Integrin αIIbβ3-dependent Calcium Signals Regulate Platelet-Fibrinogen Interactions under Flow
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m306504200
ISSN1083-351X
AutoresIsaac Goncalves, Sascha C. Hughan, Simone M. Schoenwaelder, Cindy L. Yap, Yuping Yuan, Shaun P. Jackson,
Tópico(s)Antiplatelet Therapy and Cardiovascular Diseases
ResumoPlatelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin αIIbβ3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLCγ2–/– mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLCγ2–/– mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLCγ2 in integrin αIIbβ3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLCγ2 and ADP in regulating platelet adhesion efficiency on fibrinogen. Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin αIIbβ3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLCγ2–/– mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLCγ2–/– mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLCγ2 in integrin αIIbβ3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLCγ2 and ADP in regulating platelet adhesion efficiency on fibrinogen. Platelet adhesion and aggregation at sites of vascular injury are critical for the arrest of bleeding in traumatized vessels and also for the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and stroke. The two principle adhesive ligands promoting platelet aggregation are von Willebrand factor (vWf) 1The abbreviations used are: vWf, von Willebrand factor; FITC, fluorescein isothiocyanate; PLC, phospholipase C; APB-2, 2-aminoethoxydiphenyl borate; DIC, differential interference contrast; IP3, inositol 1,4,5-trisphosphate; ITAM, immunoreceptor tyrosine-based activation motif; TXA2, thromboxane A2; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; U73122, 1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]-hexyl)-1H-pyrrole-2,5-dione; DiOC6, 3,3-dihexyl-oxacarbocyanine(3).1The abbreviations used are: vWf, von Willebrand factor; FITC, fluorescein isothiocyanate; PLC, phospholipase C; APB-2, 2-aminoethoxydiphenyl borate; DIC, differential interference contrast; IP3, inositol 1,4,5-trisphosphate; ITAM, immunoreceptor tyrosine-based activation motif; TXA2, thromboxane A2; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; U73122, 1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]-hexyl)-1H-pyrrole-2,5-dione; DiOC6, 3,3-dihexyl-oxacarbocyanine(3). and fibrinogen, with each having distinct, complementary roles in this process (1Ikeda Y. Handa M. Kawano K. Kamata T. Murata M. Araki Y. Anbo H. Kawai Y. Watanabe K. Itagaki I. Sakai K. Ruggeri Z.M. J. Clin. Invest. 1991; 87: 1234-1240Crossref PubMed Scopus (533) Google Scholar, 2Weiss H.J. Hawiger J. Ruggeri Z.M. Turitto V.T. Thiagarajan P. Hoffmann T. J. Clin. Invest. 1989; 83: 288-297Crossref PubMed Scopus (201) Google Scholar, 3Alevriadou B.R. Moake J.L. Turner N.A. Ruggeri Z.M. Folie B.J. Phillips M.D. Schreiber A.B. Hrinda M.E. McIntire L.V. Blood. 1993; 81: 1263-1276Crossref PubMed Google Scholar, 4Goto S. Ikeda Y. Saldivar E. Ruggeri Z.M. J. Clin. Invest. 1998; 101: 479-486Crossref PubMed Scopus (270) Google Scholar, 5Tsuji S. Sugimoto M. Miyata S. Kuwahara M. Kinoshita S. Yoshioka A. Blood. 1999; 94: 968-975Crossref PubMed Google Scholar). Fibrinogen acts as a bridging molecule between adjacent activated platelets and is the principle adhesive ligand promoting platelet aggregation under low and intermediate shear flow conditions (1Ikeda Y. Handa M. Kawano K. Kamata T. Murata M. Araki Y. Anbo H. Kawai Y. Watanabe K. Itagaki I. Sakai K. Ruggeri Z.M. J. Clin. Invest. 1991; 87: 1234-1240Crossref PubMed Scopus (533) Google Scholar, 2Weiss H.J. Hawiger J. Ruggeri Z.M. Turitto V.T. Thiagarajan P. Hoffmann T. J. Clin. Invest. 1989; 83: 288-297Crossref PubMed Scopus (201) Google Scholar). In addition, it also contributes to stable aggregate formation under high shear (1Ikeda Y. Handa M. Kawano K. Kamata T. Murata M. Araki Y. Anbo H. Kawai Y. Watanabe K. Itagaki I. Sakai K. Ruggeri Z.M. J. Clin. Invest. 1991; 87: 1234-1240Crossref PubMed Scopus (533) Google Scholar, 5Tsuji S. Sugimoto M. Miyata S. Kuwahara M. Kinoshita S. Yoshioka A. Blood. 1999; 94: 968-975Crossref PubMed Google Scholar, 6Ni H. Denis C.V. Subbarao S. Degen J.L. Sato T.N. Hynes R.O. Wagner D.D. J. Clin. Invest. 2000; 106: 385-392Crossref PubMed Scopus (402) Google Scholar). It is well established that the binding of fluid-phase fibrinogen to the platelet surface is dependent on the activation of integrin αIIbβ3 (GPIIb-IIIa) through intracellular signaling processes linked to various G protein-coupled and tyrosine kinase-linked receptors (7Ruggeri Z.M. Nat. Med. 2002; 8: 1227-1234Crossref PubMed Scopus (1354) Google Scholar). Platelets can also adhere to immobilized fibrinogen, a process that is important for primary platelet adhesion onto artificial surfaces, including vascular prostheses (8Salzman E.W. Lindon J. McManama G. Ware J.A. Ann. N. Y. Acad. Sci. 1987; 516: 184-195Crossref PubMed Scopus (90) Google Scholar, 9Lindon J.N. McManama G. Kushner L. Merrill E.W. Salzman E.W. Blood. 1986; 68: 355-362Crossref PubMed Google Scholar), and for normal thrombus development (5Tsuji S. Sugimoto M. Miyata S. Kuwahara M. Kinoshita S. Yoshioka A. Blood. 1999; 94: 968-975Crossref PubMed Google Scholar, 6Ni H. Denis C.V. Subbarao S. Degen J.L. Sato T.N. Hynes R.O. Wagner D.D. J. Clin. Invest. 2000; 106: 385-392Crossref PubMed Scopus (402) Google Scholar). In the case of thrombus formation, active integrin αIIbβ3 on the surface of firmly adherent platelets adsorbs soluble fibrinogen to the thrombus surface, thereby providing a reactive substrate for the recruitment of additional platelets. In contrast to soluble fibrinogen, the adhesion of platelets onto immobilized fibrinogen does not require affinity modulation of integrin αIIbβ3 (10Savage B. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 11227-11233Abstract Full Text PDF PubMed Google Scholar). As such, surface-adsorbed fibrinogen can potentially promote plateletthrombus interactions independent of initial platelet activation. This concept is supported by experimental findings demonstrating that platelet activation inhibitors have no effect on the ability of platelets to form stable adhesive interactions with a purified fibrinogen matrix under flow (11Savage B. Bottini E. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 28812-28817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 13Zaidi T.N. McIntire L.V. Farrell D.H. Thiagarajan P. Blood. 1996; 88: 2967-2972Crossref PubMed Google Scholar). This contrasts with all other platelet adhesive interactions, involving substrates such as vWf, collagen, fibronectin, and vitronectin, in which the formation of stable adhesive bonds with these surfaces is considered activation-dependent (10Savage B. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 11227-11233Abstract Full Text PDF PubMed Google Scholar, 12Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 13Zaidi T.N. McIntire L.V. Farrell D.H. Thiagarajan P. Blood. 1996; 88: 2967-2972Crossref PubMed Google Scholar, 14Nesbitt 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: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 15Mazzucato M. Pradella P. Cozzi M.R. De Marco L. Ruggeri Z.M. Blood. 2002; 100: 2793-2800Crossref PubMed Scopus (165) Google Scholar, 16Ni H. Yuen P.S. Papalia J.M. Trevithick J.E. Sakai T. Fassler R. Hynes R.O. Wagner D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2415-2419Crossref PubMed Scopus (184) Google Scholar, 17Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Once adherent to fibrinogen, platelets become activated and undergo substantial cytoskeletal remodeling, leading to platelet shape change and spreading. Integrin αIIbβ3 outside-in signals have been demonstrated to play a major role in this process (18Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 19Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar), although signaling processes downstream of this receptor per se do not appear to be sufficient for platelet spreading independent of co-stimuli such as ADP. Current evidence suggests that integrin αIIbβ3 engagement of fibrinogen induces activation of Src kinases and Syk, which promote cytoskeletal remodeling, leading to shape change and filopodial extension (20Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar, 21Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Crossref PubMed Scopus (191) Google Scholar, 22Obergfell 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 (344) Google Scholar). During this process, platelets secrete their granule contents, and the release of ADP promotes cytosolic calcium flux and lamellipodial extension via signaling pathways linked to the activation of phosphoinositide 3-kinase (23Heraud J.M. Racaud-Sultan C. Gironcel D. Albiges-Rizo C. Giacomini T. Roques S. Martel V. Breton-Douillon M. Perret B. Chap H. J. Biol. Chem. 1998; 273: 17817-17823Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Heemskerk J.W. Hoyland J. Mason W.T. Sage S.O. Biochem. J. 1992; 283: 379-383Crossref PubMed Scopus (46) Google Scholar, 25Heemskerk J.W. Vis P. Feijge M.A. Hoyland J. Mason W.T. Sage S.O. J. Biol. Chem. 1993; 268: 356-363Abstract Full Text PDF PubMed Google Scholar). In this study we have examined the mechanisms regulating platelet adhesion and activation on a fibrinogen matrix. In contrast to previous reports, our studies do not demonstrate an absolute requirement for ADP for lamellipodial extension and platelet spreading on fibrinogen. Rather, they suggest that integrin αIIbβ3 outside-in signaling linked to Src kinase-mediated phospholipase Cγ2 (PLCγ2) activation is critical for platelet spreading, whereas ADP release serves a secondary role, potentiating platelet activation. Furthermore, we demonstrate that integrin αIIbβ3-dependent calcium flux, combined with ADP release, plays an important role in sustaining platelet-fibrinogen interactions under flow. These findings challenge previous concepts of the mechanisms regulating platelet adhesion and activation on fibrinogen, defining a pivotal role for integrin αIIbβ3-dependent calcium flux in these processes. Materials—Apyrase was purified as described previously (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Human fibrinogen was purified from fresh frozen plasma according to Jakobsen et al. (27Jakobsen E. Ly B. Kierulf P. Thromb. Res. 1974; 4: 499-507Abstract Full Text PDF PubMed Scopus (27) Google Scholar). The Src kinase inhibitor PP2 was purchased from Calbiochem-Novabiochem. Probenicid, 2-aminoethoxydiphenyl borate (APB-2), and the P2Y1 antagonist A3P5PS were purchased from Sigma. The P2Y12 antagonist AR-C69931MX was obtained from Astra Zeneca. All other reagents were obtained from sources described previously (14Nesbitt 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: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Mouse Strains—C57BL/6 PLCγ2-deficient mice (PLCγ2–/–) were provided by Prof. J. Ihle (St. Jude Children's Research Hospital, Memphis, TN) (29Wang 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 (405) Google Scholar). Antibodies—PAC-1 was from BD Biosciences. The anti-PLCγ2 polyclonal antibody was obtained from Santa Cruz Biotechnology, and the anti-phosphotyrosine monoclonal antibody (PY20) was from ICN. Platelet Preparation—Washed human and murine platelets were prepared as described previously (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 30Moog S. Mangin P. Lenain N. Strassel C. Ravanat C. Schuhler S. Freund M. Santer M. Kahn M. Nieswandt B. Gachet C. Cazenave J.P. Lanza F. Blood. 2001; 98: 1038-1046Crossref PubMed Scopus (106) Google Scholar). For adhesion studies, washed platelets were resuspended in modified Tyrode's buffer (10 mm Hepes, 12 mm NaHCO3, pH 7.4, 137 mm NaCl, 2.7 mm KCl, 5 mm glucose). Static Adhesion Assays—Static adhesion assays were performed using a modified method of Yuan et al. (28Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, glass coverslips (12 mm in diameter; Lomb Scientific) were coated with fibrinogen (100 μg/ml) for 2 h at room temperature and then blocked with 10% heat-inactivated human serum pretreated with phenylmethylsulfonyl fluoride (25 μg/ml). Platelets in Tyrode's buffer (1–3 × 107/ml) supplemented with 1 mm CaCl2 were allowed to adhere to the fibrinogen matrix for the indicated time periods. Adherent platelets were fixed with 3.7% formaldehyde for 15 min, mounted onto glass slides, and imaged using differential interference contrast (DIC) microscopy or phase contrast microscopy for surface area analysis as described by Yap et al. (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Where indicated, platelets were preincubated with vehicle alone (Me2SO, 0.25% (v/v)), the Src kinase inhibitor (PP2, 1–10 μm), apyrase (1.5 units/ml, ADPase activity), the ADP receptor antagonists against P2Y1 (A3P5PS, 200 μm) or P2Y12 (AR-C69931MX, 100 nm), the IP3 receptor antagonist (APB-2, 20 μm), or the PLC inhibitor (U73122, 5–10 μm) for 10 min at 37 °C prior to the performance of adhesion assays. In other experiments, the role of TXA2 was assessed by pretreating platelets with aspirin (1 mm) for 30 min at 37 °C prior to the performance of adhesion assays. The pharmacological activity of these inhibitor(s) was confirmed as follows: PP2 abolished collagen-induced platelet aggregation (31Briddon S.J. Watson S.P. Biochem. J. 1999; 338: 203-209Crossref PubMed Scopus (82) Google Scholar); apyrase abolished ADP (25 μm)-induced platelet aggregation; aspirin blocked arachidonic-acid (1.2 mm)-induced platelet aggregation; APB-2 inhibited thrombin (0.5–1.0 units/ml)-induced platelet aggregation. Measurement of Integrin α IIb β 3 Activation—Integrin αIIbβ3 activation during platelet adhesion to fibrinogen under static conditions was assessed using the integrin αIIbβ3 activation-specific antibody PAC-1. Platelets were allowed to adhere to fibrinogen in the presence of PAC-1 (2 μg/ml) followed by fixation (3.7% formaldehyde) and incubation with a FITC-conjugated anti-mouse IgG F(ab′)2 fragment. PAC-1 immunofluorescence was visualized using confocal fluorescence microscopy (100× magnification, TCS-SP, Leica) and quantified using the Leica TCS NT software as described previously (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In some studies, platelets were preincubated with the indicated concentrations of APB-2, U73122, PP2, or apyrase alone or in combination with aspirin prior to the performance of adhesion studies. Platelet Adhesion to Fibrinogen under Flow Conditions—Flow assays were performed as described previously (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In studies examining the ability of platelets to adhere to the fibrinogen matrix, washed platelets (5 × 107/ml) were reconstituted with washed red blood cells (50% v/v; containing 0.04 units/ml apyrase and 1 unit/ml hirudin) as described previously (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Platelets were perfused through fibrinogen (100 μg/ml)-coated microcapillary tubes at a wall shear rate of 150 s–1 for 2 min at 37 °C. Platelet-matrix interactions were visualized using epifluorescence microscopy (Leica DMIRB) and video-recorded for off-line analysis. Platelet tethering was analyzed at 30, 60, and 90 s, and each platelet that interacted with the fibrinogen matrix for ≥2 frames (40 ms) was scored as "tethered." In studies examining cell displacement, DiOC6-labeled platelets were perfused through fibrinogen-coated microcapillary tubes for 5 min at 150 s–1. Platelets were considered as dislodged when exhibiting spatial displacement on the surface greater than 1 platelet diameter from their initial attachment point (12Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). Similar analysis was used for calcium-dye loaded human and murine platelets when perfused across a fibrinogen matrix (see below), except that platelet adhesion was monitored for 100 frames (0.586 frames/s) using confocal fluorescence microscopy (TCS-SP, Leica). Similar analysis was used to examine adhesion strength at high shear (1800 s–1); however, in this case, platelets were first perfused at 150 s–1 for 5 min followed by an increase in wall shear rate to 1800 s–1 for a further 60 s. Analysis of Cytosolic Calcium Flux under Static and Flow Conditions—Changes in cytosolic calcium levels were monitored according to published methods (14Nesbitt 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: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, washed platelets (1 × 109/ml) were loaded with Oregon Green 488 BAPTA-AM (1 μm) and Fura Red-AM (1.25 μm) for 30 min at 37 °C. For mouse platelets, the calcium dyes were loaded at a platelet density of 2 × 108/ml in the presence of 1.25 mm Probenicid. Dye-loaded platelets (1 × 107/ml) were then either allowed to adhere to fibrinogen under static conditions or reconstituted with red blood cells (50%) prior to perfusion through fibrinogen-coated microcapillary tubes at 150 s–1 for human platelets and 600 s–1 for mouse platelets. To examine the changes in calcium flux, sequential confocal images of adherent platelets were captured at a scan rate of 0.586 frames/s for 37.5 s at the indicated time points. Real-time platelet calcium flux was calculated from ratiometric fluorescence measurements and converted to intracellular calcium concentrations as described previously (14Nesbitt 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: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Assessment of PLCγ2 Phosphorylation—Washed platelets (1 × 108/ml), treated with vehicle alone (0.25% Me2SO) or PP2 (10 μm) for 10 min, were then applied to fibrinogen-coated dishes (100 μg/ml) for 30 min at 37 °C. Adherent cells were lysed with radioimmunoprecipitation assay buffer (10 mm Tris, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mm NaCl, 2 mm EDTA, 1 mm phenylmethylsul-fonyl fluoride, and 2 mm benzamidine). To examine the level of PLCγ2 phosphorylation in resting cells, non-adherent platelets in suspension were lysed with radioimmunoprecipitation assay buffer. All lysates were centrifuged at 15,000 × g for 5 min, and the resulting supernatant was subjected to immunoprecipitation by mixing with an α-PLCγ2 polyclonal antibody (2 μg/ml) and protein A-Sepharose beads (50% v/v slurry) for2hat4 °C.The beads were washed, and immunoprecipitated proteins were separated on a 7.5% SDS-PAGE under reducing conditions and immunoblotted using either an anti-PLCγ2 monoclonal antibody or an anti-phosphotyrosine monoclonal antibody (PY20). Statistical Analysis—Statistical significance of results was determined using one-way analysis of variance. The p values are indicated where appropriate (*, p < 0.05; **, p < 0.01). Role of ADP in Promoting Integrin α IIb β 3 Activation and Platelet Spreading on Immobilized Fibrinogen—Immobilized fibrinogen supports the adhesion and activation of platelets through engagement of integrin αIIbβ3. Previous studies have suggested an important role for ADP in promoting lamellipodial formation in fibrinogen-adherent platelets (18Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 23Heraud J.M. Racaud-Sultan C. Gironcel D. Albiges-Rizo C. Giacomini T. Roques S. Martel V. Breton-Douillon M. Perret B. Chap H. J. Biol. Chem. 1998; 273: 17817-17823Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, its role in promoting integrin αIIbβ3 activation remains less clear. To investigate this role, we examined the effect of apyrase on integrin αIIbβ3 activation during platelet adhesion to fibrinogen by performing indirect immunofluorescence studies using the activation-specific α-integrin αIIbβ3 antibody, PAC-1. As demonstrated in Fig. 1A, robust PAC-1 binding and platelet spreading was observed in control and apyrase-treated platelets following adhesion to fibrinogen. Using confocal imaging, we demonstrated that PAC-1 staining occurred predominantly at the granulomere on the apical surface of spreading platelets (Fig. 1A). Quantification of PAC-1 fluorescence on the surface of spread platelets revealed no difference between control and apyrase-treated platelets (Fig. 1B). The inability of apyrase to inhibit PAC-1 binding and spreading was unlikely to be the result of incomplete inhibition of ADP, as blocking the two major ADP purinergic receptors, P2Y1 and P2Y12, with AR-C69931MX and A3P5PS, respectively, did not inhibit these platelet responses (data not shown). These findings suggest that integrin αIIbβ3 engagement of fibrinogen can modulate the affinity status of integrin αIIbβ3 and induce lamellipodial extensions independent of ADP. To examine in further detail the relationship between ADP release and platelet spreading, time course adhesion assays were performed. As demonstrated in Fig. 1C, platelets pretreated with apyrase spread significantly slower than control platelets, with half-maximal spreading of control platelets occurring within <10 min compared with 20–30 min for apyrase-treated platelets. However, by 50 min there was no difference in spreading between control and apyrase-treated platelets. In both the fixed end point and time course adhesion assays, there was no further decrease in the rate or extent of platelet spreading when aspirin was combined with apyrase, suggesting that TXA2 did not make a significant contribution to this response. Role of ADP in Promoting Cytosolic Calcium Flux during Platelet Adhesion on Fibrinogen—The mobilization of intracellular calcium during platelet adhesion to fibrinogen is important for cytoskeletal remodeling and has been demonstrated to be ADP-dependent (32Sage S.O. Yamoah E.H. Heemskerk J.W. Cell Calcium. 2000; 28: 119-126Crossref PubMed Scopus (53) Google Scholar). To investigate the absolute requirement for ADP for fibrinogen-induced cytosolic calcium flux, confocal imaging studies were performed on adherent platelets labeled with the calcium-indicator dyes Oregon Green BAPTA-AM and Fura Red. Consistent with previous reports (26Yap 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: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 33Jen C.J. Chen H.I. Lai K.C. Usami S. Blood. 1996; 87: 3775-3782Crossref PubMed Google Scholar), platelets firmly adherent to fibrinogen elicited a sustained, oscillatory calcium response that coincided with lamellipodial extension and platelet spreading. As demonstrated in Fig. 2A, pretreating platelets with apyrase and aspirin markedly delayed the onset of the calcium response; however, by 50 min the majority of platelets exhibited a sustained calcium response and spread. Furthermore, analysis of the pattern of the cytosolic calcium response in individual platelets (Fig. 2B) revealed no difference in the frequency or magnitude of calcium oscillations between control and apyrase/aspirin-treated platelets (data not shown). In all studies, combining aspirin with apyrase had no further inhibitory effect beyond that observed with apyrase alone, which excludes a major role for TXA2 in promoting cytosolic calcium flux (data not shown). Overall, these studies define an important, albeit non-essential role for ADP in promoting cytosolic calcium flux during platelet adhesion on fibrinogen. Src Kinases are Essential for Integrin α IIb β 3 Activation and Calcium Mobilization following Platelet Adhesion to Fibrinogen—The demonstration that ADP antagonists did not eliminate platelet activation induced by immobilized fibrinogen raised the possibility that fibrinogen engagement of integrin αIIbβ3 was sufficient to induce cytosolic calcium flux through outside-in signaling processes. Src kinases play a central role in integrin signaling, and a recent study (22Obergfell 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 (344) Google Scholar) has demonstrated an important role for Src kinases in integrin αIIbβ3-dependent cytoskeletal remodeling. To examine the role of Src kinases in promoting integrin αIIbβ3 activation and calcium flux, platelets were pretreated with the Src kinase inhibitor, PP2 (34Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 6
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