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Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation
Biochimica et Biophysica Acta 1497 (2000) 227^236 www.elsevier.com/locate/bba
Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation Maria Antonietta Belisario a; *, Simona Tafuri b , Carmela Di Domenico a , Rossella Della Morte a , Caterina Squillacioti a , Antonia Lucisano b , Norma Staiano a
a
Dipartimento di Biochimica e Biotecnologie Mediche, Universita© di Napoli Federico II, Via S. Pansini, n. 5, 80131 Naples, Italy b Dipartimento di Patologia e Sanita© Animale, Universita© di Napoli Federico II, Naples, Italy Received 24 February 2000; received in revised form 1 May 2000; accepted 11 May 2000
Abstract Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet KIIb L3 binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of KIIb L3 conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADPactivated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72syk and pp125FAK . Inhibitors of platelet signalling, such as apyrase, prostaglandin E1 , cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125FAK phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125FAK phosphorylation. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Adhesion; Platelet; Protein phosphorylation; Echistatin; pp125FAK ; pp72syk
1. Introduction Integrins are involved in cell adhesion and migration which occur during physiological processes such as haemostasis, immune response, development and in£ammation [1^4]. They are important signalling
* Corresponding author. Fax: +39-081-746-3150; E-mail: [email protected]
molecules which mediate bi-directional transfer of information from the extracellular matrix to the cytoplasm and vice versa [5,6]. KIIb L3 , the major integrin expressed on platelet membrane, plays a critical role in haemostasis by mediating platelet^platelet and platelet^matrix protein interactions [1,7]. Physiological platelet agonists activate transduction pathways (inside-out signalling) leading to the conversion of KIIb L3 from a `low' to a `high' a¤nity state, which allows the integrin to be-
0167-4889 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 0 0 ) 0 0 0 6 1 - 6
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come competent to bind adhesive proteins, such as ¢brinogen and von Willebrand factor [1,8]. Following ligand engagement, KIIb L3 triggers transduction events (outside-in signalling) which, co-ordinated with other signals activated by other platelet receptors, lead to platelet aggregation [9]. Disintegrins are low molecular weight polypeptides isolated from snake venoms, which are potent inhibitors of platelet functions [10,11]. The presence in their molecules of the arginine, glycine, aspartic acid (RGD) tripeptide sequence is responsible for their antiplatelet activity, since the interaction of KIIb L3 with its natural ligand ¢brinogen is mediated, at least in part, through this recognition sequence [12]. The inhibition of KIIb L3 functions by natural and synthetic RGD-containing molecules has recently evolved as a new antiplatelet strategy [13]. In addition to bleeding problems, a major concern related to the use of competitive inhibitors of integrin functions is based on their capability to induce KIIb L3 conformational changes, which in turn result in an increased ¢brinogen-binding function of the receptor [14^16]. However, the switch of integrin conformation toward an activated state which can be induced by an RGD-peptide is not su¤cient to induce integrin-dependent outside-in signalling. Integrin clustering, provided only by multivalent ligands, has been shown to be a pre-requisite for receptor-mediated transmembrane e¡ects [17^19]. Contortrostatin, because of its dimeric structure, is the only disintegrin which was found to activate integrin-dependent protein phosphorylation in platelets [20]. It has been suggested that proteins may exhibit multivalent functions as a consequence of their immobilisation on a substrate [17]. As a result of this process, a ligand may reach a high local density and undergo conformational changes. Both these phenomena may provide either a modulation of the ligand binding properties and/or a direct mechanism for inducing adjacent integrin clustering [21,22]. In the present study, we show that the immobilisation on a plastic surface of echistatin, a monomeric disintegrin from Echis carinatus venom [23], causes an increase of its binding a¤nity for integrin(s) expressed on resting platelets. Echistatin, when in a solid-phase form, is also able to activate the early phase of occupied integrin outside-in signalling, in-
cluding pp72syk phosphorylation. Furthermore, we demonstrate that the interaction of immobilised echistatin with platelet surfaces stimulates not only signals dependent from integrin aggregation, but also those transduction events required for dense granule secretion and full platelet activation. 2. Materials and methods 2.1. Antibodies and chemicals Rabbit polyclonal (LR), mouse monoclonal (4D10) human pp72syk antibodies and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, USA); rabbit polyclonal anti-pp125FAK serum (BC3) and anti-human pp125FAK polyclonal IgG from Upstate Biotechnology (Lake Placid, NY, USA); phosphotyrosine monoclonal antibody (mAb) PT66, horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG from Sigma (St. Louis, MO, USA); anti-KIIb L3 complex (CBL130), anti-Kv (CBL490) and anti-K5 (CBL497) antibodies from Cymbus Bioscience (Southampton, UK); human plasma ¢brinogen (purity s 98%) and bisindolylmaleimide (BIS) from Calbiochem (San Diego, CA, USA); Sepharose 2B from Pharmacia Biotech (Uppsala, Sweden); apyrase, prostaglandin E1 (PGE1 ), ADP, orthovanadate, leupeptin, aprotinin, phenylmethylsulfonyl £uoride (PMSF), bovine serum albumin (BSA), cytochalasin D (CD) and indomethacin from Sigma. All the other reagents were of the highest quality available. 2.2. Preparation of platelets Citrated (0.32% w/v) blood specimens were obtained from healthy volunteers who had not taken any drugs for 9 days before bleeding. Platelet-rich plasma (PRP), obtained by blood centrifugation at 200Ug for 15 min, was further centrifuged at 1200Ug for 10 min in the presence of 1 U/ml apyrase and 1 WM PGE1 . Platelets, resuspended in HEPES bu¡er, pH 7.35, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 , 3 mM NaH2 PO4 , 5 mM glucose, 3.5 mM HEPES and 3.5 g/l BSA were gel-¢ltered on Sepharose 2B [24]. The concentration of washed platelets was assessed spectrophotometrically at
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520 nm. The platelet suspension was then diluted at the concentration required for aggregation and adhesion experiments. Platelet suspensions (2.5U108 platelets/ml), supplemented with 0.5 mg/ml ¢brinogen and 5 mM Ca2 , were tested for their ability to aggregate in response to 50 WM ADP. Aggregation was monitored at 37³C on a single channel aggregometer (Chrono-Log Aggregometer model 500-CA, Haverton, PA, USA) with continuous stirring at 900 rpm. Platelets stimulated with ADP in the absence of exogenous ¢brinogen, or platelets activated with ristocetin did not aggregate, thus indicating that the gel-¢ltration procedure yielded platelets completely separated from plasma proteins. 2.3. Platelet adhesion assay Gel-¢ltered unstimulated platelets were allowed to adhere to 6 cm diameter plates (Becton-Dickinson, NY, USA) or to 96-well polystyrene £at-bottom plates (Costar, MA, USA) pre-coated for 16 h at 4³C with substrates (10 Wg/ml). Echistatin was dissolved in 50 mM carbonate/bicarbonate bu¡er pH 9.2, whereas ¢brinogen was solubilised in phosphate bu¡er solution (PBS). Unbound sites on the plastic were blocked with 2 ml/plate or 200 Wl/well of 5 mg/ml heat-denatured BSA for 1 h at 37³C. Wells coated with BSA were used to check aspeci¢c platelet adhesion. After three washing with PBS, plates were ¢lled with 50 Wl of a platelet suspension (3U108 /ml) supplemented with 1 mM CaCl2 and left for 1 h at room temperature. Non-adherent platelets were removed and the plates were washed twice with PBS. Quantitative evaluation of the number of adherent platelets was performed in microtitre wells by measuring platelet endogenous phosphatase [25]. The plates were read at 410/630 nm on an automated plate reader (Bio-Rad model 450, Hercules, CA, USA). Platelet pre-treatments with inhibitors of platelet activation were performed as it follows: (1) 10 min with 10 U/ml apyrase or 12 nmol/ml CD or 10 nmol/ml indomethacin; (2) 1 min with 1 nmol/ml PGE1 ; 1 h with 12 nmol/ml BIS. The anti-aggregatory activity of these inhibitors was tested before their use. Adhesion experiments were also performed by adding to the platelet suspension a
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mixture of protease inhibitors (20 Wg/ml leupeptin, 100 U/ml aprotinin, and 100 WM PMSF) or hirudin (20 U/ml). In an other set of experiments, platelets were pre-incubated for 5 min with 10 Wg/ml antiKIIb L3 alone or 5 min with anti-KIIb L3 plus further 5 min with anti-Kv or anti-K5 antibodies. 2.4. Protein immunoprecipitation and Western blotting Adherent platelets were lysed with 600 Wl of ice cold RIPA bu¡er, containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 10 mM Tris^HCl, pH 7.2, 1 mM EGTA, 1 mM PMSF, 20 Wg/ml leupeptin, 100 U/ml aprotinin, and 1 mM sodium orthovanadate. Platelets in suspension were lysed by adding an equal volume of 2URIPA bu¡er. After 30 min on ice, lysates were clari¢ed by centrifugation at 14 000 rpm for 10 min at +4³C. The resulting supernatants were characterised for protein concentration by Bio-Rad DC protein assay. Equal amounts of proteins were immunoprecipitated overnight at 4³C with anti-pp125FAK (BC3) (1 Wg/100 Wg proteins) or anti-pp72syk (LR) (3 Wl of a 1:5 antibody dilution/100 Wg proteins) previously bound to protein A/G-Agarose. Beads were sedimented by brief centrifugation and washed extensively with ice-cold RIPA bu¡er. Proteins, solubilised in boiling SDS sample bu¡er (2% SDS, 5% L-mercaptoethanol, 66 mM Tris, pH 7.5, 10 mM EDTA), were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Western blotting analyses were performed as previously described [26]. To evaluate protein tyrosine phosphorylation, blots were probed with mAb PT66 (1:10 000). To monitor the loading of gel lanes, the membranes were stripped according to the Manufacturer's procedure (Amersham, Buckinghamshire, UK) and then reprobed with the anti-human pp125FAK (1:500) or anti-pp72syk (4D10) (1:200) antibodies. Immunoreactive bands were visualised by enhanced chemiluminescence kit (Amersham). Quantitisation of proteins was performed by densitometry using a Discover Pharmacia scanner equipped with a sun spark classic densitometric workstation.
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Fig. 1. Platelet adhesion to immobilised echistatin. Unstimulated (a) and ADP-activated (b) platelets (1.5U107 ) were poured on wells coated with increasing amounts of echistatin (A). Di¡erent numbers of unstimulated platelets (solid line) or platelets previously treated with 0.1 WM of soluble echistatin for 3 min (dotted line) were poured on wells coated with 0.5 Wg/well echistatin (E) or ¢brinogen (F) (B). The plates were incubated at room temperature for 1 h. Non-adherent platelets were removed and the number of adherent platelets was measured as described in Section 2. The number of platelets adhering to BSA-coated wells has been subtracted from the number of platelets adhering to echistatin- or ¢brinogen-coated wells. The results reported here are the mean values from three di¡erent experiments in which each point was tested 10-fold. Standard deviations never exceeded 10% of the mean value.
3. Results 3.1. Platelet adhesion to immobilised echistatin Gel-¢ltered human platelets showed the capability to adhere to immobilised ¢brinogen and echistatin (Fig. 1), whereas they attached to BSA-coated wells only at a very low extent. The number of adherent platelets was shown to be a function of both the amount of coating disintegrin (Fig. 1A) and the concentration of the poured platelet suspension (Fig. 1B). The percentage of platelets attached to ¢brinogen- or echistatin-coated wells was about 20 and 17%, respectively, when 0.5 Wg of protein was immobilised on plates. A similar percentage of human platelet adhesion to immobilised ¢brinogen has been previously reported [25]. When the adhesion experiments were performed with washed platelets
resuspended in EDTA-supplemented HEPES bu¡er, platelets did not attach to immobilised echistatin, thus indicating that platelet binding to immobilised echistatin requires the presence of Ca2 (data not shown). Platelet exposure to 0.1 WM echistatin for 3 min before plating completely inhibited their adhesion to echistatin- and ¢brinogen-coated wells (Fig. 1B, dotted lines). This indicates that the same integrin sequences are recognised by both soluble and immobilised echistatin. Soluble echistatin has been previously reported to bind with higher a¤nity to stimulated platelets than to resting-ones [27]. Immobilised echistatin was found to support at comparable extent the adhesion of either unstimulated or ADP-activated platelets (Fig. 1A). To rule out that a partial platelet activation, which may occur during platelet collection and isolation, was responsible for the di¡erent a¤nity
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Fig. 2. Protein tyrosine phosphorylation in platelets adhering to immobilised echistatin or ¢brinogen. Lysates containing equal amounts of proteins from platelets adhering to immobilised ¢brinogen (lane 1), immobilised echistatin (lane 2) or BSA (lane 3) or lysed platelets taken in suspension for 3 min in the presence of 0.1 WM soluble echistatin (lane 4) were either directly run on SDS^ PAGE (A) or immuno-precipitated with pp72syk (LR) antibody (B) or pp125FAK (BC3) antibody (C) and subsequently separated by electrophoresis. After Western blotting, tyrosine phosphorylated proteins were visualised by mAb PT66. The amount of pp72syk or pp125FAK loaded was equal in all lanes of each gel as it was veri¢ed on parallel immunoblots probed with the anti-pp72syk antibody (4D10) (B, lower blot) or with the polyclonal anti-human pp125FAK antibody (C, lower blot). The position of molecular markers (in kDa) and of pp72syk or pp125FAK (arrows) is indicated. The same results were obtained from three separate experiments of equal design.
properties of the two forms of echistatin, adhesion experiments were performed in the presence of the aggregation inhibitors PGE1 and apyrase. None of these inhibitors was e¡ective in reducing the number of platelets adhering to immobilised echistatin and ¢brinogen (Table 1). The possibility that platelet activation might occur because of plasmin or thrombin traces present in the platelet preparation, was also ruled out, since the extent of platelet adhesion to immobilised echistatin was unchanged by protease inhibitors (PMSF, leupeptin, aprotinin) or by hirudin (Table 1). Previous ¢ndings showed that the blockade of speci¢c platelet transduction pathways does not a¡ect platelet binding to solid-phase ¢brinogen [21,28]. Likewise, platelets pre-treated with CD, an inhibitor of actin polymerisation, or BIS, a protein kinase C (PKC) inhibitor, or indomethacin, an inhibitor of arachidonate metabolism, adhered to echistatincoated wells as well as control platelets (Table 1). These results indicate that platelet adhesion to immobilised echistatin does not require platelet activation by agonists and does not involve platelet signalling. Immobilised echistatin-platelet interaction is mainly mediated by KIIb L3 integrin. In fact, platelet adhesion to immobilised echistatin was reduced by
Table 1 E¡ect of inhibitors on platelet adhesion to immobilised echistatin or ¢brinogen Inhibitor
Number of adherent-platelets (U106 /well) Echistatin
Fibrinogen
None PGE1 Apyrase Indomethacin CD BIS Aprotinin, leupeptin, PMSF Hirudin
2.71 þ 0.26 2.65 þ 0.25 2.52 þ 0.24 2.60 þ 0.26 2.73 þ 0.24 2.80 þ 0.23 2.55 þ 0.24 2.61 þ 0.25
3.02 þ 0.28 3.10 þ 0.25 3.05 þ 0.27 3.10 þ 0.26 2.91 þ 0.28 2.95 þ 0.29 3.03 þ 0.27 2.90 þ 0.28
Gel-¢ltered platelets were treated with di¡erent platelet inhibitors at concentrations and for the incubation time reported in Section 2. Platelets were allowed to adhere to echistatin- or ¢brinogen-coated wells (0.5 Wg/well). The plates were incubated at room temperature for 1 h. Non-adherent platelets were removed and the number of adherent platelets was assessed as described in Section 2. The number of platelets adhering to BSAcoated wells (0.31U106 þ 0.02) has been subtracted from the number of platelets adhering to immobilised echistatin or ¢brinogen. Data are the mean values þ S.D. calculated from three separate experiments in which each point was tested 10-fold.
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Fig. 3. E¡ect of inhibitors on pp125FAK phosphorylation induced by platelet adhesion to immobilised echistatin or ¢brinogen. pp125FAK phosphorylation was measured in platelets allowed to adhere to plates coated with echistatin (A) or ¢brinogen (B) in the absence (lane 1) and in the presence of apyrase (lane 2), PGE1 (lane 3), indomethacin (lane 4) and cytochalasin D (lane 5). C shows the results obtained from control platelets left for 1 h at room temperature (lanes 1 and 3) and BIS-pre-treated platelets (lanes 2 and 4) adhering to echistatin (lanes 1 and 2) or ¢brinogen (lanes 3 and 4). Platelet lysates containing equal amounts of proteins were subjected to immunoprecipitation with anti-pp125FAK (BC3) antibody and subsequently separated by electrophoresis. Tyrosine phosphorylated proteins were visualised by mAb PT66 (upper blots). The blots were stripped and reprobed with polyclonal anti-human pp125FAK antibody (lower blots). The position of molecular markers (in kDa) and pp125FAK (arrow) is indicated. Similar results were obtained from four experiments of identical design.
83% in the presence of anti-KIIb L3 antibody. However, the integrin receptors Kv L3 and K5 L1 could also be involved in such interaction, since a statistically signi¢cant higher inhibition of platelet adhesion to immobilised echistatin was observed when platelets were simultaneously treated with anti-KIIb L3 plus anti-Kv or anti-K5 antibodies (data not shown). 3.2. Platelet adhesion to immobilised echistatin promotes protein tyrosine phosphorylation Several tyrosine phosphorylated proteins resulted as a consequence of platelet binding to the natural ligand ¢brinogen or to immobilised echistatin (Fig. 2A). The pattern of phosphorylated proteins in echistatin-attached platelets resembled that of ¢brinogenadherent platelets. Platelets held in suspension or platelets attached to BSA showed very low levels of total protein phosphorylation, thus indicating that platelet signalling was speci¢cally dependent upon platelet adhesion to immobilised ¢brinogen or echistatin. Lysates containing equal amount of proteins, from echistatin-, ¢brinogen- and BSA-adherent platelets, were assayed for the tyrosine phosphorylation of pp72syk and pp125FAK , two kinases activated at
di¡erent levels downstream integrin^ligand engagement. pp72syk resulted strongly phosphorylated in echistatin-attached platelets as well as in platelets adhering to ¢brinogen (Fig. 2B). pp72syk phosphorylation was observed, even though at a low extent, also in platelets adhering to BSA. This result is not surprising, as previous studies demonstrated that resting platelets exhibit a basal level of protein tyrosine phosphorylation, including pp72syk [29]. Immobilised echistatin-platelet interaction induced pp125FAK phosphorylation and the level of phosphorylation was comparable to that measured in ¢brinogen-adherent platelets (Fig. 2C). Unlike pp72syk , phosphorylated pp125FAK was completely absent in platelets adhering to BSA. Thus, we can rule out that a partial platelet activation, due to the presence of Ca2 ions in the adhesion bu¡er, might be responsible for the activation of this kinase in echistatin- or ¢brinogen-attached platelets. The lack of any protein tyrosine phosphorylation in suspended platelets exposed to soluble echistatin (Fig. 2, lane 4) con¢rmed that pp72syk and pp125FAK activating signals arise only as a consequence of the binding of platelets with the solid-phase form of the disintegrin. Preliminary experiments by light mi-
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croscopy analysis of paraformaldehyde-¢xed platelets showed that platelets adhering to immobilised ¢brinogen or echistatin, where pp125FAK resulted phosphorylated, also spread. Conversely, platelets attached to the substrates in the presence of apyrase, which prevented pp125FAK phosphorylation, did not spread (data not shown). These ¢ndings are in agreement with previously reported data [30^31]. The involvement of speci¢c transduction pathways in the echistatin-induced pp125FAK phosphorylation was investigated by using inhibitors of platelet signalling. Platelets were pre-treated with PGE1 , apyrase, indomethacin, CD, or BIS before plating. pp125FAK phosphorylation was completely abolished by all, but one, of used inhibitors (Fig. 3). In particular, PGE1 , apyrase, CD and BIS were shown to be e¡ective, thus indicating that signals activating and/ or depending on dense granule secretion, cytoskeleton assembly and PKC function are involved in echistatin-induced pp125FAK phosphorylation. The presence of phosphorylated pp125FAK in control platelets left for 1 h at room temperature (Fig. 3C, lane 3) con¢rms that the lack of pp125FAK phosphorylation in BIS-pre-treated platelets was due to the inhibition of PKC and not to an aspeci¢c impairment of platelet functions caused by the long platelet-inhibitor pre-incubation time. Indomethacin, which is known to inhibit the cyclooxygenase pathway, did not cause any reduction of echistatin-induced pp125FAK phosphorylation (Fig. 3A, lane 4). The e¡ects of platelet inhibitors on pp125FAK activation triggered by platelet adhesion to ¢brinogen (Fig. 3B) are in agreement with data previously reported by Haimovich et al. [28]. 4. Discussion Echistatin is a potent inhibitor of platelet aggregation stimulated by various agonists [23,32]. It binds with high a¤nity to the puri¢ed platelet receptor KIIb L3 : the cross-linking of the disintegrin occurs within the L3 217^302 sequence, a domain which has been implicated in many receptor functions, such as heterodimer association, activation-dependent conformational changes and ¢brinogen binding [33,34]. We have recently demonstrated that soluble echistatin inhibits pp72syk and pp125FAK phosphory-
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lation in ADP-stimulated ¢brinogen-adherent platelets [26]. In order to gain more insights into the mechanism of action of disintegrins on platelet functions, in this study we investigated the capability of immobilised echistatin to support platelet adhesion and signalling activation. Gel-¢ltered human platelets attach to echistatincoated plates, as a function of both the disintegrin coating concentration and the number of plated platelets (Fig. 1). While the binding a¤nity of soluble echistatin to resting platelets was found to be much lower than its binding a¤nity to ADP-activated platelets [27], the number of platelets adhering to immobilised echistatin in the absence of ADP is almost comparable to that measured in the presence of the agonist. Such a di¡erence in the receptor binding a¤nity of di¡erent forms (soluble or solid-phase) of the same molecule has been already observed, including for the platelet natural ligand ¢brinogen [21,34]. New binding properties can be acquired by these molecules as a consequence of immobilisation-induced conformational changes [17]. Echistatin coating on a plastic surface may allow its RGD loop and the C-terminal secondary binding sequence to become more accessible for KIIb L3 expressed on unstimulated platelets. This might be particularly true for the echistatin positively charged. C-terminal domain, which has been shown to be responsible for echistatin-induced ligand induced binding sites (LIBS) expression on puri¢ed KIIb L3 [35,36]. The interaction between echistatin and platelet receptor KIIb L3 and the subsequent activation of the receptor do not require transduction signals induced by agonists. In fact, the level of platelet adhesion to immobilised echistatin is not a¡ected by the presence of inhibitors of platelet signalling and aggregation (Table 1). Monomeric disintegrins and other RGD mimetics, which are able to modulate integrin a¤nity, fail to activate signals mediated by these receptors [15,16]. Only the disul¢de-linked dimeric disintegrin contortrostatin was shown to activate the early phase of KIIb L3 outside-in signalling [20]. This ¢nding and analogue results obtained with antibody-induced integrin crosslink [37] led to the conclusion that receptor oligomerisation is a condition necessary to initiate bio-
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chemical signals. Here, we show that platelet interaction with immobilised echistatin promotes the phosphorylation of pp72syk , a kinase involved in the early phase of clustered-integrin signalling (Fig. 2). This indicates that echistatin, when occurs in a solid-phase form, exerts the function of a multivalent ligand, allowing adjacent integrin aggregation, and thus exhibiting properties similar to those of contortrostatin. However, unlike this dimeric disintegrin, echistatin in adherent platelets activates also the tyrosine phosphorylation of the focal adhesion kinase, pp125FAK (Fig. 2), which is regulated in a pattern distinct from that of pp72syk [38]. Previously, it has been found that clustering of KIIb L3 mediated by antibodies, which recognise the activate form of the receptor, failed to trigger pp125FAK phosphorylation [39]. Thus, the presence of phosphorylated pp125FAK in platelets adhering to immobilised echistatin suggests that the disintegrin binds and clusters the resting form of KIIb L3 . Furthermore, inhibition experiments show that immobilised echistatin activates signals resembling those induced by solid-phase ¢brinogen (Fig. 3), which is known to cluster the non-active form of KIIb L3 [39]. In platelets adhering to ¢brinogen, the activation of pp125FAK requires both signals induced by the occupied integrin and signals activated by ADP secreted from platelet dense granules [31]. We found that, in platelets attached to immobilised echistatin, the phosphorylation of pp125FAK is completely abolished by the addition of apyrase, an ADP scavenger, and PGE1 , an inhibitor of secretion (Fig. 3). This indicates that immobilised echistatin, like immobilised ¢brinogen, also stimulates signals responsible for endogenous agonist secretion. Further similarities between immobilised echistatin- and ¢brinogen-activated transduction pathways are observed by using other inhibitors of intraplatelet signals (Fig. 3). For both substrates, the cytoskeleton integrity and PKC functionality are conditions necessary for pp125FAK phosphorylation, whereas the e¤ciency of cycloxygenase pathway is not required. Adhesion experiments performed in the presence of anti-KIIb L3 , without or with anti-Kv or anti-K5 subunits, show that KIIb L3 is the main receptor involved in immobilised echistatin-platelet interaction. Even
though echistatin has been shown to bind Kv L3 and K5 L1 receptors with higher a¤nity than KIIb L3 [40], KIIb L3 is a thousand-fold more abundant than Kv L3 and K5 L1 on platelet surfaces. Furthermore, the observed similarities between signal transduction pathways stimulated by solid-phase echistatin and those activated by KIIb L3 -immobilised ¢brinogen strongly suggest that KIIb L3 is the integrin responsible for signals arising in echistatin-adherent platelets. However, Kv L3 and K5 L1 which can be activated by ligandmimetic binding [16,41], may play a role in echistatin-induced pp125FAK phosphorylation, since the activation of this kinase is a general property of L3 and L1 subunit signalling [42]. Furthermore, Kv L3 and K5 L1 may contribute to echistatin-induced signalling by activating KIIb L3 , according to recent ¢ndings which demonstrate that an integrin can modulate positively or negatively the function of an other integrin [43,44]. Studies are in progress to clarify whether and how Kv L3 and K5 L1 participate to signals in echistatin-adherent platelets. Previous studies demonstrating the integrin activating property of disintegrins and other RGD-ligand mimetics [14,15], raise the question whether these compounds promote rather than prevent thrombus formation. In fact, they, inducing KIIb L3 high a¤nity state, may allow ¢brinogen-dependent aggregation without the need of agonists [14]. In addition to the property to modulate integrin a¤nity, our results highlight that the RGD polypeptide echistatin, when occurring in a solid-phase form, also induces complex transduction events which may result in full platelet activation. Our model of solid-phase echistatin may mimic an event which might also occur in the blood environment with integrin antagonist drugs. In fact, these chemicals may be associated to platelets for a relatively long time as a result of their interaction with KIIb L3 [45], thus acquiring the properties of an immobilised substrate for other circulating cells and thus promoting their activation. In conclusion, our ¢ndings suggest that a better understanding and further characterisation of the potential activating properties of RGD antagonists is crucial for the future development of anti-thrombotic drugs based on integrin blockade therapeutic strategies.
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Acknowledgements The ¢nancial support of Telethon-Italy (Grant E.738) is gratefully acknowledged. We thank the Immunohaematological and Transfusional Medicine Service of the Medicine and Surgery Faculty, University of Naples Federico II, for providing blood samples.
[16]
[17]
[18]
References [1] D.R. Phillips, I.F. Charo, R.M. Scarborough, GPIIb^IIIa the responsive integrin, Cell 65 (1991) 359^362. [2] M.E. Hemler, VLA proteins in the integrin family : structures, functions, and their role on leukocytes, Annu. Rev. Immunol. 8 (1990) 365^400. [3] L. Osborn, Leukocyte adhesion to endothelium in in£ammation, Cell 62 (1990) 3^6. [4] L.C. Plantefaber, R.O. Hynes, Changes in integrin receptors on oncogenically transformed cells, Cell 56 (1989) 281^ 290. [5] E.A. Clark, J.S. Brugge, Integrins and signal transduction pathways: the road taken, Science 268 (1995) 233^239. [6] S. Dedhar, G.E. Hannigan, Integrin cytoplasmatic interactions and bidirectional transmembrane signalling, Curr. Opin. Cell. Biol. 8 (1996) 657^669. [7] M.H. Ginsberg, X. Du, T.E. O'Toole, J.C. Loftus, Platelet integrins, Thromb. Haemost. 74 (1995) 352^359. [8] J. Hawiger, Adhesive interactions of platelets and their blockade, Ann. New York Acad. Sci. 614 (1991) 270^ 278. [9] S.J. Shattil, H. Kashiwagi, N. Pampori, Integrin signalling : the platelet paradigm, Blood 91 (1998) 2645^2657. [10] C.P. Blobel, J.M. White, Structure, function and evolutionary relationship of proteins containing a disintegrin domain, Curr. Opin. Cell. Biol. 4 (1992) 760^765. [11] R.M. Scarborough, J.W. Rose, M.A. Naughton, D.R. Phillips, L. Nannizzi, A. Arfsten, A.M. Campbell, I.F. Charo, Characterization of the integrin speci¢cities of disintegrins isolated from American pit viper venoms, J. Biol. Chem. 268 (1993) 1058^1065. [12] Z.M. Ruggeri, New insights into the mechanisms of platelet adhesion and aggregation, Semin. Hematol. 31 (1994) 229^ 239. [13] R.M. Scarborough, N.S. Kleiman, D.R. Phillips, Platelet glycoprotein IIb/IIIa antagonists. What are the relevant issues concerning their pharmacology and clinical use?, Circulation 100 (1999) 437^444. [14] X.P. Du, E.F. Plow, A.L. Frelinger, T.E. O'Toole, J.C. Loftus, M.H. Ginsberg, Ligands `activate' integrin KIIb L3 (platelet GPIIb^IIIa), Cell 65 (1991) 409^416. [15] K. Peter, M. Schwarz, J. Ylanne, B. Kohler, M. Moser, T. Nordt, P. Salbach, W. Kubler, C. Bode, Induction of ¢brin-
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[29] S.J. Shattil, M.H. Ginsberg, J.S. Brugge, Adhesive signalling in platelets, Curr. Opin. Cell. Biol. 6 (1994) 695^704. [30] B. Haimovich, L. Lipfert, J.S. Brugge, S.J. Shattil, Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands, J. Biol. Chem. 268 (1993) 15868^ 15877. [31] S.J. Shattil, B. Haimovich, M. Cunningham, L. Lipfert, J.T. Parsons, M.H. Ginsberg, J.S. Brugge, Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signalling through integrin and agonist receptors, J. Biol. Chem. 269 (1994) 14738^14745. [32] B. Savage, U.M. Marzec, B.H. Chao, L.A. Harker, J.M. Maraganore, Z.M. Ruggeri, Binding of the snake venomderived proteins applagin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb/IIIa complex inhibits receptor function, J. Biol. Chem. 265 (1990) 11766^11772. [33] J.J. Calvete, M.A. McLane, G.J. Stewart, S. Niewiarowski, Characterization of the cross-linking site of disintegrins albolabrin, bitistatin, echistatin and eristostatin on isolated human platelet integrin GPIIb/IIIa, Biochem. Biophys. Res. Commun. 202 (1994) 135^140. [34] B. Savage, Z.M. Ruggeri, Selective recognition of adhesive sites in surface-bound ¢brinogen by glycoprotein IIb-IIIa on nonactivated platelets, J. Biol. Chem. 266 (1991) 11227^ 11233. [35] P.S. Wright, V. Saudek, T.J. Owen, S.L. Harbeson, A.J. Bitonti, An echistatin C-terminal peptide activates GPIIbIIIa binding to ¢brinogen, ¢bronectin, vitronectin and collagen type I and type IV, Biochem. J. 293 (1993) 263^267. [36] C. Marcinkiewicz, S. Vijay-Kumar, M.A. McLane, S. Niewiarowski, Signi¢cance of RGD loop and C-terminal domain of echistatin for recognition of KIIb L3 and KV L3 integrins and expression of ligand-induced binding site, Blood 90 (1997) 1565^1575.
[37] M.M. Huang, L. Lipfert, M. Cunningham, J.S. Brugge, M.H. Ginsberg, S.J. Shattil, Adhesive ligand binding to integrin KIIb L3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK , J. Cell Biol. 122 (1993) 473^483. [38] J. Gao, K.E. Zoller, M.H. Ginsberg, J.S. Brugge, S.J. Shattil, Regulation of the pp72syk protein tyrosine kinase by platelet integrin KIIb L3 , EMBO J. 21 (1997) 6414^6425. [39] A.J. Pelletier, T. Kunicki, Z.M. Ruggeri, V. Quaranta, The activation state of the integrin KIIb L3 a¡ects outside-in signals leading to cell spreading and focal adhesion kinase phosphorylation, J. Biol. Chem. 270 (1995) 18133^18140. [40] M. Pfa¡, M.A. McLane, L. Beviglia, S. Niewiarowski, R. Timpl, Comparison of disintegrins with limited variation in the RGD loop in their binding to puri¢ed integrins KIIb L3 , KV L3 and K5 L1 and in cell adhesion inhibition, Cell Adhes. Commun. 2 (1994) 491^501. [41] A.J. Pelletier, T. Kunicki, V. Quaranta, Activation of the integrin Kv L3 involves a discrete cation-binding site that regulates conformation, J. Biol. Chem. 271 (1996) 1364^1370. [42] S.K. Akiyama, S.S. Yamada, K.M. Yamada, S.E. LaFlamme, Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras, J. Biol. Chem. 269 (1994) 15961^15964. [43] P. Huhtala, M.J. Humphries, J.B. McCarthy, P.M. Tremble, Z. Werb, C.H. Damsky, Cooperative signalling by K5 L1 and K4 L1 integrins regulates metalloproteinase gene expression in ¢broblasts adhering to ¢bronectin, J. Cell. Biol. 129 (1995) 867^879. [44] F. Diaz-Gonzalez, J. Forsyth, B. Steiner, M.H. Ginsberg, Trans-dominant inhibition of integrin function, Mol. Biol. Cell. 7 (1996) 1939^1951. [45] J.J. Cook, T.F. Huang, B. Rucinski, M. Strzyzewski, R.F. Tuma, J.A. Williams, S. Niewiarowski, Inhibition of platelet hemostatic plug formation by trigramin, a novel RGD-peptide, Am. J. Physiol. 256 (1989) H1038^H1043.
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Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation Maria Antonietta Belisario a; *, Simona Tafuri b , Carmela Di Domenico a , Rossella Della Morte a , Caterina Squillacioti a , Antonia Lucisano b , Norma Staiano a
a
Dipartimento di Biochimica e Biotecnologie Mediche, Universita© di Napoli Federico II, Via S. Pansini, n. 5, 80131 Naples, Italy b Dipartimento di Patologia e Sanita© Animale, Universita© di Napoli Federico II, Naples, Italy Received 24 February 2000; received in revised form 1 May 2000; accepted 11 May 2000
Abstract Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet KIIb L3 binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of KIIb L3 conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADPactivated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72syk and pp125FAK . Inhibitors of platelet signalling, such as apyrase, prostaglandin E1 , cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125FAK phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125FAK phosphorylation. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Adhesion; Platelet; Protein phosphorylation; Echistatin; pp125FAK ; pp72syk
1. Introduction Integrins are involved in cell adhesion and migration which occur during physiological processes such as haemostasis, immune response, development and in£ammation [1^4]. They are important signalling
* Corresponding author. Fax: +39-081-746-3150; E-mail: [email protected]
molecules which mediate bi-directional transfer of information from the extracellular matrix to the cytoplasm and vice versa [5,6]. KIIb L3 , the major integrin expressed on platelet membrane, plays a critical role in haemostasis by mediating platelet^platelet and platelet^matrix protein interactions [1,7]. Physiological platelet agonists activate transduction pathways (inside-out signalling) leading to the conversion of KIIb L3 from a `low' to a `high' a¤nity state, which allows the integrin to be-
0167-4889 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 0 0 ) 0 0 0 6 1 - 6
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come competent to bind adhesive proteins, such as ¢brinogen and von Willebrand factor [1,8]. Following ligand engagement, KIIb L3 triggers transduction events (outside-in signalling) which, co-ordinated with other signals activated by other platelet receptors, lead to platelet aggregation [9]. Disintegrins are low molecular weight polypeptides isolated from snake venoms, which are potent inhibitors of platelet functions [10,11]. The presence in their molecules of the arginine, glycine, aspartic acid (RGD) tripeptide sequence is responsible for their antiplatelet activity, since the interaction of KIIb L3 with its natural ligand ¢brinogen is mediated, at least in part, through this recognition sequence [12]. The inhibition of KIIb L3 functions by natural and synthetic RGD-containing molecules has recently evolved as a new antiplatelet strategy [13]. In addition to bleeding problems, a major concern related to the use of competitive inhibitors of integrin functions is based on their capability to induce KIIb L3 conformational changes, which in turn result in an increased ¢brinogen-binding function of the receptor [14^16]. However, the switch of integrin conformation toward an activated state which can be induced by an RGD-peptide is not su¤cient to induce integrin-dependent outside-in signalling. Integrin clustering, provided only by multivalent ligands, has been shown to be a pre-requisite for receptor-mediated transmembrane e¡ects [17^19]. Contortrostatin, because of its dimeric structure, is the only disintegrin which was found to activate integrin-dependent protein phosphorylation in platelets [20]. It has been suggested that proteins may exhibit multivalent functions as a consequence of their immobilisation on a substrate [17]. As a result of this process, a ligand may reach a high local density and undergo conformational changes. Both these phenomena may provide either a modulation of the ligand binding properties and/or a direct mechanism for inducing adjacent integrin clustering [21,22]. In the present study, we show that the immobilisation on a plastic surface of echistatin, a monomeric disintegrin from Echis carinatus venom [23], causes an increase of its binding a¤nity for integrin(s) expressed on resting platelets. Echistatin, when in a solid-phase form, is also able to activate the early phase of occupied integrin outside-in signalling, in-
cluding pp72syk phosphorylation. Furthermore, we demonstrate that the interaction of immobilised echistatin with platelet surfaces stimulates not only signals dependent from integrin aggregation, but also those transduction events required for dense granule secretion and full platelet activation. 2. Materials and methods 2.1. Antibodies and chemicals Rabbit polyclonal (LR), mouse monoclonal (4D10) human pp72syk antibodies and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, USA); rabbit polyclonal anti-pp125FAK serum (BC3) and anti-human pp125FAK polyclonal IgG from Upstate Biotechnology (Lake Placid, NY, USA); phosphotyrosine monoclonal antibody (mAb) PT66, horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG from Sigma (St. Louis, MO, USA); anti-KIIb L3 complex (CBL130), anti-Kv (CBL490) and anti-K5 (CBL497) antibodies from Cymbus Bioscience (Southampton, UK); human plasma ¢brinogen (purity s 98%) and bisindolylmaleimide (BIS) from Calbiochem (San Diego, CA, USA); Sepharose 2B from Pharmacia Biotech (Uppsala, Sweden); apyrase, prostaglandin E1 (PGE1 ), ADP, orthovanadate, leupeptin, aprotinin, phenylmethylsulfonyl £uoride (PMSF), bovine serum albumin (BSA), cytochalasin D (CD) and indomethacin from Sigma. All the other reagents were of the highest quality available. 2.2. Preparation of platelets Citrated (0.32% w/v) blood specimens were obtained from healthy volunteers who had not taken any drugs for 9 days before bleeding. Platelet-rich plasma (PRP), obtained by blood centrifugation at 200Ug for 15 min, was further centrifuged at 1200Ug for 10 min in the presence of 1 U/ml apyrase and 1 WM PGE1 . Platelets, resuspended in HEPES bu¡er, pH 7.35, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 , 3 mM NaH2 PO4 , 5 mM glucose, 3.5 mM HEPES and 3.5 g/l BSA were gel-¢ltered on Sepharose 2B [24]. The concentration of washed platelets was assessed spectrophotometrically at
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520 nm. The platelet suspension was then diluted at the concentration required for aggregation and adhesion experiments. Platelet suspensions (2.5U108 platelets/ml), supplemented with 0.5 mg/ml ¢brinogen and 5 mM Ca2 , were tested for their ability to aggregate in response to 50 WM ADP. Aggregation was monitored at 37³C on a single channel aggregometer (Chrono-Log Aggregometer model 500-CA, Haverton, PA, USA) with continuous stirring at 900 rpm. Platelets stimulated with ADP in the absence of exogenous ¢brinogen, or platelets activated with ristocetin did not aggregate, thus indicating that the gel-¢ltration procedure yielded platelets completely separated from plasma proteins. 2.3. Platelet adhesion assay Gel-¢ltered unstimulated platelets were allowed to adhere to 6 cm diameter plates (Becton-Dickinson, NY, USA) or to 96-well polystyrene £at-bottom plates (Costar, MA, USA) pre-coated for 16 h at 4³C with substrates (10 Wg/ml). Echistatin was dissolved in 50 mM carbonate/bicarbonate bu¡er pH 9.2, whereas ¢brinogen was solubilised in phosphate bu¡er solution (PBS). Unbound sites on the plastic were blocked with 2 ml/plate or 200 Wl/well of 5 mg/ml heat-denatured BSA for 1 h at 37³C. Wells coated with BSA were used to check aspeci¢c platelet adhesion. After three washing with PBS, plates were ¢lled with 50 Wl of a platelet suspension (3U108 /ml) supplemented with 1 mM CaCl2 and left for 1 h at room temperature. Non-adherent platelets were removed and the plates were washed twice with PBS. Quantitative evaluation of the number of adherent platelets was performed in microtitre wells by measuring platelet endogenous phosphatase [25]. The plates were read at 410/630 nm on an automated plate reader (Bio-Rad model 450, Hercules, CA, USA). Platelet pre-treatments with inhibitors of platelet activation were performed as it follows: (1) 10 min with 10 U/ml apyrase or 12 nmol/ml CD or 10 nmol/ml indomethacin; (2) 1 min with 1 nmol/ml PGE1 ; 1 h with 12 nmol/ml BIS. The anti-aggregatory activity of these inhibitors was tested before their use. Adhesion experiments were also performed by adding to the platelet suspension a
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mixture of protease inhibitors (20 Wg/ml leupeptin, 100 U/ml aprotinin, and 100 WM PMSF) or hirudin (20 U/ml). In an other set of experiments, platelets were pre-incubated for 5 min with 10 Wg/ml antiKIIb L3 alone or 5 min with anti-KIIb L3 plus further 5 min with anti-Kv or anti-K5 antibodies. 2.4. Protein immunoprecipitation and Western blotting Adherent platelets were lysed with 600 Wl of ice cold RIPA bu¡er, containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 10 mM Tris^HCl, pH 7.2, 1 mM EGTA, 1 mM PMSF, 20 Wg/ml leupeptin, 100 U/ml aprotinin, and 1 mM sodium orthovanadate. Platelets in suspension were lysed by adding an equal volume of 2URIPA bu¡er. After 30 min on ice, lysates were clari¢ed by centrifugation at 14 000 rpm for 10 min at +4³C. The resulting supernatants were characterised for protein concentration by Bio-Rad DC protein assay. Equal amounts of proteins were immunoprecipitated overnight at 4³C with anti-pp125FAK (BC3) (1 Wg/100 Wg proteins) or anti-pp72syk (LR) (3 Wl of a 1:5 antibody dilution/100 Wg proteins) previously bound to protein A/G-Agarose. Beads were sedimented by brief centrifugation and washed extensively with ice-cold RIPA bu¡er. Proteins, solubilised in boiling SDS sample bu¡er (2% SDS, 5% L-mercaptoethanol, 66 mM Tris, pH 7.5, 10 mM EDTA), were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Western blotting analyses were performed as previously described [26]. To evaluate protein tyrosine phosphorylation, blots were probed with mAb PT66 (1:10 000). To monitor the loading of gel lanes, the membranes were stripped according to the Manufacturer's procedure (Amersham, Buckinghamshire, UK) and then reprobed with the anti-human pp125FAK (1:500) or anti-pp72syk (4D10) (1:200) antibodies. Immunoreactive bands were visualised by enhanced chemiluminescence kit (Amersham). Quantitisation of proteins was performed by densitometry using a Discover Pharmacia scanner equipped with a sun spark classic densitometric workstation.
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Fig. 1. Platelet adhesion to immobilised echistatin. Unstimulated (a) and ADP-activated (b) platelets (1.5U107 ) were poured on wells coated with increasing amounts of echistatin (A). Di¡erent numbers of unstimulated platelets (solid line) or platelets previously treated with 0.1 WM of soluble echistatin for 3 min (dotted line) were poured on wells coated with 0.5 Wg/well echistatin (E) or ¢brinogen (F) (B). The plates were incubated at room temperature for 1 h. Non-adherent platelets were removed and the number of adherent platelets was measured as described in Section 2. The number of platelets adhering to BSA-coated wells has been subtracted from the number of platelets adhering to echistatin- or ¢brinogen-coated wells. The results reported here are the mean values from three di¡erent experiments in which each point was tested 10-fold. Standard deviations never exceeded 10% of the mean value.
3. Results 3.1. Platelet adhesion to immobilised echistatin Gel-¢ltered human platelets showed the capability to adhere to immobilised ¢brinogen and echistatin (Fig. 1), whereas they attached to BSA-coated wells only at a very low extent. The number of adherent platelets was shown to be a function of both the amount of coating disintegrin (Fig. 1A) and the concentration of the poured platelet suspension (Fig. 1B). The percentage of platelets attached to ¢brinogen- or echistatin-coated wells was about 20 and 17%, respectively, when 0.5 Wg of protein was immobilised on plates. A similar percentage of human platelet adhesion to immobilised ¢brinogen has been previously reported [25]. When the adhesion experiments were performed with washed platelets
resuspended in EDTA-supplemented HEPES bu¡er, platelets did not attach to immobilised echistatin, thus indicating that platelet binding to immobilised echistatin requires the presence of Ca2 (data not shown). Platelet exposure to 0.1 WM echistatin for 3 min before plating completely inhibited their adhesion to echistatin- and ¢brinogen-coated wells (Fig. 1B, dotted lines). This indicates that the same integrin sequences are recognised by both soluble and immobilised echistatin. Soluble echistatin has been previously reported to bind with higher a¤nity to stimulated platelets than to resting-ones [27]. Immobilised echistatin was found to support at comparable extent the adhesion of either unstimulated or ADP-activated platelets (Fig. 1A). To rule out that a partial platelet activation, which may occur during platelet collection and isolation, was responsible for the di¡erent a¤nity
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Fig. 2. Protein tyrosine phosphorylation in platelets adhering to immobilised echistatin or ¢brinogen. Lysates containing equal amounts of proteins from platelets adhering to immobilised ¢brinogen (lane 1), immobilised echistatin (lane 2) or BSA (lane 3) or lysed platelets taken in suspension for 3 min in the presence of 0.1 WM soluble echistatin (lane 4) were either directly run on SDS^ PAGE (A) or immuno-precipitated with pp72syk (LR) antibody (B) or pp125FAK (BC3) antibody (C) and subsequently separated by electrophoresis. After Western blotting, tyrosine phosphorylated proteins were visualised by mAb PT66. The amount of pp72syk or pp125FAK loaded was equal in all lanes of each gel as it was veri¢ed on parallel immunoblots probed with the anti-pp72syk antibody (4D10) (B, lower blot) or with the polyclonal anti-human pp125FAK antibody (C, lower blot). The position of molecular markers (in kDa) and of pp72syk or pp125FAK (arrows) is indicated. The same results were obtained from three separate experiments of equal design.
properties of the two forms of echistatin, adhesion experiments were performed in the presence of the aggregation inhibitors PGE1 and apyrase. None of these inhibitors was e¡ective in reducing the number of platelets adhering to immobilised echistatin and ¢brinogen (Table 1). The possibility that platelet activation might occur because of plasmin or thrombin traces present in the platelet preparation, was also ruled out, since the extent of platelet adhesion to immobilised echistatin was unchanged by protease inhibitors (PMSF, leupeptin, aprotinin) or by hirudin (Table 1). Previous ¢ndings showed that the blockade of speci¢c platelet transduction pathways does not a¡ect platelet binding to solid-phase ¢brinogen [21,28]. Likewise, platelets pre-treated with CD, an inhibitor of actin polymerisation, or BIS, a protein kinase C (PKC) inhibitor, or indomethacin, an inhibitor of arachidonate metabolism, adhered to echistatincoated wells as well as control platelets (Table 1). These results indicate that platelet adhesion to immobilised echistatin does not require platelet activation by agonists and does not involve platelet signalling. Immobilised echistatin-platelet interaction is mainly mediated by KIIb L3 integrin. In fact, platelet adhesion to immobilised echistatin was reduced by
Table 1 E¡ect of inhibitors on platelet adhesion to immobilised echistatin or ¢brinogen Inhibitor
Number of adherent-platelets (U106 /well) Echistatin
Fibrinogen
None PGE1 Apyrase Indomethacin CD BIS Aprotinin, leupeptin, PMSF Hirudin
2.71 þ 0.26 2.65 þ 0.25 2.52 þ 0.24 2.60 þ 0.26 2.73 þ 0.24 2.80 þ 0.23 2.55 þ 0.24 2.61 þ 0.25
3.02 þ 0.28 3.10 þ 0.25 3.05 þ 0.27 3.10 þ 0.26 2.91 þ 0.28 2.95 þ 0.29 3.03 þ 0.27 2.90 þ 0.28
Gel-¢ltered platelets were treated with di¡erent platelet inhibitors at concentrations and for the incubation time reported in Section 2. Platelets were allowed to adhere to echistatin- or ¢brinogen-coated wells (0.5 Wg/well). The plates were incubated at room temperature for 1 h. Non-adherent platelets were removed and the number of adherent platelets was assessed as described in Section 2. The number of platelets adhering to BSAcoated wells (0.31U106 þ 0.02) has been subtracted from the number of platelets adhering to immobilised echistatin or ¢brinogen. Data are the mean values þ S.D. calculated from three separate experiments in which each point was tested 10-fold.
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Fig. 3. E¡ect of inhibitors on pp125FAK phosphorylation induced by platelet adhesion to immobilised echistatin or ¢brinogen. pp125FAK phosphorylation was measured in platelets allowed to adhere to plates coated with echistatin (A) or ¢brinogen (B) in the absence (lane 1) and in the presence of apyrase (lane 2), PGE1 (lane 3), indomethacin (lane 4) and cytochalasin D (lane 5). C shows the results obtained from control platelets left for 1 h at room temperature (lanes 1 and 3) and BIS-pre-treated platelets (lanes 2 and 4) adhering to echistatin (lanes 1 and 2) or ¢brinogen (lanes 3 and 4). Platelet lysates containing equal amounts of proteins were subjected to immunoprecipitation with anti-pp125FAK (BC3) antibody and subsequently separated by electrophoresis. Tyrosine phosphorylated proteins were visualised by mAb PT66 (upper blots). The blots were stripped and reprobed with polyclonal anti-human pp125FAK antibody (lower blots). The position of molecular markers (in kDa) and pp125FAK (arrow) is indicated. Similar results were obtained from four experiments of identical design.
83% in the presence of anti-KIIb L3 antibody. However, the integrin receptors Kv L3 and K5 L1 could also be involved in such interaction, since a statistically signi¢cant higher inhibition of platelet adhesion to immobilised echistatin was observed when platelets were simultaneously treated with anti-KIIb L3 plus anti-Kv or anti-K5 antibodies (data not shown). 3.2. Platelet adhesion to immobilised echistatin promotes protein tyrosine phosphorylation Several tyrosine phosphorylated proteins resulted as a consequence of platelet binding to the natural ligand ¢brinogen or to immobilised echistatin (Fig. 2A). The pattern of phosphorylated proteins in echistatin-attached platelets resembled that of ¢brinogenadherent platelets. Platelets held in suspension or platelets attached to BSA showed very low levels of total protein phosphorylation, thus indicating that platelet signalling was speci¢cally dependent upon platelet adhesion to immobilised ¢brinogen or echistatin. Lysates containing equal amount of proteins, from echistatin-, ¢brinogen- and BSA-adherent platelets, were assayed for the tyrosine phosphorylation of pp72syk and pp125FAK , two kinases activated at
di¡erent levels downstream integrin^ligand engagement. pp72syk resulted strongly phosphorylated in echistatin-attached platelets as well as in platelets adhering to ¢brinogen (Fig. 2B). pp72syk phosphorylation was observed, even though at a low extent, also in platelets adhering to BSA. This result is not surprising, as previous studies demonstrated that resting platelets exhibit a basal level of protein tyrosine phosphorylation, including pp72syk [29]. Immobilised echistatin-platelet interaction induced pp125FAK phosphorylation and the level of phosphorylation was comparable to that measured in ¢brinogen-adherent platelets (Fig. 2C). Unlike pp72syk , phosphorylated pp125FAK was completely absent in platelets adhering to BSA. Thus, we can rule out that a partial platelet activation, due to the presence of Ca2 ions in the adhesion bu¡er, might be responsible for the activation of this kinase in echistatin- or ¢brinogen-attached platelets. The lack of any protein tyrosine phosphorylation in suspended platelets exposed to soluble echistatin (Fig. 2, lane 4) con¢rmed that pp72syk and pp125FAK activating signals arise only as a consequence of the binding of platelets with the solid-phase form of the disintegrin. Preliminary experiments by light mi-
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croscopy analysis of paraformaldehyde-¢xed platelets showed that platelets adhering to immobilised ¢brinogen or echistatin, where pp125FAK resulted phosphorylated, also spread. Conversely, platelets attached to the substrates in the presence of apyrase, which prevented pp125FAK phosphorylation, did not spread (data not shown). These ¢ndings are in agreement with previously reported data [30^31]. The involvement of speci¢c transduction pathways in the echistatin-induced pp125FAK phosphorylation was investigated by using inhibitors of platelet signalling. Platelets were pre-treated with PGE1 , apyrase, indomethacin, CD, or BIS before plating. pp125FAK phosphorylation was completely abolished by all, but one, of used inhibitors (Fig. 3). In particular, PGE1 , apyrase, CD and BIS were shown to be e¡ective, thus indicating that signals activating and/ or depending on dense granule secretion, cytoskeleton assembly and PKC function are involved in echistatin-induced pp125FAK phosphorylation. The presence of phosphorylated pp125FAK in control platelets left for 1 h at room temperature (Fig. 3C, lane 3) con¢rms that the lack of pp125FAK phosphorylation in BIS-pre-treated platelets was due to the inhibition of PKC and not to an aspeci¢c impairment of platelet functions caused by the long platelet-inhibitor pre-incubation time. Indomethacin, which is known to inhibit the cyclooxygenase pathway, did not cause any reduction of echistatin-induced pp125FAK phosphorylation (Fig. 3A, lane 4). The e¡ects of platelet inhibitors on pp125FAK activation triggered by platelet adhesion to ¢brinogen (Fig. 3B) are in agreement with data previously reported by Haimovich et al. [28]. 4. Discussion Echistatin is a potent inhibitor of platelet aggregation stimulated by various agonists [23,32]. It binds with high a¤nity to the puri¢ed platelet receptor KIIb L3 : the cross-linking of the disintegrin occurs within the L3 217^302 sequence, a domain which has been implicated in many receptor functions, such as heterodimer association, activation-dependent conformational changes and ¢brinogen binding [33,34]. We have recently demonstrated that soluble echistatin inhibits pp72syk and pp125FAK phosphory-
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lation in ADP-stimulated ¢brinogen-adherent platelets [26]. In order to gain more insights into the mechanism of action of disintegrins on platelet functions, in this study we investigated the capability of immobilised echistatin to support platelet adhesion and signalling activation. Gel-¢ltered human platelets attach to echistatincoated plates, as a function of both the disintegrin coating concentration and the number of plated platelets (Fig. 1). While the binding a¤nity of soluble echistatin to resting platelets was found to be much lower than its binding a¤nity to ADP-activated platelets [27], the number of platelets adhering to immobilised echistatin in the absence of ADP is almost comparable to that measured in the presence of the agonist. Such a di¡erence in the receptor binding a¤nity of di¡erent forms (soluble or solid-phase) of the same molecule has been already observed, including for the platelet natural ligand ¢brinogen [21,34]. New binding properties can be acquired by these molecules as a consequence of immobilisation-induced conformational changes [17]. Echistatin coating on a plastic surface may allow its RGD loop and the C-terminal secondary binding sequence to become more accessible for KIIb L3 expressed on unstimulated platelets. This might be particularly true for the echistatin positively charged. C-terminal domain, which has been shown to be responsible for echistatin-induced ligand induced binding sites (LIBS) expression on puri¢ed KIIb L3 [35,36]. The interaction between echistatin and platelet receptor KIIb L3 and the subsequent activation of the receptor do not require transduction signals induced by agonists. In fact, the level of platelet adhesion to immobilised echistatin is not a¡ected by the presence of inhibitors of platelet signalling and aggregation (Table 1). Monomeric disintegrins and other RGD mimetics, which are able to modulate integrin a¤nity, fail to activate signals mediated by these receptors [15,16]. Only the disul¢de-linked dimeric disintegrin contortrostatin was shown to activate the early phase of KIIb L3 outside-in signalling [20]. This ¢nding and analogue results obtained with antibody-induced integrin crosslink [37] led to the conclusion that receptor oligomerisation is a condition necessary to initiate bio-
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chemical signals. Here, we show that platelet interaction with immobilised echistatin promotes the phosphorylation of pp72syk , a kinase involved in the early phase of clustered-integrin signalling (Fig. 2). This indicates that echistatin, when occurs in a solid-phase form, exerts the function of a multivalent ligand, allowing adjacent integrin aggregation, and thus exhibiting properties similar to those of contortrostatin. However, unlike this dimeric disintegrin, echistatin in adherent platelets activates also the tyrosine phosphorylation of the focal adhesion kinase, pp125FAK (Fig. 2), which is regulated in a pattern distinct from that of pp72syk [38]. Previously, it has been found that clustering of KIIb L3 mediated by antibodies, which recognise the activate form of the receptor, failed to trigger pp125FAK phosphorylation [39]. Thus, the presence of phosphorylated pp125FAK in platelets adhering to immobilised echistatin suggests that the disintegrin binds and clusters the resting form of KIIb L3 . Furthermore, inhibition experiments show that immobilised echistatin activates signals resembling those induced by solid-phase ¢brinogen (Fig. 3), which is known to cluster the non-active form of KIIb L3 [39]. In platelets adhering to ¢brinogen, the activation of pp125FAK requires both signals induced by the occupied integrin and signals activated by ADP secreted from platelet dense granules [31]. We found that, in platelets attached to immobilised echistatin, the phosphorylation of pp125FAK is completely abolished by the addition of apyrase, an ADP scavenger, and PGE1 , an inhibitor of secretion (Fig. 3). This indicates that immobilised echistatin, like immobilised ¢brinogen, also stimulates signals responsible for endogenous agonist secretion. Further similarities between immobilised echistatin- and ¢brinogen-activated transduction pathways are observed by using other inhibitors of intraplatelet signals (Fig. 3). For both substrates, the cytoskeleton integrity and PKC functionality are conditions necessary for pp125FAK phosphorylation, whereas the e¤ciency of cycloxygenase pathway is not required. Adhesion experiments performed in the presence of anti-KIIb L3 , without or with anti-Kv or anti-K5 subunits, show that KIIb L3 is the main receptor involved in immobilised echistatin-platelet interaction. Even
though echistatin has been shown to bind Kv L3 and K5 L1 receptors with higher a¤nity than KIIb L3 [40], KIIb L3 is a thousand-fold more abundant than Kv L3 and K5 L1 on platelet surfaces. Furthermore, the observed similarities between signal transduction pathways stimulated by solid-phase echistatin and those activated by KIIb L3 -immobilised ¢brinogen strongly suggest that KIIb L3 is the integrin responsible for signals arising in echistatin-adherent platelets. However, Kv L3 and K5 L1 which can be activated by ligandmimetic binding [16,41], may play a role in echistatin-induced pp125FAK phosphorylation, since the activation of this kinase is a general property of L3 and L1 subunit signalling [42]. Furthermore, Kv L3 and K5 L1 may contribute to echistatin-induced signalling by activating KIIb L3 , according to recent ¢ndings which demonstrate that an integrin can modulate positively or negatively the function of an other integrin [43,44]. Studies are in progress to clarify whether and how Kv L3 and K5 L1 participate to signals in echistatin-adherent platelets. Previous studies demonstrating the integrin activating property of disintegrins and other RGD-ligand mimetics [14,15], raise the question whether these compounds promote rather than prevent thrombus formation. In fact, they, inducing KIIb L3 high a¤nity state, may allow ¢brinogen-dependent aggregation without the need of agonists [14]. In addition to the property to modulate integrin a¤nity, our results highlight that the RGD polypeptide echistatin, when occurring in a solid-phase form, also induces complex transduction events which may result in full platelet activation. Our model of solid-phase echistatin may mimic an event which might also occur in the blood environment with integrin antagonist drugs. In fact, these chemicals may be associated to platelets for a relatively long time as a result of their interaction with KIIb L3 [45], thus acquiring the properties of an immobilised substrate for other circulating cells and thus promoting their activation. In conclusion, our ¢ndings suggest that a better understanding and further characterisation of the potential activating properties of RGD antagonists is crucial for the future development of anti-thrombotic drugs based on integrin blockade therapeutic strategies.
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Acknowledgements The ¢nancial support of Telethon-Italy (Grant E.738) is gratefully acknowledged. We thank the Immunohaematological and Transfusional Medicine Service of the Medicine and Surgery Faculty, University of Naples Federico II, for providing blood samples.
[16]
[17]
[18]
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