Title: SHIP1 and Lyn Kinase Negatively Regulate Integrin αIIbβ3 Signaling in Platelets
Abstract: Integrin αIIbβ3 plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin αIIbβ3 signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin αIIbβ3-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P3 metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P3 metabolism down-regulates the stability of integrin αIIbβ3-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin αIIbβ3 adhesive and signaling function. Integrin αIIbβ3 plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin αIIbβ3 signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin αIIbβ3-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P3 metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P3 metabolism down-regulates the stability of integrin αIIbβ3-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin αIIbβ3 adhesive and signaling function. Integrins are a large family of heterodimeric transmembrane receptors mediating cell-cell and cell-matrix interactions. Adhesive events mediated through these receptors are fundamental to a broad range of physiological processes, including embryogenesis, inflammation, immunity, and hemostasis (1Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Google Scholar, 2Hynes R.O. Cell. 1987; 48: 549-554Google Scholar, 3Hynes R.O. Cell. 1992; 69: 11-25Google Scholar). A common feature of all integrins is their ability to respond to environmental cues, leading to changes in receptor conformation/clustering and a corresponding alteration in integrin adhesive function. The responsiveness of integrins is due in part to their ability to respond and transmit signals across the plasma membrane in a bi-directional manner, via inside-out and outside-in signaling processes. A major pathway involved in integrin bi-directional signaling involves the activation of phosphoinositide 3-kinases (PI 1The abbreviations used are: PI or PtdIns, phosphatidylinositol; SHIP1, Src homology 2 (SH2) domain-containing inositol 5′-phosphatase-1; FGN, fibrinogen; DIC, differential interference contrast; PWB, platelet wash buffer; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; TXA2, thromboxane A2; PECAM-1, platelet and endothelial cell adhesion molecule-1; Ab, antibody; HPLC, high performance liquid chromatography. 3-kinases). These enzymes phosphorylate membrane inositol phospholipids at the 3-OH position, and are classified into 3 distinct classes based on their primary structure, mode of regulation, and in vitro substrate specificity (reviewed in Ref. 4Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Google Scholar). The Class I PI 3-kinases are activated by a diverse array of cell surface receptors and principally phosphorylate the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), generating phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). This lipid is in turn rapidly metabolized by one or more 5-phosphatases producing phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have important second messenger functions within the cell, recruiting, and activating a wide range of pleckstrin homology (PH) domain-containing signaling proteins involved in the regulation of cell proliferation, survival, cytoskeletal remodeling, and glucose metabolism (for review see Refs. 5Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. Cell Signal. 2001; 13: 377-387Google Scholar and 6Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google Scholar). An integrin whose adhesive function is tightly regulated by PI 3-kinases is the platelet integrin, αIIbβ3. This receptor plays a key role in regulating a variety of functional platelet responses, including platelet spreading, aggregation, clot retraction and microvesiculation, and as such, has an indispensable role in hemostasis (7Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar, 8Phillips D.R. Nannizzi-Alaimo L. Prasad K.S. Thromb. Haemost. 2001; 86: 246-258Google Scholar). The precise molecular basis by which PI 3-kinase lipid products induce integrin αIIbβ3 activation has not been clearly defined, although several target enzymes, including AKT (PKB) (9Watanabe N. Nakajima H. Suzuki H. Oda A. Matsubara Y. Moroi M. Terauchi Y. Kadowaki T. Koyasu S. Ikeda Y. Handa M. Blood. 2003; 102: 541-548Google Scholar, 10Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Google Scholar), PLCγ2 (9Watanabe N. Nakajima H. Suzuki H. Oda A. Matsubara Y. Moroi M. Terauchi Y. Kadowaki T. Koyasu S. Ikeda Y. Handa M. Blood. 2003; 102: 541-548Google Scholar, 11Gratacap M.P. Payrastre B. Viala C. Mauco G. Plantavid M. Chap H. J. Biol. Chem. 1998; 273: 24314-24321Google Scholar, 12Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Google Scholar), and Rap1b (13Woulfe D. Jiang H. Mortensen R. Yang J. Brass L.F. J. Biol. Chem. 2002; 277: 23382-23390Google Scholar) are likely to be involved. PI 3-kinases also play an important role in integrin αIIbβ3 outside-in signaling, regulating functional responses such as shear-resistant platelet adhesion, spreading, and irreversible platelet aggregation (14Yap C.L. Anderson K.E. Hughan S.C. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99: 151-158Google Scholar, 15Mazzucato M. Pradella P. Cozzi M.R. De Marco L. Ruggeri Z.M. Blood. 2002; 100: 2793-2800Google Scholar, 16Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Google Scholar). The involvement of PI 3-kinases in integrin αIIbβ3 bi-directional signaling is well established, however the mechanisms negatively regulating integrin αIIbβ3 signaling have not been so clearly delineated. One possible candidate is the SH2 domain-containing inositol 5-phosphatase (SHIP1). SHIP1 has been demonstrated to be a key negative regulator of PI 3-kinase signaling in a variety of cell types (17Huber M. Helgason C.D. Scheid M.P. Duronio V. Humphries R.K. Krystal G. EMBO J. 1998; 17: 7311-7319Google Scholar, 18Liu Q. Oliveira-Dos-Santos A.J. Mariathasan S. Bouchard D. Jones J. Sarao R. Kozieradzki I. Ohashi P.S. Penninger J.M. Dumont D.J. J. Exp. Med. 1998; 188: 1333-1342Google Scholar, 19Liu Q. Sasaki T. Kozieradzki I. Wakeham A. Itie A. Dumont D.J. Penninger J.M. Genes Dev. 1999; 13: 786-791Google Scholar, 20Deuter-Reinhard M. Apell G. Pot D. Klippel A. Williams L.T. Kavanaugh W.M. Mol. Cell. Biol. 1997; 17: 2559-2565Google Scholar). It hydrolyzes the 5-position phosphate from PtdIns(3,4,5)P3 (21Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Google Scholar, 22Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar) and has recently been demonstrated to play an important role in PtdIns(3,4,5)P3 metabolism in platelets (23Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Google Scholar, 24Giuriato S. Pesesse X. Bodin S. Sasaki T. Viala C. Marion E. Penninger J. Schurmans S. Erneux C. Payrastre B. Biochem. J. 2003; 376: 199-207Google Scholar). Studies in thrombin-stimulated platelets have demonstrated that SHIP1 phosphorylation is regulated downstream of integrin αIIbβ3 (25Giuriato S. Payrastre B. Drayer A.L. Plantavid M. Woscholski R. Parker P. Erneux C. Chap H. J. Biol. Chem. 1997; 272: 26857-26863Google Scholar), raising the possibility that this enzyme participates in integrin αIIbβ3 outside-in signaling. However, one problem with such a hypothesis is that integrin αIIbβ3 has not previously been demonstrated to promote formation of PtdIns(3,4,5)P3 in platelets (26Gironcel D. Racaud-Sultan C. Payrastre B. Haricot M. Borchert G. Kieffer N. Breton M. Chap H. FEBS Lett. 1996; 389: 253-256Google Scholar). In fact, it has been suggested that integrin αIIbβ3 primarily stimulates PtdIns(3,4)P2 accumulation through the action of a Class II PI 3-kinase (possibly PI 3-kinase C2α), a signaling pathway that is not regulated by SHIP1 (27Zhang J. Banfic H. Straforini F. Tosi L. Volinia S. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 14081-14084Google Scholar, 28Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Google Scholar). In this study, we have examined the possibility that SHIP1 acts as a negative regulator of integrin αIIbβ3 outside-in signaling. Using SHIP1–/– mouse platelets, we have demonstrated that integrin αIIbβ3 can induce substantial amounts of PtdIns(3,4,5)P3 accumulation. Enhanced PtdIns(3,4,5)P3 accumulation was associated with accelerated calcium flux and an increased rate of platelet spreading. Moreover, under flow conditions, SHIP1 played an important role in negatively regulating the stability of integrin αIIbβ3-dependent adhesion contacts. Our studies have also identified an important role for the Src kinase family member, Lyn, in regulating SHIP1 phosphorylation and PtdIns(3,4,5)P3 accumulation. Our findings define an important role for SHIP1 and Lyn as negative regulators of integrin αIIbβ3 signaling. Materials—Apyrase was purified from potatoes according to the method of Molnar and Lorand (29Molnar J. Lorand L. Arch. Biochem. Biophys. 1961; 93: 353-363Google Scholar). Human fibrinogen (FGN) was purified from fresh frozen plasma according to Jakobsen et al. (30Jakobsen E. Ly B. Kierulf P. Thromb. Res. 1974; 4: 499-507Google Scholar). LY294002 and the Src kinase inhibitor PP2 was purchased from Calbiochem-Novabiochem Corp. Probenecid and acetylsalicylic acid (aspirin) were purchased from Sigma. 5,5′-Dimethyl-BAPTA, AM, (DM-BAPTA), Oregon Green 488 BAPTA-1, AM, and Fura Red, AM, were from Molecular Probes Inc. (Eugene, OR). The integrin αIIbβ3 antagonist GPI562 was synthesized and characterized in our laboratory according to the methods of Kottirsch et al. (31Kottirsch G. Zerwes H.-G. Cook N.S. Taparelli C. Bioorg. Med. Chem. Lett. 1997; 7: 727-732Google Scholar) and Choudhri et al. (32Choudhri T.F. Hoh B.L. Zerwes H.G. Prestigiacomo C.J. Kim S.C. Connolly Jr., E.S. Kottirsch G. Pinsky D.J. J. Clin. Investig. 1998; 102: 1301-1310Google Scholar). All other reagents were obtained from sources described previously (33Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Google Scholar, 34Yuan Y. Kulkarni S. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Google Scholar). Mouse Strains—129/C57Bl/6J SHIP1+/+ and SHIP1–/– mice (18Liu Q. Oliveira-Dos-Santos A.J. Mariathasan S. Bouchard D. Jones J. Sarao R. Kozieradzki I. Ohashi P.S. Penninger J.M. Dumont D.J. J. Exp. Med. 1998; 188: 1333-1342Google Scholar) were obtained from The Jackson Laboratory. C57Bl Lyn+/+ and Lyn–/– mice (35Hibbs M.L. Tarlinton D.M. Armes J. Grail D. Hodgson G. Maglitto R. Stacker S.A. Dunn A.R. Cell. 1995; 83: 301-311Google Scholar) were generated at the Ludwig Institute for Cancer Research (Melbourne, Australia). Antibodies—The anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology (Lake Placid, NY). Anti-SHIP1 polyclonal serum was a kind gift from Dr. Peter Parker (Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute). Platelet and Red Cell Preparation—Human blood was obtained from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Blood was anticoagulated with acid-citrate-dextrose (ACD) anticoagulant (13 mm sodium citrate, 1 mm citric acid, 20 mm dextrose, and 10 mm theophylline). Washed human platelets and red cells were prepared as previously described (33Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Google Scholar, 34Yuan Y. Kulkarni S. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Google Scholar). Human washed platelets were kept at 37 °C in platelet-washing buffer (PWB) (4.3 mm K2HPO4, 4.3 mm Na2HPO4, 24.3 mm NaH2PO4, pH 6.5, 113 mm NaCl, 5.5 mm glucose, 0.5% bovine serum albumin) containing theophylline (10 mm) and apyrase (0.04 units/ml), and then resuspended in Tyrode's buffer (10 mm Hepes, 12 mm NaHCO3, pH 7.4, 137 mm NaCl, 2.7 mm KCl, 5 mm glucose) prior to use in adhesion studies. Mouse platelets were isolated from blood collected from the inferior vena cava of anesthetized animals (60 mg/kg sodium pentobarbitone), anticoagulated with a combination of low molecular weight heparin (40 units/ml) and ACD. To obtain an optimal amount of platelet-rich plasma (PRP), blood was mixed with 300 μl of PWB and then centrifuged at 250 × g for 2 min. PRP was removed and a further 300 μl of PWB added to the remaining blood, which was centrifuged again at 250 × g for 2 min. This step was repeated, and platelets from the pooled PRP were pelleted by centrifugation at 2,000 × g for 1 min and resuspended in Tyrode's buffer for adhesion studies. Static Adhesion Assays—Static adhesion assays were performed using a modified method of Yuan et al. (36Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Google Scholar). Briefly, glass coverslips (12 mm in diameter; Lomb Scientific, Australia) were coated with fibrinogen (FGN) (100 μg/ml) for 2 h at room temperature. The uncoated glass surface was blocked with 5% heat-inactivated human serum pretreated with phenylmethylsulfonyl fluoride (50 μg/ml). Platelets (human at 2 × 107/ml; mouse at 3 × 107/ml) in Tyrode's buffer supplemented with 1 mm CaCl2 (or 1 mm EGTA and 2 mm MgCl2) were allowed to adhere to the fibrinogen matrix for the indicated period of time. Non-adherent platelets were removed by gentle washing, then adherent platelets fixed with 3.7% formaldehyde, and imaged using differential interference contrast (DIC) or phase contrast microscopy for surface area analysis as described previously (33Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Google Scholar). In control studies, non-stimulated platelets were fixed while suspended in Tyrode's buffer. The increase in surface area of adherent platelets was expressed in pixels following subtraction of the surface area of resting platelets. Where indicated, platelets were preincubated with vehicle alone (0.25% Me2SO), the PI 3-kinase inhibitors LY294002 (20 μm) or wortmannin (100 nm), the Src kinase inhibitor PP2 (10 μm) for 10 min at 37 °C prior to the performance of adhesion assays. To examine the role of ADP in platelet spreading on fibrinogen, platelet adhesion was performed in the presence of apyrase (1 units/ml, ADPase activity), while the role of TXA2 was assessed by pretreating platelets with aspirin (1 mm) for 30 min at 37 °C. The pharmacological activity of these inhibitors was confirmed as follows: PP2 (10 μm) abolished collagen (10 μg/ml)-induced platelet aggregation; apyrase (1 unit/ml) abolished ADP (25 μm)-induced platelet aggregation; aspirin (1 mm) blocked arachidonic acid (1.2 mm)-induced platelet aggregation; LY294002 (20 μm), and wortmannin (100 nm) inhibited thrombin (1 unit/ml)-induced PI 3-kinase lipid production. Mouse Platelet Adhesion to Fibrinogen under Flow Conditions—Flow assays were performed as described (33Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Google Scholar). Mouse whole blood anticoagulated with low molecular weight heparin (40 units/ml) was perfused over fibrinogen-coated (100 μg/ml) glass microcapillary tubes at a wall shear rate of 600 s–1 for 5 min. Platelet-matrix interactions were visualized using DIC microscopy (Leica DMIRB, Leica, Germany) and video-recorded for off-line analysis. Platelet tethering was quantitated in 5 random fields at the indicated times during perfusion, and normalized to platelet count (expressed as number of platelets per mm2, divided by the total number of platelets perfused over the 5-min period). Platelet count in blood from SHIP1–/– mice tended to be lower than that of SHIP1+/+ littermates (SHIP1–/–, 460 ± 111 × 109/liter; compared with SHIP1+/+, 590 ± 151 × 109/liter; n = 3, p = 0.296), although hematocrit was unaltered. In studies examining stability of platelet contacts, stationary adhesion was defined as an adherent platelet that did not move more than one cell diameter over a 10-s observation period. A tethered cell was classified as one which interacted with the matrix surface for a minimum of 40 ms. Analysis of Cytosolic Calcium Flux under Static and Flow Conditions—Washed mouse platelets were loaded with calcium dyes and changes in cytosolic calcium levels were monitored according to published methods (16Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Google Scholar, 37Goncalves I. Hughan S.C. Schoenwaelder S.M. Yap C.L. Yuan Y. Jackson S.P. J. Biol. Chem. 2003; 278: 34812-34822Google Scholar). Briefly, mouse platelets (2 × 108/ml in PWB) were loaded with Oregon Green 488 BAPTA-AM (1 μm) and Fura Red-AM (1.25 μm) for 30 min at room temperature in the presence of 1.25 μm Probenecid. Dye-loaded platelets were resuspended in Tyrode's buffer (3 × 107/ml) containing 1.25 mm Probenecid, and then allowed to adhere to fibrinogen-coated (100 μg/ml) glass coverslips under static conditions in the presence of CaCl2 (1 mm), or EGTA and MgCl2 (1 mm and 2 mm, respectively). For studies under flow conditions, platelets (1 × 108/ml) were reconstituted with packed red blood cells (50% hematocrit) in Tyrode's buffer supplemented with 1 mm CaCl2, then perfused through fibrinogen (100 μg/ml)-coated microcapillary tubes at 600 s–1. To examine changes in calcium flux, sequential confocal images of adherent platelets were captured at a scan rate of 0.568 frames/s for 200 frames at the indicated time points. Real-time platelet calcium flux was analyzed from ratiometric fluorescence measurements and converted to intracellular calcium concentrations as described (14Yap C.L. Anderson K.E. Hughan S.C. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99: 151-158Google Scholar, 16Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Google Scholar). SHIP1 Immunoprecipitation and Western Blot Analysis—Washed human or mouse platelets (3 × 107/ml) in 1 mm CaCl2 supplemented Tyrode's buffer were treated with vehicle (0.25% Me2SO) or PP2 (10 μm) for 10 min, then applied onto a fibrinogen (100 μg/ml)-coated 10-cm dish for 30 min at 37 °C. Non-adherent platelets were removed and adherent cells lysed with radioimmunoprecipitation assay buffer (10 mm Tris, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 158 mm NaCl, 2 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 2 mm benzamidine). To examine the level of SHIP1 tyrosine phosphorylation in resting cells, non-stimulated platelets suspended in Tyrode's buffer were lysed. All lysates were centrifuged at 15,000 × g for 5 min at 4 °C, and SHIP1 immunoprecipitated from the resultant supernatant using a specific anti-SHIP1 polyclonal Ab and protein A-Sepharose beads. The beads were washed, then boiled in reducing conditions (50 mm Tris, pH 6.8, 4 mm EDTA, 2 mm Na3VO4, 10% glycerol, 4% SDS, 10% β-mercaptoethanol, 0.002% bromphenol blue), and immunoprecipitated SHIP1 was subjected to 7.5% SDS-PAGE and immunoblotted using an anti-phosphotyrosine Ab (4G10). The membrane was then stripped and re-probed with the anti-SHIP1 antibody to examine SHIP1 protein loading. Western blots were quantified by densitometry (GelPro software), and SHIP1 tyrosine phosphorylation was expressed as a percentage of control platelets. HPLC-based Phospholipid Analysis—Human or mouse platelets were washed twice with phosphate-free Tyrode's buffer containing the platelet activation inhibitor theophylline (10 mm), then labeled with 0.3 mCi/ml inorganic [32P]H3PO4 for 2 h at 37 °C. Unincorporated [32P]H3PO4 was removed by washing platelets twice with phosphate-free Tyrode's buffer in the presence of theophylline (10 mm) and then resuspended in theophylline-free Tyrode's buffer containing 1 mm CaCl2. 32P-labeled platelets were allowed to adhere to a fibrinogen (100 μg/ml)-coated glass dish for 60 min at room temperature. Non-adherent platelets were removed, adherent cells lysed, then lipids extracted and separated by HPLC analysis according to the method of Stephens et al. (38Stephens 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: 105-114Google Scholar). In other studies, mouse platelets were suspended at 3 × 108/ml in Tyrode's buffer containing 1 mm CaCl2, and then stimulated with 1 unit/ml thrombin (or saline as a vehicle control) for 10 min. Control and thrombin-stimulated platelets were lysed and lipids extracted as detailed above. Phosphoinositide peaks co-eluting with commercially available PtdIns(3,4)P2 and PtdIns(3,4,5)P3 standards were integrated and expressed as a percentage of total phospholipid applied. Statistical Analysis—Statistical significance was analyzed using a Student's t test, using Prism software (GraphPad Software for Science, San Diego, CA). Adhesion-dependent PtdIns(3,4,5)P3 Formation in SHIP1–/– Platelets—To investigate the potential involvement of SHIP1 in integrin αIIbβ3 signal transduction, changes in the cellular levels of 3-phosphorylated phosphoinositides were examined in control (SHIP1+/+) and SHIP1-deficient (SHIP1–/–) platelets. In initial studies, lipid profiles of human and wild-type mouse platelets were examined in fibrinogen-adherent platelets (an integrin αIIbβ3-specific ligand), to determine if there were significant species differences. In both cases, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 were barely detectable in resting platelets; however, increases in PtdIns(3,4)P2 were readily observed in spreading platelets (see Fig. 1, A–C). Notably, there was also a very small increase in the cellular levels of PtdIns(3,4,5)P3 in both mouse and human platelets (Fig. 1, A and C). Time course adhesion studies (10–90 min) did not reveal any clear substrate-product relationship between the two lipid species (data not shown). In control studies we confirmed that the increases in PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels were dependent on PI 3-kinases as they were abolished by pretreating platelets with LY294002 (Fig. 1A). Resting SHIP1–/– platelets displayed a slightly elevated basal PtdIns(3,4,5)P3 level, compared with matched SHIP1+/+ platelets (Fig. 1, B and C). Following 60 min of adhesion to fibrinogen, PtdIns(3,4,5)P3 levels increased significantly in SHIP–/– platelets (>10-fold higher levels than in SHIP1+/+) (Fig. 1C), representing ∼6% of the levels of PtdIns(4,5)P2 (data not shown). This increase in PtdIns(3,4,5)P3 was specific as there was no significant increase in the levels of other phosphoinositides (data not shown). Reduced PtdIns(3,4,5)P3 metabolism in SHIP1–/– platelets is typically associated with a corresponding reduction in the cellular levels of PtdIns(3,4)P2 compared with SHIP1+/+ platelets (23Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Google Scholar, 24Giuriato S. Pesesse X. Bodin S. Sasaki T. Viala C. Marion E. Penninger J. Schurmans S. Erneux C. Payrastre B. Biochem. J. 2003; 376: 199-207Google Scholar), a finding confirmed in thrombin-stimulated SHIP1–/– platelets (Fig. 1D) (24Giuriato S. Pesesse X. Bodin S. Sasaki T. Viala C. Marion E. Penninger J. Schurmans S. Erneux C. Payrastre B. Biochem. J. 2003; 376: 199-207Google Scholar). Interestingly, in fibrinogen-adherent SHIP1–/– platelets, there was no significant decrease in PtdIns(3,4)P2 levels. In fact, there was a slight elevation in PtdIns(3,4)P2 levels in SHIP1–/– platelets, although this difference was not statistically significant (Fig. 1C). To provide further evidence that integrin αIIbβ3 can regulate the cellular levels of PtdIns(3,4,5)P3, we pretreated SHIP1–/– platelets with an antagonist of integrin αIIbβ3 prior to thrombin stimulation. These studies demonstrated that inhibition of ligand binding to integrin αIIbβ3 prevented PtdIns(3,4,5)P3 accumulation by up to 70% (data not shown). Overall, these studies support a potentially important role for SHIP1 in regulating integrin αIIbβ3-dependent PtdIns(3,4,5)P3 accumulation in platelets. Enhanced Platelet Spreading and Cytosolic Calcium Flux in SHIP1–/– Platelets—Adhesion of human and wild-type mouse platelets to a fibrinogen matrix is associated with the generation of a sustained oscillatory calcium response that promotes lamellipodial extension and platelet spreading (37Goncalves I. Hughan S.C. Schoenwaelder S.M. Yap C.L. Yuan Y. Jackson S.P. J. Biol. Chem. 2003; 278: 34812-34822Google Scholar, 39Wonerow P. Pearce A.C. Vaux D.J. Watson S.P. J. Biol. Chem. 2003; 278: 37520-37529Google Scholar). PI 3-kinases play an important role in this process by promoting calcium release from internal stores (14Yap C.L. Anderson K.E. Hughan S.C. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99: 151-158Google Scholar, 16Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Google Scholar). To examine whether dysregulated metabolism of PI 3-kinase lipid products resulted in alterations in calcium signaling, cytosolic calcium flux was examined in adherent platelets using a confocal-based dual-dye ratiometric assay. In contrast to a previous report in platelets (23Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Google Scholar), but in agreement with reports in other cell types (17Huber M. Helgason C.D. Scheid M.P. Duronio V. Humphries R.K. Krystal G. EMBO J. 1998; 17: 7311-7319Google Scholar, 18Liu Q. Oliveira-Dos-Santos A.J. Mariathasan S. Bouchard D. Jones J. Sarao R. Kozieradzki I. Ohashi P.S. Penninger J.M. Dumont D.J. J. Exp. Med. 1998; 188: 1333-1342Google Scholar, 40Huber M. Helgason C.D. Damen J.E. Liu L. Humphries R.K. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11330-11335Google Scholar, 41Nakamura K. Malykhin A. Coggeshall K.M. Blood. 2002; 100: 3374-3382Google Scholar) no difference in resting calcium levels was observed between SHIP1+/+ or SHIP1–/– platelets (data not shown). However, the onset of an oscillatory calcium response occurred significantly more rapidly in SHIP1–/– compared with SHIP1+/+ platelets (Fig. 2A). Significantly, there was no difference in the peak level of cytosolic calcium or the overall pattern of cytosolic calcium flux in SHIP1–/– platelets (data not shown). SHIP1–/– platelets typically spread more rapidly on the fibrinogen substrate and extended more prominent filopodia and lamellae (Fig. 2, B and C). In fact, whereas 30 min of