Annexin V relocates to the platelet cytoskeleton ... - Wiley Online Library

Eur. J. Biochem. 267, 4720±4730 (2000) q FEBS 2000

Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin Eleni Tzima, Patrick J. Trotter, Margaret A. Orchard and John H. Walker School of Biochemistry and Molecular Biology, University of Leeds, UK

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca21 -ionophore activation. The time-dependence of annexin V incorporation into the detergent-extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re-associated with the actin filaments when re-polymerization was induced in vitro. Using a bifunctional crosslinking reagent we have identified an 85-kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca21], however, inclusion of an extract from thrombin-stimulated platelets lowered the [Ca21] requirement for the binding of annexin V to F-actin to physiological levels. We also show that GST±annexin V mimics the physiological binding of annexin V to membranes, and that this GST±annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and g- but not b-actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely g-actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to g-actin in an activation-dependent manner forms a strong association with the platelet cytoskeleton. Keywords: annexin V; platelet; cytoskeleton; actin. Stimulation of human platelets with the physiological agonist thrombin initiates a complex series of events, which include dramatic changes in the organization of the cytoskeleton. These changes are initiated by the dissociation of the actin cytoskeleton and its subsequent remodelling by a process involving polymerization and crosslinking of actin bundles, which fill the developing filopodia [1±3]. These newly formed actin filaments interact with phosphorylated myosin [4], an association that generates the tension necessary for the centralization of granules and the retraction of filopodia with the externally bound clot. It is now becoming clear that in addition to generating shape change and tension the cytoskeleton plays an important role in regulating aspects of membrane function. For example, following activation, several signal transduction mediators bind to the cytoskeleton. These include tyrosine kinases such as pp60c±src [5±9] and pp62c±yes [9], small GTPbinding proteins such as Rap 1B [10,11] and p21ras GAP [9], phosphoinositide 3-kinase [6] and the protein tyrosine phosphatase SH-PTP1 [12]. The platelet cytoskeleton therefore Correspondence to J.H. Walker, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. Fax: 1 44 113 2333167, Tel.: 1 44 113 2333119. Abbreviations: GST, glutathione S-transferase; CE, cytochalasin E; DMS, dimethylsuberimidate; DNase I, deoxyribonuclease I; PtdIns, phosphatidylinositol; DAG, diacylglycerol; GAP, GTPase-activating protein. (Received 10 December 1999, revised 27 March 2000, accepted 30 May 2000)

appears to play an important role in cell signalling events, possibly in localizing signalling molecules to the site of action [13]. In addition, the membrane cytoskeleton stabilizes the plasma membrane [14] and may play a role in the regulation of membrane channels and pumps [15,16]. The reorganization of the cytoskeleton may therefore be important in regulating the function of a variety of platelet processes. Interestingly, many changes in cytoskeletal organization, including the depolymerization of actin filaments by gelsolin [17] and the shape changes induced by activation of myosin light chain kinase [4] require calcium. Members of the highly conserved, calcium-binding family of annexins have been shown to copurify with cytoskeletal proteins [18±20], suggesting that they may play a role in cytoskeletal function. Annexin II has been shown to bind to the cytoskeleton of chromaffin culture cells [21] and to bind to actin at mM calcium, an observation that suggests that annexin II and actin may interact in vivo [22±26]. In addition, annexin I [27,28] and the plant annexins p34 and p35 have been shown to bind directly to Factin [29]; annexin IV has been reported to associate with the cytoskeleton of rabbit enterocytes in a calcium-dependent manner [30]; annexin VI binds the membrane cytoskeletal protein calspectrin [31], and annexin I has been reported to bind to the cytoskeletal regulatory protein profilin and thus regulate membrane-cytoskeleton organization [32,33]. The molecular interactions of annexin V and cytoskeletal components remain less well characterized, although pure annexin V appears to bind reversibly to the cytoskeleton [34], has been reported to bind to F-actin in high calcium, and to associate with both

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Annexin V associates with the platelet cytoskeleton (Eur. J. Biochem. 267) 4721

cytoskeletal filaments in the human glioma cell line GL15 [35] and with actin rich regions in epithelial cells of both the small intestine and kidney proximal tubular cells [36]. We have previously shown that following physiological activation annexin V in platelets relocates from the cytosol to the plasma membrane [37±39] and that several phosphoproteins were found to coprecipitate with membrane associated annexin V [40,41]. These findings and the evidence that annexin V relocates to the cytoplasmic side of the plasma membrane of activated platelets where it associates with actin [42] suggest that annexin V may play a role at the membrane± cytoskeleton interface. In this paper we demonstrate that annexin V binds to the detergent-extracted cytoskeleton of activated platelets, we characterize the cytoskeletal interactions of annexin V including the role of calcium in this interaction, and show that annexin V binds to actin filaments following physiological increases in [Ca21] as well as to g-actin, which is the minor isoform of actin in platelets.

M AT E R I A L S A N D M E T H O D S Materials All chemicals were purchased from Sigma unless otherwise indicated. ECL reagents were purchased from Amersham International plc. Recombinant annexin V was expressed as a GST±annexin V fusion protein using the plasmid pGEX 2TH in Escherichia coli strain N842 (a gift from F. R. Marie, Institut Cochin de GeÂneÂtique MoleÂculaire, Paris) and pure recombinant annexin V was prepared by cleaving the GST±annexin V fusion protein. Rabbit polyclonal antiserum specific for human annexin V was purchased from Alexis Corporation (Bingham, UK) Ltd. A monoclonal antibody which recognizes all actin isoforms (C4 clone) was purchased from Boehringer Mannheim, UK, the monoclonal antibody against b-actin was purchased from Sigma and the antibody against g-actin was a gift from C. Chapponier, University of Geneva, Switzerland [43]. Platelet actin was purchased from Cytoskeleton (Denver, USA). Preparation and activation of platelets Platelets from healthy volunteers were prepared as previously described [44] and suspended in Hepes-buffered Tyrode's solution (129 mm NaCl, 8.9 mm NaHCO3, 2.8 mm KCl, 0.8 mm KH2PO4, 0.8 mm MgCl2, 5.6 mm dextrose, 10 mm Hepes-NaOH pH 7.4). The number of platelets and contaminating white blood cells were determined using a Coulter S Plus counter (courtesy of The Haematology Department, The General Infirmary, Leeds, UK). Platelets (1 109 cells) were activated by incubation with the calcium ionophore A23187 (5 mm) in the presence of 1 mm CaCl2 for 10 min at 37 8C or by incubation with 0.5 U´mL21 thrombin in the presence of 1 mm CaCl2 for 2 min or as indicated. Depolymerization of the cytoskeleton was achieved either by incubating platelets with 5 1025 m cytochalasin E for 10 min at 37 8C or subfractionating in the presence of 2 mg´mL21 DNase I. In certain experiments, platelets were preincubated in 1 mm CaCl2 or 1 mm EGTA for 5 min at 37 8C and were subsequently activated with thrombin. Sedimentation of total cytoskeletons from detergent lysed platelets Detergent-insoluble cytoskeletons were isolated by using a modified method of Fox [45]. A suspension (500 mL) of

washed platelets (1 109 mL21) was pelleted by centrifugation at 8000 g for 3 min in a microfuge and resuspended in 1000 mL of ice-cold Triton X-100 lysis buffer, containing 1% (v/v) Triton X-100, 100 mm KCl, 5 mm EGTA, 1 mm NaN3, 10 mm Pipes pH 7.4, and the protease inhibitors: 1 mm phenylmethanesulfonyl fluoride, 1 mg´mL21 leupeptin, 50 mm benzamidine, 1 mm NaVO4. Where indicated, CaCl2 was added to give a specific free [Ca21] as predicted by the Metlig computer program [46,47]. Cytoskeletons were isolated by centrifugation at 200 000 g for 2.5 h in a Beckman TL-centrifuge at 4 8C. The cytoskeletons were solubilized by the addition of SDS sample buffer to the pellet and samples were analysed for annexin V by Western blotting.

Chemical crosslinking of membranes and cytoskeleton fractions Cytoskeletons or membranes from unstimulated or stimulated platelets were incubated for 1 h at room temperature in a final volume of 100 mL in buffer C (0.2 m triethanolamine, 1 mm CaCl2 pH 8.0), in either the presence or absence of the bifunctional reagent DMS added to a final concentration of 5 mm. The crosslinked material was then sedimented by centrifugation at 200 000 g and the pellet resuspended in 0.25 mL Buffer A (100 mm KCl, 5 mm EGTA, 1 mm NaN3, 10 mm Pipes, pH 7.4 including 1 mg´mL21 leupeptin, 1 mm phenylmethanesulfonyl fluoride, 50 mm benzamidine, 1 mm NaVO4). The sample was then taken up in sample buffer containing 10% (v/v) mercaptoethanol and 2% (w/v) SDS. Annexin V-protein and actin-protein complexes were identified by Western blotting.

Preparation of the polymerized actin-rich fraction Cytoskeletons from thrombin-stimulated platelets were solubilized with 1 mL of buffer containing 0.6 m KI, 100 mm Pipes, 100 mm KCl, 1 mg´mL21 leupeptin, 1 mm phenylmethanesulfonyl fluoride, 50 mm benzamidine, 1 mm NaVO4, pH 6.5, for 50 min at 4 8C under gentle shaking and then centrifuged at 200 000 g for 30 min at 4 8C. The supernatant (KI soluble) was collected and dialysed overnight at 4 8C against a buffer containing 10 mm Pipes, 2 mm MgCl2, 1 mg´mL21 leupeptin, 1 mm phenylmethanesulfonyl fluoride, 50 mm benzamidine, 1 mm NaVO4, pH 7.4 and CaCl2 was added to give a free [Ca21] of 0.8 mm, 8.8 mm or 1 mm. The insoluble material containing the re-polymerized actin and actin-binding proteins was recovered by centrifugation at 200 000 g for 5 min at 4 8C, washed twice with dialysis buffer and finally resuspended in sample buffer.

Actin binding assays Co-sedimentation assays using pure annexin V and pure platelet F-actin were performed as described previously [42] with the minor modification that annexin V and F-actin were incubated in 5 mm EGTA or in the presence of increasing free [Ca21]. When extracts from resting and thrombin-stimulated platelets were included, Triton X-100-soluble material was isolated as described above and incubated with or without 1.1 mm F-actin. Supernatants were carefully aspirated, pellets were resuspended in the original assay volume and equal aliquots of both the supernatants and pellets were then analysed by SDS/PAGE.

4722 E. Tzima et al. (Eur. J. Biochem. 267)

Isolation of platelet membranes Platelets were activated as described above, ruptured by freezethawing, and membranes were sedimented by centrifugation at 200 000 g for 15 min at 4 8C. For subcellular fractionation into cytosolic and membrane-associated material, the supernatant (supernatant a) was removed and the pellet resuspended in 0.25 mL of Buffer A. This procedure was repeated a further five times to produce six supernatants in total representing the EGTA-elutable material. The washed pellet was then resuspended in 0.25 mL of Buffer B [Buffer A 1 1% (v/v) Triton X-100] and centrifuged at 200 000 g for 15 min. The supernatant representing tightly bound membrane material (supernatant b) was decanted and the final pellet, which constitutes the Triton X-100-insoluble fraction, was resuspended in 0.25 mL of buffer B. All samples were analysed for annexin V by Western blotting. Binding of recombinant annexin V to platelet membranes Platelet membranes (isolated from 2 109 cells) were incubated in 1 mL of buffer A containing 8.8 mm Ca21 in the absence (lane a), or the presence of 0.2 mg (Fig. 7, lane b), 2 mg (Fig. 7, lane c), 10 mg (Fig. 7, lane d), 20 mg (Fig. 7, lane e), 40 mg (Fig. 7, lane g) or 60 mg annexin V (Fig. 7, lane h) for 1 h at 37 8C. Some membrane fractions were also incubated in the absence (Fig. 7, lane j) or the presence (Fig. 7, lane i) of 60 mg GST±annexin V for 30 min prior to incubation with 20 mg annexin V. The binding was terminated by centrifugation at 200 000 g for 15 min at 4 8C. The supernatant was removed and the pellet resuspended in 0.25 mL buffer A. This procedure was repeated a further four times to produce a washed pellet which was then resuspended in 0.25 mL buffer B and centrifuged at 200 000 g for 15 min. Samples were then analysed for annexin V by western blotting. Isolation of GST±annexin V bound complexes Washed platelet membranes were incubated with GST or GST±annexin V (60 mg) in the presence of 8.8 mm calcium for 1 h at 37 8C. The binding was terminated by centrifugation at 200 000 g for 15 min. The supernatant was removed and the pellet resuspended in 0.25 mL buffer A at 4 8C. The resuspended pellet was again centrifuged at 200 000 g for 15 min. This procedure was repeated a further four times to produce a washed pellet which was then resuspended in 0.25 mL buffer B and centrifuged at 200 000 g for 15 min. The Triton X-100 extractable material was then incubated with 300 mL of glutathione-agarose for 150 min at 4 8C. Glutathione fusion proteins and complexing proteins were sedimented by centrifugation for 2 min at 8500 g and washed six times in NaCl/Pi containing 1% (v/v) Triton X-100, 10% (v/v) glycerol and 1 mm phenylmethanesulfonyl fluoride. Immunoprecipitation of membrane-associated annexin V Immunoprecipitation of annexin V was performed with a polyclonal antiserum raised against bovine annexin V. This antiserum was specific to a single immunoreactive band when various tissues were screened by western blotting (data not shown). No immunoreactivity was seen with preimmune serum isolated from the rabbit prior to inoculation with annexin V. A Triton X-100-soluble fraction from stimulated platelets was prepared as described above. This supernatant was transferred to protein A±Sepharose 4B (50 mg) beads to which the relevant

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IgG fraction (10 mL serum) had previously been bound by incubation overnight at 4 8C. The Triton X-100 lysate was incubated with the beads for 3 h at room temperature. The beads were sedimented by centrifugation for 2 min at 8000 g and washed six times in immunoprecipitation buffer [150 mm NaCl, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 20 mm Hepes/NaOH pH 7.4]. Identification of annexin V by electrophoresis and Western blot analysis Proteins were separated by SDS/PAGE using a 10% (w/v) polyacrylamide gel and a discontinuous buffer system as described by Laemmli [48]. Separated proteins were silver stained by the method of Gottlieb [49]. Western blot analysis was performed as previously described [37]. Annexin V immunoreactivity was quantitated using an Apple One Scanner and the nihimage densitometry application program version 1.52.

R E S U LT S Binding of annexin V to the cytoskeleton of activated platelets To determine whether annexin V associates with the cytoskeleton of platelets that had been activated with a physiological agonist, washed platelets were activated with 0.5 U´mL21 thrombin in the presence of calcium. Immunoblotting analysis of the Triton X-100-insoluble and -soluble fractions has revealed that in unstimulated platelets annexin V is recovered in the soluble fraction (Fig. 1A, 0 s). Upon stimulation with thrombin, there is an increase in the amount of annexin V that is recovered along with the Triton X-100-insoluble fraction that is enriched in actin filaments (Fig. 1A, 30 s to 10 min). To correlate the annexin V±cytoskeleton association with different phases of platelet activation, detergent-resistant fractions were prepared at different intervals after inducing platelet activation with 0.5 U´mL21 thrombin, and the time-course of the incorporation of annexin V into the Triton-insoluble residue was investigated. As thrombin-induced platelet activation proceeds, an elevation in the amount of Triton-insoluble annexin V can be demonstrated (Fig. 1A). To determine the percentage of annexin V in the cytoskeleton fraction relative to the total amount of annexin V, quantitative analyses of the immunoblots probed with rabbit anti-(annexin V) Ig were performed by comparative densitometry of each band. Under our experimental conditions, 18.7 ^ 1.1% (n 4) of total annexin V was detected in platelets stimulated with 0.5 U´mL21 thrombin for 2 min and 2.1 ^ 0.1% (n 4) in resting platelets. Consequently, a ninefold increase in the amount of annexin V in the cytoskeleton fraction of activated platelets was observed as compared with resting platelets (Fig. 1B). With prolonged thrombin activation, the amount associated with the cytoskeleton fraction increased to 39 ^ 0.6% (n 4) by 10 min. To test whether an increase in intracellular [Ca21] can cause the relocation of annexin V to the cytoskeleton, platelets were activated with the ionophore A23187 in the presence of 1 mm CaCl2, as described in Materials and methods. The amount of annexin V that is incorporated into the cytoskeleton when platelets are activated with the ionophore A23187 is illustrated in Fig. 1C. Platelets were activated in the presence of calcium for 10 min, then the cytoskeleton was isolated by extraction in 1% Triton X-100 in the presence of protease inhibitors and centrifugation. The extraction was carried out in the presence of

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Fig. 1. Association of annexin V with the cytoskeleton of stimulated platelets. (A) Incorporation of annexin V into the cytoskeletal fraction during platelet activation with the physiological agonist thrombin. Resting or thrombin-activated platelets (0.5 U´mL21) were lysed at diferent times after the addition of thrombin with Triton X-100-containing buffer in 5 mm EGTA and centrifuged to separate the detergent-soluble (s) and detergent-insoluble (p) fractions as described in Materials and methods. Fractions were subjected to SDS/PAGE, electroblotted and blots were probed with rabbit anti-(annexin V) Ig and peroxidase activity was detected using the ECL detection method. As loaded on the gels, pellet samples are twice more concentrated than supernatant samples. (B) Histogram showing the changes in the amount of annexin V in the TX-100-soluble and -insoluble fractions calculated from the immunoblot analyses. Values represent the percentage of the amount of annexin V in the cytoskeleton fraction (mean ^ SEM, n 4) as compared with the total amount of annexin V in platelets. (C) Non-stimulated (NS) or A23187-treated platelets (5 mm, 10 min in the presence of 1 mm CaCl2) were lysed with Triton X-100containing buffer in the presence of increasing free [Ca21] and centrifuged to separate the detergent-soluble (s) and detergent-insoluble (p) fractions as described in Materials and methods. Fractions were subjected to SDS/PAGE, electroblotted and blots were probed with rabbit anti-(annexin V) Ig and peroxidase activity was detected using the ECL detection method. As loaded on the gels, pellet samples are twice more concentrated than supernatant samples.

EGTA or increasing free [Ca21], as described in Materials and methods. The cytoskeletal fraction was subjected to SDS/PAGE and Western blot analysis. The cytoskeleton fraction from resting platelets did not show any detectable amounts of annexin V (Fig. 1C, NS/EGTA), consistent with the subcellular fractionation studies performed before [38], in which annexin V is found in the cytosol when platelets are lysed and subfractionated in EGTA. After A23187 stimulation and extraction of the cytoskeletons in the presence of EGTA (Fig. 1C, A23187/ EGTA), cytoskeletons showed low but detectable amounts of annexin V. Extraction of cytoskeletons in the presence of increasing free [Ca21] induced increasing association of

annexin V with the cytoskeleton fractions (Fig. 1C, A23187/ 0.2 mm to 1 mm; maximum signal was reached when extraction was carried out in 8.8 mm free [Ca21], which is within the physiological range. The efficiency of the extraction procedure was assessed using the integral membrane protein CD9 as a marker for a plasma membrane component; the isolated Triton X-100-insoluble (cytoskeleton) fraction was free of membrane/phospholipid contamination (not shown). This is in agreement with previous studies, which show that 1 mL of 1% Triton X-100 is sufficient to cause solubilization of membrane components and result in a highly enriched cytoskeleton fraction from a platelet suspension containing 5 108 cells [50].


Fig. 2. Effect of extracellular calcium on the association of annexin V with the cytoskeleton of thrombin-activated platelets. (A) Platelets were preincubated with 1 mm EGTA or 1 mm CaCl2 and subsequently activated with 0.5 U´mL21 thrombin for 2 min. Cytoskeletons were isolated by lysis in a Triton X-100-containing buffer in 5 mm EGTA and centrifuged to separate the detergent-soluble (s) and detergent-insoluble (p) fractions as described in Materials and methods.(B) Thrombin-activated platelets (0.5 U´mL21, 2 min in 1 mm CaCl2) were lysed with Triton X-100containing buffer in the presence of 5 mm EGTA or 8.8 mm free Ca21 and centrifuged to separate the detergent-soluble (s) and detergent-insoluble (p) fractions as described in Materials and methods. As loaded on the gels, pellet samples are twice more concentrated than supernatant samples.

Role of extracellular calcium on the translocation of annexin V to the platelet cytoskeleton In order to determine if the relocation of annexin V to the platelet cytoskeleton is dependent on influx of extracellular calcium, the association of annexin V with the cytoskeleton in response to thrombin was compared in the presence and absence of extracellular calcium. Experiments were performed in which relocation of annexin V to platelet cytoskeletons was induced by thrombin in the presence or absence of the extracellular Ca21 chelating agent EGTA. As shown in Fig. 2A, chelation of extracellular Ca21 by addition of 1 mm EGTA prior to addition of thrombin reduced the magnitude of the amount of annexin V that relocated to platelet cytoskeletons to 10.1 ^ 0.6% (n 3), whereas when platelets were activated in the presence of 1 mm extracellular Ca21, the amount of Triton X-100-insoluble annexin V was 17.1 ^ 0.8% (n 3, P , 0.002) (Fig. 2A). The effect of Ca21 on the association of annexin V with the platelet cytoskeleton was investigated even further by lysing platelets in the presence of 8.8 mm free Ca21. Washed platelets were activated with thrombin in the presence of calcium and cytoskeletons were isolated by extraction in 1% Triton X-100 in the presence of protease inhibitors and 5 mm EGTA or 8.8 mm free Ca21 and centrifugation, as described in

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Fig. 3. Association of annexin V with the platelet cytoskeleton: effect of F-actin depolymerizing agents. (A) Platelets were preincubated with 5 1025 m cytochalasin E for 15 min and subsequently activated with 0.5 U´mL21 thrombin for 2 min in 1 mm CaCl2. Cytoskeletons were isolated by lysis in a Triton X-100-containing buffer in 5 mm EGTA and centrifuged to separate the detergent-soluble (s) and detergent-insoluble (p) fractions as described in Materials and methods.(B) Platelets were activated with thrombin as above and cytoskeletons were isolated by lysis in a Triton X-100-containing buffer in 5 mm EGTA in the presence of 2 mg´mL21 DNase I. Supernatant (s) and cytoskeleton (p) fractions were separated by centrifugation.All fractions were subjected to SDS/PAGE, electroblotted and blots were probed with rabbit anti-(annexin V) Ig and peroxidase activity was detected using the ECL detection method. As loaded on the gels, pellet samples are twice as concentrated than supernatant samples.

Materials and methods. The soluble and cytoskeletal fractions were subjected to SDS/PAGE and Western blot analysis to quantify annexin V (Fig. 2B). While the association of annexin V in cytoskeletons isolated in EGTA and calcium were qualitatively similar, considerably more annexin V was associated with the cytoskeleton when the extraction was performed in the presence of calcium (20.8 ^ 1.0%, n 3) than in the presence of EGTA (18.8 ^ 0.9%, n 3, P , 0.003). This is paralleled by the finding described previously that platelets activated with A23187 and lysed in the presence of increasing calcium concentrations show increased association of annexin V with the cytoskeleton (Fig. 1C). Taken together, these results demonstrate the calcium-dependency of the association of the annexin V with the detergent-resistant cytoskeleton of platelets that have been activated either by thrombin or by ionophore.

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annexin V in the Triton X-100-insoluble fraction was also reduced. The differential effect of CE and DNase I on the recovery of annexin V in the Triton X-100-insoluble fraction suggests that annexin V is selectively associated with the membrane-bound actin filaments in platelets. Annexin V and actin form an 85-kDa complex

Fig. 4. Crosslinking of annexin V to actin. (A) Triton X-100-insoluble fractions from activated platelets. (A) were prepared in the absence (lanes a) or the presence of 5 mm DMS (lanes b) as described in Materials and methods. Fractions from untreated platelets (NS) are included as a control.(B) Triton X-100-soluble fractions from activated platelets (A) were prepared from control membranes (lanes a) or membranes incubated with 5 mm DMS (lanes b) as described in Materials and methods. Fractions from nonstimulated platelets (NS) are included as a control. The formation of high molecular mass complexes was visualized by SDS/PAGE and Western blotting against annexin V and actin as indicated. The arrows indicate the 85 kDa immunoreactive complex.

Effect of depolymerization of the cytoskeleton on the association of annexin V with the Triton X-100-insoluble fraction of thrombin-activated platelets Because several other proteins in addition to actin filaments are inherently insoluble in Triton X-100 [51], the effect of depolymerization of the actin filaments on the Triton X-100insolubility of annexin V was examined. Two depolymerizing agents were used: cytochalasin E (CE) and DNase I. Addition of CE to intact cells inhibits the activation-induced actin polymerization in platelets, but does not affect the small pool of actin filaments that is membrane bound [52]. Figure 3 shows that CE had little effect on the recovery of annexin V in the Triton X-100-insoluble fraction. In contrast to CE, DNase I depolymerizes both cytoplasmic and membrane-bound filaments [51]. When DNase I was added to the lysis buffer at concentrations shown previously to depolymerize < 40% of the total actin filament content and release < 20% of integrin GPIb from the Triton X-100-insoluble fraction [51], the recovery of

The experiments described so far have shown an association of annexin V with the cytoskeleton of activated platelets; it is, however, unknown whether this recovery of annexin V with the cytoskeleton is due to its binding to actin. We have previously shown that annexin V can be crosslinked to actin in the membrane fraction of activated platelets [42]. In order to investigate further the association of annexin V with actin and its role on the isolation of annexin V with the cytoskeleton of activated platelets, a series of crosslinking experiments were performed to identify the Triton X-100 resistant components which are targets for annexin V action. The bifunctional crosslinker DMS was added to an isolated Triton X-100insoluble fraction as described in the Materials and methods section. A single high molecular mass band of 85 kDa, showing immunoreactivity against annexin V, was observed in the presence (Fig. 4A, lane b) but not the absence of DMS (Fig. 4A, lane a). This has an identical mobility to the immunoreactive complex previously identified when annexin V binds tightly to EGTA washed membranes (Fig. 4B, lane b) and [42]. When cytoskeleton fractions containing annexin V were assayed for the crosslinking of actin to other proteins, three high molecular mass bands of 85, 100 and 105 kDa were observed in the presence (Fig. 4A, lane b) but not the absence of DMS (Fig. 4A, lane a). The actin-containing 85 kDa complex identified here has an identical mobility to that recognized by antibodies against annexin V (Fig. 4A, lane b), a finding which is consistent with the annexin V binding protein being actin. The 85 kDa complex also has an identical mobility to the complex identified in platelet membranes when crosslinked fractions were examined for immunoreactivity against actin (Fig. 4B, lane b). These results clearly demonstrate that in activated platelets, annexin V binds to a protein with an identical molecular mass to actin, and that actin in these fractions binds to a protein with an identical molecular mass to annexin V. As expected, no crosslinking occurs in unstimulated platelets (Fig. 4A,B). The above experiments provide evidence which suggests that actin is the annexin V binding protein. A series of in vitro binding studies were then performed to investigate if pure annexin V and pure actin associate to form an 85-kDa complex. The addition of DMS produced an 85-kDa complex, which had an identical mobility on SDS/PAGE to the endogenous 85-kDa complex described above (data not shown). Furthermore, this complex had an identical mobility on 2D electrophoresis to the endogenous 85-kDa complex isolated from platelet membranes (data not shown), thereby demonstrating that, by the criteria of molecular mass and isoelectric point, the endogenous complex consists of annexin V and actin. Interaction of annexin V with actin filaments To test the possible interaction of annexin V with the actin filament system of the cytoskeleton, a repolymerized actinrich fraction was prepared from the Triton X-100-insoluble material of stimulated platelets as described in Materials and methods. As shown in Fig. 5A, the annexin V present in the Triton X-100-insoluble material was also recovered in the


q FEBS 2000 Fig. 5. Binding of annexin V to actin filaments. (A) Annexin V interacts with a re-polymerized actin-rich fraction from activated platelets. Triton X-100-insoluble material was isolated from activated platelets in the presence of increasing free [Ca21], as described previously. Solubilization of the Triton X-100-insoluble material was achieved with KI. Induction of actin polymerization was initiated as described in Materials and methods. Supernatant (s) and insoluble (p) fractions were subjected to SDS/ PAGE, electroblotted and blots were probed with rabbit anti-(annexin V) Ig. Peroxidase activity was detected using the ECL detection method. As loaded on the gels, pellet samples are four times more concentrated than supernatant samples. (B) Binding of annexin V to F-actin in a cosedimentation assay. Annexin V and F-actin were mixed in the presence of EGTA (5 mm) or increasing free [Ca21], incubated for 1 h at room temperature and then centrifuged at 200 000 g for 15 min. Equivalent amounts of supernatants (s) and pellets (p) were separated by SDS/PAGE and stained with Coomassie blue. The composition of the samples analysed is indicated at the top of each lane. (C) Actin binding assay of extracts from resting or activated platelets.Extracts from nonstimulated (NS) or thrombin-activated platelets were incubated with 1.1 mm F-actin in 5 mm EGTA or 8.8 mm free [Ca21], and were then centrifuged at 200 000 g for 15 min. Equivalent amounts of supernatants (s) and pellets (p) were separated by SDS/PAGE, electroblotted and probed with rabbit anti-(annexin V) Ig. Peroxidase activity was detected using the ECL detection method.

repolymerized actin fraction, indicating that annexin V directly interacts with actin or associates to actin-binding proteins. Direct binding of annexin V to F-actin in the presence of different [Ca21] was investigated using a cosedimentation assay of purified proteins. Annexin V did not sediment in the absence of actin in EGTA or high Ca21 [42]. Annexin V failed to cosediment with actin in the presence of EGTA or calcium concentrations within the physiological range; cosedimentation with actin was only observed in the presence of 1 mm [Ca21] (Fig. 5B). Densitometric data for bound annexin V in the presence of 1 mm Ca21 revealed that annexin V bound to actin with an apparent molar ratio of 1 : 1 (annexin V:actin). A possible reason for the high Ca21 requirement observed for the binding of annexin V to F-actin in vitro might be that, in vivo, a cofactor is required to facilitate binding at physiological [Ca21], whereas in its absence the Ca21 requirement for interaction reaches nonphysiological levels. To test this possibility, an extract containing annexin V was isolated from nonstimulated and thrombin-stimulated platelets and was examined for its binding by the addition of exogenous F-actin, as described in Materials and methods. In the absence of added F-actin, annexin V remained in the supernatant, for both nonstimulated and thrombin-stimulated platelets (data not shown). However, annexin V from thrombin-stimulated platelets coprecipitated with exogenous F-actin, whereas annexin V from nonstimulated platelets remained in the supernatant (Fig. 5C). When the calcium requirement for the interaction

of annexin V with the actin filaments was examined, it was found that it occurs at calcium concentrations as low as 1 mm (data not shown), with maximal binding occurring at 8.8 mm. Isolation of GST±annexin V bound complexes A series of experiments using GST±annexin V was performed to investigate further the role of actin in the relocation of annexin V. We have previously shown that when annexin V is added to isolated platelet membranes it binds tightly to membranes in a manner that is analogous to the binding seen by endogenous annexin V following physiological stimulation [14] (Fig. 6A). When membranes were incubated with GST± annexin V prior to incubation with annexin V (Fig. 6A, lane i), it was apparent that GST±annexin V bound to the membranes, and competitively inhibited the subsequent association of annexin V (compare Fig. 6A, lanes i and j). These results demonstrate that GST±annexin V binds to membranes in an analogous way to endogenous annexin V, and shows that GST±annexin V and annexin V bind to the same component(s) on platelet membranes. The identity of these membrane components was investigated further by binding GST±annexin V to membranes, and subsequently isolating GST±annexin V and its complexing proteins using glutathionine-agarose as described in the Materials and methods section. When the material which copurified with GST±annexin V was visualized by silver staining it was apparent that major

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Fig. 6. Isolation of GST±annexin V bound complexes. (A) GST±annexin V inhibits the binding of annexin V to platelet membranes. Platelet membranes were incubated in the absence of annexin V (lane a), or the presence of 0.005 mm (lane b), 0.05 mm (lane c), 1 mm (lane d), 1.4 mm (lane e), 2 mm (lane g) or 4 mm annexin V (lane h) for 1 h at 37 8C. The membranes were subsequently washed five times in buffer A as described in Materials and methods. The annexin V which remained associated with membranes was assayed by SDS/PAGE and Western blotting. In lane i, the membranes were first incubated with GST±annexin V before the addition of 0.5 mm annexin V. Control membranes were incubated in the absence of GST±annexin V (lane j). Standards of pure annexin V were also subjected to by SDS/PAGE and Western blotting (1, 2, 5, 8 and 12 ng). (B) Membranes which had been preincubated with either GST (lanes a) or GST±annexin V (lanes b) were washed and then solubilized with 1% Triton X-100 and incubated with glutathione-agarose as described in Materials and methods. Silver staining, immunoreactivity against GST, immunoreactivity against all actin isoforms, immunoreactivity against b-actin, and immunoreactivity against g-actin of glutathione-bound proteins is shown. Free GST (at 27 kDa) indicates breakdown of GST±annexin V subsequent to membrane binding and affinity isolation.

bands of 27 and 62 kDa, and minor bands of 20, 43, 85 and 105 kDa were seen in material isolated from membranes which were incubated in the presence of GST±annexin V (Fig. 6B, lane b), but not in membranes which were incubated in the presence of GST alone (Fig, 6B, lane a). Western blotting using a monoclonal antibody against GST demonstrates that the two major proteins of 27 and 62 kDa observed in silver stained gels correspond to GST and GST±annexin V, respectively (Fig. 6B, lane b). In addition, no immunoreactivity against GST was seen in the control fraction (Fig. 6B, lane a), demonstrating that the association of GST±annexin V with membranes is not due to the existence of GST±binding proteins in this fraction. The possibility that the 43 kDa protein was actin was investigated by immunoblotting for actin. An antibody that recognizes all actin isoforms (Fig. 6B, lane b) also recognizes the 43 kDa

Fig. 7. Immunoprecipitation of annexin V. Triton X-100 extractable material containing endogenous annexin V was isolated from platelets as described in the Materials and methods section. Pre-immune serum (lanes a) or antisera specific for annexin V (lanes b) conjugated to protein-G beads was then added. Sedimented material was then extracted in sample buffer and visualized by SDS/PAGE and Western blotting against all actin isoforms, b- and g-actin.

protein. Surprisingly, antibodies against the b-isoform of actin did not crossreact despite the fact that the b-isoform is the major isoform of actin in platelets (Fig. 6B, lane b). The actin copurifying with annexin V was recognized by an antibody specific to the minor g-isoform of actin (Fig. 6B, lane b). These observations demonstrate that membrane associated annexin V interacts with platelet actin, and that this association is specific for the g isoform of actin. Immunoprecipitation of annexin V The experiments using GST±annexin V affinity purification suggested that annexin V binds to actin, and in particular to the g-isoform of actin. To confirm such binding, experiments were designed to determine whether the annexin V antibodies could immunoprecipitate g-actin from the Triton X-100-extractable material. When antiserum specific for annexin V was added to the Triton extract, g-actin sedimented in conjunction with annexin V (Fig. 7). In contrast, when the immunoprecipitated sample was examined by Western blotting for the presence of b-actin, no immunoreactivity was seen. Immunoprecipitation using the preimmune serum showed no immunoreactivity for b- or g-actin (Fig. 7). Thus, the coprecipitation of annexin V and g-actin suggests an interaction of annexin V with g- but not b-actin.

DISCUSSION The work presented here demonstrates that activation of platelets with the physiological agonist thrombin results in


the relocation of annexin V from the cytosol to the platelet cytoskeleton. The appearance of annexin V in the cytoskeleton fraction as soon as platelets are activated and its increased incorporation during activation with thrombin (Fig. 1A,B) suggest it may play an important role in the dynamic processes of platelet stimulation. As platelets change their shape from disc to sphere within 20 s and the secretion of ATP from dense bodies is normally maximal within 1 minute [53], it is possible that shape change and/or platelet secretion are the processes during which annexin V±cytoskeleton interaction starts taking place. The ability of the calcium ionophore A23187 to cause association of annexin V with the platelet cytoskeleton suggests that relocation of annexin V is a calcium-dependent event. Maximal association of annexin V with the cytoskeleton is seen when calcium levels are 8.8 mm (Fig. 1C), suggesting that we are observing a physiological event. Results with the physiological agonist thrombin confirm this hypothesis. Interestingly, it has previously been shown that activation of platelets by different activators results in different interactions between the membrane and the cytoskeleton [54,55]. This is reflected by the significant difference in the association of annexin V with the cytoskeleton between receptor and nonreceptor-mediated activation. It is possible that annexin V requires the synergistic action of protein kinase C and Ca21 (triggered by agonists such as thrombin) for optimal association with the cytoskeleton. Similar to the results presented here for annexin V, Rotman and coworkers have previously shown that the association of a-actinin with the cytoskeleton of platelets depends on the modes of activation: when platelets are activated by a receptormediated agonist there is a high degree of association of a-actinin with the cytoskeleton. When platelets are activated via a nonreceptor-mediated mechanism, there is only a small amount of incorporation of this protein in the cytoskeleton [55]. The observation that chelation of extracellular Ca21 levels reduces but does not abolish the recovery of annexin V with the platelet cytoskeleton suggests that release of Ca21 from intracellular stores is enough to cause relocation of annexin V to the cytoskeleton (Fig. 2A). Although the initial transient increase in [Ca21] from the intracellular stores is enough to relocate intracellular annexin V to the platelet cytoskeleton, this relocation is further enhanced by influx of calcium from extracellular stores (Fig. 2B). It is important to note here that the thrombin-induced rise in cytoplasmic Ca21 is biphasic, depending upon the release of intracellular stores for the early rapid spike and upon the extracellular stores for the later slower one [56]. It seems therefore that the relocation of annexin V to the cytoskeleton of thrombin-stimulated platelets is calciumdependent in both the early rise in [Ca21] due to release of intracellular stores, and the later rise due to influx from the extracellular milieu. The calcium±dependent association of annexin V with the cytoskeleton is similar to that described for GPIIb-IIIa [57±59]; the association of GPIIb-IIIa, however, is also aggregation-dependent, whereas that of annexin V is not (not shown). The association of annexin V with the platelet cytoskeleton is also independent of the stimulation-induced actin polymerization (Fig. 3A). When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, certain proteins and signalling molecules associate with the Triton X-100-insoluble cytoskeleton, such as phosphatidylinositol (PtdIns)-4- and -3-kinases, PtdIns [4]P-5-kinase, diacylglycerol (DAG) kinase, phospholipase C, the tyrosine kinases pp60c-src and pp62c-yes and the

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p21ras GTPase-activating protein (GAP) [5,6,9]. These proteins are not directly associated with newly polymerized actin, as blockage of actin polymerization by cytochalasins, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of these proteins. Depolymerization of the membrane cytoskeleton with DNase I, however, significantly decreases the sedimentability of these proteins with the Triton X-100 fraction. Similarly, translocation of annexin V to the cytoskeleton in thrombin-stimulated platelets is a reversible and specific process mediated by its interaction with the actin filaments or with some actin-binding proteins. This is demonstrated by the evidence that cytoskeletal annexin V was solubilized when actin filaments of both the membrane and cytosolic cytoskeleton were depolymerized by the action of DNase I (Fig. 3B), and spontaneously re-associated with the actin when repolymerization was induced in vitro (Fig. 5A). The crosslinking of annexin V to actin in the membrane and cytoskeleton fractions strongly supports the hypothesis that annexin V binds to actin (Fig. 4A,B). The binding which we have observed in this study is similar to that previously observed by Giambanco and coworkers who reported that pure annexin V, when added to a bovine brain cytoskeletal preparation, also binds to a actin [34]. Although annexin V was found to bind to the platelet cytoskeleton following stimulation and was crosslinked to actin, an interaction between purified annexin V and F-actin was only reconstituted in vitro in the presence of high [Ca21] (Fig. 5B). It is important to note that actin filaments in cells differ from purified F-actin in vitro by being modulated by actin binding proteins that alter filament association through crosslinking and bundling and filament length through capping, severing and actin monomer sequestration [60]. It is therefore possible that a cofactor is required to facilitate binding of annexin V to F-actin at physiological [Ca21], whereas in its absence the Ca21 requirement for interaction reaches nonphysiological levels. Indeed, the inclusion of an extract from thrombin-stimulated platelets lowered the Ca21 requirement for the binding of annexin V to F-actin to physiological levels (Fig. 5C). Interestingly, binding of purified GST±annexin V to pure G-actin did not occur even in high [Ca21] (not shown), however, binding of GST±annexin V to the Triton X-100soluble fraction has shown that GST±annexin V retains a specific isoform of actin on glutathione beads (Fig. 6B). In support of this, antibodies against annexin V induce the cosedimentation of both platelet annexin V and g- but not b-actin (Fig. 7). In mammalian nonmuscle cells, the predominant isoforms of actin are cytoplasmic b- and g-actin. Although there have been reports on differential functions and/or subcellular localizations [61±65], differences in isoform distribution and function have been minimally explored. There have been a limited number of reports of actin binding proteins preferring one isoform of actin over another. Ezrin [66], profilin [67], thymosin b-4 and l-plastin [68] have been shown to interact more strongly with b- and g-actin than with rabbit skeletal a-actin. The mechanism behind this isoform selectivity is unclear, though the differences in charged residues at the amino terminus of actin have been suggested as a possible mechanism. This is the first report of a possible selective binding to the g-isoform of actin. The binding of annexin V to actin at the plasma membrane is of particular interest in light of previous reports showing that actin filaments coisolate with the platelet plasma membrane [69]. Davies found that platelet membrane preparations always contain actin, which is resistant to extraction with 0.6 m

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potassium iodide and hence is tightly associated with the membrane [70]. Zucker-Franklin provided ultrastructural evidence for submembranous actin containing filaments that insert into the inner side of the plasma membrane [71]. More recently, Qingqi and Stratcher have shown that platelet membrane actin may be partially embedded into the lipid bilayer and disulfide linked to integral membrane proteins [72]. We propose that following an interaction of the ligand (e.g. thrombin) with its receptor, annexin V forms a strong association with the already organized cytoskeleton and this association is increased during the progressive stages of activation. In light of the changes in the submembrane area that follow platelet activation, the localization of annexin V positions this protein at a site where complex cytoskeletal and membrane modifications take place within the activated platelet.

14.

15.

16.

17.

18.

ACKNOWLEDGEMENTS This work was funded by a BHF Grant (PG/96108). A special thanks to Dr C. Chapponier for the gift of the anti g-actin antibody and Dr F. Russo-Marie for providing the plasmid for the expression of recombinant GST±annexin V. We would also like to thank all the blood donors.

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