A Model of Platelet Aggregation Involving Multiple Interactions of Thrombospondin-1, Fibrinogen, and GPIIbIIIa Receptor*

Arnaud BonnefoyDagger §, Roy Hantgan||, Chantal LegrandDagger , and Mony M. Frojmovic§**

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thrombospondin-1 (TSP) may, after secretion from platelet alpha  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thrombospondin-1 (TSP)1 represents 20-30% of the glycoproteins stored in human platelet alpha -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 alpha vbeta 3 and alpha IIbbeta 3 (GPIIbIIIa), through its cryptic RGDA sequences (12). However, the interaction of TSP with GPIIbIIIa is controversial (13-17).

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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 alpha IIbbeta 3 (GPIIbIIIa) than for alpha vbeta 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).

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-1. These studies were compared with beads prepared with 5% Fg receptor occupancy that yield maximal kinetics of aggregation, as previously reported (34).

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 (alpha ), 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).

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 alpha  by modifying our equations as previously described (34).

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).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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).



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Fig. 1.   Kinetics of FITC-TSP binding to Fg-beads. Fg-beads () or BSA-beads (open circle ) (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 (open circle ) 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.

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-1. However, Fg-beads preincubated for 20 min with 50-200 nM TSP before shear, aggregated dose-dependently. Aggregation efficiencies (alpha ) 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 (alpha  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.

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.



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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 (open circle ) 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*.

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).



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Fig. 6.   Binding isotherm of FITC-TSP to GPIIbIIIa*-beads or Fg-GPIIbIIIa*-beads. GPIIbIIIa*-beads (open circle ) 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*.

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-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).



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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 (open circle ) 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.

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-1 was slowed down, corresponding to a decrease of alpha  of ~30%, from 16.9 ± 0.1% to 12.0 ± 1.2% (Fig. 8).



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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 (open circle ) or 180 nM TSP (), and sheared at 300 s-1. Beads prepared with 5% Fg receptor occupancy (black-down-triangle ) 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*.

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-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 (alpha ) 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 alpha  at all Fg receptor occupancies tested (p < 0.01 to p < 0.07), with mean increases ranging from 30 to 61% (Table I).


                              
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Table I
Aggregation of AFP by Fg with varying Fg receptor occupancy: effect of the addition of TSP on the aggregation efficiency (alpha ) at a fixed shear rate
AFP (40,000/µl) were sheared at 300 s-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.

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, alpha  decreased 15-fold when increasing the shear rate from 300 to 2000 s-1. The addition of TSP induced a significant increase of alpha  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).


                              
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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 (alpha )
AFP (40,000/µl) were incubated 30 min in BAT buffer supplemented with CaCl2 (1 mM) and with Fg (20 nM), or with Fg (20 nM) together with TSP (80 nM), preincubated for 15 min at room temperature to reach 20% GPIIbIIIa receptor occupancy. The platelets were then sheared in a microcouette at 300, 1000, or 2000 s-1. Aggregation efficiencies were calculated from aggregation curves. Means ± S.E. of four to eleven experiments.

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-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 alpha  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 alpha  were reduced by >80% and >90%, respectively. TSP-beads did not aggregate either with Fg-beads or GPIIbIIIa*-beads (results not shown).



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Fig. 9.   Binding isotherm of FITC-TSP to TSP-beads. FITC-TSP (0-500 nM) was incubated with TSP-beads () or BSA-beads (open circle ) (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.



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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

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 (alpha ) 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 alpha  = 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 alpha  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 Aalpha 241-476 (4), Aalpha 113-126 and Bbeta 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).

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-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).



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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 s-1), 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.



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Fig. 12.   Model for the participation of TSP in platelet aggregation mediated by Fg-GPIIbIIIa* interactions. In absence of TSP (shaded square), Fg (black bar) directly cross-bridges GPIIbIIIa* (white oval with indentation) molecules on different platelets (A); TSP could form cross-bridges between Fg bound to GPIIbIIIa*, either as a monomer (B) or multimer of n repeating units (C). TSP-(n)-TSP interactions could cross-bridging platelets via receptors (black oval) other than Fg bound to GPIIbIIIa* (D). TSP could also participate in macromolecular structures generating inter- and intra-platelet cross-links favoring cluster formation on and between platelets (E).

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 alpha IIbbeta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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