Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.
Lefkovits J, Plow EF, Topol EJ (1995) Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med 332: 1553–1559.
[3]
O’Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, et al. (1994) Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 124: 1047–1059.
[4]
Hughes PE, Diaz-Gonzalez F, Leong L, Wu C, McDonald JA, et al. (1996) Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem 271: 6571–6574.
[5]
Lu C, Takagi J, Springer TA (2001) Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state. J Biol Chem 276: 14642–14648.
[6]
Vinogradova O, Velyvis A, Velyviene A, Hu B, Haas T, et al. (2002) A structural mechanism of integrin alpha(IIb)beta(3) “inside-out” activation as regulated by its cytoplasmic face. Cell 110: 587–597.
[7]
Li R, Mitra N, Gratkowski H, Vilaire G, Litvinov R, et al. (2003) Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations. Science 300: 795–798.
[8]
Luo BH, Springer TA (2004) Takagi (2004) A specific interface between integrin transmembrane helices and affinity for ligand. PLoS Biol 2: e153.
[9]
Petrich BG, Marchese P, Ruggeri ZM, Spiess S, Weichert RA, et al. (2007) Talin is required for integrin-mediated platelet function in hemostasis and thrombosis. J Exp Med 204: 3103–3111.
[10]
Ma YQ, Qin J, Wu C, Plow EF (2008) Kindlin-2 (Mig-2): a co-activator of beta3 integrins. J Cell Biol 181: 439–446.
[11]
Qin J, Vinogradova O, Plow EF (2004) Integrin bidirectional signaling: a molecular view. PLoS Biol 2: e169.
[12]
Cho MJ, Liu J, Pestina TI, Steward SA, Thomas DW, et al. (2003) The roles of alpha IIb beta 3-mediated outside-in signal transduction, thromboxane A2, and adenosine diphosphate in collagen-induced platelet aggregation. Blood 101: 2646–2651.
[13]
Li Z, Delaney MK, O’Brien KA, Du X (2010) Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 30: 2341–2349.
[14]
Gong H, Shen B, Flevaris P, Chow C, Lam SC, et al. (2010) G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 327: 340–3.
[15]
Deshmukh L, Meller N, Alder N, Byzova T, Vinogradova O (2011) Tyrosine phosphorylation as a conformational switch: a case study of integrin beta3 cytoplasmic tail. J Biol Chem 286: 40943–40953.
[16]
Shattil SJ (2009) The beta3 integrin cytoplasmic tail: protein scaffold and control freak. J Thromb Haemost 7 Suppl 1210–213.
[17]
Shattil SJ (2005) Integrins and Src: dynamic duo of adhesion signaling. Trends Cell Biol 15: 399–403.
[18]
Flevaris P, Stojanovic A, Gong H, Chishti A, Welch E, et al. (2007) A molecular switch that controls cell spreading and retraction. J Cell Biol 179: 553–565.
[19]
Naik UP, Patel PM, Parise LV (1997) Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain. J Biol Chem 272: 4651–4654.
[20]
Naik UP, Naik MU (2003) Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen. Blood 102: 1355–1362.
[21]
Naik MU, Naik UP (2003) Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen. Blood 102: 3629–3636.
[22]
Rantala JK, Pouwels J, Pellinen T, Veltel S, Laasola P, et al. (2011) SHARPIN is an endogenous inhibitor of beta1-integrin activation. Nat Cell Biol 13: 1315–1324.
[23]
Liu J, Jackson CW, Gruppo RA, Jennings LK, Gartner TK (2005) The beta3 subunit of the integrin alphaIIbbeta3 regulates alphaIIb-mediated outside-in signaling. Blood 105: 4345–4352.
[24]
Resendiz JC, Kroll MH, Lassila R (2007) Protease-activated receptor-induced Akt activation–regulation and possible function. J Thromb Haemost 5: 2484–2493.
[25]
Flevaris P, Li Z, Zhang G, Zheng Y, Liu J, et al. (2009) Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 113: 893–901.
[26]
Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, et al. (1998) A dual thrombin receptor system for platelet activation. Nature 394: 690–694.
[27]
Kahner BN, Kato H, Banno A, Ginsberg MH, Shattil SJ, et al. (2012) Kindlins, integrin activation and the regulation of talin recruitment to alphaIIbbeta3. PLoS One 7: e34056.
[28]
Wang R, Shattil SJ, Ambruso DR, Newman PJ (1997) Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex. J Clin Invest 100: 2393–2403.
[29]
Derrick JM, Shattil SJ, Poncz M, Gruppo RA, Gartner TK (2001) Distinct domains of alphaIIbbeta3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an alphaIIbbeta3 interacting peptide. Thromb Haemost 86: 894–901.
[30]
Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, et al. (1998) Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci U S A 95: 6642–6646.
[31]
Xiang B, Zhang G, Liu J, Morris AJ, Smyth SS, et al. (2010) A G(i) -independent mechanism mediating Akt phosphorylation in platelets. J Thromb Haemost 8: 2032–2041.
[32]
Coller BS (1980) Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia. Blood 55: 169–178.
[33]
Kieffer N, Fitzgerald LA, Wolf D, Cheresh DA, Phillips DR (1991) Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells. J Cell Biol 113: 451–461.
[34]
Gartner TK, Amrani DL, Derrick JM, Kirschbaum NE, Matsueda GR, et al. (1993) Characterization of adhesion of “resting”. and stimulated platelets to fibrinogen and its fragments.Thromb Res 71: 47–60.
[35]
Liu J, Joglekar M, Ware J, Fitzgerald ME, Lowell CA, et al. (2008) Evaluation of the physiological significance of botrocetin/ von Willebrand factor in vitro signaling J Thromb Haemost. 6: 1915–1922.
[36]
Liu J, Pestina TI, Berndt MC, Jackson CW, Gartner TK (2005) Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an alphaIIbbeta3- and aggregation-independent manner. Blood 106: 2750–2756.
[37]
Liu J, Pestina TI, Berndt MC, Steward SA, Jackson CW, et al. (2004) The roles of ADP and TXA in botrocetin/VWF-induced aggregation of washed platelets. J Thromb Haemost 2: 2213–2222.
[38]
Kouns WC, Wall CD, White MM, Fox CF, Jennings LK (1990) A conformation-dependent epitope of human platelet glycoprotein IIIa. J Biol Chem 265: 20594–20601.
[39]
Coller BS (1985) A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest 76: 101–108.
[40]
Thomas DW, Mannon RB, Mannon PJ, Latour A, Oliver JA, et al. (1998) Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2. J Clin Invest 102: 1994–2001.
Cho MJ, Liu J, Pestina TI, Steward SA, Jackson CW, et al. (2003) AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets. J Thromb Haemost 1: 363–373.
[43]
Derrick JM, Taylor DB, Loudon RG, Gartner TK (1997) The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3. Biochem J 325: 309–313.
