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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Physiology, University of
Cambridge, Cambridge, United Kingdom; the Departments of Biochemistry
and Human Biology, University of Maastricht; the Laboratory of
Haematology, Haemostasis Laboratory, Department of Internal
Medicine, Academic Hospital Maastricht; and the Department of
Haematology, St Radbout Hospital, Nijmegen, The Netherlands.
Effects of the occupation of integrin
Integrin Stimulation of human platelets with various agonists results in an
elevation in the [Ca++]i that consists of 2 components; release of Ca++ from the intracellular stores
and activation of Ca++ entry through plasma membrane
channels.6 Store-mediated Ca++ entry (SMCE) is
the main mechanism responsible for Ca++ influx in human
platelets7; however, the mechanism by which the filling
state of the Ca++ stores is communicated to the plasma
membrane is poorly understood. Current hypotheses fall into 2 main
categories: indirect coupling and direct coupling.8
Recently, the direct coupling model has received support from studies
indicating that the mechanism of activation of SMCE shares properties
with the activation of secretion.9-11 In different cell
types, including human platelets, the reorganization of the actin
cytoskeleton appears to play a pivotal role in the activation of SMCE,
possibly by mediating translocation of the Ca++ stores to
the plasma membrane to facilitate the coupling
process.9,11
It has been reported that integrin Materials
Platelet preparation
Measurement of [Ca++]i Fluorescence was recorded from 1.5-mL aliquots of magnetically stirred washed platelet suspension (108 cells/mL) at 37°C using a Cairn Research Spectrophotometer (Cairn Research, Faversham, Kent, United Kingdom) with excitation wavelengths of 340 and 380 nm and emission at 500 nm. Changes in [Ca++]i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al.16 Measurements on platelets from patients with Glanzmann thrombocytopenia and from matched controls were performed using an SLM Aminco 8100 spectrophotometer (Aminico, Rochester, NY) as described previously.17Determination of [Ca++]i elevation Agonist-evoked [Ca++]i elevation was measured as the integral of the rise in [Ca++]i above basal levels for 11/2 minutes after the addition of the agonist in the presence of external Ca++. Ca++ entry in TG-induced store-depleted platelets was estimated using the integral of the rise in [Ca++]i15,17 for 21/2 minutes after the addition of CaCl2.Measurement of F-actin content The F-actin content of resting and activated platelets was determined as previously described.11 Briefly, washed platelets (2 × 108 cells/mL) were activated in HBS. Samples of platelet suspension (200 µL) were transferred to 200 µL ice-cold 3% (wt/vol) formaldehyde in phosphate-buffered saline (PBS) for 10 minutes. Fixed platelets were permeabilized by incubation for 10 minutes with 0.025% (vol/vol) Nonidet P-40 detergent dissolved in PBS. Platelets were then incubated for 30 minutes with fluorescein isothiocyanate-labeled phalloidin (1 µM) in PBS supplemented with 0.5% (wt/vol) BSA. After incubation, the platelets were collected by centrifugation in an MSE Micro-Centaur Centrifuge (MSE Scientific Instruments, Crawley, Sussex, United Kingdom) for 90 seconds at 3000g and resuspended in PBS. Staining of 2 × 107 cells/mL was measured using a Perkin-Elmer (Norwalk, CT) Fluorescence Spectrophotometer. Samples were excited at 496 nm, and emission was at 516 nm.Analysis of cytoskeleton-associated p60src Human platelet fractionation was carried out according to a procedure published previously.18 Briefly, activated and control platelets (2 × 109 cells/mL) were immediately lysed with an equal volume of 2 × Triton buffer (2% Triton X-100, 2 mM EGTA, 100 mM Tris-HCl, pH 7.2, 100 µg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, and 2 mM Na3VO4) at 4°C for 30 minutes. Platelet lysate was centrifuged at 16 000g for 5 minutes. The supernatant was removed, and the pellet (cytoskeleton-rich fraction) was solubilized into the original volume in Laemmli's buffer,19 boiled for 5 minutes, and subjected to Western blotting using the anti-p60src monoclonal antibody GD11.Western blotting Proteins were electrophoresed on 7.5% sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred for 2 hours at 0.8 mA/cm2 in a semidry blotter (Hoefer Scientific, Newcastle, Staffordshire, United Kingdom) onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (wt/vol) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein-binding sites. Membranes were then incubated for 1 hour with anti-p60src monoclonal antibody (GD11) diluted 1:500 in TBST. The primary antibody was removed, and blots were washed 6 times for 5 minutes each with TBST. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated ovine antimouse IgG antibody diluted 1:10 000 in TBST, washed 6 times in TBST, and exposed to enhanced chemiluminescence reagents for 1 minute. Blots were then exposed to preflashed photographic film. The density of bands on the film was measured using a Quantimet 500 densitometer (Leica, Milton Keynes, United Kingdom).Statistical analysis Values stated are mean ± SE of the number of observations (n) indicated. Analysis of statistical significance was performed using Student t test. For multiple comparison, one-way analysis of variance combined with the Dunnett test was used. P < .05 was considered statistically significant.
Fibrinogen inhibits ADP-evoked Ca++ elevations Fura-2-loaded aspirin-treated human platelets were used to assess Ca++ responses evoked by different agonists. As previously reported,20 in the presence of 1 mM external Ca++, 5 µM ADP evoked a transient increase in [Ca++]i. The addition of fibrinogen 45 seconds before stimulation with ADP decreased the [Ca++]i elevation in a concentration-dependent manner (Figure 1A; n = 5). Fibrinogen significantly decreased the ADP-evoked Ca++ elevation (see "Materials and methods") by 17% ± 6%, 41% ± 1%, and 61% ± 4% at 0.1, 0.3, and 1 mg/mL, respectively (Figure 1A; P < .05; n = 5). As shown in Figure 1B, in the presence of 1 mM external Ca++, 5 µM ADP evoked a peak [Ca++]i rise of 321 ± 22 nM (n = 7), which, in the presence of 1 mg/mL fibrinogen, was reduced to 213 ± 33 nM (P < .05; n = 7 ). When experiments were performed in the presence of 5 µM PPACK to inactivate traces of thrombin that might have been generated, the same results were obtained (data not shown). RGDS has been reported to inhibit the binding of fibrinogen with its receptor.21 Under our experimental conditions, the addition of RGDS (100 µM) did not alter the elevation in [Ca++]i evoked by ADP, thrombin, or vasopressin (data not shown). However, the addition of RGDS (100 µM) 45 seconds before fibrinogen reversed the effect of fibrinogen. In the presence of both RGDS and fibrinogen, treatment of platelets with 5 µM ADP evoked a peak [Ca++]i elevation of 285 ± 37 nM, similar to that reported under control conditions (Figure 1B; P > .05; n = 7). Similar results were obtained using abciximab (ReoPro, a chimeric antibody against IIb 3). Addition of abciximab (5 µM) 45 seconds before fibrinogen completely reversed its inhibitory effect
(Figure 1C; n = 5).
Effect of fibrinogen and RGDS peptide on thrombin-evoked Ca++ elevations Figure 2 shows that the addition of fibrinogen (1 mg/mL) 45 seconds before the agonist resulted in substantial inhibition of the elevation in [Ca++]i evoked by thrombin (0.1 U/mL) in medium containing 1 mM Ca++. The initial peak [Ca++]i elevation above basal level after treatment with agonist was decreased significantly from 351 ± 58 to 194 ± 60 nM (P < .01; n = 7). Fibrinogen significantly reduced the integral of the thrombin-evoked elevation in [Ca++]i by 44% ± 4% (P < .001). As with ADP, RGDS reversed the inhibition of the thrombin-evoked elevation in [Ca++]i by fibrinogen. In the presence of RGDS (100 µM), fibrinogen only slightly reduced the initial peak of the rise in [Ca++]i evoked by thrombin from 351 ± 58 to 272 ± 22 nM (Figure 2A; P > .05;n = 7).
Fibrinogen inhibits receptor-operated Ca++ entry To address the role of fibrinogen in modulating Ca++ signaling, we studied its effect on ADP-evoked release of Ca++ from the internal stores. Although the preincubation of platelets with high concentrations of EGTA (5 mM) induces dissociation of theIIb3 complex,
reducing the ability of this receptor to bind fibrinogen,22,23 more recent studies have demonstrated
that incubation in the presence of lower concentrations of EGTA (500 µM) does not destroy the complex.1 Using flow cytometry,
we have found that under our experimental conditions, the incubation of
platelets for 30 seconds in the presence of 100 µM EGTA did not
interfere with the binding of fibrinogen to the
IIb3 complex (data not shown). In the
absence of external Ca++ (100 µM EGTA added), fibrinogen
was without effect on the ADP-induced rise in
[Ca++]i, suggesting no effect on the release
of Ca++ from the intracellular stores (Figure
3A). To further investigate this issue,
we monitored ADP-evoked Ca++ release in a medium containing
external Ca++ and SKF96365, a nonselective cation channel
blocker.24 In a medium containing 1 mM Ca++,
treatment of human platelets with ADP (5 µM) in the presence of
SKF96365 (50 µM) evoked a Ca++ elevation similar to that
observed in the absence of external Ca++, indicating that
SKF96365 abolished Ca++ entry under our conditions (data
not shown). As shown in Figure 3B, in the presence of SKF96365, the
addition of fibrinogen (1 mg/mL) to the platelet suspension 45 seconds
before ADP did not alter ADP-induced Ca++ mobilization
(P > .05; n = 6). These findings strongly suggest that
the effect of fibrinogen on Ca++ signaling is a selective
inhibition of Ca++ entry over internal release.
To assess whether fibrinogen might mediate these effects by
Ca++ channel blockage, we investigated the effect of
fibrinogen on Ca++ entry mediated by activation of
P2x1 receptors, which act as cation
channels.25 As shown in Figure 3C, the addition of
fibrinogen 45 seconds before the P2x1-selective agonist,
Fibrinogen reduces store-mediated Ca++ entry To directly address the specific inhibitory role of fibrinogen on Ca++ entry, we investigated its effect on SMCE. In a medium containing 1 mM Ca++, TG, a specific inhibitor of the Ca++-ATPase of the internal stores (the sarcoplasmic/endoplasmic-reticulum Ca++-ATPase, or SERCA),26,27 evoked a transient increase in [Ca++]i in platelets mediated by the release of Ca++ from the internal stores and Ca++ entry (Figure 4A). The addition of fibrinogen (1 mg/mL) 45 seconds before TG (200 nM) significantly decreased the elevation in [Ca++]i by 25% ± 5% (Figure 4A; P < .01; n = 5). As shown in Figure 4A, the addition of RGDS (100 µM) before fibrinogen reversed the fibrinogen-induced inhibition of TG-evoked Ca++ entry.
Another index for SMCE is the rapid elevation in [Ca++]i after the addition of CaCl2 to suspensions of store-depleted platelets. Cells were treated with TG (200 nM) in a Ca++-free medium, and, 3 minutes later, CaCl2 (300 µM) was added to initiate Ca++ entry. Consistent with the results obtained in the presence of 1 mM Ca++, the addition of fibrinogen (1 mg/mL) before TG significantly reduced the Ca++ entry by 61% ± 6% (Figure 4B; P < .01; n = 6). As shown in Figure 4B, this effect of fibrinogen was completely blocked by RGDS. Cytosolic Ca++ concentration is a result of the balance
between the mechanisms involved in Ca++ intake and removal
from the cytosol. Ca++ release from the intracellular
stores and Ca++ entry are responsible for rises in
[Ca++]i, whereas the sequestration of
Ca++ in the internal stores Effect of fibrinogen on Ca++ elevations in preactivated platelets To further investigate the role of fibrinogen in Ca++ entry, we examined the effect of the addition of fibrinogen after platelet activation. As shown in Figure 5A, the addition of fibrinogen (FG; 1 mg/mL) 45 seconds before ADP (5 µM) clearly reduced the ADP-induced Ca++ elevation (FG ± control). Addition of fibrinogen 10 seconds after platelet activation had no effect on the ADP-induced response. The lack of effect of fibrinogen after platelet activation was confirmed using TG to activate the platelets. In both a Ca++-free medium and the presence of 1 mM external Ca++, the addition of fibrinogen (1 mg/mL) to TG-pretreated platelets did not alter store depletion-induced Ca++ entry (Figure 5B-C). In contrast, the addition of 100 µM lanthanum, a nonspecific cation channel blocker, caused a rapid decrease in [Ca++]i over the same time course (Figure 5C), indicating that Ca++ entry had been inhibited. Together, these results indicate that fibrinogen is able to inhibit agonist-evoked and store-regulated Ca++ entry, an effect that can be reversed by the RGDS peptide. The lack of effect of fibrinogen on previously activated platelets indicates that it affects the activation but not the maintenance of SMCE.
Fibrinogen does not modify platelet Ca++ signaling in platelets from patients with Glanzmann thrombasthenia The addition of 1 mM fibrinogen 45 seconds before stimulation with 5 mM ADP had little effect on the elevation in [Ca++]i in platelets from patients homozygous for Glanzmann thrombasthenia, reducing this to 94.3% ± 5.7% of control (P > .1; n = 8). In platelets from healthy controls prepared in parallel, fibrinogen reduced the ADP-evoked rise in [Ca++]i to 72.9% ± 4.7% of control (P < .001; n = 7). The integral of the ADP-evoked rise in [Ca++]i was reduced to 71.5% ± 2.0% of control (P < .001; n = 7) in platelets from healthy controls compared with 93.5% ± 8.5% of control (P > .2; n = 8) in platelets from patients with Glanzmann thrombasthenia.Role of fibrinogen in reorganization of the actin cytoskeleton in human platelets It has previously been shown that the treatment of human platelets with ADP or TG induces actin polymerization.15,29 Because reorganization of the actin cytoskeleton has been reported to play a key role in the activation and maintenance of SMCE in different cell types, including platelets,9-11 we tested whether the actin cytoskeleton was involved in the inhibitory effect of fibrinogen on Ca++ entry in platelets. Treatment of platelets with TG (1 µM) or ADP (5 µM) in the presence of 1 mM external Ca++ enhanced F-actin content by 30% ± 3% and 34% ± 4%, respectively. In the presence of fibrinogen (1 mg/mL), TG- and ADP-induced actin polymerization was significantly inhibited by 66% ± 1% and 88% ± 2%, respectively (Figure 6A; P < .01; n = 6). As shown in Figure 6A, fibrinogen was without significant effect on the actin filament content of unstimulated platelets. To investigate whether the effect of fibrinogen is mediated by a reduction in [Ca++]i elevation, we loaded platelets with the Ca++ chelator dimethyl BAPTA to prevent [Ca++]i rises.30 In dimethyl BAPTA-loaded human platelets, we found similar results. Treatment of platelets with 1 µM TG or 5 µM ADP enhanced actin filament content by 51% ± 10% and 32% ± 6%, respectively. The responses observed after platelet stimulation with TG or ADP were significantly reduced in the presence of fibrinogen (1 mg/mL) by 88% ± 5% and 72% ± 5%, respectively (Figure 6B; P < .05; n = 6).
Because fibrinogen did not alter [Ca++]i elevation stimulated by TG or ADP when added after the activation of platelets, we investigated the effect of fibrinogen on the actin filament content of preactivated platelets. A series of experiments was carried out under conditions identical to those used for [Ca++]i measurements. Treatment of human platelets with TG (1 µM) or ADP (5 µM) enhanced F-actin content by 38% ± 7% and 42% ± 6%, respectively. When fibrinogen (1 mg/mL) was added to activated platelets, no significant changes were detected in TG- or ADP-evoked actin polymerization, which remained enhanced over basal levels by 43% ± 7% and 39% ± 2%, respectively (Figure 6C; P > .05; n = 6). These results suggest that actin polymerization is independent of [Ca++]i elevation and might be the target for fibrinogen-mediated modulation of Ca++ signaling. To further investigate the possibility that fibrinogen modulates
Ca++ entry by interfering with actin polymerization, we
examined the effect of CD and JP on fibrinogen-induced inhibition of
SMCE. CD is a widely used inhibitor of actin
polymerization,31 whereas JP induces stabilization of the
actin filaments.32 We have previously reported that
treatment with 10 µM CD for 40 minutes abolishes TG-induced actin
polymerization without having any effect on the actin filament content
in resting cells.15 Treatment of human platelets with 10 µM JP for 60 seconds had no effect either on the actin filament
content of resting platelets or on TG-induced actin polymerization
(data not shown). As reported above, the addition of fibrinogen 45 seconds before TG reduced SMCE by 60%. A similar effect was observed
when cells were treated with a combination of CD and fibrinogen
(58% ± 7%, Figure 7, n = 4). In
contrast, the preincubation of human platelets for 60 seconds with 10 µM JP impaired the effect of fibrinogen and, on the basis that the binding affinity of JP for F-actin (Kd = 15
nM)32 is greater than that of CD
(Kd = 50 nM),33 also impaired the
combined effect of fibrinogen and CD (Figure 7, n = 4). Treatment of
platelets for 60 seconds with JP (10 µM) did not alter SMCE (data not
shown). These results clearly suggest that fibrinogen modulates SMCE in platelets by a mechanism involving the inhibition of actin
polymerization.
Translocation of p60src to the cytoskeletal fraction in store-depleted platelets Tyrosine kinases have been shown to be involved in the activation of SMCE in several cell types, including human platelets.34-36 Previous studies have shown that ADP induces association of the tyrosine kinase p60src to the actin cytoskeleton in human platelets,37 an important process for phosphorylation of its substrates during platelet activation.18 To investigate the effect of fibrinogen in the cytoskeletal association of p60src induced by ADP, Western immunoblot analysis was performed on the cytoskeletal fraction of resting and ADP-activated platelets. Platelets heavily loaded with dimethyl BAPTA were used for this study to eliminate the Ca++-dependent association of p60src with the cytoskeleton. When the cytoskeletal fraction was probed for the presence of the protein tyrosine kinase p60src, a small amount of this protein was detected and was associated with the cytoskeleton of resting platelets (Figure 8). Treatment of platelets with ADP (5 µM) for 3 minutes enhanced the amount of p60src associated with the cytoskeleton by 4.5 ± 0.3-fold (Figure 8). As shown in Figure 8, in the presence of fibrinogen (1 mg/mL), no change was detected in the association of p60src with the cytoskeleton in resting platelets; however, the ADP-evoked response was almost completely abolished (there was only a 1.1 ± 0.1-fold increase over basal levels; P < .001;n = 4).
Integrin In the present study, we demonstrate that the effect of fibrinogen is
selectively mediated by the inhibition of Ca++ entry rather
than Ca++ release from the intracellular stores. Our
studies performed in the absence of external Ca++, in which
EGTA (100 µM) was added at the time of experiment to avoid uncoupling
of the integrin We found that the effect of fibrinogen was specifically mediated by
binding to its receptor, integrin In human platelets, a major mechanism for Ca++ influx is
SMCE, where depletion of the intracellular stores induces
Ca++ entry across the plasma membrane. Our results
demonstrate for the first time that the occupation of integrin
Recently, it has been reported that the actin cytoskeleton might play a
key role in the activation of SMCE in platelets and other
cells.9-11 In human platelets, actin polymerization is
also important for the maintenance of SMCE, an observation supporting the conformational coupling model.11 We further
investigated the role of the integrin
Recent studies have shown that tyrosine kinases of the Src family associate with the platelet cytoskeleton on stimulation with different agonists, a process that has been shown to be independent of platelet aggregation44 and that is important for phosphorylation of their substrates.18 In addition, we have recently found that Ca++ store depletion stimulates translocation of the tyrosine kinase p60src to the actin cytoskeleton,45 a process that might be essential for SMCE activation.46 In the present study, we show that ADP-evoked translocation of p60src is independent of [Ca++]i mobilization, as demonstrated using cells loaded with dimethyl BAPTA, a Ca++ chelator. In addition, we have found that the effect of ADP on p60src translocation is blocked in the presence of fibrinogen. In agreement with previous studies44 showing that actin reorganization is required for p60src cytoskeletal association, fibrinogen-induced inhibition of the cytoskeletal association of p60src could be a consequence of its inhibitory role in actin polymerization in human platelets. In summary, we have shown that fibrinogen binding to the integrin
Submitted September 8, 2000; accepted December 21, 2000.
Supported in part by The Wellcome Trust (grant 046115) and by grants from the Junta de Extremadura, Spain (J.A.R.) and The Dr Saal van Zwanenbergstichting (E.M.Y.M.).
J.A.R. and E.M.Y.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stewart O. Sage, Dept of Physiology, University of Cambridge, Downing St, Cambridge CB2 3EG, United Kingdom; e-mail: sos10{at}cam.ac.uk.
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© 2001 by The American Society of Hematology.
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  N. Prevost, J. V. Mitsios, H. Kato, J. E. Burke, E. A. Dennis, T. Shimizu, and S. J. Shattil Group IVA cytosolic phospholipase A2 (cPLA2{alpha}) and integrin {alpha}IIb{beta}3 reinforce each other's functions during {alpha}IIb{beta}3 signaling in platelets Blood, January 8, 2009; 113(2): 447 - 457. [Abstract] [Full Text] [PDF]
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 G. E. Woodard, G. M. Salido, and J. A. Rosado Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1323 - C1331. [Abstract] [Full Text] [PDF]
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 H. Miajlovic, A. Loughman, M. Brennan, D. Cox, and T. J. Foster Both Complement- and Fibrinogen-Dependent Mechanisms Contribute to Platelet Aggregation Mediated by Staphylococcus aureus Clumping Factor B Infect. Immun., July 1, 2007; 75(7): 3335 - 3343. [Abstract] [Full Text] [PDF]
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 H. A. Praetorius, J. Praetorius, S. Nielsen, J. Frokiaer, and K. R. Spring {beta}1-Integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling Am J Physiol Renal Physiol, November 1, 2004; 287(5): F969 - F978. [Abstract] [Full Text] [PDF]
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 W. S. Nesbitt, S. Giuliano, S. Kulkarni, S. M. Dopheide, I. S. Harper, and S. P. Jackson Intercellular calcium communication regulates platelet aggregation and thrombus growth J. Cell Biol., March 31, 2003; 160(7): 1151 - 1161. [Abstract] [Full Text] [PDF]
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 W. S. Nesbitt, S. Kulkarni, S. Giuliano, I. Goncalves, S. M. Dopheide, C. L. Yap, I. S. Harper, H. H. Salem, and S. P. Jackson Distinct Glycoprotein Ib/V/IX and Integrin alpha IIbbeta 3-dependent Calcium Signals Cooperatively Regulate Platelet Adhesion under Flow J. Biol. Chem., January 18, 2002; 277(4): 2965 - 2972. [Abstract] [Full Text] [PDF]
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IIb
3 modulates store-mediated calcium
entry in human platelets
Top
Abstract
Introduction
and mainly the plasma membrane
Ca++ ATPase
2 and pp125FAK in concanavalin A-stimulated platelets.
Thromb Haemost.
1999;81:124-130