Identification of a Unique Co-operative Phosphoinositide 3-Kinase Signaling Mechanism Regulating Integrin αIIbβ3 Adhesive Function in Platelets
2007; Elsevier BV; Volume: 282; Issue: 39 Linguagem: Inglês
10.1074/jbc.m704358200
ISSN1083-351X
AutoresSimone M. Schoenwaelder, Akiko Ono, Sharelle A. Sturgeon, Siew Mei Chan, P Mangin, Mhairi J. Maxwell, Shannon Turnbull, Megha Mulchandani, Karen E. Anderson, Gilles Kauffenstein, Gordon W. Rewcastle, Jackie D. Kendall, Christian Gachet, Hatem Hassan Salem, Shaun P. Jackson,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPhosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110β and p110γ) have been implicated in Gi-dependent integrin αIIbβ3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for Gi-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin αIIbβ3 activation in p110γ-/- platelets was not associated with alterations in the levels of PI(3,4)P2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110γ principally regulated integrin αIIbβ3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin αIIbβ3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y12/Gi-dependent integrinαIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways. Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110β and p110γ) have been implicated in Gi-dependent integrin αIIbβ3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for Gi-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin αIIbβ3 activation in p110γ-/- platelets was not associated with alterations in the levels of PI(3,4)P2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110γ principally regulated integrin αIIbβ3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin αIIbβ3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y12/Gi-dependent integrinαIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways. The phosphoinositide (PI) 2The abbreviations used are: PIphosphoinositidefMLPformylmethionylleucylphenylalaninePI(3,4)P2phosphatidylinositol 3,4-bisphosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-trisphosphateMAP kinasemitogen-activated protein kinaseHPLChigh performance liquid chromatography. 3-kinases are a well defined family of lipid kinases that participate in a broad range of signaling processes downstream of growth factor, antigen, hormone, and adhesion receptors (1Wymann M.P. Marone R. Curr. Opin. Cell Biol. 2005; 17: 141-149Crossref PubMed Scopus (179) Google Scholar, 2Huang E.J. Reichardt L.F. Annu. Rev. Biochem. 2003; 72: 609-642Crossref PubMed Scopus (1982) Google Scholar). They are classified into several distinct groups (types I-III), based on their primary structure, mode of regulation, and substrate specificity (3Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar, 4Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (764) Google Scholar). The most intensely studied members of the PI 3-kinase superfamily are the type I PI 3-kinases, due to their involvement in the regulation of fundamental cell processes, including proliferation, glucose metabolism, survival, and migration (1Wymann M.P. Marone R. Curr. Opin. Cell Biol. 2005; 17: 141-149Crossref PubMed Scopus (179) Google Scholar). PI 3-kinases principally transduce signals through the catalytic generation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2); second messengers that facilitate the recruitment of pleckstrin homology domain-containing signaling proteins to the plasma membrane (5Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Crossref PubMed Scopus (580) Google Scholar). The type I enzymes are divided into two subtypes, Ia and Ib; type Ia isoforms include p110α, -β, and -δ and type Ib includes a single isoform p110γ. p110α, -β, and -δ share common regulatory subunits (p85α, p85β, p55α, p55γ, p50α) and are classically regulated by tyrosine kinase-linked receptors, although G protein-coupled receptor-mediated activation of p110β has also been demonstrated (6Maier U. Babich A. Nurnberg B. J. Biol. Chem. 1999; 274: 29311-29317Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 7Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). PI 3-kinase p110γ contains structurally distinct p84 and p101 subunits that are typically regulated by G protein-coupled receptors, particularly Gβγi subunits (4Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (764) Google Scholar). phosphoinositide formylmethionylleucylphenylalanine phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,4,5-trisphosphate mitogen-activated protein kinase high performance liquid chromatography. There is a growing body of evidence demonstrating important functional specialization of individual type I PI 3-kinase isoforms. For example, p110α plays a key role in promoting cell growth and survival in response to growth factor and oncogenic stimulation (8Zhao J.J. Roberts T.M. Sci. STKE. 2006; : pe52PubMed Google Scholar, 9Zhao J.J. Cheng H. Jia S. Wang L. Gjoerup O.V. Mikami A. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16296-16300Crossref PubMed Scopus (181) Google Scholar). Moreover, gain-of-function mutations in p110α have oncogenic potential (10Wang J. Kuropatwinski K. Hauser J. Rossi M.R. Zhou Y. Conway A. Kan J.L. Gibson N.W. Willson J.K. Cowell J.K. Brattain M.G. Mol. 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In addition, individual family members may also amplify the signals other PI 3-kinases, i.e. the initial production of a small amount of 3-phosphoinositides may enhance subsequent activation of other family members. This may involve PI(3,4,5)P3 binding to p85 (35Rameh L.E. Chen C.S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar) or through recruitment of pleckstrin homology domain containing proteins that modulate PI 3-kinase activity. These mechanisms are not mutually exclusive and it is possible that multiple co-operative signaling processes operate in the cell. In the current study we have investigated the mechanisms underlying signaling co-operativity between p110γ and p110β in platelets. Our studies indicate that p110β predominately regulates Gi-dependent integrin αIIbβ3 activation through a classical lipid kinase-dependent mechanism, involving Rap1 and AKT, whereas p110γ appears to regulate integrin αIIbβ3 principally through a non-catalytic signaling mechanism. Moreover, inhibiting the kinase function of PI 3-kinases, in combination with p110γ deletion, led to a much greater defect in platelet adhesive function than inhibition of the catalytic function of PI 3-kinases alone. These studies demonstrate the existence of a co-operative PI 3-kinase signaling mechanism, involving the non-catalytic and catalytic function of p110γ and p110β, respectively. Furthermore, they demonstrate that this signaling mechanism is the predominant pathway utilized by the Gi-coupled P2Y12 receptor to regulate integrin αIIbβ3 adhesive function. These findings have potentially important implications for the antithrombotic potential of isoform-selective PI 3-kinase inhibitors. Materials—The synthesis and characterization of p110β (TGX221), p110δ (d-010), p110γ (AS252424), and p110α selective inhibitors (PIK-75) were as reported previously (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar, 31Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Hirsch E. Ruckle T. Camps M. Rommel C. Jackson S.P. Chilvers E.R. Stephens L.R. Hawkins P.T. Blood. 2005; 106: 1432-1440Crossref PubMed Scopus (257) Google Scholar, 36Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 37Sadhu C. Dick K. Treiberg J. Sowell G. Kesicki E. Oliver A. 2003Google Scholar). Oregon Green-labeled fibrinogen was from Molecular Probes (Eugene, OR). All other reagents were from sources previously described (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar, 38Maxwell M.J. Westein E. Nesbitt W.S. Giuliano S. Dopheide S.M. Jackson S.P. Blood. 2007; 109: 566-576Crossref PubMed Scopus (176) Google Scholar, 39Maxwell M.J. Yuan Y. Anderson K.E. Hibbs M.L. Salem H.H. Jackson S.P. J. Biol. Chem. 2004; 279: 32196-32204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Mouse Strains—All procedures involving the use of mice were approved by the Alfred Medical Research and Education Precinct (AMREP) animal ethics committee (AEC) (Melbourne, Australia). p110γ-deficient mice (p110γ-/-) (AEC number E/0275/2004/M) and p110δ deficient mice (p110δ-/-) (AEC number E/0299/2004/M) were from sources previously described (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar). P2Y1-deficient mice (P2Y1-/-) (AEC number E/0299/2004/M) were generated as described previously (40Leon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. J. Clin. Investig. 1999; 104: 1731-1737Crossref PubMed Scopus (403) Google Scholar). Preparation of Washed Platelets—Preparation of washed murine platelets was performed as described previously (39Maxwell M.J. Yuan Y. Anderson K.E. Hibbs M.L. Salem H.H. Jackson S.P. J. Biol. Chem. 2004; 279: 32196-32204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Measurement of Integrin αIIbβ3 Activation—Integrin αIIbβ3 activation was assessed by measurement of Oregon Green-labeled fibrinogen binding, as described previously (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar). Background (nonspecific) Oregon Green-labeled fibrinogen binding was determined on ADP-stimulated platelets pre-treated with GPI562 (100 μm). Specific Oregon Green-labeled fibrinogen binding was determined by subtracting nonspecific fluorescence readings from total fluorescence obtained from ADP-stimulated platelets. HPLC-based Phospholipid Analysis—Washed mouse platelets were labeled with 0.5 mCi/ml inorganic [32P]H3PO4, as described previously (39Maxwell M.J. Yuan Y. Anderson K.E. Hibbs M.L. Salem H.H. Jackson S.P. J. Biol. Chem. 2004; 279: 32196-32204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and stimulated with ADP (10 μm) for the indicated time periods. Lipids were extracted and separated by HPLC according to a modified method of Stephens et al. (41Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). PI peaks co-eluting with commercially available PI(3,4)P2 and PI(3,4,5)P3 standards were integrated and normalized to the total lipid applied. Measurement of Rap1 Activation—The level of GTP-bound Rap1 in mouse platelet lysates was measured as described previously (28Woulfe D. Jiang H. Mortensen R. Yang J. Brass L.F. J. Biol. Chem. 2002; 277: 23382-23390Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 29Lova P. Paganini S. Hirsch E. Barberis L. Wymann M. Sinigaglia F. Balduini C. Torti M. J. Biol. Chem. 2003; 278: 131-138Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Measurement of AKT Activation—AKT activation was measured by Western blot analysis, using a phosphoserine-473 AKT polyclonal antibody to detect active AKT, and an AKT polyclonal antibody (Cell Signaling Technology, MA) to determine total AKT, as described previously (42Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Crossref PubMed Scopus (207) Google Scholar). PI 3-Kinase Lipid Kinase Assays—Lipid kinase assays were performed to determine the level of PI 3-kinase inhibition achieved in platelets following preincubation of mouse whole blood with vehicle/wortmannin, or following administration (intravenous bolus) of vehicle/wortmannin in mice. Platelets were isolated from whole blood as described above, lysed, and PI 3-kinase immunoprecipitated from platelet lysates using an anti-p85 polyclonal antibody (Upstate Biotechnology). Kinase assays were performed on immunoprecipitated PI 3-kinase according to a previously described method (43Foukas L.C. Daniele N. Ktori C. Anderson K.E. Jensen J. Shepherd P.R. J. Biol. Chem. 2002; 277: 37124-37130Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Immunoprecipitations were also were performed in the absence of the anti-p85 polyclonal antibody, and the resulting PI 3-kinase activity subtracted as background. Platelet Aggregation Studies—For aggregation studies, washed mouse platelets (2.0 × 108/ml) were preincubated with vehicle alone, LY294002 (25 μm), TGX221 (0.5 μm), D-010 (5 μm), or AS252424 (1-10 μm) prior to stimulation with the indicated concentrations of ADP in the presence of calcium (1 mm) and fibrinogen (500 μg/ml). All aggregations were initiated by stirring the suspensions at 600 rpm for 10 min at 37 °C in a four-channel automated platelet analyzer (AggRAM, Haem). The extent of platelet aggregation was defined as the percentage change in optical density as measured by the automated platelet analyzer. In Vitro Thrombus Formation under Flow—Flow-based thrombus formation assays on a bovine fibrillar type I collagen matrix were performed as described previously (44Mangin P. Yap C.L. Nonne C. Sturgeon S.A. Goncalves I. Yuan Y. Schoenwaelder S.M. Wright C.E. Lanza F. Jackson S.P. Blood. 2006; 107: 4346-4353Crossref PubMed Scopus (126) Google Scholar). Anticoagulated whole blood (100 μg/ml lepirudin) collected from p110γ+/+ or p110γ-/- mice was labeled with DiOC6 (1 μm) and preincubated with vehicle (Me2SO) or wortmannin (400 nm) (10 min, 37 °C) prior to perfusion through fibrillar type I collagen-coated microcapillary tubes (2.5 mg/ml) at 1800 s-1 for up to 2.5 min. Thrombus formation was observed using an inverted Leica DMIRB microscope (Leica Microsystems, Wetzlar, Germany) and a ×63, 1.2 numeric aperture water objective. Images were acquired using a Dage-MTI charge-coupled device camera 300 ETRCX (Dage-MTI, Michigan City, IN). Analysis of thrombi volume and height was performed after 2.5 min (where thrombi reached a maximal volume in vehicle-treated p110γ+/+ platelets), as described previously. Total thrombus volume and maximum height of thrombi in a given field (covering 25,058.89 μm2) were calculated using Metamorph 6. Electrolytic Model of Occlusive Thrombus Formation—An in vivo electrolytic model of occlusive thrombus formation was performed as described previously (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar, 44Mangin P. Yap C.L. Nonne C. Sturgeon S.A. Goncalves I. Yuan Y. Schoenwaelder S.M. Wright C.E. Lanza F. Jackson S.P. Blood. 2006; 107: 4346-4353Crossref PubMed Scopus (126) Google Scholar). p110γ+/+ and p110γ-/- mice were administered (intravenous bolus) vehicle (10% Me2SO) or the indicated concentrations of wortmannin, prior to induction of electrolytic injury. Blood flow was monitored for 60 min, and changes in blood flow recorded offline for analysis. The total amount of blood flowing through the injured artery following vascular injury was determined by calculating the area under the blood flow curve, corrected for body weight. Tail Bleeding Studies in Mice—Tail bleeding time was measured in anesthetized (pentobarbitone 60 mg/kg, intraperitoneal), ventilated p110γ+/+ and p110γ-/- mice, with body temperature maintained at 37 °C throughout the experiment (16Jackson S.P. Schoenwaelder S.M. Goncalves I. Nesbitt W.S. Yap C.L. Wright C.E. Kenche V. Anderson K.E. Dopheide S.M. Yuan Y. Sturgeon S.A. Prabaharan H. Thompson P.E. Smith G.D. Shepherd P.R. Daniele N. Kulkarni S. Abbott B. Saylik D. Jones C. Lu L. Giuliano S. Hughan S.C. Angus J.A. Robertson A.D. Salem H.H. Nat. Med. 2005; 11: 507-514Crossref PubMed Scopus (534) Google Scholar, 44Mangin P. Yap C.L. Nonne C. Sturgeon S.A. Goncalves I. Yuan Y. Schoenwaelder S.M. Wright C.E. Lanza F. Jackson S.P. Blood. 2006; 107: 4346-4353Crossref PubMed Scopus (126) Google Scholar). Tail bleeding was measured before and 10 min after administration of vehicle or wortmannin (0.1 mg/kg). Incisions 5-mm long and 1-mm deep were made, 10 mm from the tip of the tail, and bleeding was monitored by blotting the edge of the incision with a tissue every 30 s until it had ceased (=tail bleeding time). Histology—Carotid arteries were paraffin-embedded, processed, and stained using the Carstair stain, as described previously (44Mangin P. Yap C.L. Nonne C. Sturgeon S.A. Goncalves I. Yuan Y. Schoenwaelder S.M. Wright C.E. Lanza F. Jackson S.P. Blood. 2006; 107: 4346-4353Crossref PubMed Scopus (126) Google Scholar). Using this staining technique, fibrin appears red, platelets appear blue/purple, red blood cells appear yellow, and collagen staining in the vessel wall appears blue. Statistical Analysis—Data are presented as mean ± S.E. Average S.E. for carotid blood flow over time was calculated from repeated measures of analysis variance. When comparing matched values between two treatment groups, an unpaired Student's t test with two-way analysis of variance was used (Prism software, GraphPAD Software for Science, San Diego, CA) (not significant; p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Relative Roles of Type I PI 3-Kinase Isoforms in Gi-dependent Integrin αIIbβ3 Activation—ADP is an important platelet agonist stimulating integrin αIIbβ3 activation through two purinergic receptors, P2Y1 and P2Y12 (45Gachet C. Pharmacol. Ther. 2005; 108: 180-192Crossref PubMed Scopus (96) Google Scholar). P2Y1 is a Gq-coupled receptor that induces activation of phospholipase Cβ and transient calcium mobilization, whereas P2Y12 is a Gi-coupled receptor linked to the inhibition of adenylyl cyclase and activation of PI 3-kinase (45Gachet C. Pharmacol. Ther. 2005; 108: 180-192Crossref PubMed Scopus (96) Google Scholar). ADP binding to P2Y1 stimulates transient integrin αIIbβ3 activation, whereas P2Y12 amplifies and sustains integrin αIIbβ3 activation, necessary for stable platelet aggregation. To examine the contribution of PI 3-kinases to integrin αIIbβ3 activation over a broad range of ADP concentrations, fibrinogen binding studies were performed on murine platelets in the presence of the pan-PI 3-kinase inhibitors, LY294002 or wortmannin. As demonstrated in Fig. 1, stimulation of platelets with increasing concentrations of ADP led to a dose-dependent increase in fibrinogen binding (Fig. 1). This increase was specific to integrin αIIbβ3 as it was completely eliminated by an anti-integrin αIIbβ3 receptor antagonist (data not shown). Pretreating platelets with LY294002 decreased fibrinogen binding by 75-85% over a broad range of ADP concentra
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