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Related Collections Signal Transduction Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 November 2000, Vol. 96, No. 10, pp. 3439-3446 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY FcRIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation Marie-Pierre Gratacap, Jean-Pascal Hérault, Cécile Viala, Ashraf Ragab, Pierre Savi, Jean-Marc Herbert, Hugues Chap, Monique Plantavid, and Bernard Payrastre From Institut Fédératif de Recherche en Immunologie Cellulaire et Moléculaire, Université Paul Sabatier and Centre Hospitalo - Universitaire de Toulouse, Institut National de la Santé et de la Recherche médicale, Unité 326, Hôpital Purpan, 31059 Toulouse Cedex, France; and Sanofi-Synthelabo, Route d'Espagne, 31036 Toulouse Cedex, France.     Abstract Top Abstract Introduction Materials and methods Results Discussion References FcRIIA, the only Fc receptor present in platelets, is involved in heparin-associated thrombocytopenia (HIT). Recently, adenosine diphosphate (ADP) has been shown to play a major role in platelet activation and aggregation induced by FcRIIA cross-linking or by sera from HIT patients. Herein, we investigated the mechanism of action of ADP as a cofactor in FcRIIA-dependent platelet activation, which is classically known to involve tyrosine kinases. We first got pharmacologic evidence that the ADP receptor coupled to Gi was required for HIT sera or FcRIIA clustering-induced platelet secretion and aggregation. Interestingly, the signaling from this ADP receptor could be replaced by triggering another Gi-coupled receptor, the 2A-adrenergic receptor. ADP scavengers did not significantly affect the tyrosine phosphorylation cascade initiated by FcRIIA cross-linking. Conversely, the Gi-dependent signaling pathway, initiated either by ADP or epinephrine, was required for FcRIIA-mediated phospholipase C activation and calcium mobilization. Indeed, concomitant signaling from Gi and FcRIIA itself was necessary for an efficient synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger playing a critical role in the process of phospholipase C2 activation. Altogether, our data demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet secretion and aggregation induced by FcRIIA. (Blood. 2000;96:3439-3446) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References There is now growing evidence that FcRIIA, the subclass of Fc receptors predominantly expressed in platelets,1 is able to transduce an activating signal that contributes to the rapid destruction of these cells during immune thrombocytopenia. This is well documented in the case of important venous and arterial thrombotic complications occurring in heparin-induced thrombocytopenia (HIT) and in some autoimmune diseases.2-5 Moreover, a number of platelet-activating monoclonal antibodies directed against several major platelet membrane glycoproteins, such as CD9, CD36, GPIb, or the integrin IIb3, require an intact immunoglobulin (Ig) G Fc domain for platelet activation, implying that binding to FcRIIA, of the same or an adjacent platelet, is crucial in the activation process.6 The specific clustering of FcRIIA is also sufficient per se to trigger platelet activation.7 Addition of the nonactivating specific anti-FcRIIA monoclonal antibody IV-3, followed by addition of F(ab')2 fragments of a secondary antimouse antibody to immune cells or platelets, is now a classical model widely used to study the signaling pathways evoked by this receptor.8 The cytosolic tail of FcRIIA bears 3 tyrosine residues. Two of them belong to a specific sequence closely related to the immunoreceptor-tyrosine based activation motif (ITAM) found in the cytoplasmic domains of several Ig gene family receptors, including B-cell receptor complex.7,9,10 Upon clustering, FcRIIA becomes rapidly phosphorylated on tyrosines, potentially through a protein tyrosine kinase of the Src family.9 A role for Lyn in this phosphorylation has, for instance, been suggested in neutrophils.11 Subsequently, tyrosine phosphorylated FcRIIA plays a role as a docking protein and specifically recruits SH2 domain-containing signaling proteins, including the tyrosine kinase Syk.7,9,12 The 2 SH2 domains of Syk interact with the 2 phosphotyrosines of the ITAM-like motif of FcRIIA, leading, at least in vitro, to the stimulation of its tyrosine kinase activity.12 In platelets, this event is required for tyrosine phosphorylation and activation of phospholipase C2 (PLC2, a key enzyme in the early activation process initiated by FcRIIA.13,14 Recent data suggest that the early steps of platelet activation by FcRIIA clustering may be modulated by an important cofactor secreted by platelets. Indeed, an interesting observation by Hérault et al,15 confirmed by Polgàr et al,16 indicates that secreted adenosine diphosphate (ADP) is required for platelet secretion and aggregation induced by HIT sera and FcRIIA cross-linking. This major observation suggests that ADP receptor antagonists may be effective as therapeutic agents for prevention or treatment of HIT. At least 3 distinct ADP receptors17 are present on the platelet surface. The role of the ionotropic P2X1 purinergic receptor is not yet clearly understood in these cells. Conversely, both the P2Y1 purinergic receptor coupled to Gq and a not yet identified P2 receptor, negatively coupled to adenylyl cyclase, are required for ADP-induced aggregation.18 Evidence is now accumulating that the P2Y1 purinergic receptor coupled to Gq mediates Ca++ mobilization and shape change, whereas the other P2 receptor activates Gi proteins.18 The Gi-coupled ADP receptor seems to be involved in potentiating the effects of other platelet agonists,19 including the thrombin receptor-activating peptide (TRAP).20,21 Here, we investigated the molecular mechanisms by which ADP plays its crucial role as coactivator following FcRIIA cross-linking, a model classically known to involve tyrosine kinases.7,9 Our data indicate that both Gi-dependent and tyrosine kinase-dependent signaling pathways were required for platelet secretion and aggregation induced by FcRIIA clustering or HIT sera. A key target of these converging signaling pathways appeared to be phosphatidylinositol 3,4,5-trisphosphate (PtdIns[3,4,5]P3), a lipid second messenger playing a critical role in the early steps of PLC2 activation and the subsequent calcium mobilization and phosphatidic acid (PtdOH) production.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Reagents The anti-FcRII monoclonal antibody (moAb IV.3), the rabbit polyclonal antibody against the linker for activation of T cells (LAT), and the monoclonal antiphosphotyrosine 4G10 antibody were purchased from Upstate Biotechnology Inc. The specific F(ab')2 fragments were from Jackson Immunoresearch Laboratories, and the rabbit polyclonal anti-PLC2 antibody was from Santa Cruz Biotechnology Inc. [32P]orthophosphate, 5-hydroxy[14C]tryptamine (56.0 mCi/mmol), and enhanced chemiluminescence (ECL) Western blotting reagents were from Amersham International. AR-C69931MX was a generous gift from Dr J. Turner (ASTRA Charnwood, UK). Thin-layer chromatography (TLC) plates were from Merck (Nogent-sur-Marne, France), and all other reagents were purchased from Sigma (Saint Quentin-Fallavier, France) unless otherwise indicated. HIT serum samples HIT was identified in patients whose platelet count was below 100 × 109/L or who experienced a 50% decrease in platelet count for no apparent reason other than heparin administration. These sera were aliquoted and stored at 80°C until use. Prior to use in any HIT assay, the sera were heated at 56°C for 1 hour and centrifuged to remove any residual thrombin activity. Sera were designated as positive or negative based on their ability to promote platelet aggregation in an HIT aggregation system and with regard to the so-called "SRA" assay as described previously.22 Preparation and activation of platelets Human blood platelet concentrates were obtained from the local blood bank (Etablissement de Transfusion Sanguine, Toulouse, France). Platelets were prepared essentially as described previously.23 Briefly, they were washed in a washing buffer (pH 6.5) containing 140-mmol/L NaCl, 5-mmol/L KCl, 5-mmol/L KH2PO4, 1-mmol/L MgSO4, 10-mmol/L HEPES, 5-mmol/L glucose, and 0.35% bovine serum albumin (wt/vol). The same buffer containing 1-mmol/L CaCl2 was added to the final suspension, and pH was adjusted to 7.4. For inositol lipid analysis, platelets were labeled with 0.5-mCi/mL of [32P]orthophosphate during 60 minutes in a phosphate-free washing buffer (pH 6.5) at 37°C. [32P]-labeled platelets were then washed once in the same buffer and finally resuspended at a final concentration of platelets of 1000 × 109/L (pH 7.4). Cross-linking of the low-affinity receptor for IgG, FcRIIA, was performed by preincubation of platelets for 1 minute with the monoclonal antibody IV.3 (2 µg/mL) followed by stimulation for different periods by addition of antimouse IgG F(ab')2 (30 µg/mL) at 37°C under gentle shaking as described previously.8 For activation of platelets by HIT serum, 400 µL of platelet suspension was incubated with heparin (0.5 IU) and HIT sera (80 µL). When indicated, 1-IU/mL apyrase, 5-mmol/L creatine phosphate (CP), 40-IU/mL creatine phosphokinase (CPK), 500-µmol/L A3P5PS, 50-µmol/L adenosine triphosphate (ATP)S, or 1-µmol/L epinephrine was added 1 minute before stimulation at 37°C. Calcium flux measurements Platelets were prepared as described above with slight modifications. Platelet-rich plasma was incubated for 30 minutes at 37°C with 1-µmol/L Fura 2-acetoxymethylester, washed, and the final platelet concentration adjusted to 300 × 109 cells/L in the stimulation buffer. The fluorescence excitation wavelengths and the emission wavelength were 340 nm, 380 nm, and 510 nm, respectively. Platelets were preincubated and stirred for 1 minute at 37°C in the presence of 1-mmol/L EGTA and were stimulated by FcRIIA cross-linking in the presence or absence of inhibitors and epinephrine as indicated. The changes in fluorescence were recorded using a PTI Deltascan spectrofluorometer. Lipid extraction and analysis Reactions were stopped by addition of chloroform/methanol (vol/vol), and lipids were extracted following a Bligh and Dyer modified procedure.24,25 Lipids were first resolved by TLC using chloroform/acetone/methanol/acetic acid/water (80/30/26/24/14, vol/vol). The spots corresponding to PtdIns(3,4,5)P3 were then scraped off, deacylated by 20% methylamine, and analyzed by high-performance liquid chromatography on a Whatman Partisphere 5 SAX column (Whatman International Ltd, United Kingdom) as described previously.25 For PtdOH quantification, lipids were resolved by TLC using CHCl3/CH3OH/HCl 10N (87/13/0.5, vol/vol) as described previously.26 Platelet aggregation and 5-hydroxytryptamine secretion studies Aggregation was monitored by a turbidimetric method using a dual-channel Payton aggregometer (Payton Assoc, Scarborough, ON) with continuous stirring at 900 rev/min at 37°C (500 × 109 platelets/L). Secretion of 5-hydroxytryptamine was performed as described previously.27 Briefly, platelets loaded with 5-hydroxy[14C]tryptamine were preincubated or not with different ADP inhibitors: A3P5PS (500 µM), CP (5 mmol/L), and CPK (40 IU/ML) for 1 minute and stimulated by FcRIIA cross-linking during 3 minutes in the presence of 5-µmol/L imipramin. Incubations were stopped by addition of 3% formaldehyde, 0.1-mol/L ethylenediaminetetraacetic acid (EDTA), cooling on ice, and centrifugation. The 5-hydroxy[14C]tryptamine released from platelet-dense granules was determined by liquid-scintillation counting.25 Gel electrophoresis and immunoblotting Proteins were resuspended in electrophoresis sample buffer containing 100-mmol/L Tris-HCl (pH 6.8), 15% (vol/vol) glycerol, 25-mmol/L dithiothreitol, and 3% sodium dodecyl sulfate (SDS), boiled for 5 minutes, separated on 7.5% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto a nitrocellulose membrane (Gelman Sciences). The nitrocellulose was blocked for 60 minutes at room temperature with 1% (wt/vol) milk powder and 1% (wt/vol) bovine serum albumin in a TBST buffer containing 10-mmol/L Tris-HCl (pH 7.5), 150-mmol/L NaCl, and 0.05% (wt/vol) Tween 20 as reported previously.25 Immunodetection was achieved using the relevant antibody, peroxidase-conjugated secondary antibody, and the ECL system. Immunoprecipitation For PLC2 and LAT immunoprecipitations, reactions were stopped by addition of 1 volume of ice-cold 2 × lysis buffer containing 80-mmol/L Tris-HCl (pH 7.4), 200-mmol/L NaCl, 200-mmol/L NaF, 20-mmol/L EDTA, 80-mmol/L Na4P2O7, 4-mmol/L Na3VO4, 2% Triton X-100 (vol/vol), and 10 µg/mL each of aprotinin and leupeptin. After gentle shaking during 20 minutes at 4°C and centrifugation (12 000g for 10 minutes at 4°C), the soluble fraction was collected and precleared for 30 minutes at 4°C with protein A-Sepharose CL4B. The precleared suspensions were incubated overnight at 4°C with the anti-PLC2 antibody or anti-LAT antibody, and immune complexes were then precipitated by addition of 10% (wt/vol) protein A-Sepharose CL4B for 1 hour at 4°C and centrifugation (6000g for 5 minutes at 4°C). The immunoprecipitates were washed once in 1 × lysis buffer and twice in a washing buffer containing 10-mmol/L Tris-HCl (pH 7.4), 100-mmol/L NaCl, 100-µmol/L Na3VO4, and 1 µg/mL each of aprotinin and leupeptin. Immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and analyzed by Western blotting. For FcRIIA immunoprecipitation, reactions were stopped by addition of 1 volume of ice-cold 2 × RIPA buffer containing 2-mmol/L Na3VO4, 10-mmol/L EDTA, 20-mmol/L Tris (pH 7.4), 320-mmol/L NaCl, 0.2% SDS, 2% sodium deoxycholate, 2% NP-40, and 10 µg/mL each of aprotinin and leupeptin. After gentle shaking during 20 minutes at 4°C and centrifugation (12 000g for 10 minutes at 4°C), the soluble fraction was collected. The suspensions were then incubated for 1 hour at 4°C with the MoAb IV.3, and immune complexes were then precipitated by addition of 10% (wt/vol) pansorbin for 30 minutes at 4°C and centrifugation (6000g for 5 minutes at 4°C). The immunoprecipitates were washed 3 times in 1 × RIPA buffer. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and analyzed by Western blotting.     Results Top Abstract Introduction Materials and methods Results Discussion References Requirement of a Gi-dependent pathway initiated either by ADP or epinephrine for FcRIIA-mediated platelet secretion and aggregation As previously shown,15,16 platelet aggregation induced by FcRIIA clustering (Figure 1A) or by addition of HIT sera (Figure 1B) were both fully inhibited in the presence of the 2 unrelated ADP scavengers, apyrase or CP-CPK. In agreement with these results, ATPS, an antagonist of ADP platelet receptors, strongly inhibited platelet aggregation. In the presence of A3P5PS, a specific platelet P2Y1 purinergic receptor antagonist,28-30 aggregation was not significantly affected. Conversely, the selective antagonist of the ADP receptor coupled to Gi, AR-C69931MX,31 strongly inhibited FcRIIA-mediated platelet aggregation. Epinephrine, known to selectively activate Gi proteins via the 2-adrenergic receptor, could overcome the inhibitory effect of CP-CPK on FcRIIA and HIT sera-induced platelet aggregation (Figure 1, right panels). It is noteworthy that epinephrine per se does not induce platelet shape change, calcium mobilization, inositol trisphosphate formation, fibrinogen binding, and aggregation,32,33 although it potentiates platelet aggregation induced by other agonists.32 Altogether, these results strongly suggest that the not yet identified platelet ADP receptor coupled to Gi was essential for the coactivation effect of ADP. Moreover, serotonin secretion was also strongly inhibited by ADP scavengers (Figure 2) but not affected by A3P5PS. Again, epinephrine did not induce secretion per se but was able to replace ADP as a coactivator of FcRIIA to obtain secretion (Figure 2). View larger version (15K): [in this window] [in a new window]   Figure 1. The inhibitory effect of ADP scavengers on platelet aggregation induced by FcRIIA cross-linking or by HIT sera is reversed by epinephrine. Human platelet suspensions were preincubated (1 minute, 37°C) with 500-mol/L A3P5PS, 0.1 µmol/L AR-C69931MX, 1-IU/mL apyrase, 50-µmol/L ATPS, 5-mmol/L CP, and 40-IU/mL CPK or CP-CPK plus 0.5-µmol/L or 1-mol/L epinephrine and then stimulated by FcRIIA cross-linking (A) or by 80 µL of sera from HIT patients (B) as indicated in "Materials and methods." Aggregation was assessed using a Chrono-Log dual-channel aggregometer with stirring at 900 rev/min (5 × 108 cells/mL). View larger version (14K): [in this window] [in a new window]   Figure 2. The inhibitory effects of ADP scavengers on serotonine secretion evoked by FcRIIA is reversed by epinephrine. Platelets were previously loaded with 5-hydroxy[14C]tryptamine, and secretion was determined as described in "Materials and methods." A3P5PS, CP-CPK, and CP-CPK and 1-µmol/L epinephrine were added 1 minute before stimulation by FcRIIA cross-linking. The effect of 1-µmol/L epinephrine alone was also assessed. Data are the mean of 2 independent experiments. (A) Stimulation by FcRIIA cross-linking; (B) stimulation by HIT sera. These results suggest that an early step of the signal transduction cascade initiated by FcRIIA was regulated by concomitant signaling from Gi. Optimal FcRIIA-mediated PLC activation and Ca++ signaling require a Gi-dependent pathway initiated either by ADP receptor or by 2-adrenergic receptor PLC activation, one of the key biochemical events related to platelet secretion, was first investigated. In 32P-labeled platelets, the production of 32P-PtdOH is a good marker of PLC activation.34,35 As shown in Figure 3A,C, FcRIIA-mediated PtdOH synthesis was strongly inhibited in the presence of CP-CPK. A3P5PS had a weak inhibitory effect probably due, at least in part, to the inhibition of the Gq-dependent activation of PLC via the P2Y1 ADP receptor. Increasing concentrations of A3P5PS, up to 1.1 mmol/L, did not significantly amplify this effect (not shown). This is consistent with the fact that 10 µmol/L of ADP alone was able to induce a very modest production of PtdOH (not shown) as previously observed.36 In agreement with these results, Ca++ mobilization (Figure 4) was also strongly inhibited in the presence of CP-CPK (72% ± 8% of inhibition, n = 4) whereas A3P5PS had a weak but significant inhibitory effect (36% ± 13% of inhibition, n = 3). Nevertheless, the partial inhibition of PLC and Ca++ mobilization by the P2Y1 antagonist A3P5PS was not sufficient to lead to a detectable decrease in platelet secretion and aggregation (Figures 1 and 2). View larger version (28K): [in this window] [in a new window]   Figure 3. CP-CPK inhibits FcRIIA-mediated PtdOH production. 32P-labeled platelets were incubated or not with CP-CPK, A3P5PS, or CP-CPK and 1-µmol/L epinephrine (1 minute, 37°C) as described in "Materials and methods" and activated by FcRIIA cross-linking during different periods of time (A) or during 2 minutes (B). Increasing concentrations of CP-CPK (C) or AR-C69931MX (D) were also tested. The inhibitory effects of these compounds were overcome in a dose-dependent manner by epinephrine (C,D). Lipids were immediately extracted, PtdOH was separated by TLC, and the radioactivity incorporated into PtdOH was quantified by PhosphorImager analysis. Data are representative of 2 independent experiments with very similar results (A), mean ± standard errors of 6 independent experiments (B), or representative of 3 independent experiments (C,D). **Significant difference (P < .01) according to Student t test. View larger version (26K): [in this window] [in a new window]   Figure 4. Epinephrine overcomes the inhibitory effect of CP-CPK on FcRIIA-mediated Ca++ mobilization. Platelets were loaded with 1-µmol/L Fura-2 as described in "Materials and methods" and stimulated by FcRIIA cross-linking in the absence (A) or in the presence of A3P5PS (B) or CP-CPK (C). In (D), platelets were stimulated by FcRIIA cross-linking in the absence (i) or in the presence of CP-CPK (ii) or CP-CPK and epinephrine (iii). In (iv), platelets were stimulated by 1-µmol/L epinephrine alone. The variations in fluorescence, reflecting changes in intracellular Ca++ concentration, were monitored using a PTI Deltascan spectrofluorometer. Data are representative of 2 to 4 independent experiments. Interestingly, as with CP-CPK, AR-C69931MX was able to strongly inhibit the production of PtdOH. In agreement with the results shown in Figures 1 and 2, epinephrine was able to overcome, in a dose-dependent manner, the inhibitory effects of CP-CPK or AR-C69931MX on FcRIIA-mediated PtdOH production (Figure 3B-D). The inhibitory effect of CP-CPK on calcium mobilization was also significantly overcome by epinephrine (Figure 4D). FcRIIA stimulates the tyrosine phosphorylation of a set of proteins, including PLC2, independently of ADP and Gi pathway It is now well established that FcRIIA cross-linking induces the activation of PLC2 through a mechanism involving its tyrosine phosphorylation.9,37 The contribution of the Gi pathway on tyrosine phosphorylation of PLC2 following FcRIIA clustering was therefore evaluated. Figure 5A indicates that the whole pattern of phosphotyrosyl proteins in platelets stimulated by FcRIIA cross-linking was not significantly affected by the ADP scavenger CP-CPK. Furthermore, the tyrosine phosphorylation of PLC2 was not impaired by addition of ADP scavengers (Figure 5B). In addition, Figure 5C,D shows that the rapid tyrosine phosphorylation of FcRIIA itself as well as the tyrosine phosphorylation of LAT, a docking protein recently identified in platelets,38 were not significantly affected by the ADP scavenger. View larger version (36K): [in this window] [in a new window]   Figure 5. ADP is not significantly involved in FcRIIA-mediated protein tyrosine phosphorylation. Platelets were preincubated in the absence or in the presence of A3P5PS, CP-CPK, and CP-CPK and 1 µmol/L epinephrine and stimulated by FcRIIA cross-linking. The effect of 1-µmol/L epinephrine alone was also assessed. (A) Immunoblotting of platelet total protein extracts with the antiphosphotyrosine antibody 4G10. PLC2 (B), FcRIIA (C), and LAT (D) were immunoprecipitated and submitted to immunoblotting with 4G10 antibody. As a loading control, the nitrocellulose membranes were stripped and reprobed with anti-PLC2 antibody and anti-LAT antibody (lower panels). (C) Immunoprecipitations performed with nonimmune serum as a control (C,D). Con, control. Data are representative of 3 to 4 independent experiments. These results indicate that the tyrosine kinase pathway initiated by FcRIIA cross-linking is independent of ADP. ADP is required for an optimal production of PtdIns(3,4,5)P3 in FcRIIA-stimulated platelets and can be replaced by epinephrine Recently, we have shown that the early synthesis of PtdIns(3,4,5)P3 is absolutely required for the activation of the tyrosine phosphorylated PLC2 upon FcRIIA cross-linking.25 The role of ADP in the synthesis of this particular phosphoinositide was therefore investigated. Figure 6A shows that PtdIns(3,4,5)P3 production was strongly inhibited by the ADP scavenger CP-CPK (68.2% ± 12% of inhibition, n = 4). The P2Y1 antagonist A3P5PS was a weak inhibitor of this production (Figure 6A) even at high concentrations, up to 1.1 mmol/L (not shown). Conversely, AR-C69931MX was as efficient as CP-CPK to inhibit PtdIns(3,4,5)P3 production (Figure 6B,C). As already described, ADP39,40 or epinephrine alone was only able to induce the production of trace amounts of PtdIns(3,4,5)P3. However, again, epinephrine could overcome, in a dose-dependent manner, the inhibitory effects of CP-CPK or AR-C69931MX (Figure 6B,C), indicating that this 2-adrenergic receptor agonist could replace ADP as a coactivator of FcRIIA-mediated PtdIns(3,4,5)P3 production. View larger version (23K): [in this window] [in a new window]   Figure 6. The inhibitory effect of ADP scavengers on FcRIIA-mediated PtdIns(3,4,5)P3 synthesis is reversed by epinephrine. 32P-labeled platelets were incubated with or without A3P5PS, CP-CPK, and CP-CPK and 1-µmol/L epinephrine (1 minute, 37°C) and activated by FcRIIA cross-linking (A). The effect of 1-µmol/L epinephrine alone was assessed. Increasing concentrations of CP-CPK (B) and AR-C69931MX (C) were tested, and the inhibitory effects of these compounds were overcome in a dose-dependent manner by epinephrine (B,C). After 2 minutes of stimulation, lipids were extracted, and the radioactivity incorporated in PtdIns(3,4,5)P3 was determined as described in "Materials and methods." Data are expressed as a percentage of PtdIns(3,4,5)P3 produced compared with control (100% being the production obtained by FcRIIA clustering at 2 minutes) and are means ± standard errors of 3 independent experiments (A) or representative. *,***Significant difference (P < .05) and (P < .001), respectively, according to Student t test. To further confirm the role of ADP as a cofactor in FcRIIA-mediated phosphatidylinositol (PI) 3-kinase activation, we used the PI 3-kinase inhibitor wortmannin at a low concentration (10 nmol/L) to strongly, but not completely, inhibit the production of PtdIns(3,4,5)P3 (78% of inhibition) (Figure 7). Under these conditions, platelet aggregation was fully inhibited (Figure 7A) and the production of PtdOH was inhibited by 63.8 ± 10% (Figure 7C). It is important to note that wortmannin does not affect tyrosine phosphorylation of PLC2 25 and that the remaining production of PtdIns(3,4,5)P3 did not induce sufficient activation of PLC2 to obtain platelet secretion (not shown) and aggregation (Figure 7A). Interestingly, addition of exogenous ADP to 10-nmol/L wortmannin-treated platelets could overcome the FcRIIA-mediated PtdIns(3,4,5)P3 production (Figure 7B), PtdOH synthesis (Figure 7C), and platelet aggregation (Figure 7A). This effect of ADP was dose-dependent (Figure 7). The intracellular production of PtdIns(3,4,5)P3 and PtdOH is correlated with the intensity of platelet aggregation (Figure 7A-C). As expected, this effect of ADP was no longer observed with a dose of wortmannin (50 nmol/L) capable of irreversibly inhibiting most PI 3-kinase copies, subsequently blocking the production of PtdIns(3,4,5)P3 (Figure 7B). Although ADP by itself was a very weak activator of PtdIns(3,4,5)P3 production (maximum, 1.4-fold increase upon 10 µmol/L ADP), these results indicate that it is a crucial FcRIIA cofactor for the production of this lipid second messenger, subsequent PtdOH synthesis, calcium mobilization, and platelet aggregation. Moreover, epinephrine could again replace ADP to overcome the inhibitory effect of low doses of wortmannin (not shown), indicating that a Gi pathway is required for this mechanism. Finally, when exogenous ADP was added during FcRIIA-mediated normal platelet activation, we observed a clear potentiation of PtdIns(3,4,5)P3 production in a dose-dependent manner (1.8- and 2.2-fold increase at 5- and 10-µmol/L ADP, respectively). View larger version (17K): [in this window] [in a new window]   Figure 7. ADP overcomes the inhibitory effect of low doses of wortmannin on PtdIns(3,4,5)P3 production, PtdOH synthesis, and platelet aggregation. Human platelet suspensions were preincubated with or without 10-nmol/L wortmannin (2 minutes, 37°C) and stimulated by FcRIIA cross-linking in the absence (left panel) or in the presence (right panel) of 5-µmol/L (i) or 10-µmol/L (ii) ADP (A). Aggregation was assessed using a Chrono-Log dual-channel aggregometer with stirring at 900 rev/min. (B,C) 32P-labeled platelets were incubated with 10 or 50-nmol/L wortmannin (2 minutes, 37°C) and activated by FcRIIA cross-linking in the presence or in the absence of 5-µmol/L or 10-µmol/L ADP as indicated. After 2 minutes of stimulation, lipids were extracted and the radioactivity incorporated in PtdIns(3,4,5)P3 (B) or PtdOH (C) was determined as described in "Materials and methods." Data are expressed as a percentage of PtdIns(3,4,5)P3 or PtdOH produced compared with control (100% being the production obtained by FcRIIA clustering at 2 minutes) and are means ± standard errors of 3 independent experiments.     Discussion Top Abstract Introduction Materials and methods Results Discussion References ADP is required for platelet activation and aggregation induced by FcRIIA cross-linking using either specific antibodies or sera from HIT patients.16 Besides the important therapeutic developments, these findings raise a number of crucial questions relating to the molecular mechanism of agonist-induced platelet activation. Several recent reports have also shown that, although ADP is a weak agonist per se, it plays an important role as coactivator of other platelet agonists such as collagen, TRAP, or thrombin.19-21,41,42 Moreover, according to the agonist used, ADP can play a role at different stages of platelet activation. It is involved in the stabilization of thrombin or TRAP-induced human platelet aggregation21 but also plays a critical role in the very early steps of FcRIIA-dependent platelet activation.16 ADP can also potentiate platelet secretion independently of aggregation.43 These results strongly suggest that specific signaling pathways initiated by ADP receptors may modulate or amplify intracellular biochemical events initiated by other platelet agonists. Here we show that A3P5PS, a P2Y1 ADP receptor antagonist, had no effect on FcRIIA-mediated secretion and aggregation. Conversely, AR-C69931MX, a selective antagonist of the ADP receptor coupled to Gi,31 was a potent inhibitor of FcRIIA-mediated platelet secretion (not shown) and aggregation. Interestingly, triggering of another receptor selectively coupled to Gi, the 2A-adrenergic receptor,18,32,33 could overcome the inhibitory effect of ADP scavengers. These observations indicated that a Gi-dependent pathway either initiated by ADP or epinephrine participated in FcRIIA-induced platelet activation. One of the first intracellular signaling events required for FcRIIA-mediated platelet activation is tyrosine phosphorylation of the ITAM-like motif of this receptor. We found that this phosphorylation as well as the phosphorylation of other downstream targets, including the adaptor molecule LAT or PLC2, were not significantly affected by ADP scavengers. Another early major event of this activation cascade is the stimulation of enzymes of the phosphoinositide metabolism. Interestingly, the PtdOH production and the calcium mobilization, reflecting PLC activation, were both strongly inhibited by ADP scavengers. The selective block of the P2Y1 receptor had a weak effect on the production of PtdOH. This effect may correspond, at least partially, to the slight activation of PLC observed upon ADP addition by several groups.36 Interestingly, AR-C69931MX inhibited the PtdOH production following FcRIIA clustering as efficiently as CP-CPK, indicating that the major cofactor effect of ADP was actually due to its receptor negatively coupled to adenylyl cyclase. Moreover, addition of epinephrine could restore this inhibitory effect of CP-CPK on PtdOH production and calcium mobilization in a dose-dependent manner. PLC2 is activated downstream of tyrosine kinases and is an effector of Syk in FcRIIA-mediated platelet activation.7,9,37 Recently, we demonstrated that the PI 3-kinase product PtdIns(3,4,5)P3 was absolutely required for activation of the tyrosine phosphorylated PLC2 following FcRIIA clustering.25 It is interesting to note that, in contrast to the situation in B lymphocytes where BTK, a Tec family tyrosine kinase activated through PtdIns(3,4,5)P3 production, is involved in the phosphorylation and the activation of PLC2,44 our results strongly suggest that PLC2 is not a major substrate for Tec family kinases in platelets stimulated through FcRIIA, as also observed following activation of glycoprotein VI in mice platelets.45 These discrepancies may reflect a difference in the role of BTK between cell types or agonist-dependent activation and highlight a critical role of PtdIns(3,4,5)P3 as a direct cofactor of PLC2 activation possibly by allowing an adequate localization of this enzyme at the membrane-cytoskeleton interface. Thus, calcium mobilization and activation of protein kinase C, 2 events required for secretion, inside-out activation of the integrin IIb3 and aggregation, are also indirectly regulated by PtdIns(3,4,5)P3 production. Interestingly, the production of PtdIns(3,4,5)P3 was strongly inhibited by ADP scavengers and AR-C69931MX, whereas the P2Y1 receptor antagonist, A3P5PS, had only a weak inhibitory effect. It is noteworthy that ADP or epinephrine alone are very weak activators of PtdIns(3,4,5)P3 production.39,40 Again, epinephrine could overcome the inhibitory effect of CP-CPK or AR-C69931MX in a dose-dependent manner. These results strongly suggest that concomitant signaling from tyrosine kinases, initiated by FcRIIA cross-linking, and Gi-dependent pathway, initiated either by ADP or epinephrine, were required to reach a sufficient level of PtdIns(3,4,5)P3. This is consistent with several reports that have placed a wortmannin-sensitive PI 3-kinase as a key signaling molecule in FcRIIA-mediated platelet activation.12,25,46 To confirm the role of ADP in the regulation of the amount of PtdIns(3,4,5)P3, we have used low doses of wortmannin (10 nmol/L) capable of irreversibly inhibiting a large number of, but not all, PI 3-kinase copies. Under these conditions, 22% of the PtdIns(3,4,5)P3 produced upon FcRIIA cross-linking remained, but this production was not sufficient to allow platelet aggregation. Interestingly, addition of ADP could restore, in a dose-dependent manner, the production of PtdIns(3,4,5)P3, allowing sufficient PLC activation for platelet aggregation. In contrast, ADP, at any concentration used, was unable to overcome the inhibitory effect of a higher dose of wortmannin (50 nmol/L), which blocked most PI 3-kinase copies and fully inhibited PtdIns(3,4,5)P3 production. These results clearly demonstrate that ADP can up-regulate the rapid FcRIIA-mediated PtdIns(3,4,5)P3 production in human platelets. This effect of ADP could be replaced by the activation of the 2-adrenergic receptor, indicating that a Gi-dependent pathway was involved early on in the production of PtdIns(3,4,5)P3. How can ADP signaling via Gi regulate the level of PtdIns(3,4,5)P3 production? Type IA PI 3-kinase has been shown to transiently associate with FcRIIA,12 possibly through the interaction of its adaptor subunit p85 with the docking protein Cbl,46 and may therefore account for the rapid production of PtdIns(3,4,5)P3. This isoform of PI 3-kinase is activated by tyrosine kinase-dependent mechanisms and specific protein-protein interactions.47 Interestingly, a synergistic effect involving subunits of G proteins and phosphotyrosyl peptide has been recently described in the activation of the heterodimeric PI 3-kinase p85-p110 but not of p85-p110.48 The subunits may also activate p110 independently of the p85 subunits in vitro.49 These results suggest that 2 different types of membrane receptors, one activating the tyrosine kinase pathway and the other activating GTP-binding proteins, may cooperate for the production of PtdIns(3,4,5)P3. Among the p85 subunits, p85 is preferentially expressed in human platelets. The p110 catalytic subunit is also present,39 and we recently observed, by immunoblotting experiments, the expression of p110 (not shown). Thus, a p85-p110 form of PI 3-kinase may exist in platelets and could require synergistic activation via of Gi and a tyrosine-phosphorylated docking protein. The subunits of Gi may also directly involve type IB PI 3-kinase (p110),49 which is less sensitive to wortmannin than type IA.39 However, because ADP or epinephrine alone are poor activators of PtdIns(3,4,5)P3 synthesis, this hypothesis becomes invalidated. Moreover, we cannot exclude the possibility that unidentified isoforms of PI 3-kinase, weakly sensitive to low concentrations of wortmaninn, might be involved in this mechanism. At last, although we did not observe an accumulation of PtdIns(3,4)P2 in the presence of CP-CPK, ADP might also modulate the degradation of PtdIns(3,4,5)P3 through specific phosphatases such as the 5-phosphatase SHIP1.45,50 In conclusion, our results demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet activation and aggregation induced by FcRIIA and suggest that antagonists of the ADP receptor coupled to Gi may be effective as therapeutic agents for prevention or treatment of HIT. Based on our results and recent reports, we propose that concomitant signaling through Gi and either Gq18,51 or tyrosine kinases, according to the primary agonist used, might be a general mechanism by which platelet activation and aggregation occurs.     Acknowledgments The authors thank Drs P. Raynal, F. Gaits, J. Ragab, C. Trumel, G. Mauco, S. Giuriato, and K. Missy for stimulating discussions and C. Greenland for correcting the English.     Footnotes Submitted November 1, 1999; accepted July 6, 2000. Supported by grants from Association pour la Recherche sur le Cancer, European Union Biomed 2 Program BMH4-CT-97 2609, and Région Midi-Pyrénées. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Bernard Payrastre, INSERM U326, Hôpital Purpan, 31059 Toulouse, France.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Anderson CL, Chacko GW, Osborne JM, Brandt JT. The Fc receptor for immunoglobulin G (Fc gamma RII) on human platelets. Semin Thromb Hemost. 1995;21:1-9[Medline] [Order article via Infotrieve]. 2. Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7[Medline] [Order article via Infotrieve]. 3. Arnout J. The pathogenesis of the antiphospholipid syndrome: a hypothesis based on parallelisms with heparin-induced thrombocytopenia. Thromb Haemost. 1996;75:536-541[Medline] [Order article via Infotrieve]. 4. Horne MK, Alkins BR. Platelet binding of IgG from patients with heparin-induced thrombocytopenia. J Lab Clin Med. 1996;127:435-442[Medline] [Order article via Infotrieve]. 5. Hoylaerts MF, Thys C, Arnout J, Vermylen J. Recurrent arterial thrombosis linked to autoimmune antibodies enhancing von Willebrand factor binding to platelets and inducing Fc gamma RII receptor-mediated platelet activation. Blood. 1998;91:2810-2817[Abstract/Free Full Text]. 6. Horsewood P, Hayward CP, Warkentin TE, Kelton JG. Investigation of the mechanisms of monoclonal antibody-induced platelet activation. Blood. 1991;78:1019-1026[Abstract/Free Full Text]. 7. Chacko GW, Duchemin AM, Coggeshall KM, Osborne JM, Brandt JT, Anderson CL. Clustering of the platelet Fc gamma receptor induces noncovalent association with the tyrosine kinase p72syk. J Biol Chem. 1994;269:32435-32440[Abstract/Free Full Text]. 8. Yanaga F, Poole A, Asselin J, et al. Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc gamma-IIA receptor. Biochem J. 1995;311:471-478. 9. Huang MM, Indik Z, Brass LF, Hoxie JA, Schrei-ber AD, Brugge JS. Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII. J Biol Chem. 1992;267:5467-5473[Abstract/Free Full Text]. 10. Gibbins J, Asselin J, Farndale R, Barnes M, Law CL, Watson SP. Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets. J Biol Chem. 1996;271:18095-18099[Abstract/Free Full Text]. 11. Ibarrola I, Vossebeld PJ, Homburg CH, Thelen M, Roos D, Verhoeven AJ. Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa. Biochim Biophys Acta. 1997;1357:348-358[Medline] [Order article via Infotrieve]. 12. Chacko GW, Brandt JT, Coggeshall KM, Anderson CL. Phosphoinositide 3-kinase and p72syk noncovalently associate with the low affinity Fc gamma receptor on human platelets through an immunoreceptor tyrosine-based activation motif. Reconstitution with synthetic phosphopeptides. J Biol Chem. 1996;271:10775-10781[Abstract/Free Full Text]. 13. Blake RA, Asselin J, Walker T, Watson SP. Fc gamma receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor. FEBS Lett. 1994;342:15-18[Medline] [Order article via Infotrieve]. 14. Daniel JL, Dangelmaier C, Smith JB. Evidence for a role for tyrosine phosphorylation of phospholipase C gamma 2 in collagen-induced platelet cytosolic calcium mobilization. Biochem J. 1994;302:617-622. 15. Herault JP, Lale A, Savi P, Pflieger AM, Herbert JM. In vitro inhibition of heparin-induced platelet aggregation in plasma from patients with HIT by SR 121566, a newly developed Gp IIb/IIIa antagonist. Blood Coagul Fibrinolysis. 1997;8:206-207[Medline] [Order article via Infot