. We reproduced the experiment using Fg bound to
activated fixed platelets (Fg-AFP) instead of Fg-GPIIbIIIa*-beads (Fig. 7B). As explained under "Experimental Procedures," only
~5% of surface-expressed activated GPIIbIIIa* on AFP remained
occupied by Fg molecules in the presence of 1 µM Ro
44-9883. Thus, with all free GPIIbIIIa* blocked by Ro 44-9883, no
aggregation of Fg-AFP occurred in the absence of TSP, but added TSP
induced 12.5 ± 7.1% aggregation by 120 s of shear at 300 s
1, with a related aggregation efficiency of 1.9 ± 0.2%. The TSP/Fg ratio measured at the surface of the platelets was
the same as on Fg-GPIIbIIIa*-beads (10-15%), corresponding here to a
TSP surface density of 4-5 molecules/µm2
(n = 3).
View larger version (15K):
[in a new window]
Fig. 7.
Aggregation of Fg-GPIIbIIIa*-beads or
Fg-activated fixed platelets (Fg-AFP) by soluble TSP.
Fg-GPIIbIIIa*-beads (10,000/µl) (A) or Fg-AFP
(40,000/µl) (B) were incubated for 30 min at room
temperature without ( ) or with (
) 180 or 235 nM TSP,
respectively, in BAT buffer supplemented with 1 mM
CaCl2 and then sheared at 300 s
1 in presence
of 1 µM Ro 44-9883. Results, expressed as percentage of
aggregation, are the means ± S.E. of three experiments.
Inset, schematic representation describing TSP-mediated
aggregation of Fg-GPIIbIIIa*-beads or Fg-AFP in presence of Ro
44-9883.Black bar, Fg; shaded square,
TSP; white oval with indentation, GPIIbIIIa*;
shaded bar, Ro 44-9883.
1
was slowed down, corresponding to a decrease of
of ~30%, from 16.9 ± 0.1% to 12.0 ± 1.2% (Fig.
8).
View larger version (12K):
[in a new window]
Fig. 8.
Effect of the addition of TSP on aggregation
efficiency of Fg-GPIIbIIIa*-beads at 0.5% receptor occupancy.
GPIIbIIIa*-beads (7000/µl) where incubated in BAT buffer, 1 mM CaCl2, with 0.15 nM of FITC-Fg
for 30 min at room temperature to reach 0.5% receptor occupancy. Beads
were then incubated 30 min at room temperature with buffer ( ) or 180 nM TSP (
), and sheared at 300 s
1. Beads
prepared with 5% Fg receptor occupancy (
) were sheared in parallel
as a control for maximal aggregation. Means ± S.E. of three
experiments are shown. Inset, schematic representation
describing the assay. Black bar, Fg; shaded
square, TSP; white oval with indentation,
GPIIbIIIa*.
1. AFP were incubated with Fg or
Fg plus TSP (molar ratio of 1:4) to yield Fg-GPIIbIIIa* occupancies of
3-6%, 20, and 35% with about 10-15% of Fg occupied by TSP.
Aggregation efficiencies (
) for AFP sheared with Fg only, increased
from 9 to 20% with increasing Fg receptor occupancy from 3-6% to
35% (Table I). The addition of TSP
induced a significant increase of
at all Fg receptor occupancies
tested (p < 0.01 to p < 0.07), with
mean increases ranging from 30 to 61% (Table I).
Aggregation of AFP by Fg with varying Fg receptor occupancy: effect of
the addition of TSP on the aggregation efficiency () at a fixed
shear rate
1 in BAT buffer
supplemented with 1 mM CaCl2, after a 30-min
preincubation with Fg or a mixture of Fg and TSP, with varying the
incubation time and the concentrations to reach increasing GPIIbIIIa*
occupancy on AFP. Aggregation efficiencies were calculated from
aggregation curves. Means ± S.E. of three to eleven experiments.
decreased
15-fold when increasing the shear rate from 300 to 2000 s
1. The addition of TSP induced a significant increase of
at both 300 and 2000 s
1 (p < 0.1),
and an increase of 30% was also observed at 1000 s
1
(p < 0.3) (Table
II).
Aggregation of AFP by Fg with varying shear rate at a fixed
receptor occupancy: effect of the addition of TSP on the aggregation
efficiency ()
1. Aggregation efficiencies were
calculated from aggregation curves. Means ± S.E. of four to
eleven experiments.
1 in the microcouette (Fig.
10A). TSP-beads coaggregated
at all shear rates tested. The extent of aggregation after 120 s
of shear increased with increasing shear rate from 100 to 300 s
1 (59 ± 3 and 77 ± 3%, respectively) but
decreased at higher shear rates (67 ± 0.4 and 49 ± 3% at
1000 and 2000 s
1, respectively). The calculated related
indicated that the high efficiency of TSP-beads coaggregation at
100 s
1 (22.2 ± 4.9%) decreased by about 10- and
40-fold at 1000 and 2000 s
1, respectively (Fig.
10B). When TSP-beads were sheared at 300 s
1 in
the presence of 8 mM EDTA, both PA (Fig.
10A) and
were reduced by >80% and >90%,
respectively. TSP-beads did not aggregate either with Fg-beads or
GPIIbIIIa*-beads (results not shown).
View larger version (14K):
[in a new window]
Fig. 9.
Binding isotherm of FITC-TSP to
TSP-beads. FITC-TSP (0-500 nM) was incubated with
TSP-beads ( ) or BSA-beads (
) (nonspecific binding) for 1 h
at room temperature in the dark. Results, expressed as the number of
FITC-TSP molecules bound per bead, are the means ± S.E. of three
experiments.
View larger version (19K):
[in a new window]
Fig. 10.
Coaggregation of TSP-beads. TSP-beads
(10,000/µl) in BAT buffer supplemented with 1 mM
CaCl2 were sheared with varying the shear rate from 100 to
2000 s 1. TSP-beads were also sheared at 300 s
1 in the presence of 8 mM EDTA (preincubated
with the beads 1 min before the shear). Results, expressed as
percentages of aggregated platelets (A) or aggregation
efficiencies (B), are the means ± S.E. of at least
three separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) of 14 ± 2.1%
at a shear rate of 300 s
1. Aggregation of
GPIIbIIIa*-beads by TSP only occurred after preincubation of the beads
with Fg. With added Ro 44-9883, the RGD mimetic, to block the
cross-bridges between GPIIbIIIa* bound Fg and free GPIIbIIIa*,
aggregation occurred through Fg-TSP-Fg cross-bridges with
= 18.1 ± 4.8% at 300 s
1 similar to that
obtained with Fg-beads at comparable TSP surface densities (~30-60
TSP/µm2). This experiment reproduced with AFP gave
comparable results; aggregation by TSP at 300 s
1, with
added Ro 44-9883, only occurred in the presence of receptor-bound Fg,
with
lower than seen for Fg-GPIIbIIIa*-beads, expected for the
10-fold lower density of Fg and TSP in this system. These experiments
clearly demonstrate that TSP can induce aggregation of beads or AFP by
directly cross-bridging two Fg presented on two particles, at a
physiological shear rate (300 s
1). A recombinant fragment
encompassing residues 1-174 of TSP, TSP18, previously shown to inhibit
the secretion-dependent phase of platelet aggregation (6),
inhibited aggregation of Fg-beads by TSP but did not disaggregate the
formed aggregates. This is an indication for (i) the involvement of the
amino-terminal part of the TSP molecule in TSP-Fg interactions and (ii)
a strong TSP-Fg interaction (off-rate very low), as also observed in
the binding of FITC-TSP to Fg-GPIIbIIIa*-beads, where TSP18 was not
able to displace the bound FITC-TSP but inhibited any further binding of FITC-TSP (results not shown). There are several putative binding sites on Fg and TSP that could stabilize or reinforce their mutual interactions, including three sites on Fg: the A
241-476 (4), A
113-126 and B
243-252 (5); and at least two sites on TSP: TSP
1-174 (6), potentially the same as TSP 151-164 (41) and TSP 385-522
(42).
1) tested, with 10-15% of
receptor-bound Fg decorated by TSP. We expect that TSP-Fg associations
on the cell surface would accelerate the kinetics of aggregation
(i.e. aggregation efficiency) by (i) increasing the length
of the bridges, which will both increase the surface area available for
cross-bridging (Fig. 11) and decrease the actual distance required for cell-cell interactions, thereby increasing the collision frequency, (ii) increasing the number of
bridges between the platelets, and (iii) concentrating adhesive molecules in multivalent adhesive structures (clusters) that will favor
firm platelet to platelet adhesion (see below).
View larger version (25K):
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Fig. 11.
Schematic diagram, adapted from Goldsmith
et al. (52) (not drawn to scale) illustrating the area
available for cross-bridging with GPIIbIIIa*-Fg-GPIIbIIIa* and/or with
GPIIbIIIa*-Fg-TSP-Fg-GPIIbIIIa* cross-bridges on the surface of
activated platelets of 1.13-µm equivalent
spherical radius (53). Surface areas were calculated as published
(52), using the following estimated molecular lengths: GPIIbIIIa, 10 nm
(54), fibrinogen, 47 nm (55), and TSP, 54 nm (length of a single chain)
(56). As shown, GPIIbIIIa*-Fg-GPIIbIIIa* cross-bridges
(total length of 67.5 nm) give a maximum surface area for cross-linking
of 0.20 µm2 (gray area), compared with 0.56 µm2 (2.8×, hatched area) in presence of
GPIIbIIIa*-Fg-TSP-Fg-GPIIbIIIa* cross-bridges (total length
of 169 nm).
In addition to Fg-TSP-Fg interactions, we have shown that
TSP-TSP interactions can also participate in cross-bridging
to drive particle aggregation at varying shear rates. The beads
coaggregated at all shear rates, with a 10-fold decrease in efficiency
for a 10-fold increase in shear rate (100-1000 s1),
similar to the 5-fold decrease previously reported for Fg-mediated aggregation of ADP-activated platelets driven by GPIIbIIIa*-Fg cross-bridges (37). The maximal aggregation efficiency at 300 s
1 (10.4 ± 1.2%) was only two to three times lower
than for Fg-mediated aggregation of particles or platelets with
surface-bound GPIIbIIIa* (Refs. 34 and 37 and this study).
Our work demonstrates the direct contribution of TSP in reinforcing the
inter-platelet interactions in physiological flow conditions. The
participation of TSP is thought to be maximal during the early phase of
the platelet secretion process when high concentrations of TSP may
accumulate at the contact of activated platelets with Fg already bound
to a significant number of GPIIbIIIa*. Expected TSP-mediated
cross-bridges are modelled in Fig. 12:
The GPIIbIIIa*-Fg-GPIIbIIIa* cross-bridges (Fig.
12A) will cohabitate with
GPIIbIIIa*-Fg-TSP-Fg-GPIIbIIIa* cross-bridges (Fig.
12B), with possible participation of TSP(n)
interactions (Fig. 12C). Direct TSP or
TSP(n) cross-bridges might also form, involving TSP
ligands/receptors other than GPIIbIIIa*-bound Fg CD36, CD47 (?
in Fig. 12D). This latter model may appear controversial
because platelets from patients with Glanzmann's thrombastenia, which lack GPIIbIIIa, have been shown to express normal levels of TSP at the
cell surface upon thrombin activation, with no aggregation detected in
an aggregometer (23, 47). However, this apparent contradiction with our
model may rather arise from the facts that (i) micro-aggregates of <10
platelets observed in one Glanzmann's thrombastenia patient by phase
contrast microscopy may not be detected by aggregometry (48) and (ii)
electron microscopy studies revealed that upon thrombin activation, TSP
was not distributed normally on the platelet surface of one
Glanzmann's thrombastenia patient, with decreased size and number of
TSP clusters (49). It is therefore conceivable that in absence of
GPIIbIIIa (and membrane-bound Fg), TSP may not be in an "efficient
clustered" conformation to support platelet cross-bridging. Our model
of coaggregation of TSP-beads is expected to circumvent these
experimental artifacts because (i) our experimental setting detects
micro-aggregates as small as doublets and (ii) we used TSP-beads with
high TSP-surface density (2347 ± 424 molecules/µm2)
that possibly mimic the concentrated TSP found in clusters.
|
Finally our model suggests that macromolecular TSP/Fg/GPIIbIIIa* associations could also form, with TSP involved in both inter- and intra-platelet cross-bridges (Fig. 12E). The physiological relevance of such associations is supported by previous electron microscopy studies showing colocalization of TSP, Fg, CD36, and GPIIbIIIa in clusters on the surface of activated platelets (43, 44, 50, 51).
A role for TSP has been reported in intra-platelet costimulatory
signaling resulting in an enhanced affinity/avidity of GPIIbIIIa (25).
We have additionally demonstrated that TSP also provides an
extracellular amplification system of platelet aggregation via
inter-platelet cross-bridges possibly involving several molecules on
the surface of activated platelets. This amplification system, which is
characterized by an acceleration of the platelet aggregation, may be of
crucial importance in hemostasis, especially as a platelet colliding
with a thrombogenic surface (damaged vessel wall, activated platelet,
or endothelium) in flowing blood, is expected to be activated, secrete,
and adhere within milliseconds. Further studies will be required to
look more precisely at the role of TSP in experimental thrombosis
models. It is suggested that henceforth TSP is to be considered as a
potential target for developing new antithrombotic drugs, with the aim
of preventing undue thrombus growth while minimally affecting primary hemostasis.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully thank Professors Theo van de Ven (Chemistry, McGill University) and Harry Goldsmith (Medicine, McGill University) for helpful discussions and suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, and the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of salary support from Sanofi-Thrombose, the International Council for Canadian Studies, the Heart and Stroke Foundation of Quebec, and from the Société Française d'Hématologie, with travel money from the Quebec-France exchange program of FRSQ-INSERM, which supported exchange between our two laboratories.
** To whom correspondence should be addressed: Dept of Physiology, McGill University, 3655 Drummond Ave., #1137, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-4326; Fax: 514-398-7452; E-mail: mony@med.mcgill.ca.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M010091200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TSP, thrombospondin-1;
Fg, fibrinogen;
GPIIbIIIa*, activated
IIb
3 integrin;
AFP, activated fixed
platelet;
FITC, fluorescein isothiocyanate;
BSA, bovine serum albumin;
FITC-TSP, FITC-labeled TSP;
FITC-TSP18, FITC-labeled TSP18;
FITC-Fg, FITC-labeled Fg;
Fg-bead, Fg-coated bead;
TSP-bead, TSP-coated bead;
BSA-bead, BSA-coated bead;
GPIIbIIIa*-bead, GPIIbIIIa*-coated
bead.
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