Eur J Biochem 270, 2959–2970 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03670.x Glycoprotein Ib-mediated platelet activation A signalling pathway triggered by thrombin ´ ´ Frederic Adam1, Marie-Claude Guillin1,2 and Martine Jandrot-Perrus1 INSERM E0348, Faculte´ Xavier Bichat, Paris; 2He´matologie et Immunologie, Hoˆpital Bichat, AP-HP, Paris, France Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs) On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations We present here new evidence in favour of a thrombin receptor function for GPIb We have selected conditions in which thrombin– GPIb interactions were enhanced by thrombin immobilization Activation was studied independently of PAR cleavage by using active-site-blocked thrombin We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin aIIbb3-dependent platelet–platelet interactions The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins Inhibition of platelet–platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin Platelet activation by a-thrombin is an important event in normal haemostasis as well as in pathological arterial thrombosis [1,2] Traces of the enzyme formed at sites of vessel injury are sufficient to activate platelets, thereby contributing to thrombus formation and also to the coagulation cascade by exposure of procoagulant phospholipids on the platelet surface and release of factor V Thrombin interaction with platelets is characterized by a combination of a hormone-like binding step and a proteolytic step [3] Thrombin cleaves the two protease-activated receptors, PAR1 and PAR4, in human platelets [4], the activation of which triggers successively outside-in and inside-out signalling processes resulting in integrin aIIbb3 activation, fibrinogen binding, and platelet aggregation Glycoprotein Ib (GPIb), a major component of the platelet membrane, is involved in platelet adhesion to von Willebrand factor (vWF) on injured vascular wall [5] It is expressed as a complex consisting of its disulfide-linked subunits, GPIba and GPIbb, noncovalently associated with GPIX and GPV in a : : : stoichiometry [6] The complex associates with the cytoskeleton through the interaction of the GPIba intracellular domain with actinbinding protein [7,8] and with the signalling molecule 14-3-3f assumed to play a role in the activation pathway triggered by vWF [9] GPIb is also a high-affinity binding site for thrombin [10], but its role in thrombin-induced platelet activation has been overshadowed by the identification of PARs However, PARs activation does not take into account all the aspects of thrombin-induced platelet responses, and the role of GPIb is the object of renewed interest [11,12] GPIb takes part in platelet activation induced by low thrombin concentrations [11–13] and binds to thrombin independently of its catalytic activity [14–20] Platelets from patients with Bernard–Soulier syndrome (BSS), which are deficient in GPIb [21,22], or in which the extracellular domain of GPIba has been removed by proteolysis [23–29], show decreased sensitivity to thrombin, together with an increased lag time before platelet responses These observations suggest that thrombin binding to GPIb primes the activation of PAR1 The aim of this study was to examine whether the binding of thrombin to GPIb triggers an alternative plateletactivation pathway We hypothesized that GPIb-coupled signals evoked by thrombin may be undetectable by conventional methods because of their low intensity relative to the PARs-coupled signals We have therefore developed a model based on the immobilization of catalytically inactivated thrombin to study events enhanced by agonist immobilization and independent of PARs activation by blockade of thrombin proteolytic activity We demonstrate that platelets interact with immobilized, active-site-blocked Correspondence to M Jandrot-Perrus, INSERM E0348, ´ Faculte Xavier Bichat, BP 416, 75870 Paris cedex 18, France Fax: + 33 44 85 62 17, Tel.: + 33 44 85 62 16, E-mail: mjandrot@bichat.inserm.fr Abbreviations: PAR, protease-activated receptor; GPIb, glycoprotein Ib; vWF, von Willebrand factor; BSS, Bernard–Soulier syndrome; PI3-kinase, phosphoinositide 3-kinase; PGE1, prostaglandin E1; FITC, fluorescein isothiocyanate; MbCD, methyl-b-cyclodextrin; PPACK, D-Phe-Pro-Arg chloromethyl ketone dihydrochloride; PKC, protein kinase C (Received April 2003, revised May 2003, accepted 14 May 2003) Keywords: glycoprotein Ib; platelets; signalling; thrombin Ó FEBS 2003 2960 F Adam et al (Eur J Biochem 270) thrombin This interaction is GPIb-dependent, requires the integrity of cholesterol-enriched membrane microdomains, and triggers activation processes, including shape modifications, dense-granule release, activation of protein kinases and phosphoinositide 3-kinases (PI3-kinases), and aIIbb3dependent platelet–platelet interactions Materials and methods Materials BSA, N-ethylmaleimide, staurosporin, fura 2-AM, prostaglandin E1 (PGE1), grade VII apyrase, MRS 2179, 2-MeSAMP, fluorescein isothiocyanate (FITC)-phalloidin, methyl-b-cyclodextrin (MbCD), cholesterol-water soluble, p-nitrophenyl phosphate, dimethylsulfoxide (Sigma Chemical Co., St Louis, MO, USA), human PAR1 agonist peptide SFLLRN (Neosystem, Strasbourg, France), H-DPhe-Arg-pNa (S2238) (Biogenic S.A., Maurin, France), D-Phe-Pro-Arg chloromethyl ketone dihydrochloride (PPACK), H-Arg-Gly-Asp-Ser-OH (RDGS), wortmannin, bisindolylmaleimide I (GF109203X), LY294002 (Calbiochem Novabiochem Corp., La Jolla, CA, USA), aspirin (AspegicÒ; Synthelabo, Meudon la Foret, France), ˆ poly(vinylidene difluoride) membrane (Millipore, Bedford, MA, USA), heparin (Sanofi, Gentilly, France), sodium [51Cr]chromate (CIS Bio International, Gif-sur-Yvette, France), ECL, 5-hydroxy[14C]tryptamine (Amersham, Les Ulis, France), ReoProÒ (Centocor B.V., Leiden, the Netherlands), anti-phosphotyrosine mAb PY20 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and antiGPIb mAb SZ2 (Immunotech, Marseille, France) were purchased as indicated The anti-GPIb mAb Bx-1 directed against the macroglycopeptide part of the molecule was kindly provided by P Nurden (UMR5533 CNRS, Pessac, France), and purified glycocalicin was a gift from K J Clemetson (Theodor Kocher Institute, Berne, Switzerland) Human a-thrombin was purified as previously reported [30] Thrombin active site was blocked using the irreversible inhibitor PPACK The absence of free PPACK in the solution was controlled PPACK-thrombin (2 lM) was devoid of measurable amidolytic and coagulant activity It did not induce any platelet responses (aggregation, secretion, shape change) at concentrations up to 150 nM and for incubation times up to 15 Inactive Ser205Ala thrombin mutant was a gift from Dr B Le Bonniec (INSERM U428, Paris, France) Purified vWF [31] was a gift from ´ Dr N Ajzenberg (Hematologie et Immunologie, Hopital ˆ Bichat, AP-HP, Paris, France) The two single-stranded DNA oligonucleotides, HD1 and HD22, with the sequences 5¢-GGTTGGTGTGG TTGG-3¢ and 5¢-AGTCCGTGGTAGGGCAGGTTGG GGTGACT-3¢, were synthesized by Life Technologies (Cergy Pontoise, France) HD1 binds to exosite and inhibits fibrinogen cleavage [32], and HD22 binds with high affinity to the heparin-binding site of thrombin [33] was obtained by centrifugation at 120 g for 15 at room temperature The patient with BSS has previously been described, and the molecular defect consists of a GPIX mutation, N45S, [35] identical with the defect described by Clemetson et al [36] in another patient BSS platelet-rich plasma was obtained after sedimentation of the blood at room temperature The remaining red blood cells were eliminated from the patient’s platelet-rich plasma by a 75 s centrifugation at 1000 g In some cases, platelet-rich plasma was incubated for 30 at 37 °C in the presence of 20 lCiỈmL)1 51Cr or 0.6 lM 5-hydroxy[14C]tryptamine Platelets were washed twice in washing buffer (36 mM acid citric, mM glucose, mM KCl, mM MgCl2, 103 mM NaCl, mM CaCl2, pH 6.5) containing 3.5 mgỈmL)1 BSA, 75 mmL)1 apyrase and 100 nM PGE1, and recovered by centrifugation at 1200 g for 12 at room temperature Washed platelets were resuspended in Hepes-buffered modified Tyrode solution (137 mM NaCl, mM KCl, 0.3 mM NaH2PO4, mM MgCl2, 5.5 mM glucose, mM Hepes, 12 mM NaHCO3, pH 7.3), containing 3.5 mgỈmL)1 BSA Platelet number was adjusted to · 108 plateletsỈmL)1 for the controls, and to 1.5 · 108 plateletsỈmL)1 for the patient because of their large size Protein immobilization Microtitre plates (Immulon II; Dynex Technologies, Worthing, UK) were coated overnight at °C with thrombin (25 lg in 100 lL NaCl/Tris, 20 mM Tris, 150 mM NaCl, pH 8) in the absence or presence of PPACK (20 lM) or with vWF (5 lg in NaCl/Pi, pH 7.5) Plates were saturated with mgỈmL)1 BSA and washed with NaCl/Tris The amount of thrombin immobilized, quantified using 125I-thrombin, was 0.45 ± 0.05 lg per well and was not modified by the presence of PPACK (0.55 ± 0.19 lg per well) Thrombin immobilized in the absence of PPACK was still active, with a rate of S-2238 hydrolysis of  0.9 lmolỈmin)1, whereas thrombin immobilized in the presence of PPACK was fully inactivated, as indicated by the absence of measurable S-2238 hydrolysis In addition, PPACK-inactivated thrombin (up to 500 nM) failed to induce aggregation when added to a platelet suspension in the aggregometer Platelet binding to immobilized proteins Platelet binding was measured in static conditions on the microtitre plates coated with thrombin, PPACK-thrombin, or vWF using 51Cr-labelled platelets (3 · 108 per mL) After incubation at room temperature, wells were washed with the reaction buffer Attached platelets were quantified by c-counting of the wells (CobraTM II auto-gammaÒ; Packard BioScience Company) Alternatively, platelet adhesion was quantified by measuring phosphatase activity with p-nitrophenyl phosphate [37] Similar results were obtained with both methods whatever the experimental conditions used Dense-granule release Preparation of platelets Blood from consenting healthy human donors was collected on 15% (v/v) acid/citrate/dextrose Platelets were isolated essentially as previously described [34] Platelet-rich plasma Platelets (3 · 108 per mL) were incubated for different times on immobilized active thrombin or PPACK-thrombin After addition of 0.2 vol 0.1 M ice-cold EDTA and centrifugation at 1600 g for 10 at °C, 5-hydroxy[14C]tryptamine was Ó FEBS 2003 GPIb-dependent thrombin-induced platelet activation (Eur J Biochem 270) 2961 quantified in the supernatants by scintillation counting Secretion was expressed as the percentage of the total content of 5-hydroxy[14C]tryptamine in nonactivated platelets Cholesterol depletion and repletion Cholesterol was depleted by incubating platelets (3 · 108 per mL) for 10 at 37 °C in washing buffer containing 3.5 mgỈmL)1 BSA, 75 mU apyrase, 100 nM PGE1 and 6–10 mM MbCD Cholesterol-depleted platelets were centrifuged at 1200 g for 12 at room temperature and resuspended in Hepes-buffered modified Tyrode solution containing mgỈmL)1 BSA For the cholesterol repletion, cholesterol-depleted platelets were centrifuged at 1200 g for 12 at room temperature and resuspended for 30 at 37 °C in washing buffer, without BSA, containing a mixture of cholesterol and MbCD in a ratio of mM/10 mM Cholesterol-repleted platelets were centrifuged and resuspended in Hepes-buffered modified Tyrode solution [38] Protein tyrosine phosphorylation Platelets bound to immobilized active thrombin or PPACKthrombin were lysed with 6% (w/v) SDS in buffer [20 mM Tris/HCl, pH 6.8, 30 mM NaCl, mM EDTA, mM N-ethylmaleimide, 10% (v/v) 2-mercaptoethanol, 1.2 M urea] containing 20 mM vanadate (Na2VO4) and mM phenylarsineoxide Platelet lysates were heated for at 100 °C Samples were adjusted for their platelet content before analysis Proteins were separated by SDS/PAGE and transferred to poly(vinyl difluoride) membranes Membranes were blocked with 1.5% (w/v) BSA in NaCl/Tris, incubated with the anti-phosphotyrosine mAb PY20 (1 lgỈmL)1), and visualized using an anti-mouse peroxidase-coupled antibody and chemiluminescence Fluorescence and confocal microscopy Platelets were incubated for 30 at room temperature on coverslips coated with BSA (10 mgỈmL)1), active thrombin, PPACK-thrombin or vWF After being washed, platelets were fixed with 2% paraformaldehyde for 30 at 37 °C, permeabilized with 0.1% Triton X-100 and incubated with FITC-labeled phalloidin (2 lM) to detect actin filaments After 30 of incubation and washing, images were collected using a Leica DM IRB fluorescence microscope ´ and treated with the Archimed Pro software (Micromecanique, Evry, France) Confocal images were acquired using a Zeiss LSM-510 inverted confocal microscope with a PlanApochromat 63x/1.4 oil DIC objective (Zeiss) FITCphalloidin was excited by the 488 nm line of an argon laser, and fluorescence measured at >505 nm Zeiss confocal software Windows NT controlled the scanner module and performed image analysis Statistical analysis Statistical analysis was performed using Student’s two-tailed unpaired t test (PRISM, GraphPad Software Inc.) All data are presented as means ± SEM Results Platelet binding to immobilized proteins As expected, immobilized active thrombin induced tight platelet binding (11.5 ± 0.3% and 22.9 ± 0.5% after 30 and 60 incubation, respectively), and binding to immobilized vWF reached 6.3 ± 0.2% after 30 incubation, whereas binding to immobilized BSA did not exceed 0.6 ± 0.1% whatever the incubation time Stable attachment to immobilized active-site-blocked PPACK-thrombin was significantly reduced compared with active thrombin (P < 0.001) but well above nonspecific binding to immobilized BSA, with 6.8 ± 0.4% and 14.9 ± 0.6% bound platelets after 30 and 60 incubation, respectively To confirm that residual catalytic activity of PPACK-thrombin, undetectable by conventional methods, was not responsible for cell attachment, we substituted the inactive thrombin mutant S205A for PPACK-thrombin and obtained similar results (7.9 ± 0.9% and 14.3 ± 0.5% after 30 and 60 incubation, respectively) Thrombin presents two important functional, positively charged domains remote from the catalytic site, exosites and Platelet binding to immobilized PPACKthrombin was: (a) dose-dependently inhibited by heparin (IC50 0.4 mL)1; plateau at 90% inhibition for concentrations >1 mL)1) and also decreased by  90% in the presence of 500 nM DNA aptamer HD22 which binds to exosite [33]; (b) inhibited by  30% in the presence of hirudin (0.1 mL)1) or DNA aptamer HD1 (500 nM) which binds to exosite [32] These results indicate that positively charged thrombin domains participate in platelet binding to immobilized PPACK-thrombin in agreement with previous reports [39,40] Morphological changes of platelets bound to immobilized PPACK-thrombin Platelets stably attached to immobilized active or activesite-blocked thrombin or to vWF were labelled using FITC-phalloidin and observed by fluorescence and confocal microscopy Large aggregates were observed almost covering the surface of active thrombin, the limits of individual platelets being difficult to observe (Fig 1A) Aggregates  22 lm thick were observed by confocal microscopy by measuring the transversal section of aggregates, indicating that several layers of platelets were stacked Aggregates were also observed at the surface of PPACK-thrombin, but their size was significantly smaller than on active thrombin and often limited to a few platelets In addition, their thickness did not exceed lm, which is compatible with the superposition of two platelets The platelets had undergone a shape change: their size increased, with signs of spreading (lamellipodia) and activation (filopodia) Platelet junctions and stress fibres were observed (Fig 1B) For comparison, platelets bound to immobilized vWF as a single layer Spreading was very intense, characterized by a Ôchinese hat-likeÕ shape: very thin at the periphery ( lm) and thicker ( lm) at the centre 2962 F Adam et al (Eur J Biochem 270) Ó FEBS 2003 Fig Analysis of platelet binding to immobilized thrombin by fluorescence and confocal microscopy Platelets (3 · 108 per mL) pretreated with buffer (indicated by /), SZ2 (10 lgỈmL)1) or ReoPro (3 lgỈmL)1) were incubated for 30 on BSA, immobilized active thrombin, PPACK-thrombin or vWF Bound platelets were fixed, incubated with FITC-labelled phalloidin as described in Materials and methods, and analyzed by fluorescence microscopy (A) or confocal microscopy (B) Ó FEBS 2003 GPIb-dependent thrombin-induced platelet activation (Eur J Biochem 270) 2963 Stable platelet binding to immobilized inactive thrombin is GPIb-dependent We examined the role of GPIb in tight platelet attachment to immobilized PPACK-thrombin by using SZ2, an antiGPIba mAb, the epitope of which partly covers the thrombin-binding site [29] SZ2 dose-dependently decreased platelet binding to immobilized PPACK-thrombin, the inhibition being almost complete at a SZ2 concentration of 10 lgỈmL)1 (Fig 2A) In contrast, the binding of platelets to immobilized active thrombin was not decreased by SZ2 Another anti-GPIb mAb, Bx-1, directed against the macroglycopeptide domain, did not modify stable platelet binding to immobilized inactivated or active thrombin, indicating that the inhibitory effect of SZ2 was specific (not shown) The role of GPIb in platelet attachment to immobilized PPACK-thrombin was further investigated using platelets from a BSS patient Binding of BSS platelet to immobilized PPACK-thrombin was reduced by more than 90% compared with the binding of control platelets (Fig 2B), whereas binding of BSS platelets to active thrombin was preserved In addition, lM purified glycocalicin, the extracellular domain of GPIba, completely inhibited the tight binding of platelets to immobilized PPACK-thrombin (data not shown) Together, these results indicate that GPIb plays a crucial role in the tight attachment of platelets to immobilized inactivated thrombin In agreement with the results of platelet quantification, microscopic examination showed that platelet coverage and aggregation at the surface of immobilized active thrombin or vWF were not notably reduced in the presence of SZ2 SZ2 had a restraining effect on platelet adhesion to immobilized vWF because of its partial effect on vWFinduced platelet activation [29] In contrast, SZ2 inhibited the formation of platelet aggregates at the surface of PPACK-thrombin: the number, size and thickness of aggregates were markedly decreased, and mostly isolated platelets were observed (Fig 1A) Platelet binding to immobilized PPACK-thrombin involves integrin aIIbb3-dependent platelet–platelet contacts Microscopic analysis indicated that binding of platelets to PPACK-thrombin resulted in platelet activation We thus examined the contribution of aIIbb3 integrin to platelet– platelet interactions EDTA, RGDS and ReoPro inhibited the stable attachment of platelets to both immobilized active thrombin and PPACK-thrombin (Table 1) In addition, platelets from a patient with Glanzmann thrombasthenia were found to bind poorly (4.5-fold less than control platelets) to immobilized PPACK-thrombin (data not shown) The size of the aggregates was reduced by ReoPro (Fig 1A) or RGDS, and the thickness limited to a single layer of platelets However, platelets bound to PPACK-thrombin in the presence of ReoPro were spread and showed signs of activation such as filopodia (Fig 1B) This suggests that GPIb interaction with immobilized PPACK-thrombin triggers pathways resulting in aIIbb3 activation, which is in turn involved in platelet–platelet interactions that stabilize platelet attach- Fig Role of GPIb in platelet binding to immobilized active or PPACK-thrombin (A) Platelets (3 · 108 per mL) were preincubated for 15 with the anti-GPIb mAb Bx-1 (5 lgỈmL)1) (open bar) or SZ2 (10 lgỈmL)1) (filled bar) After 30 of incubation on BSA, immobilized active thrombin or PPACK-thrombin, bound platelets were quantified and expressed as the percentage of platelets deposited in the wells Results are means ± SEM from three different experiments performed in triplicate (B) Control platelets (open bar) or BSS platelets (filled bar) were incubated for 30 on immobilized active thrombin or PPACK-thrombin, and the number of platelets bound was measured Results represent means ± SEM from one experiment performed in triplicate ***Significantly different from control platelets (P < 0.001) ment The blockade of integrin aIIbb3 also largely reduced the size and thickness (6 lm) of platelet aggregates formed at the surface of immobilized active thrombin Platelet adhesion to immobilized vWF was also decreased by ReoPro, but in contrast with the observation made on PPACK-thrombin, platelets were round with only a few filopodia (Fig 1B), indicating that vWF interaction with aIIbb3 and/or avb3 plays a major role in spreading Ó FEBS 2003 2964 F Adam et al (Eur J Biochem 270) Table Binding of platelets to immobilized active and PPACKthrombin is inhibited by EDTA, RGDS peptide and ReoPro Control platelets (3 · 108 per mL) were preincubated for at 37 °C in the presence of mM EDTA, 300 lM RGDS peptide or lgỈmL)1 ReoPro After 30 of incubation on immobilized active thrombin or PPACK-thrombin, the percentage of platelets bound was determined as above Results are presented as means ± SEM from four different experiments performed in triplicate Bound platelets (%) Active thrombin Control EDTA RGDS ReoPro PPACK-thrombin 11.1 2.1 0.9 0.6 7.1 1.1 0.7 0.6 ± ± ± ± 0.4 0.5 0.1 0.1 ± ± ± ± 0.2 0.3 0.2 0.1 Signals activated on platelet interaction with immobilized, inactivated thrombin The capacity of immobilized thrombin to induce platelet secretion was analyzed by measuring 5-hydroxy[14C]tryptamine release 5-Hydroxy[14C]tryptamine was very slowly secreted upon platelet incubation with immobilized PPACK-thrombin at a level reaching 6.4% after 60 min, whereas it was rapidly and efficiently released on immobilized active thrombin (Fig 3) Leakage of 5-hydroxy[14C] tryptamine, measured by incubating platelets on immobilized BSA, was limited to 0.3% Dense-granule secretion induced by immobilized PPACK-thrombin was fully inhibited in the presence of 10 lgỈmL)1 SZ2 We investigated whether secreted secondary agonists, ADP and thromboxane A2, participated in platelet stable binding induced by immobilized PPACK-thrombin The ADP scavenger, apyrase, dose-dependently inhibited the binding; maximum inhibition was obtained with 0.5 mL)1 apyrase which decreased the binding by 60% Fig Dense-granule release induced by immobilized active or PPACKthrombin Platelets (3 · 108 per mL) were incubated for 15, 30 or 60 with immobilized BSA, active or PPACK-thrombin Activation was stopped by the addition of mM EDTA and centrifugation Supernatants were assayed for 5-hydroxy[14C]tryptamine by scintillation counting Dense-granule secretion was expressed as the percentage of the total content of 5-hydroxy[14C]tryptamine present in a lysate of nonactivated platelets Each point represents the mean ± SEM from four different experiments performed in triplicate Fig Role of ADP and ADP receptors on platelet binding to immobilized PPACK-thrombin (A) Platelets (3 · 108 per mL) were preincubated for at 37 °C with increasing concentrations of apyrase After 30 of incubation with immobilized PPACKthrombin, the percentage of bound platelets was determined Results are presented as the percentage of inhibition by apyrase of platelet binding to PPACK-thrombin, and each point represents the mean ± SEM from three experiments performed in triplicate (B) Platelets (3 · 108 per mL) were preincubated for 10 at 37 °C with lM MRS2179 (white bar) or 500 lM 2-MeSAMP (black bar) After 30 of incubation with immobilized PPACK-thrombin, the percentage of bound platelets was determined Results are presented as the percentage of inhibition by ADP receptor inhibitors of platelet binding to PPACK-thrombin and are expressed as means ± SEM from four different experiments performed in triplicate (Fig 4A) These data support a role for ADP secretion in aIIbb3 integrin-dependent platelet–platelet interactions induced via GPIb interaction with immobilized PPACKthrombin ADP-induced activation involves two major receptors [41]: P2Y1 coupled to Gq [42] and P2Y12 coupled to Gi [43] Selective antagonists of P2Y1 (MRS 2179) and P2Y12 (2-MeSAMP) reduced platelet binding by 40% and 60%, respectively (Fig 4B) Inhibition of platelet cyclooxygenase and thromboxane A2 production by aspirin had a lesser inhibitory effect than apyrase, reducing platelet binding to immobilized PPACK-thrombin by 40% Agents that increase intracellular cAMP concentration activate a cAMP-dependent kinase involved in the inhibition of platelet activation and in the phosphorylation of the Ó FEBS 2003 GPIb-dependent thrombin-induced platelet activation (Eur J Biochem 270) 2965 Table Effect of different signalling inhibitors on platelet binding to immobilized active thrombin or PPACK-thrombin Platelets (3 · 108 per mL), preincubated for 15 at 37 °C with solvent (dimethylsulfoxide) or inhibitors at the indicated concentrations, were added to wells coated with immobilized active thrombin or PPACK-thrombin After 30 min, the percentage of platelets bound was determined Results are presented as means ± SEM of inhibition (%) determined in four different experiments performed in triplicate Inhibition of platelet binding (%) Active thrombin PP1 (10 lM) GF109203X (10 lM) Staurosporin (1 lM) LY294002 (50 lM) PPACK-thrombin 0.0 54.2 94.7 26.5 62.3 89.7 46.9 67.7 ± ± ± ± 7.3 3.7*** 1.5*** 7.9** ± ± ± ± 3.5*** 3.2*** 2.3*** 9.1*** ** (0.001 < P < 0.01); *** (P < 0.001) vs values obtained in the absence of inhibitor cytoplasmic domain of GPIbb [44–47] Binding experiments were performed in the presence of 100 nM PGE1 to increase cAMP concentration PGE1 inhibited by 60% (P < 0.001) platelet binding induced by PPACK-thrombin In contrast, PGE1 had only a slight inhibitory effect on platelet binding induced by active thrombin, in agreement with its low inhibitory effect on PARs-triggered platelet aggregation (data not shown) To gain further insight into the GPIb-dependent signalling pathway, we conducted studies in the presence of inhibitors acting on different signalling enzymes or the same concentration of solvent (dimethylsulfoxide) (Table 2) The tight platelet binding induced by immobilized PPACK6 thrombin was inhibited by  60% in the presence of PP1, a specific inhibitor of Src-family kinases Binding was greatly reduced by the protein kinase C (PKC) inhibitor GF109203X and also by staurosporin but to a lesser extent PI3-kinase inhibitors had a very potent inhibitory effect: 50 lM LY294002 reduced binding by 68%, and wortmannin dose-dependently decreased binding, full inhibition being achieved at concentrations above 25 nM (Fig 5) These results contrast with those obtained when platelet binding was induced by immobilized active thrombin PP1 had no effect, wortmannin and LY294002 only slightly reduced the binding The greatest inhibition was produced by the PKC inhibitors, but here staurosporin was more potent than GF109203X Together, these results indicate that the signalling pathway triggered by GPIb interaction with active-site-blocked thrombin differs from the signalling cascade coupled to PAR activation by active thrombin Platelet protein tyrosine phosphorylation is induced by immobilized PPACK-thrombin To determine whether the interaction of immobilized PPACK-thrombin with GPIb is coupled to the activation of protein-tyrosine kinases, we analyzed tyrosyl-phosphorylated proteins in platelets tightly bound to the inactive thrombin surface and compared the results with those obtained on the active thrombin surface Samples were normalized for the number of platelets before analysis (Fig 6) Fig Effect of inhibition of PI3-kinases on platelet binding to immobilized active or PPACK-thrombin Platelets (3 · 108 per mL) were preincubated for 15 at 37 °C with increasing amounts of wortmannin (from a 1-mM solution in dimethylsulfoxide) After 30 of incubation with immobilized active thrombin (s) or PPACK-thrombin (d), bound platelets were quantified Results are expressed as means ± SEM from three different experiments performed in triplicate Several proteins were tyrosyl-phosphorylated in platelets bound to PPACK-thrombin The main phosphorylated proteins migrated with an apparent molecular mass of 130/115 and 51/50 kDa doublets, 105, 78 and 65 kDa The global pattern of protein tyrosine phosphorylation induced by immobilized active thrombin resembled the pattern observed with PPACK-thrombin (Fig 6A,B) PP1 reduced tyrosine phosphorylation induced by PPACKthrombin but it had no effect on that induced by active thrombin (Fig 6C) In contrast, staurosporin completely inhibited phosphorylation in platelets bound to active thrombin as in those bound to PPACK-thrombin, whereas GF109203X had no effect on phosphorylation in platelets bound to active thrombin but inhibited the tyrosine phosphorylation induced by immobilized PPACKthrombin Wortmannin completely inhibited phosphorylation in platelets bound to PPACK-thrombin but had no effect on that occurring in platelets bound to active thrombin (Fig 6A,B) Experiments were also performed in the presence of ReoPro to determine whether protein tyrosine phosphorylation occurred upstream or downstream of platelet–platelet interactions ReoPro did not modify the pattern of proteins phosphorylated in platelets bound to active thrombin (Fig 6D) Minimal modifications of phosphorylation in platelets bound to PPACKthrombin were observed, with only a slight decrease in the intensity of bands at 105 and 130 kDa, a larger decrease of bands at 50, 90 and 180 kDa, and the appearance of one band at 40 kDa These results indicate that most of the protein tyrosine phosphorylations take place downstream of PI3-kinases, Src family kinases and PKC activation, but upstream of aIIbb3 engagement after platelet activation by immobilized inactive thrombin, in agreement with the morphological changes shown in Fig Ó FEBS 2003 2966 F Adam et al (Eur J Biochem 270) Fig Platelet protein tyrosine phosphorylation induced by immobilized active or PPACK-thrombin and inhibition by different signalling inhibitors or aIIbb3 integrin inhibitor Platelets (3 · 108 per mL) were preincubated for 15 at 37 °C with buffer (control), 100 nM wortmannin, lM staurosporin, 10 lM GF109203X, 10 lM PP1 or at room temperature with lgỈmL)1 ReoPro After 30 of incubation on active thrombin (B and left hand side of C and D) or PPACK-thrombin (A and right hand side of C and D), bound platelets were lysed as described in Materials and methods ÔBasalÕ corresponds to platelets incubated on immobilized BSA in the absence of inhibitors Samples were normalized for their platelet content before analysis After protein separation by SDS/PAGE (7.5% gel), tyrosyl-phosphorylated proteins were identified by immunoblotting with PY20 and chemiluminescence Effect of cholesterol depletion on platelet binding to immobilized proteins Lipid rafts are implicated in the recruitment of specialized proteins for intracellular signal transduction We thus analyzed their role in the tight platelet binding induced by immobilized active thrombin, PPACK-thrombin, and vWF MbCD (10 mM) reduced platelet binding to PPACK-thrombin by 83.7 ± 2.6%, binding to active thrombin by 29.8 ± 2.2%, and binding to vWF by 63.7 ± 0.7% A lower MbCD concentration (6 mM) was used for controlled cholesterol depletion and repletion experiments as described [38] These conditions emphasized the differences observed between the different immobilized ligands Platelet binding was inhibited by 68.5 ± 3.5% on PPACK-thrombin, by 6.5 ± 2.5% on active thrombin, and by 23.5 ± 1.5% on vWF Cholesterol repletion restored full platelet binding to active thrombin and vWF and 82.5 ± 2.5% of binding to PPACK-thrombin Together, these results indicate that integrity of the lipid rafts is required for platelet activation by immobilized PPACKthrombin but not by active thrombin Lipid rafts appear to play some role in platelet adhesion to immobilized vWF, but their integrity is not an absolute requirement Discussion Two studies using different experimental models have recently provided evidence for a role for GPIb as a true thrombin receptor Both models used active-site-blocked thrombin as a key reagent to differentiate between GPIbmediated and PARs-mediated responses, as this thrombin form is able to bind GPIb but is unable to activate PARs Ramakrishnan et al [11] have shown that proteolytically inactive thrombin can induce in vitro and in vivo activation of mouse platelets via the GPIb–IX complex if GPV has been removed by proteolysis or knock-out At the same time, Soslau et al [12] observed that GPIb-dependent activation of human platelets by proteolytically inactive thrombin required the presence of polymerizing fibrin We describe here a third model of GPIb-dependent activation of human platelets by active-site-blocked thrombin, which does not require GPV removal or the presence of polymerizing fibrin but requires immobilization of thrombin on a solid support Our hypothesis was that immobilization of the GPIb-ligand thrombin would favour receptor crosslinking and consequently would amplify the signals A comparable mechanism is probably involved both in the model used by Soslau et al [12], in which cross-linking of Ó FEBS 2003 GPIb-dependent thrombin-induced platelet activation (Eur J Biochem 270) 2967 GPIb may be facilitated by thrombin bound to the surface of polymerizing fibrin, and in GPV –/– mouse platelets, in which dimerization of GPIb may be favoured by GPV removal [11] As shown in these three independent studies ([11,12] and the present study), the GPIb-bound thrombin does not need to be catalytically active to elicit platelet responses We show here that platelet interaction with immobilized active-site-blocked thrombin results in shape change, dense-granule secretion, and integrin aIIbb3-dependent platelet–platelet interactions These responses are linked to the activation of signalling enzymes and require the integrity of lipid rafts Antagonists of aIIbb3 did not prevent attachment, spreading and activation of a few platelets but significantly reduced the size of the platelet aggregates, indicating that aIIbb3 activation occurs downstream of the GPIb interaction with immobilized PPACKthrombin and is involved in platelet–platelet interactions, in agreement with the observations of Ramakrishnan et al [11] using GPV null mouse platelets In contrast, platelet activation and spreading on immobilized vWF was almost completely inhibited by ReoPro, as expected from the well-established participation of aIIbb3 in irreversible platelet adhesion on immobilized vWF [48] Together, these observations indicate that events coupled to GPIb interaction with immobilized PPACK-thrombin and vWF are different We thus propose a two-step model for the tight binding of platelets triggered by immobilized inactive thrombin: the first step corresponds to the highaffinity interaction of GPIb with immobilized PPACKthrombin and results in weak reversible binding which, in the second step, is stabilized by aIIbb3-dependent platelet– platelet interactions Fibrinogen necessary for this interaction may be provided by granule secretion, as indicated by the observation that granule secretion takes place slowly but significantly in our model Granule secretion also occurred during GPIb-dependent activation by thrombin in the presence of polymerizing fibrin [12] In that study, the authors observed that an additional unidentified receptor distinct from aIIbb3 was involved because platelet aggregation was not inhibited by RGDS In our study, such an additional receptor is not required because of the use of inactivated thrombin, which is unable to form fibrin The proposed model implies the interaction of platelet GPIb with an immobilized ligand We provide some evidence that the signalling pathway and consequences of adhesion to active-site-blocked thrombin are different from those of platelets adherent to immobilized vWF The important part played in platelet spreading by the RGD sequence in vWF may be the reason for these differences We cannot exclude the possibility that other ligands of GPIb could trigger the same signalling pathway as immobilized PPACK-thrombin Indeed, Navdaev & Clemetson [49] recently reported that immobilized echicetin induced platelet spreading, exocytosis of a-granule markers, and activation of aIIbb3 However, neither ADP antagonists nor PP1 inhibited platelet activation by immobilized echicetin, in contrast with their substantial inhibitory effect on platelet activation by immobilized PPACK-thrombin Further studies are required to determine more precisely the specific signalling pathways triggered by the different GPIb ligands, but the important contribution of this study is the demonstration that GPIb acts as a thrombin receptor As the granule content is secreted in a GPIb-dependent manner, we hypothesized that secondary platelet agonists may be involved in aIIbb3-dependent platelet–platelet interactions We show here that, in human as in mouse platelets, ADP plays a central role in platelet responses triggered by GPIb interaction with active-site-blocked thrombin The inhibitory effect of the P2Y1 and P2Y12 antagonists indicates that secreted ADP activates both receptors with a preference for P2Y12, as observed in the activation of GPV-depleted mouse platelets by inactivated thrombin [11] Thus, in our model, ADP secretion takes place downstream of GPIb binding to thrombin and upstream of aIIbb3 activation GPIb plays an important role in platelet activation by low thrombin concentrations and therefore at the start of the haemostasis process when trace amounts of thrombin are produced It is thus tempting to speculate that pharmacological agents able to regulate thrombin binding to GPIb would have antithrombotic effects Despite the fact that P2Y12 antagonists not directly interfere with thrombin binding to GPIb, they appear to largely block the consequences of this interaction (this study and [11]) It is therefore likely that inhibition in vivo of the GPIb-dependent potentiation of thrombininduced platelet activation by thienopyridines contributes to their antithrombotic effect cAMP appears to be an important modulator of platelet activation by immobilized PPACK-thrombin for PGE1inhibited stable platelet binding Increased cAMP concentrations may counteract ADP activation of the Gi-coupled P2Y12 receptor Alternatively, we cannot exclude the possibility that phosphorylation of the GPIbb intracellular domain by a cAMP-dependent protein kinase A [44–47] may block its thrombin receptor function The characteristics of the responses coupled to GPIb cross-linking by immobilized active-site-blocked thrombin in human platelets (ADP secretion, aIIbb3-dependent platelet–platelet interactions, downregulation by cAMP) are thus very similar to the characteristics of the GPIb-coupled responses induced by inactive thrombin in GPV-depleted mouse platelets [11], the major difference being that, in our model, GPV does not need to be removed to unmask the GPIb thrombin receptor function Platelet responses elicited by immobilized active thrombin and PPACK-thrombin show major differences Platelet recruitment by active thrombin was greater, consistent with the contribution of PAR activation In addition, the signals initiated by thrombin binding to GPIb were different from those linked to PAR activation, as observed by the use of inhibitors acting on signalling enzymes, combined with the study of phosphorylated proteins First, PI3-kinase activation appears to be of major importance for the GPIb-coupled responses but not for PAR activation The major involvement of PI3kinases in GPIb-mediated platelet–platelet interactions may be related to the importance of ADP receptor activation in the stabilization of these interactions [50] Secondly, the GPIb-dependent signal involves the Src family kinases, in contrast with the PAR-coupled signal Lastly, the signals generated by thrombin via the GPIb pathway and via PAR imply PKC activation, but the Ó FEBS 2003 2968 F Adam et al (Eur J Biochem 270) observation that staurosporin and GF109203X had different effects on these pathways is in favour of the involvement of different PKC isoforms Concerning the chronology of the GPIb-coupled signalling events, PI3-kinases, Src family kinases and PKC activation appear to precede tyrosine-kinase activation induced by the GPIb-dependent pathway because inhibitors of these signalling proteins reduced protein tyrosylphosphorylation However, activation of protein tyrosine kinases in PPACK-thrombin-activated platelets precedes aIIbb3 engagement, as ReoPro had only a limited effect on the phosphorylation pattern, in agreement with the morphological changes observed in these conditions The assumption that protein tyrosyl-phosphorylations occur at a late stage of platelet activation is supported by the observation that immobilized active and inactivated thrombin produce similar patterns of phosphorylated proteins whereas the mechanisms of activation involved in each case are different The dramatic effect of raft disruption by MbCD on the ability of platelets to adhere to immobilized PPACK-thrombin suggests that GPIb and/or the proteins involved in the coupled signalling pathway could be localized in or near the lipid rafts Shrimpton et al [51] recently reported that a fraction of the GPIb–IX–V complex resides within the lipid rafts Indeed, PI3-kinase is very important in the GPIb-coupled pathway and its activity is highly dependent on the rafts [38] Furthermore, the Src-related protein tyrosine kinases that we showed to be important in the GPIb pathway are also tightly associated with lipid rafts [52] In contrast, the lack of effect of MbCD on platelet activation induced by immobilized active thrombin suggests that activation of PARs and their G-protein-coupled signalling pathway occurs outside the rafts An interesting point is that lipid raft disruption had only a moderate effect on platelet adhesion to immobilized vWF This suggests that events coupled to GPIb differ depending on whether it is ligated by PPACK-thrombin or vWF It is well established that only a small proportion of the GPIb expressed at the platelet surface is able to bind thrombin, whereas all GPIb copies are competent to bind vWF Our observation suggests that the small number of GPIb molecules able to bind thrombin may be associated with lipid rafts This hypothesis, currently under investigation, is supported by the previous observation that enrichment in cholesterol increased the number of high-affinity thrombin-binding sites on platelets, whereas cholesterol depletion had the opposite effect [53] Acknowledgements We acknowledge Mrs Laurence Venisse for her 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258, 11840–11845  ... to thrombin and upstream of aIIbb3 activation GPIb plays an important role in platelet activation by low thrombin concentrations and therefore at the start of the haemostasis process when trace... PPACKthrombin and is involved in platelet? ? ?platelet interactions, in agreement with the observations of Ramakrishnan et al [11] using GPV null mouse platelets In contrast, platelet activation and... prevent attachment, spreading and activation of a few platelets but significantly reduced the size of the platelet aggregates, indicating that aIIbb3 activation occurs downstream of the GPIb interaction