A Critical Role for Phospholipase Cγ2 in αIIbβ3-mediated Platelet Spreading
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305077200
ISSN1083-351X
AutoresPeter Wonerow, Andrew C. Pearce, David J. Vaux, Steve P. Watson,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe interaction of fibrinogen with the integrin αIIbβ3 plays a crucial role in platelet adhesion and platelet activation leading to the generation of intracellular signals that nucleate the reorganization of the cytoskeleton. Presently, we have only a limited understanding of the signaling cascades and effector proteins through which changes in the cytoskeletal architecture are mediated. The present study identifies phospholipase Cγ2 (PLCγ2) as an important target of the Src-dependent signaling cascade regulated by αIIbβ3. Real time phasecontrast microscopy is used to show that formation of filopodia and lamellapodia in murine platelets on a fibrinogen surface is dramatically inhibited in the absence of PLCγ2. Significantly, the formation of these structures is mediated by Ca2+ elevation and activation of protein kinase C, both directly regulated by PLC activity. With the involvement of Syk, SLP-76, and Btk, αIIbβ3-induced PLCγ2 activation partly overlaps with the pathway used by the collagen receptor glycoprotein VI. Important differences, however, exist between the two signaling cascades in that activation of PLCγ2 by αIIbβ3 is unaltered in murine platelets, which lack the FcR γ-chain or the adaptor LAT, but is abolished in the presence of cytochalasin D. Therefore, PLCγ2 plays not only a crucial role in activation of αIIbβ3 by collagen receptors but also in αIIbβ3-mediated responses. The interaction of fibrinogen with the integrin αIIbβ3 plays a crucial role in platelet adhesion and platelet activation leading to the generation of intracellular signals that nucleate the reorganization of the cytoskeleton. Presently, we have only a limited understanding of the signaling cascades and effector proteins through which changes in the cytoskeletal architecture are mediated. The present study identifies phospholipase Cγ2 (PLCγ2) as an important target of the Src-dependent signaling cascade regulated by αIIbβ3. Real time phasecontrast microscopy is used to show that formation of filopodia and lamellapodia in murine platelets on a fibrinogen surface is dramatically inhibited in the absence of PLCγ2. Significantly, the formation of these structures is mediated by Ca2+ elevation and activation of protein kinase C, both directly regulated by PLC activity. With the involvement of Syk, SLP-76, and Btk, αIIbβ3-induced PLCγ2 activation partly overlaps with the pathway used by the collagen receptor glycoprotein VI. Important differences, however, exist between the two signaling cascades in that activation of PLCγ2 by αIIbβ3 is unaltered in murine platelets, which lack the FcR γ-chain or the adaptor LAT, but is abolished in the presence of cytochalasin D. Therefore, PLCγ2 plays not only a crucial role in activation of αIIbβ3 by collagen receptors but also in αIIbβ3-mediated responses. The integrin αIIbβ3 mediates platelet aggregation in cell suspensions and supports adhesion to fibrinogen and von Willebrand factor (vWf) 1The abbreviations used are: vWf, von Willebrand factor; 2-ABP, 2-aminoethoxydiphenylborate; BSA, bovine serum albumin; FAK, focal adhesion kinase; FcR γ-chain, Fc receptor γ-chain; Fura 2-AM, Fura 2-acetoxymethylester; PRP, platelet-rich plasma; GPVI, glycoprotein VI; PKC, protein kinase C; PBS, phosphate-buffered saline; PLCγ2, phospholipase Cγ2; BAPTA, 1,2-bis(2-aminophenoyl)ethane-N, N,N′,N′-tetraacetic acid.-coated surfaces. On resting platelets, the integrin is in a low affinity conformation that does not support binding to either adhesion molecule at their normal plasma concentrations. An increase in affinity of αIIbβ3, leading to fibrinogen and vWf binding, is mediated by inside-out signals from G protein-coupled and tyrosine kinase-linked agonists, and thereby promotes aggregation. In addition, binding to αIIbβ3 induces outside-in signals that lead to reorganization of the cytoskeleton and synergize with other agonists to mediate activation. The central role of the integrin αIIbβ3 in thrombosis and hemostasis is highlighted by the severe bleeding disorders in patients with Glanzmann thrombasthenia, which lack functional integrin. One of the earlier events to occur following ligation of αIIbβ3 is the activation of the tyrosine kinase Syk via one or more Src kinases (1Miranti C.K. Leng L. Maschberger P. Brugge J.S. Shattil S.J. Curr. Biol. 1998; 8: 1289-1299Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 2Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar). This leads to tyrosine phosphorylation of the adaptor molecule SLP-76, which is constitutively associated with a second adaptor SLAP-130 (3Obergfell A. Judd B.A. del Pozo M.A. Schwartz M.A. Koretzky G.A. Shattil S.J. J. Biol. Chem. 2001; 276: 5916-5923Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 4Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), also known as Fyn-binding protein or adhesion- and degranulation-promoting adapter protein (5da Silva A.J. Li Z. de Vera C. Canto E. Findell P. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7493-7498Crossref PubMed Scopus (234) Google Scholar, 6Griffiths E.K. Penninger J.M. Curr. Opin. Immunol. 2002; 14: 317-322Crossref PubMed Scopus (56) Google Scholar). Together with proteins of the Vav GTPase exchange family, the adapter Nck, and the actin-binding protein VASP, this cascade has been shown to lead to activation of phosphoinositol 3-kinase and phosphorylation of FAK, and subsequent reorganization of the cytoskeleton in αIIbβ3-transfected Chinese hamster ovary cells (7Phillips D.R. Nannizzi-Alaimo L. Prasad K.S. Thromb. Haemostasis. 2001; 86: 246-258Crossref PubMed Scopus (126) Google Scholar, 8Payrastre B. Missy K. Trumel C. Bodin S. Plantavid M. Chap H. Biochem. Pharmacol. 2000; 60: 1069-1074Crossref PubMed Scopus (99) Google Scholar, 9Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Crossref PubMed Scopus (192) Google Scholar). Evidence that this cascade mediates reorganization of the cytoskeleton in platelets by αIIbβ3 has been provided using kinase inhibitors and murine cells deficient in Src kinases, Syk and SLP-76 (2Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar, 3Obergfell A. Judd B.A. del Pozo M.A. Schwartz M.A. Koretzky G.A. Shattil S.J. J. Biol. Chem. 2001; 276: 5916-5923Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Judd B.A. Myung P.S. Leng L. Obergfell A. Pear W.S. Shattil S.J. Koretzky G.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12056-12061Crossref PubMed Scopus (73) Google Scholar). Recently, we have shown that αIIbβ3 as well as the receptor for vWF, the glycoprotein (GP) Ib-IX-V complex, stimulate tyrosine phosphorylation of PLCγ2 (11Wonerow P. Obergfell A. Wilde J.I. Bobe R. Asazuma N. Brdicka T. Leo A. Schraven B. Horejsi V. Shattil S.J. Watson S.P. Biochem. J. 2002; 364: 755-765Crossref PubMed Scopus (89) Google Scholar, 12Marshall S.J. Asazuma N. Best D. Wonerow P. Salmon G. Andrews R.K. Watson S.P. Biochem. J. 2002; 361: 297-305Crossref PubMed Scopus (36) Google Scholar). PLCγ2 is known to be a major target of signaling by the collagen receptor GPVI in platelets. GPVI exists in a complex with the Fc receptor γ-chain (FcR γ-chain), which contains one copy of an immunoreceptortyrosine-based activation motif (13Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (261) Google Scholar). GPVI activates platelets through tyrosine phosphorylation of the FcR γ-chain immunoreceptor tyrosine-based activation motif by the Src kinases Lyn and Fyn and recruitment of Syk (14Briddon S.J. Watson S.P. Biochem. J. 1999; 338: 203-209Crossref PubMed Scopus (82) Google Scholar, 15Ezumi Y. Shindoh K. Tsuji M. Takayama H. J. Exp. Med. 1998; 188: 267-276Crossref PubMed Scopus (186) Google Scholar, 16Quek L.S. Pasquet J.M. Hers I. Cornall R. Knight G. Barnes M. Hibbs M.L. Dunn A.R. Lowell C.A. Watson S.P. Blood. 2000; 96: 4246-4253Crossref PubMed Google Scholar). Syk regulates a cascade that involves the adapters LAT, Gads, and SLP-76, the Tec family kinase Btk, and phosphatidylinositol 3-kinase (17Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Crossref PubMed Scopus (104) Google Scholar, 18Gross B.S. Melford S.K. Watson S.P. Eur. J. Biochem. 1999; 263: 612-623Crossref PubMed Scopus (57) Google Scholar, 19Gross B.S. Lee J.R. Clements J.L. Turner M. Tybulewicz V.L. Findell P.R. Koretzky G.A. Watson S.P. J. Biol. Chem. 1999; 274: 5963-5971Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Functional activation of PLCγ2 downstream of this cascade is crucial and absolutely necessary for platelet activation by collagen (20Wang D. Feng J. Wen R. Marine J.C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). In contrast, phosphorylation of PLCγ2 downstream of GPIb-IX-V does not lead to a functional activation of the phospholipase suggesting that PLCγ2 activity is not required for GPIb-IX-V-mediated signals (12Marshall S.J. Asazuma N. Best D. Wonerow P. Salmon G. Andrews R.K. Watson S.P. Biochem. J. 2002; 361: 297-305Crossref PubMed Scopus (36) Google Scholar). The role of PLCγ2 downstream of the fibrinogen receptor αIIbβ3 is not known. In the present study, we show that PLCγ2 is activated downstream of the fibrinogen receptor αIIbβ3 and that this plays a critical role in spreading through the mobilization of calcium and activation of protein kinase C. In addition, we also identify a number of additional proteins that are regulated downstream of the integrin-regulated signaling cascade but demonstrate important differences with the cascade used by GPVI. The present study expands the role of the PLCγ2 in platelet activation by demonstrating a central role in remodeling of the cytoskeleton by immunoreceptor tyrosine-based activation motif and integrin receptors. Antibodies and Reagents—PLCγ2 and anti-Syk polyclonal antibodies were from previously described sources (16Quek L.S. Pasquet J.M. Hers I. Cornall R. Knight G. Barnes M. Hibbs M.L. Dunn A.R. Lowell C.A. Watson S.P. Blood. 2000; 96: 4246-4253Crossref PubMed Google Scholar). Polyclonal rabbit anti-FAK antibody was from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Fibrinogen depleted of plasminogen and vWf were from Kordia Laboratory Supplies, Leiden, NL. 2-aminoethoxydiphenylborate (2-ABP) was from Tocris (Ellisville, MO), the PLC inhibitor U71322 and the PKC inhibitor Ro 31-8220 were from Calbiochem. All other reagents were from Sigma or previously named sources (11Wonerow P. Obergfell A. Wilde J.I. Bobe R. Asazuma N. Brdicka T. Leo A. Schraven B. Horejsi V. Shattil S.J. Watson S.P. Biochem. J. 2002; 364: 755-765Crossref PubMed Scopus (89) Google Scholar, 19Gross B.S. Lee J.R. Clements J.L. Turner M. Tybulewicz V.L. Findell P.R. Koretzky G.A. Watson S.P. J. Biol. Chem. 1999; 274: 5963-5971Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21Pasquet J.M. Gross B. Quek L. Asazuma N. Zhang W. Sommers C.L. Schweighoffer E. Tybulewicz V. Judd B. Lee J.R. Koretzky G. Love P.E. Samelson L.E. Watson S.P. Mol. Cell. Biol. 1999; 19: 8326-8334Crossref PubMed Google Scholar). Animals—C57B1/6 mice deficient in FcR γ-chain were obtained as previously described (22Park S.Y. Arase H. Wakizaka K. Hirayama N. Masaki S. Sato S. Ravetch J.V. Saito T. Eur. J. Immunol. 1995; 25: 2107-2110Crossref PubMed Scopus (47) Google Scholar). Mice deficient in the adapter LAT were obtained as previously described and bred from heterozygotes on a B6 background (23Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (227) Google Scholar). Mice deficient in PLCγ2 were generated as described (20Wang D. Feng J. Wen R. Marine J.C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar) and bred from heterozygotes on a B6 background. Wild type littermates were used as controls. Preparation of Human Platelets—Human blood was taken from drug-free volunteers on the day of the experiment and drawn into sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation of the blood samples at 200 × g for 20 min. Platelets were isolated from PRP by centrifugation at 800 × g for 10 min in the presence of prostacyclin (0.1 μg/ml). The pellet was resuspended in modified Tyrode's-HEPES buffer (134 mm NaCl, 0,34 mm Na2HPO4, 2,9 mm KCl, 12 mm NaHCO3, 20 mm HEPES, 5 mm glucose, 1 mm MgCl2, pH 7.3) containing 0.1 μg/ml prostacyclin. The platelets were recentrifuged at 800 × g for 10 min and resuspended at 5 × 108 cells/ml in Tyrode's-HEPES buffer. Preparation of Mouse Platelets—Blood (750–1000 μl) was taken into 100 μl of acid citrate dextrose by cardiac puncture under terminal CO2 narcosis. PRP was obtained by centrifugation of the blood samples at 300 × g for 10 min at room temperature. PRP was centrifuged at 1000 × g in the presence of prostacyclin (0.1 μg/ml) for 6 min at room temperature. The pellet was resuspended in a modified Tyrode's-HEPES buffer to the required concentration and left for 30 min at room temperature prior to stimulation. Adhesion Assays—Surfaces (Petri dishes, 6 well plates, coverslips) were coated with fibrinogen (200 μg/ml) or BSA (5 mg/ml) overnight at 4 °C. Surfaces were washed twice with PBS, blocked with denatured BSA (5 mg/ml) for 1 h, and washed again twice with PBS before use in the spreading experiments. Platelets did not adhere or become activated to surfaces coated with BSA. To study tyrosine phosphorylation events in response to ligation to αIIbβ3, platelets (5 × 108) were incubated for 30 min in dishes coated with fibrinogen or BSA in the presence of 2 units/ml apyrase and 10 μm indomethacin (24Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). Dishes coated with fibrinogen were washed twice with PBS to remove non-adherent cells. Platelets adherent to fibrinogen or in suspension over BSA were lysed in ice-cold immunoprecipitation buffer and lysates were subjected to immunoprecipitation assays or used directly for SDS-PAGE. Immunoprecipitation and Immunoblotting—Resting and convulxin-stimulated platelets (5 × 108/ml, 500 μl) were lysed by adding an equal volume of ice-cold IP buffer (2% (v/v) Nonidet P-40, 20 mm Tris, 300 mm NaCl, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, pH 7.3). Samples were pre-cleared for 1 h at 4 °C with protein A- or G-Sepharose (50% (w/v) in Tris-buffered saline plus Tween 20 (TBS-T: 20 mm Tris, 137 mm NaCl, 0.1% (v/v) Tween 20, pH 7.6). Antibodies were added and samples were rotated overnight at 4 °C. The Sepharose pellet was washed sequentially in lysis buffer and TBS-T before addition of Laemmli sample buffer. Proteins were separated by SDS-PAGE on 10% gels and electrically transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 10% (w/v) BSA dissolved in TBS-T. Antibodies were diluted in TBS-T containing 2% (w/v) BSA and incubated with polyvinylidene difluoride membranes for 1 h at room temperature. Membranes were washed in TBS-T after each incubation and developed using an enhanced chemiluminescence system (ECL, Amersham Biosciences, Cardiff, United Kingdom). Measurement of Platelet Cytosolic Ca2 + Concentration—Platelets isolated from PRP were resuspended in modified Tyrode's-HEPES buffer to a concentration of 3 × 108 cells/ml and incubated with Fura 2-acetoxymethylester (Fura 2-AM, 3 μm, 1 h, 30 °C) (Molecular Probes, Eugene, OR). After being washed in Tyrode's-HEPES buffer, platelets were resuspended at 2 × 107 cells/ml in the presence of 2 units/ml apyrase and 10 μm indomethacin. Platelets were allowed to spread on a fibrinogen-coated coverslip and real time calcium imaging was performed using Openlab software (Improvision, Coventry, UK). Microscopy—Platelets (1.5 × 107 in 0.5 ml of Tyrode's-HEPES buffer) were added to fibrinogen-coated coverslips and incubated for 30 min at 37 °C. Non-adherent platelets were washed away and attached platelets were fixed with 3.7% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in PBS. Platelets were stained for F-actin with rhodamine-conjugated phalloidin and viewed under an inverted fluorescence microscope using Openlab imaging software (Improvision). Platelet spreading was observed in real time at 37 °C and recorded using time-lapse laser scanning phase-contrast microscopy. The system consisted of a Bio-Rad Microradiance laser scanning instrument (Bio-Rad) attached to a Zeiss Axiovert inverted microscope equipped with a Solent Scientific environmental chamber; a Ph3 Plan-apochromat 63 × 1.4 NA objective was used and the images were acquired using Laser-sharp 2000 (version 4.2) software. Determination of Phosphatidic Acid Production—Platelets were suspended in Tyrode's-HEPES without phosphate and were labeled with [32P]orthophosphate (0.5 mCi/ml) for 1 h at 30 °C. Platelets were washed twice and resuspended in Tyrode's buffer at a concentration of 5 × 108/ml. Platelets were incubated for 30 min in 6-well plates (5 × 108/well) coated with fibrinogen or over BSA in the presence of 2 units/ml apyrase and 10 μm indomethacin. Dishes coated with fibrinogen were washed twice with PBS to remove non-adherent cells. Platelets adherent to fibrinogen or in suspension over BSA were lysed in a buffer containing 100 mm EDTA, 5 n HCl, and 1% Nonidet P-40. Phospholipids were extracted by addition of 400 μl of chloroform-methanol-HCl (100/200/1, v/v/v), and [32P]phosphatidic acid was separated by thin-layer chromatography. Thin-layer chromatography plates were exposed to Kodak PhosphorScreen and phosphatidic acid signals were quantified using Molecular Imager FX and Quantity One Software version 4 for Macintosh (Bio-Rad) Statistical analyses were performed using Student's t test. Differential Effects of Cytochalasin D on α IIb β 3 and GPVI-induced Phosphorylation—It has been shown previously that tyrosine phosphorylation by αIIbβ3 is regulated through activation of a Src family kinase and that it is modulated by disruption of actin polymerization using cytochalasin D (2Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar). Thus αIIbβ3-mediated tyrosine phosphorylation events can be divided into those that are mediated upstream and downstream of actin polymerization. We have compared the effect of the actin polymerization inhibitor cytochalasin D and the Src kinase inhibitor PP2 on signals mediated by αIIbβ3 and GPVI. Stimulation of αIIbβ3 was achieved by incubation of platelets over a fibrinogen-coated matrix for 30 min. GPVI was activated with the snake toxin convulxin under stirring conditions in suspension using an incubation time of 30 s, a time point known to be the peak for tyrosine phosphorylation induced by this agonist. αIIbβ3 and GPVI induce overlapping patterns of increases in tyrosine phosphorylation as measured using whole cell lysates. Both stimuli induced marked increases in tyrosine-phosphorylated bands of 70–80 kDa, which co-migrate with Syk, Btk, and SLP-76. The constitutively phosphorylated bands between 50 and 60 kDa co-migrate with Src family kinases. There is also a marked increase in a band of 130 kDa and a number of minor bands around this molecular mass in response to the two agonists. In contrast, a doublet of tyrosine-phosphorylated proteins with a molecular mass between 28 kDa and 32 kDa is regulated downstream of αIIbβ3 but not by GPVI (Fig. 1A, arrows). Convulxin stimulates marked tyrosine phosphorylation of proteins of 36 and 12 kDa, which co-migrate with LAT and the FcR γ-chain, respectively (Fig. 1A, arrows). Both signaling cascades are strongly dependent on the presence of functionally active Src kinases. Incubation of platelets with 20 μm PP2 causes a complete loss of inducible tyrosine phosphorylation following convulxin stimulation and markedly inhibits the response to fibrinogen, although tyrosine phosphorylation of a 130-kDa band is preserved (Fig. 1A, arrow). Cytochalasin D has a distinct effect on the degree of phosphorylation by αIIbβ3 and GPVI. Whereas the response to GPVI is only slightly inhibited by 10 μm cytochalasin D, there is a marked reduction in tyrosine phosphorylation of all bands by αIIbβ3 showing that it has a much greater dependence on the reorganization of the cytoskeleton (Fig. 1A). As a prelude to comparing the signaling cascades regulated by αIIbβ3 and GPVI, we investigated some of the proteins that undergo increases in tyrosine phosphorylation following incubation of platelets over a fibrinogen-coated matrix for 30 min through immunoprecipitation with specific antibodies and Western blotting with the antiphosphotyrosine antibody, 4G10. In agreement with studies from other groups, platelet spreading over fibrinogen leads to a substantial phosphorylation of the integrin β3 subunit (Fig. 1C), tyrosine kinase Syk, focal adhesion kinase (FAK) (Fig. 1B), adapter proteins SLP-76 (Fig. 1C) and SLAP-130 as well as the guanine nucleotide exchange factor Vav1 and the ubiquitin-regulator c-Cbl (not shown). In addition, we identified a number of proteins that have been previously shown to be phosphorylated downstream of GPVI, namely the FcR γ-chain, the Tec family tyrosine kinase Btk (Fig. 1C), as well as PLCγ2 (Fig. 1B). The adapter molecules Grb2 and Gads were also recruited to signaling complexes downstream of αIIbβ3 and formed associations with unidentified tyrosine-phosphorylated proteins of 32, 55, and 150 kDa, and 50, 76, and 130 kDa, respectively (not shown). There was, however, one important omission in the proteins that undergo tyrosine phosphorylation downstream of αIIbβ3 relative to GPVI, namely the adapter LAT. Whereas LAT is one of the major tyrosine-phosphorylated proteins downstream of GPVI we were not able to detect an increase in LAT phosphorylation after αIIbβ3 stimulation (Fig. 1B). These data demonstrate that platelet adhesion to fibrinogen leads to tyrosine phosphorylation of PLCγ2 and identifies many similarities but also important differences with the events regulated downstream of GPVI. The effect of cytochalasin D and PP2 on phosphorylation of Syk, LAT, FAK, and PLCγ2 by αIIbβ3 and GPVI was explored following their immunoprecipitation and Western blotting for phosphotyrosine. Tyrosine phosphorylation of Syk induced by αIIbβ3 and convulxin was reduced in the presence of cytochalasin D (Fig. 1B) and abolished in the presence of PP2 (not shown). In contrast, cytochalasin D had a differential effect on tyrosine phosphorylation of a band of 12 kDa, which co-precipitates with Syk and was shown through immunoprecipitation studies to be the FcR γ-chain (not shown). Tyrosine phosphorylation of FcR γ-chain by αIIbβ3 was abolished by treatment with cytochalasin D, whereas it was only slightly inhibited in convulxin-stimulated cells (Fig. 1B, arrows). This demonstrates that tyrosine phosphorylation of FcR γ-chain is dependent on actin polymerization in response to αIIbβ3. As already discussed, αIIbβ3 does not cause phosphorylation of LAT. In contrast LAT is strongly phosphorylated downstream of GPVI and this phosphorylation is reduced following cytochalasin D treatment (Fig. 1B) and abolished after inhibition of Src kinases with PP2 (not shown). In contrast αIIbβ3 stimulation induces a robust, cytochalasin D-sensitive phosphorylation of FAK, whereas convulxin stimulation only causes minimal tyrosine phosphorylation of FAK (Fig. 1B) even at times up to 30 min (not shown). Cytochalasin D had a differential effect on the regulation of tyrosine phosphorylation of PLCγ2 by αIIbβ3 and GPVI. Tyrosine phosphorylation of PLCγ2by αIIbβ3 was almost abolished in the presence of cytochalasin D, whereas the response to convulxin was inhibited by ∼50% (Fig. 1B). PP2 completely blocked tyrosine phosphorylation of PLCγ2 by both receptors (not shown). The differential effect of cytochalasin D on tyrosine phosphorylation of FcR γ-chain and PLCγ2 by αIIbβ3 and GPVI further distinguishes the two signaling cascades. Thus, activation of Syk by αIIbβ3 is independent of phosphorylation of FcR γ-chain and signals from the kinase are not mediated through tyrosine phosphorylation of LAT. In contrast, Syk is regulated downstream of FcR γ-chain by GPVI (25Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar) and signals are associated with robust phosphorylation of LAT. The differential role of FcR γ-chain in these two pathways agrees with previous work from the Shattil group (26Obergfell A. Buensuceso C. Shattil S.J. Blood. 100. The American Society of Hematology, Philadelphia2002: 122aGoogle Scholar) showing that activation of Syk by αIIbβ3 is through a Src kinase-dependent pathway but does not require prior phosphorylation of an immunoreceptor tyrosinebased activation motif-containing protein. Phosphorylation of PLCγ2 Downstream of α IIb β 3 Is Independent of the Adapter LAT and Fc Receptor γ-Chain—Previously, we have reported that tyrosine phosphorylation of Syk and PLCγ2 through GPVI is abolished in FcR γ-chain-deficient murine platelets, whereas tyrosine phosphorylation of PLCγ2 is markedly reduced in LAT-deficient cells (21Pasquet J.M. Gross B. Quek L. Asazuma N. Zhang W. Sommers C.L. Schweighoffer E. Tybulewicz V. Judd B. Lee J.R. Koretzky G. Love P.E. Samelson L.E. Watson S.P. Mol. Cell. Biol. 1999; 19: 8326-8334Crossref PubMed Google Scholar, 25Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar). We have now investigated the role of these two proteins in tyrosine phosphorylation of Syk and PLCγ2 by αIIbβ3. Adhesion of murine platelets to fibrinogen induced a marked increase in tyrosine phosphorylation of Syk and PLCγ2, similar to that seen in human platelets. This increase in phosphorylation was not altered in platelets deficient in FcR γ-chain or LAT (Fig. 2). Similarly, tyrosine phosphorylation of the adapter SLP-76 by fibrinogen was unaltered in platelets deficient in FcR γ-chain or LAT (Fig. 2B). As with human platelets, FcR γ-chain coprecipitated with Syk upon stimulation by αIIbβ3. This association was preserved in LAT-deficient platelets (Fig. 2A, arrow). These results confirm that neither FcR γ-chain nor LAT are required for tyrosine phosphorylation of Syk and PLCγ2 by αIIbβ3 in contrast to signals from GPVI. α IIb β 3 Stimulates Activation of PLCγ2, Calcium Elevation, and Spreading—The functional consequence of PLCγ2 phosphorylation downstream of αIIbβ3 was investigated by measurement of phosphatidic acid and intracellular calcium, two indirect markers of PLC activity. Platelets spread on a fibrinogen-coated surface had a 2.4 ± 0.1-fold increase in the production of phosphatidic acid relative to cells exposed to a BSA-coated surface (p = 0.005, Fig. 3A). The increase in phosphatidic acid was inhibited strongly in the presence of cytochalasin D (1.5 ± 0.1-fold) and reduced to basal levels using the PLC inhibitor U-73122 (0.96 ± 0.13-fold). PP2 also caused a complete inhibition of phosphatidic acid accumulation, reducing the level below that of cells exposed to BSA (0.57 ± 0.11-fold), strongly suggesting that the increase is mediated via PLCγ rather than PLCβ isoforms (Fig. 3A). To further clarify the role of PLCγ2 downstream of αIIbβ3 we determined the production of phosphatidic acid in PLCγ2-deficient murine platelets. As seen in human platelets, spreading of murine platelets on fibrinogen caused an increase in the production of phosphatidic acid (1.5 ± 0.08-fold) relative to cells exposed to a BSA-coated surface (1.0 ± 0.05-fold). This increase was inhibited following cytochalasin D treatment (0.90 ± 0.12-fold) and in PLCγ2- (1.13 ± 0.10-fold) deficient platelets (Fig. 3B). These data suggest that PLCγ2 is the main enzyme responsible for the increase in phosphatidic acid following spreading of platelets on fibrinogen. The increase in formation of phosphatidic acid was associated with a sustained increase in intracellular calcium, as measured using the calcium reporter Fura 2 (delivered as Fura 2-AM). All of the platelets that had undergone partial or complete spreading on fibrinogen had elevated levels of intracellular calcium. Moreover, the increase in calcium and spreading (measured by monitoring fluorescence at 380 nm) occurred in parallel as illustrated in Fig. 4B suggesting that they are causally related. The calcium increase was observed 2–3 min after the initial contact with the fibrinogen-coated surface and is accompanied by the formation of filopodia and lamellipodia. The increase in intracellular calcium and spreading were substantially inhibited by cytochalasin D and PP2 although they had only minimal effects on attachment of the platelets to the fibrinogen-coated surfaces (Fig. 4A). Calcium elevation and spreading was also strongly inhibited by 2-ABP an inositol 1,4,5-trisphosphate receptor antagonist. The increase in intracellular calcium was specific for αIIbβ3 because it was
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