1Division of Hematology, Brigham and Womens Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts; and 2Institut National de la Santé et de la Recherche Médicale Unité 428, Unité de Formation et de Recherche des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris, and 3Centre National de la Recherche Scientifique Unité Mixte de Recherche 7131, Hôpital Broussais, Paris, France
Submitted 1 December 2004 ; accepted in final form 10 May 2005
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ABSTRACT |
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Agonist-induced platelet activation also triggers inside-out signals, which convert the fibrinogen receptor, the integrin IIb
3, into its active, fibrinogen-bound form. Engagement of activated
IIb
3 by linking fibrinogen is necessary for platelet aggregation, which induces subsequent outside-in signals to enhance platelet activation. Although platelet actin assembly and shape change per se do not require the participation of
IIb
3, actin remodeling is influenced by outside-in signals coming from this ligated receptor (13). Key signaling intermediates involved in integrin-induced remodeling of the actin cytoskeleton are the small Rho family of GTPases (23), which induce actin assembly and the formation of focal adhesions. They also involve the phosphorylatable adaptor protein pleckstrin, recently shown to be important for integrin-induced cell spreading (34), and the tyrosine kinases Src and Syk, which activate downstream signaling intermediates such as the adaptor proteins SLP-76 and Cbl, the tyrosine kinase focal adhesion kinase (FAK), and the guanine nucleotide exchange factor Vav (27, 28). Cbl likely links integrin signaling to phosphoinositide (PI)3-kinase (36), known to be important for platelet spreading, and Vav is a guanine nucleotide exchange factor for Rac (24), a small GTPase that induces lamellipodia formation in activated platelets (2, 19). Finally, FAK is an important component of focal adhesions, which are linked to actin stress fibers in a Rho-dependent manner (7, 40).
One protein that may be implicated in platelet actin remodeling is cofilin (8). Cofilin belongs to the actin-depolymerizing factor/cofilin family of small (1821 kDa) actin-binding proteins that accelerate actin filament turnover by increasing filament end numbers (9). Human platelets express nonmuscle cofilin isoform 1 (31). Cofilin binds to or near the pointed ends of actin filaments, where it can sever filaments and/or promote actin monomer release (6, 33). Both of these functions accelerate actin filament turnover because newly released monomers are recycled for rounds of polymerization at filament barbed ends and because severing of actin filaments increases the number of barbed ends available for binding of the Arp2/3 complex, which amplifies the actin polymerization response (15, 21, 25). Cofilin is negatively regulated through phosphorylation on Ser3 (1, 3), and it has been established in platelets that dephosphorylation of cofilin accompanies activation by agonists including thrombin and phorbol esters (8). Herein we establish a central role for sustained signals generated by IIb
3 in maintaining cofilin in its active dephosphorylated form in aggregating platelets.
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MATERIALS AND METHODS |
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Development of anti-phosphocofilin antibody. Peptide MApSGVAVSDGV (500 µg) was injected subcutaneously in multiple sites into rabbits. After 2 wk, rabbits were boosted with injections of Freunds incomplete adjuvant containing the phosphopeptide and serum was collected. Sepharose 2B affinity columns were prepared according to the manufacturers instructions. Rabbit anti-phosphocofilin serum was passed three times over the column, and bound antibodies were eluted with 2.5 M glycine, pH 2.7, in tubes containing sufficient 1 M phosphate buffer to return the pH to >6. Fractions containing the IgG antibodies were dialyzed overnight in Tris-buffered saline and subsequently tested for phosphospecificity on dot blots against phosphorylated and nonphosphorylated cofilin peptides (Fig. 1A). Rabbits were maintained and treated as approved by the Harvard Medical Area Standing Committee on Animals and according to National Institutes of Health standards as set forth in the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals.
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SDS-PAGE and immunoblotting.
Platelets were lysed with 0.1% Triton X-100 in (in mM) 60 PIPES, 25 HEPES, 10 EGTA, and 2 MgCl2, pH 6.9 (PHEM buffer) containing protease inhibitors and 2 µM phalloidin. F-actin was isolated by centrifugation at 100,000 g for 30 min at 4°C in a Beckman Optima TL ultracentrifuge (Palo Alto, CA) as described previously (12). Triton X-100-insoluble and -soluble fractions were lysed in SDS-PAGE sample buffer containing 5% -mercaptoethanol. After being boiled for 5 min, platelet proteins were separated on 15% polyacrylamide gels and transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). Membranes were incubated for 1 h in Tris-buffered saline containing 0.05% Tween 20 and 5% dry milk. Membranes were then incubated overnight with either 5 µg/ml rabbit anti-phosphocofilin antibody or 2 µg/ml chicken anti-cofilin antibody. Detection was performed with an enhanced chemiluminescence system (Pierce, Rockford, IL).
Cofilin phosphorylation was evaluated by nonequilibrium isoelectric focusing as described previously (29, 30). Gels contained 10% polyacrylamide, 30% Triton X-100, 11.5 M urea, and 3% ampholyte, pH 310 (Amersham Biosciences). The cathode (lower) and anode (upper) chambers of the electrophoresis apparatus contained 100 mM NaOH and 20 mM phosphoric acid, respectively. After electrophoresis, gels were equilibrated in SDS-PAGE sample buffer before transfer onto an Immobilon-P membrane as above.
Immunofluorescence. Resting platelets or platelets activated in suspension with 1 U/ml thrombin for 1 or 5 min at 37°C were fixed with an equal volume of 6.8% paraformaldehyde for 10 min, deposited on glass coverslips, and permeabilized with a solution containing 0.1% Triton X-100, 500 nM FITC-phalloidin, and 80 µg/ml anti-phosphocofilin antibody for 1 h at room temperature. Coverslips were washed three times in phosphate-buffered saline containing 1% BSA and incubated in a solution containing 1:200 goat anti-rabbit Cy3-conjugated secondary antibodies for 30 min at room temperature. Coverslips were mounted onto slides, viewed, and photographed on a Nikon epifluorescence microscope (Melville, NY).
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RESULTS |
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Cofilin activation in thrombin-stimulated platelets. We further determined the amount of phosphocofilin in resting platelets using nonequilibrium isoelectric focusing in which the phosphorylated and nonphosphorylated forms of cofilin were resolved by charge difference (Fig. 2A; Refs. 29, 30). Densitometric analysis revealed that 87% (SD 7; n = 4) of cofilin is phosphorylated on Ser3 in resting platelets. Dephosphorylation was observed in platelets stimulated for 1 min with 1 U/ml thrombin under nonaggregating conditions.
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Cofilin incorporation in active platelet cytoskeleton.
The dephosphorylation of cofilin correlates temporally with a transient increase in its association with the actin cytoskeleton (Fig. 3). In resting platelets, <5% of cofilin is associated with the cytoskeleton. After 12 min of thrombin stimulation under nonaggregating conditions, 40% of cofilin associates with the Triton X-100-insoluble fraction. Cofilin subsequently dissociates from the cytoskeleton in a time course that correlates with its rephosphorylation. Phosphocofilin is not detected in the cytoskeletal fractions of resting and activated platelets, confirming in vitro findings that phosphorylated cofilin does not bind to F-actin (33).
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Platelet treatment with 300 µg/ml RGDS peptide or with 50 nM wortmannin rescued both the decrease of the F-actin content and the incorporation of the Arp2/3 complex in the actin cytoskeleton observed after 5 min of stimulation, although not to the levels obtained in nonstirred platelets (Fig. 7). The data indicate that IIb
3 outside-in signals modulate platelet actin dynamics by decreasing the F-actin content and/or increasing filament turnover.
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DISCUSSION |
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Platelets contain 28 µM cofilin, 5% of the estimated molar concentration of actin. We showed that 90% of cofilin is phosphorylated on Ser3 in resting platelets, suggesting a slow turnover of actin filaments in circulating platelets. This extent of phosphorylation is much greater than that reported earlier (8). The most likely explanation for this difference is the metabolic radiolabeling approach used in the earlier study. The rate of cofilin phosphorylation/dephosphorylation in resting platelets may be slow to maintain cofilin in an inactive, phosphorylated state. Nevertheless, the rate of phosphorylation exceeds dephosphorylation in the resting platelet. After platelet stimulation by thrombin, cofilin dephosphorylation is rapid, reaching a maximal extent of 75% in 12 min. Cofilin dephosphorylation parallels its incorporation into F-actin but temporally follows the peak of maximal actin filament assembly. These findings indicate that in platelets, cofilin activity is not primarily involved in the initial boost of actin assembly, a process dominated by calcium-activated gelsolin, but accelerates the turnover of actin filaments under the control of
IIb
3.
Our findings show that cofilin becomes maximally rephosphorylated at Ser3 2 min after stimulation with thrombin under nonaggregating conditions, exceeding resting levels by 510 min. However, cofilin activity is extended by signals coming from the IIb
3 integrin. When
IIb
3 signaling is disrupted by RGDS peptide or wortmannin, cofilin is rapidly rephosphorylated. This finding was directly confirmed by using platelets isolated from a patient with Glanzmann thrombasthenia, which express only 23% of normal
IIb
3 levels and failed to delay cofilin rephosphorylation. Cofilin phosphorylation is effected by LIM kinase and can be reversed by the Slingshot phosphatase (1, 10, 26, 37, 39). Rho and its downstream effector, Rho-activated kinase, are upstream in the LIM kinase activation pathway, and Rac has been shown to link to LIM kinase through p21-activated kinase (10). Both Rac and Rho are activated during thrombin stimulation (2, 16, 23), and such activation would be expected to lead to cofilin phosphorylation and inactivation in platelets. LIM kinase is expressed in megakaryocytes and is likely to be responsible for cofilin phosphorylation in platelets (22).
Our findings also indicate that IIb
3 signals modulate cytoskeletal remodeling and actin turnover and decrease stable incorporation of actin and the Arp2/3 complex in the cytoskeleton. Cofilin, maintained in its active, dephosphorylated form in platelets stimulated under aggregating conditions, is likely to be intimately involved in these events. Active, dephosphorylated cofilin binds ADP-bound actin subunits, severs actin filaments, and releases actin subunits from the pointed ends of filaments, which can then be recycled by profilin and
4-thymosin for new rounds of polymerization (6, 15, 33). Integrin signaling is usually associated with the formation of focal adhesions and stable actin stress fibers. The activation of cofilin after
IIb
3 engagement implies that actin filament dynamics are essential for these events. To form stable adhesion sites, platelets may need to recycle actin filaments. Previous studies have suggested that
IIb
3 regulates the assembly of actin filaments, not their turnover (13, 14). The most likely explanation for the discrepancy is the lysis buffers used in the earlier experiments, which do not contain enough calcium chelators to block gelsolin severing activity.
In summary, we have characterized the kinetics of cofilin activation/inactivation as platelets convert from their resting to their activated shape. Cofilin activity is under the control of both the initial trigger of activation and the integrin IIb
3. Our findings suggest that cofilin activity is not essential in platelets for the initial polymerization of actin filaments that follows stimulation and leads to shape change, but for actin remodeling mediated by outside-in signals. It remains to be determined exactly how increased actin filament dynamics contribute to platelet spreading and function. Presumably, actin filament turnover is required to continue platelet spreading on the subendothelium to optimally cover breaches in the vascular wall.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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