Phosphatidylinositol 3,4,5-Trisphosphate-dependent Stimulation of Phospholipase C-γ2 Is an Early Key Event in FcγRIIA-mediated Activation of Human Platelets
1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês
10.1074/jbc.273.38.24314
ISSN1083-351X
AutoresMarie‐Pierre Gratacap, Bernard Payrastre, Cécile Viala, Gérard Mauco, Monique Plantavid, Hugues Chap,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoPlatelets express a single class of Fcγ receptor (FcγRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcγRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-γ2 (PLC-γ2), and calcium signaling. Here, we show that in response to FcγRIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCγ2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin αIIb/β3 engagement. In contrast, the stable association of PLCγ2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCγ2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCγ2 induced by FcγRIIA cross-linking. Platelets express a single class of Fcγ receptor (FcγRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcγRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-γ2 (PLC-γ2), and calcium signaling. Here, we show that in response to FcγRIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCγ2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin αIIb/β3 engagement. In contrast, the stable association of PLCγ2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCγ2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCγ2 induced by FcγRIIA cross-linking. low affinity Fcγ receptor IIA immunoreceptor tyrosine-based activation motif high affinity IgE receptor Src homology-2 phosphoinositide 3-kinase phospholipase C phosphatidylinositol phosphatidylinositol 4-phosphate 5)P2, phosphatidylinositol 4,5-bisphosphate 4)P2, phosphatidylinositol 3,4-bisphosphate 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate phosphatidic acid thin-layer chromatography high performance liquid chromatography thrombin-receptor activating peptide 1,4-piperazinediethanesulfonic acid. In addition to specific interactions involving their Fab domains, immunoglobulins G can interact with various membrane receptors by their Fc region. These so-called Fc receptors are coded by a heterogeneous family of at least eight genes forming a complex locus on chromosome 1. Based on both functional and structural criteria, these proteins can be differentiated into three groups (I–III) displaying various expression patterns in cells of the immune system (1Ravetch J.V. Kinet J.-P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1282) Google Scholar, 2Van de Winkel J.G.J. Capel P.J.A. Immunol. Today. 1993; 14: 215-221Abstract Full Text PDF PubMed Scopus (631) Google Scholar). Platelets possess a single class of Fcγ receptors, FcγRIIA.1 As reviewed by Anderson et al. (3Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemostasis. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar), clustering of FcγRIIA induces shape change, secretion, and aggregation, which are typical platelet responses contributing to their hemostatic function. In addition, there is a close link between secretion and aggregation, since released ADP was shown recently to be very critical for platelet aggregation evoked by FcγRIIA cross-linking (4Polgar J. Eichler P. Greinacher A. Clemetson K.J. Blood. 1998; 91: 549-554Crossref PubMed Google Scholar). The precise role of platelet FcγRIIA is still obscure, although it explains how these cells are activated by specific antibodies directed against various membrane antigens, by immune complexes or by aggregated IgG (3Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemostasis. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar). On a pathophysiological point of view, platelet FcγRIIA might involve an hemostatic response at inflammatory sites displaying IgG deposits, and there is increasing evidence for a direct involvement of FcγRIIA in heparin-associated thrombocytopenia occurring in patients under heparin therapy (3Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemostasis. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar). FcγRIIA is a 40-kDa polypeptide bearing two IgG-like domains in its extracellular region, a single transmembrane segment, and an ITAM, also known as Reth motif, in the cytoplasmic tail (5Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1167) Google Scholar). ITAM sequences contain two variably spaced tyrosine residues; they are present in Fcγ receptors, FcεR, various subunits of both T cell and B cell antigen receptors, and the γ-chain of Fc receptor in platelets (6Gibbins J. Asselin J. Farndale R. Barnes M. Law C-L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar); and they support the paradigm of immune cell activation (7Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1955) Google Scholar, 8Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 9Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 10Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2229) Google Scholar, 11Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1995; 8: 394-401Crossref Scopus (170) Google Scholar, 12Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar). Indeed, clustering of membrane receptors present in cells of the immune system promotes the phosphorylation, presumably by Src kinases, of the two tyrosine residues of ITAM. This results in the specific anchoring to ITAM sequences of tyrosine kinases bearing two SH2 domains,i.e. either Syk or ZAP-70. These two tyrosine kinases then induce a complex set of signaling events including calcium mobilization as well as activation of PI 3-kinase and mitogen-activated protein kinases, the latter ones via an upstream cascade involving Grb2-Sos and the small GTPase Ras (7Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1955) Google Scholar, 8Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 9Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 10Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2229) Google Scholar, 11Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1995; 8: 394-401Crossref Scopus (170) Google Scholar, 12Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar). Phosphoinositide metabolism plays a key role during the stimulation of immune cell receptors and involves two types of enzymes, PLC and PI 3-kinase. Among various isoforms identified so far, PLC-γ2 is abundant in hematopoietic cells, it is activated downstream of tyrosine kinases, and it promotes both calcium mobilization and activation of protein kinase C via the two second messengers produced upon hydrolysis of PtdIns(4,5)P2 (13Lee S.B. Rhee S.G. Curr. Opin. Cell Biol. 1995; 7: 183-189Crossref PubMed Scopus (283) Google Scholar, 14Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar). On the other hand, the heterodimeric IA class PI 3-kinase is also regulated by tyrosine kinases and promotes the accumulation of D3-phosphoinositides, which are considered as potential second messengers (15Rittenhouse S.E. Blood. 1996; 88: 4401-4414Crossref PubMed Google Scholar, 16Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar, 17Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (833) Google Scholar). Two recent studies have shown specific interactions between PLC-γ1 and PtdIns(3,4,5)P3, but depending on the authors this might involve either the N-terminal PH domain or the SH2 domains of the protein (18Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (484) Google Scholar, 19Bae Y.S. Cantley L.G. Chen C.-S. Kim S.-R. Kwon K.-S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). However, no direct link between PLC-γ2, which is inactive toward the products of PI 3-kinase, and PI 3-kinase itself has been demonstrated so far in cells activated via FcγRIIA cross-linking. Besides the fact that FcγRIIA might play a key role in the hemostatic and inflammatory function of platelets, these cells are interesting to consider in so far they contain a single class of Fcγ receptor (FcγRIIA), in contrast to neutrophils or monocytes, for instance (1Ravetch J.V. Kinet J.-P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1282) Google Scholar,3Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemostasis. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar). Clustering of platelet FcγRIIA promotes phosphorylation of the two tyrosine residues of the ITAM motif, which is followed by the classical set of signaling events occurring under similar conditions,i.e. various tyrosine phosphorylations and calcium mobilization, through activation of PLCγ2 (3Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemostasis. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar, 20Anderson G.P. Anderson C.L. Blood. 1990; 76: 1165-1172Crossref PubMed Google Scholar, 21Huang M.-M. Indik Z. Brass L.F. Hoxie J.A. Schreiber A.D. Brugge J.S. J. Biol. Chem. 1992; 267: 5467-5473Abstract Full Text PDF PubMed Google Scholar, 22Chacko G.W. Duchemin A.-M. Coggeshall K.M. Osborne J.M. Brandt J.T. Anderson C.L. J. Biol. Chem. 1994; 269: 32435-32440Abstract Full Text PDF PubMed Google Scholar). Moreover, Chacko et al. (23Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) provided evidence that PI 3-kinase associates transiently with FcγRIIA upon platelet receptor clustering, probably via Syk. The present study was thus undertaken in order to determine possible changes of phosphoinositide metabolism occurring upon clustering of platelet FcγRIIA. In addition, taking advantage of the use of two specific and unrelated inhibitors (wortmannin and LY294002), we have focused our interest on signaling events occurring downstream of PI 3-kinase. Our present data unravel a causal relationship between PI 3-kinase and PLC-γ2, which might also function in the signaling cascade evoked by other membrane receptors involved in the immune response. Anti-FcγRIIA monoclonal antibody IV.3 and mouse anti-phosphotyrosine 4G10 antibody were purchased from Upstate Biotechnology, Inc. Anti-mouse IgG F(ab′)2 fragments were from Jackson Immunoresearch Laboratories, rabbit polyclonal anti-PLC-γ2 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 Pharmacia Biotech, whereas [γ-32P]ATP (3,000 Ci/mmol) was from NEN Life Science Products. DiC16-PtdIns(3,4,5)P3and diC16-PtdIns(3,4)P2 were from Matreya Inc. PtdIns, PtdIns(4)P, PtdIns(4,5)P2, crude brain phosphoinositides, phosphatidylserine, thrombin, wortmannin, and RGDS were purchased from Sigma, and TLC plates from Merck. All other reagents were obtained from Sigma unless otherwise indicated. Platelets were isolated from concentrates obtained from the local blood bank (Etablissement de Transfusion Sanguine, Toulouse, France) essentially as described previously (24Cazenave J.P. Hemmendinger S. Beretz A. Sutter-Bay A. Launay J. Ann. Biol. Clin. 1983; 41: 167-175PubMed Google Scholar). They were washed in washing-buffer (pH 6.5) containing 140 mm NaCl, 5 mm KCl, 5 mm KH2PO4, 1 mmMgSO4, 10 mm Hepes, 5 mm glucose, 0.35% (w/v) bovine serum albumin. The same buffer plus 1 mm CaCl2 was added to the final suspension, and pH was adjusted to 7.4. In experiments dealing with inositol lipid analysis, platelets were labeled with 0.5 mCi/ml [32P]orthophosphate during 60 min 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 suspended at a final concentration of 1 × 109 cells/ml (pH 7.4). Cross-linking of FcγRIIA was achieved by preincubation of platelets for 1 min with the monoclonal antibody IV.3 (2 μg/ml) followed by addition of anti-mouse IgG F(ab′)2 (30 μg/ml) at 37 °C under gentle shaking as described previously (25Yanaga F. Poole A. Asselin J. Blake R. Schieven G.L. Clark E.A. Law C.-L. Watson S.P. Biochem. J. 1995; 311: 471-478Crossref PubMed Scopus (134) Google Scholar). Reactions were stopped by addition of chloroform/methanol (1/1, v/v) containing 0.4 nHCl, and lipids were immediately extracted following the modified procedure of Bligh and Dyer (26Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar, 27Sultan C. Breton M. Mauco G. Grondin P. Plantavid M. Chap H. Biochem. J. 1990; 269: 831-834Crossref PubMed Scopus (42) Google Scholar). For PtdIns(4)P and PtdIns(4,5)P2, lipids were immediately deacylated by 20% methylamine and analyzed by HPLC on a Whatman Partisphere 5 SAX column (Whatman International Ltd., UK) as described previously (27Sultan C. Breton M. Mauco G. Grondin P. Plantavid M. Chap H. Biochem. J. 1990; 269: 831-834Crossref PubMed Scopus (42) Google Scholar). For PtdIns(3,4)P2 and PtdIns(3,4,5)P3quantification, lipids were first resolved by thin-layer chromatography (TLC) using chloroform/acetone/methanol/acetic acid/water (80/30/26/24/14, v/v). The spots corresponding to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 were then scraped off, deacylated, and analyzed by HPLC as described above. For PtdOH quantification, lipids were resolved by TLC using chloroform/methanol/10 N HCl (87/13/0.5, v/v) as described previously (28Cohen P. Broekman M.J. Verkleij A. Lisman J.W.W. Derksen A. J. Clin. Invest. 1971; 50: 762-772Crossref PubMed Scopus (65) Google Scholar). The spots corresponding to PtdOH were directly quantified by PhosphorImager analysis. Aggregation was monitored using a Chrono-log dual channel aggregometer with stirring at 900 rev/min at 37 °C (5 × 108 platelets/ml). 5-Hydroxytryptamine secretion was determined as described previously (29Holmsen H. Day H.J. J. Lab. Clin. Med. 1970; 75: 840-855PubMed Google Scholar). Briefly, platelets labeled with 5-hydroxy[14C]tryptamine were preincubated or not with increasing concentrations of wortmannin or LY294002 for 15 min and stimulated by FcγRIIA cross-linking during 3 min in presence of 5 μm imipramine. Incubations were stopped by addition of 3% formaldehyde and 0.1 m EDTA, cooling on ice, and centrifugation. The radioactivity of 5-hydroxy[14C]tryptamine released from platelet dense granules was determined by liquid-scintillation counting. Proteins were resuspended in electrophoresis sample buffer containing 100 mm Tris-HCl, pH 6.8, 15% (v/v) glycerol, 25 mmdithiothreitol, and 3% SDS, boiled for 5 min, separated on 7.5% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Gelman Sciences). The nitrocellulose was blocked for 60 min at room temperature with 1% (w/v) milk powder, 1% (w/v) bovine serum albumin in a buffer containing 10 mmTris-HCl, pH 7.5, 150 mm NaCl, and 0.05% (v/v) Tween 20. Immunodetection was achieved using the relevant antibody, peroxidase-conjugated secondary antibody, and ECL system. The various bands were quantified by densitometric analysis measuring the pixel volume in each area (Gel Doc 1000, Bio-Rad). Reactions were stopped by addition of twice-concentrated ice-cold lysis buffer containing 80 mmTris-HCl, pH 7.4, 200 mm NaCl, 200 mm NaF, 20 mm EDTA, 80 mmNa4P2O7, 4 mmNa3VO4, 2% (v/v) Nonidet P-40, and 10 μg/ml each of aprotinin and leupeptin. After gentle shaking during 20 min at 4 °C and centrifugation (12,000 × g for 10 min at 4 °C), the soluble fraction was collected and precleared for 30 min at 4 °C with protein A-Sepharose CL4B. The precleared suspensions were then incubated overnight at 4 °C with the adequate antibody, and immune complexes were then precipitated by addition of 10% (w/v) protein A-Sepharose CL4B for 1 h at 4 °C and centrifugation (6,000 × g for 5 min at 4 °C). The immunoprecipitates were washed once in lysis buffer and twice in washing buffer containing 10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 100 μmNa3VO4, 1 μg/ml each of aprotinin and leupeptin. Immunoprecipitated proteins were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting. Reaction was stopped and the cytoskeleton immediately extracted by adding one volume of ice-cold twice-concentrated cytoskeleton buffer containing 100 mmTris-HCl, pH 7.4, 20 mm EGTA, 2 mmNa3VO4, 4 μg/ml each of aprotinin and leupeptin, 2 mm phenylmethylsulfonyl fluoride, and 2% (v/v) Triton X-100 as described previously (30Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 31Guinebault C. Payrastre B. Racaud-Sultan C. Mazarguil H. Breton M. Mauco G. Plantavid M. Chap H. J. Cell Biol. 1995; 129: 831-842Crossref PubMed Scopus (224) Google Scholar). After 10 min at 4 °C, the cytoskeleton was pelleted by centrifugation (12,000 × g for 10 min at 4 °C), washed once in cytoskeleton buffer with 0.5% Triton X-100, and once with the same buffer without Triton X-100. Cytoskeleton was then immediately suspended in electrophoresis sample buffer (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). After stimulation, platelets were centrifuged (3,000 × g for 30 s) and suspended in 20 mm Pipes buffer (pH 6.8) containing 150 mmKCl, 2 mm EDTA, and 30 μg/ml saponin. After 3 min at room temperature under shaking, supernatant and pellet fractions were separated by centrifugation (12,000 × g for 40 s). The pellet was suspended in Laemmli's sample buffer, and PLC-γ72 was probed by Western blotting using a specific antibody as described above. [32P]PtdIns(3)P, [32P]PtdIns(3,4)P2, and [32P]PtdIns(3,4,5)P3 were prepared by incubating crude brain phosphoinositides (60 μg) mixed with phosphatidylserine (120 μg) in 50 mm Tris-HCl, pH 7.4(1 μg of phosphoinositides/μl), vortexed, and sonicated (20 kHz for 3 × 10 s). Lipid vesicles were then incubated with immunoprecipitated PI 3-kinase in the presence of 30 μCi [γ-32P]ATP (3,000 Ci/mmol), 50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1.5 mm dithiothreitol, 0.5 mm EDTA, 5 mm MgCl2, and 100 μm ATP (33Payrastre B. Gironcel D. Plantavid M. Mauco G. Breton M. Chap H. FEBS Lett. 1994; 341: 113-118Crossref PubMed Scopus (11) Google Scholar). After 30 min at 37 °C, reaction was stopped by addition of acidified chloroform/methanol and the lipids were immediately extracted as described above. Radiolabeled phosphoinositides were dried under a nitrogen stream and suspended in HNE buffer containing 30 mm Hepes, pH 7.0, 100 mm NaCl, and 1 mm EDTA. After sonication, HNE buffer supplemented with 0.5% (v/v) Nonidet P-40 was added to obtain a final concentration of 0.02% (v/v) Nonidet P-40 (34Rameh L.E. Chen C.-S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar). Thirty μl of this suspension were added to immunoprecipitated PLC-γ2 resuspended in 30 μl of HNE buffer supplemented with 0.02% (v/v) Nonidet P-40. After 45 min of incubation at room temperature under gentle shaking, samples were washed twice with 1 ml of HNE buffer supplemented with 0.2% (v/v) Nonidet P-40 (34Rameh L.E. Chen C.-S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar). Lipids that remained associated with the immune complex were extracted as described above and resolved by TLC using chloroform/acetone/methanol/acetic acid/water (20/30/26/24/14, v/v) (35Matsuo T. Hazeki K. Hazeki O. Katada T. Ui M. Biochem. J. 1996; 315: 505-512Crossref PubMed Scopus (31) Google Scholar). Platelet suspensions were adjusted to 5 × 109 cells/ml in 132 mm NaCl, 2.8 mm KCl, 0.86 mmMgCl2, 8.9 mm NaHCO3, 2 mm Hepes, 5.6 mm glucose, 12.2 mmNa3 citrate, and 10 mm Tris, pH 7.1 as described (36Kucera G.L. Rittenhouse S.E. J. Biol. Chem. 1990; 265: 5345-5348Abstract Full Text PDF PubMed Google Scholar). Then 100 μl of platelet suspension was mixed with 400 μl of buffer containing 120 mm KCl, 4 mmMgCl2, 25 mm NaCl, 1 mmNaH2PO4, 1 mm EGTA, 0.269 mm CaCl2 and 15 mm Hepes, pH 7.1. Platelets were incubated with 10 nm wortmannin or Me2SO for 15 min at 37 °C and then permeabilized during 3 min at 37 °C with 20 μg/ml saponin in the presence of 40 μCi [γ-32P]ATP (100 μm) with or without 15 μm diC16-PtdIns(3,4,5)P3 or diC16-PtdIns(3,4)P2. The F(ab′)2 fragments were added 1 min after monoclonal antibody IV.3. The incubation was stopped after 1 min by addition of chloroform/methanol (v/v), and lipids were extracted and resolved by TLC for PtdOH quantification as described above. Cross-linking of platelet FcγRIIA has been shown to induce a transient association of PI 3-kinase to the ITAM sequences present in the cytoplasmic tail of the receptor; however, its consequences on a possible in vivo activation of the enzyme were not emphasized (23Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Therefore, using an HPLC technique, we have first measured the time course of D3-phosphoinositide synthesis during FcγRIIA-mediated activation of 32P-labeled platelets. We found that PtdIns(3,4,5)P3 was rapidly produced, whereas PtdIns(3,4)P2 accumulated with a slower time course (Fig. 1 A). In addition, we observed a rapid drop in the substrate of PLC, PtdIns(4,5)P2, followed by its resynthesis (Fig. 1 B). This sharp decrease in PtdIns(4,5)P2 was concomitant with the production of PtdOH (Fig. 1 C), an event considered to reflect PLC activation in platelets. In these cells, the main part of diacylglycerol produced by PLC is converted into PtdOH by a diacylglycerol kinase, the contribution of phospholipase D being relatively minor (37Van der Meulen J. Haslam R.J. Biochem. J. 1990; 271: 693-700Crossref PubMed Scopus (60) Google Scholar, 38Huang R. Kucera G.L. Rittenhouse S.E. J. Biol. Chem. 1991; 266: 1652-1655Abstract Full Text PDF PubMed Google Scholar). In agreement with this, the production of PtdOH evoked by FcγRIIA cross-linking was abolished by the PLC-specific inhibitor U73122 (data not shown). Finally, the radioactivity of phosphatidylinositol 4-phosphate (PtdIns(4)P) did not change significantly over the whole period of platelet stimulation by FcγRIIA cross-linking (Fig. 1 D). To determine whether PI 3-kinase was required for FcγRIIA-mediated physiological responses, wortmannin and LY294002, two unrelated PI 3-kinase inhibitors, were used in platelet secretion and aggregation assays. As reported previously (23Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), platelet aggregation induced by FcγRIIA cross-linking was fully inhibited by low doses of wortmannin, whereas, in similar conditions, platelet aggregation induced by TRAP became reversible and thrombin-induced aggregation was not significantly affected (Fig. 2 A). Similar data were obtained with LY294002, which was able to fully inhibit FcγRIIA-mediated aggregation (95 ± 5% inhibition at 25 μm). Interestingly, platelet secretion evoked by FcγRIIA cross-linking was also strongly inhibited by wortmannin or LY294002 in a dose-dependent manner (Fig. 2 B). These results clearly indicated a critical role for PI 3-kinase in the early steps of FcγRIIA-dependent platelet aggregation. Under these conditions (10 nm wortmannin), the synthesis of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 was totally suppressed, while no change was observed for the other phosphoinositides (data not shown). However, interestingly, PtdOH production was almost abolished in platelets challenged with FcγRIIA cross-linking, whereas thrombin-induced accumulation of PtdOH remained insensitive to wortmannin (Fig. 3,A and B). Inhibition of PtdOH and D3-phosphoinositide production displayed very similar dose-response curves using the two inhibitors of PI 3-kinase, with IC50of 4 nm and 2 μm for wortmannin and LY294002, respectively (Fig. 3 C). These values are comparable to those determined for inhibition of serotonin secretion (6 nm and 5 μm, see Fig. 2 B). Finally, myrecithine, a natural flavonoid with inhibitory activity toward PI 3-kinase (39Agullo G. Gamet-Payrastre L. Manenti S. Viala C. Remesy C. Chap H. Payrastre B. Biochem. Pharmacol. 1997; 53: 1649-1657Crossref PubMed Scopus (519) Google Scholar), also blocked PtdOH formation. 2M.-P. Gratacap, B. Payrastre, C. Viala, G. Mauco, M. Plantavid, and H. Chap, unpublished observations. Specific inhibition of PI 3-kinase thus appeared to secondarily block PLC activation evoked by FcγRIIA cross-linking, at a variance with the signaling pathway evoked by thrombin. As a main difference between thrombin and FcγRIIA cross-linking, the former activates PLC-β2 and -β3, which are regulated by heterotrimeric G-proteins and are present in significant amounts in platelets (40Bristol J.A. Rhee S.G. Trends Endocrinol. Metab. 1994; 5: 402-406Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 41Banno Y. Nakashima S. Ohzawa M. Nozawa Y. J. Biol. Chem. 1996; 271: 14989-14994Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In contrast, PLC-γ2 appears as an essential intermediate in the signaling pathway evoked downstream of FcγRIIA, which involves its tyrosine phosphorylation presumably by Syk (25Yanaga F. Poole A. Asselin J. Blake R. Schieven G.L. Clark E.A. Law C.-L. Watson S.P. Biochem. J. 1995; 311: 471-478Crossref PubMed Scopus (134) Google Scholar, 42Blake R.A. Schieven G.L. Watson S.P. FEBS Lett. 1994; 353: 212-216Crossref PubMed Scopus (127) Google Scholar). Indeed, inhibition of Syk by a specific inhibitor, piceatannol, was found to inhibit secretion, aggregation, and PtdOH production induced by FcγRIIA cross-linking, the same effects being reproduced with the specific PLC inhibitor U73122 (data not shown). This led us to explore the effect of wortmannin on the tyrosine phosphorylation of PLC-γ2. We first investigated the whole pattern of phosphotyrosyl proteins in platelets stimulated by FcγRIIA cross-linking. As shown in Fig. 4 A, this was not affected even at a high concentration of wortmannin (100 nm). In contrast to thrombin, FcγRIIA cross-linking promoted its own transient tyrosine phosphorylation, as reported by others (43Blake R.A. Asselin J. Walker T. Watson S.P. FEBS Lett. 1994; 342: 15-18Crossref PubMed Scopus (26) Google Scholar, 44Robinson A. Gibbins J. Rodriguez-Linares B. Finan P.M. Wilson L. Kellie S. Findell P. Watson S.P. Blood. 1996; 88: 522-530Crossref PubMed Google Scholar). Again, this was insensitive to wortmannin. PLC-γ2 also displayed a transient tyrosine phosphorylation in response to FcγRIIA cross-linking. This reached a maximum at 1 min and decreased rapidly thereafter (Fig. 4, B, panel a, and C). Surprisingly neither wortmannin (10 nm) nor LY294002 (10 μm) altered the early phase of PLC-γ2 tyrosine phosphorylation, but they abolished the secondary dephosphorylation step (Fig. 4 B, panels b and c, and C). A clearer interpretation of the latter data became possible when we observed the same effects with the tetrapeptide RGDS, which inhibits platelet aggregation by competing for fibrinogen binding to integrin αIIb/β3or in the absence of shaking that also prevented aggregation (Fig. 5 A). It thus appeared that dephosphorylation of PLC-γ2 is a relatively late signaling event occurring downstream of integrin αIIb/β3engagement, which was shown previously to acti
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