A Mitogen-activated Protein Kinase-dependent Signaling Pathway in the Activation of Platelet Integrin αIIbβ3
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m106129200
ISSN1083-351X
AutoresZhenyu Li, Xiaodong Xi, Xiaoping Du,
Tópico(s)Cell Adhesion Molecules Research
ResumoWe have recently shown that the platelet integrin αIIbβ3 is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin αIIbβ3 is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin αIIbβ3. Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation. We have recently shown that the platelet integrin αIIbβ3 is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin αIIbβ3 is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin αIIbβ3. Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation. von Willebrand factor glycoprotein Ib-IX MAPK, mitogen-activated protein kinase extracellular signal-regulated kinase thromboxane A2 Chinese hamster ovary platelet-rich plasma 8-(4-chlorophenylthio)-cGMP N-(β-d-glucopyranosyl)-N 2-acetyl-S-nitroso-d,l-penicillaminamide mitogen-activated protein kinase/extracellular signal-regulated kinase kinase The integrin αIIbβ3 mediates platelet adhesion, spreading, and aggregation and thus plays a critical role in thrombosis and hemostasis (1Ginsberg M.H. Du X. O'Toole T.E. Loftus J.C. Thromb. Haemostasis. 1995; 74: 352-359Crossref PubMed Scopus (65) Google Scholar). In normal circulating platelets, the integrin αIIbβ3 is in a resting state with a low affinity for its ligands such as fibrinogen and von Willebrand factor (vWF).1 At sites of vascular injury, exposure of platelets to soluble agonists (such as thrombin and ADP) or to matrix-bound adhesive proteins (such as collagen and vWF) induces platelet activation. A common consequence of platelet activation is the activation of the ligand binding function of the integrin αIIbβ3 (2Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (479) Google Scholar, 3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). Under high shear rate flow conditions, such as in stenotic atherosclerotic arteries, initial platelet adhesion and activation are dependent on the interaction between subendothelium-bound vWF and its receptor, the glycoprotein Ib-IX (GPIb-IX) complex (4Weiss H.J. Turitto V.T. Baumgartner H.R. J. Lab. Clin. Med. 1978; 92: 750-764PubMed Google Scholar, 5Sakariassen K.S. Bolhuis P.A. Sixma J.J. Nature. 1979; 279: 636-638Crossref PubMed Scopus (465) Google Scholar, 6Sakariassen K.S. Nievelstein P.F. Coller B.S. Sixma J.J. Br. J. Haematol. 1986; 63: 681-691Crossref PubMed Scopus (175) Google Scholar, 7Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 8Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 9Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 10Lopez J.A. Andrews R.K. Afshar-Kharghan V. Berndt M.C. Blood. 1998; 91: 4397-4418Crossref PubMed Google Scholar). GPIb-IX not only mediates the physical adherence of platelets to the site of vascular injury but also initiates signal transduction, leading to activation of ligand binding function of the platelet integrin αIIbβ3 (11De Marco L. Girolami A. Russell S. Ruggeri Z.M. J. Clin. Invest. 1985; 75: 1198-1203Crossref PubMed Scopus (113) Google Scholar, 12De Marco L. Girolami A. Zimmerman T.S. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7424-7428Crossref PubMed Scopus (96) Google Scholar, 13Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (236) Google Scholar, 14Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 15Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (117) Google Scholar). In addition, GPIbα binds thrombin and is required for the low dose thrombin-induced integrin activation and platelet aggregation (16Okumura T. Jamieson G.A. Thromb. Res. 1976; 8: 701-706Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 17Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar, 18Yamamoto N. Kitagawa H. Tanoue K. Yamazaki H. Thromb. Res. 1985; 39: 751-759Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 19Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 20De Marco L. Mazzucato M. Masotti A. Fenton J.d. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 21Mazzucato M. Marco L.D. Masotti A. Pradella P. Bahou W.F. Ruggeri Z.M. J. Biol. Chem. 1998; 273: 1880-1887Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 22Ramakrishnan V. DeGuzman F. Bao M. Hall S.W. Leung L.L. Phillips D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1823-1828Crossref PubMed Scopus (153) Google Scholar). The importance of GPIb-IX pathway in platelet function is manifested in Bernard-Soulier syndrome, in which genetic deficiency in GPIb-IX resulted in defects in platelet adhesion and activation (10Lopez J.A. Andrews R.K. Afshar-Kharghan V. Berndt M.C. Blood. 1998; 91: 4397-4418Crossref PubMed Google Scholar). The mechanism of GPIb-IX-mediated integrin activation is not fully understood. However, we have recently shown that the cGMP-dependent protein kinase (protein kinase G (PKG)) is an important stimulatory mediator in the GPIb-IX-induced activation of integrin αIIbβ3. 2Z. Li, X. Xi, M. Gu, R. Ye, and X. Du, submitted for publication.2Z. Li, X. Xi, M. Gu, R. Ye, and X. Du, submitted for publication.Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine kinase activated by many extracellular stimuli including growth factors and hormones. Four distinct subgroups within the MAPK family have been described, including the extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal kinases, ERK5/big MAP kinase (BMK1), and p38 group of protein kinases. At least three subgroups of these MAP kinases, ERK1/ERK2 (23Papkoff J. Chen R.H. Blenis J. Forsman J. Mol. Cell. Biol. 1994; 14: 463-472Crossref PubMed Google Scholar), p38 (24Saklatvala J. Rawlinson L. Waller R.J. Sarsfield S. Lee J.C. Morton L.F. Barnes M.J. Farndale R.W. J. Biol. Chem. 1996; 271: 6586-6589Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), and c-Jun NH2-terminal kinase (25Bugaud F. Nadal-Wollbold F. Levy-Toledano S. Rosa J.P. Bryckaert M. Blood. 1999; 94: 3800-3805Crossref PubMed Google Scholar), have been identified in platelets and shown to be activated when platelets are stimulated by different agonists such as thrombin and collagen. The prototype MAPK pathway, ERK pathway, consists of a cascade of protein kinases, Raf1, MEK1, and ERK1/ERK2, which sequentially activate a downstream kinase. Raf-1 activation involves Ras and 14-3-3 protein (26Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (534) Google Scholar). The functions of ERK MAPK pathway in platelets have not been fully understood. ERK has been shown to phosphorylate and activate cytoplasmic phospholipase A2, which is a rate-limiting enzyme in synthesis of thromboxane A2 (TXA2) (27Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar). However, it was reported that ERK pathway is not required in the cytoplasmic phospholipase A2 activation in platelets stimulated by thrombin, as MEK1 inhibitor PD98059 did not abolish thrombin-induced arachidonic acid release (28Borsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar). It has been reported that ERK pathway is not required for primary platelet response to high doses of collagen and thrombin (28Borsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar), although MEK1 inhibitor PD98059 has been shown in other studies to inhibit platelet aggregation induced by low doses of collagen and ADP (29Borsch-Haubold A.G. Pasquet S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The interpretation of these data has been complicated by the report suggesting that PD98059 may directly inhibit cyclooxygenases in TXA2 synthesis pathway, which is important in platelet aggregation induced by low dose collagen and ADP (29Borsch-Haubold A.G. Pasquet S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar).In this study, we have examined the roles of ERK pathway in GPIb-IX-dependent platelet activation using a combination of molecular biology and pharmacological approaches. We show that ERK pathway is important in GPIb-IX-dependent integrin activation signaling. We further show that activation of PKG is sufficient to activate ERK pathway and is necessary for GPIb-IX-mediated activation of ERK. These data, combined with our recent finding that PKG mediates GPIb-IX-dependent platelet activation,2 delineate a novel signaling pathway of platelet activation in which ligand binding to GPIb-IX sequentially activates PKG pathway and ERK MAP kinase pathway leading to integrin activation.DISCUSSIONIn this study we found that 1) ligand binding to GPIb-IX activates ERK pathway, which is important in mediating GPIb-IX-induced activation of integrin αIIbβ3, and 2) the ERK pathway is downstream from PKG in the GPIb-IX-induced integrin activation. Together with our recent finding that GPIb-IX-induced platelet activation requires the cGMP-PKG pathway, these data delineate a novel integrin activation signaling pathway in which ligand binding to GPIb-IX induces activation of PKG, which stimulates ERK pathway, leading to activation of integrin αIIbβ3(Fig. 7).The elements of ERK MAPK pathway are abundant in platelets. Previous studies indicate that ERK pathway is stimulated during platelet activation induced by platelet agonists such as ADP, collagen, and thrombin (23Papkoff J. Chen R.H. Blenis J. Forsman J. Mol. Cell. Biol. 1994; 14: 463-472Crossref PubMed Google Scholar, 28Borsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar). However, understanding the roles of MAPK in platelet activation has been complicated by the report that the commonly used inhibitor of ERK pathway, PD98059, also inhibits cyclooxygenases and, thus, TXA2 synthesis (29Borsch-Haubold A.G. Pasquet S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In this study, we took advantage of our newly reconstituted integrin activation model in CHO cells (15Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (117) Google Scholar) and examined the effects of dominant negative mutants of Raf-1 and MEK1 on GPIb-IX-mediated integrin activation in this system. We conclude that ERK pathway is important in integrin activation via the GPIb-IX signaling pathway. This conclusion is supported by the following findings. 1) Dominant negative mutants of MAP kinase pathway, Raf301 and MEK1 M97K, inhibited GPIb-IX-mediated activation of integrin αIIbβ3 in a reconstituted CHO cell expression model; 2) MEK inhibitors PD98059 and U0126 inhibited vWF and low dose thrombin-induced, integrin-dependent platelet aggregation in platelets in a cyclooxygenase-independent manner; and 3) vWF binding to GPIb-IX induced phosphorylation of ERK2 at the Thr-Glu-Tyr sequence, indicating that the ERK MAPK pathway is activated. The results obtained in the CHO cell model using recombinant DNA technology and results obtained in platelets using various MEK1 inhibitors and biochemical assays are highly consistent. Inhibition of integrin activation by the dominant negative mutants of MAPK pathway excludes the possible nonspecific effects of pharmacological agents, and results obtained in human platelets indicate that the reconstituted CHO cell model appropriately reflects the role of MAPK pathway in platelets. Furthermore, our data indicate that the inhibitory effect of MEK inhibitor PD98059 is not likely to result from its effects on cyclooxygenases because the inhibitory effect of PD98059 was seen when cyclooxygenases were already inhibited by high concentrations of aspirin (Fig. 4). Furthermore, aspirin had no inhibitory effect on vWF-induced integrin activation in CHO cells (not shown), vWF-induced the first wave of platelet aggregation (Fig. 4), or low-dose thrombin-induced platelet aggregation (Fig. 5), all of which were inhibited by PD98059. Consistent with our results, it was previously shown that ristocetin-induced production of TXA2 was inhibited by anti-integrin monoclonal antibodies, suggesting that integrin activation precedes vWF-induced TXA2 production (35Ruan C.G. Du X.P. Xi X.D. Castaldi P.A. Berndt M.C. Blood. 1987; 69: 570-577Crossref PubMed Google Scholar). Thus, MAPK pathway stimulates GPIb-IX-dependent integrin activation and platelet aggregation in a TXA2-independent manner.We have recently shown that the PKG pathway plays an important role in GPIb-IX-mediated activation of the platelet integrin αIIbβ3.2 Here we show that expression of recombinant PKG in the CHO cell model significantly enhances GPIb-IX-induced ERK pathway activation, and inhibition of PKG abolished GPIb-IX-induced ERK pathway activation. Furthermore, activation of PKG by cGMP analogs is sufficient to activate ERK pathway. Thus we conclude that the ERK pathway is downstream from PKG in the GPIb-IX-signaling pathway. Although the classic MAPK activation pathway is mediated by the receptor tyrosine kinases via Ras, it has been shown that the ERK pathway is also regulated by multiple cellular signals, including PKG (36Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (99) Google Scholar, 37Hood J. Granger H.J. J. Biol. Chem. 1998; 273: 23504-23508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Komalavilas P. Shah P.K. Jo H. Lincoln T.M. J. Biol. Chem. 1999; 274: 34301-34309Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). However, there have been controversies about whether PKG activates or inhibits MAPK pathway. In one study, ERK activity was reportedly reduced after prolonged incubation (30 min) with cGMP analogs in baby hamster kidney cells (36Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (99) Google Scholar), However, others found that PKG activated MAPK pathway in endothelial cells and smooth muscle cells (37Hood J. Granger H.J. J. Biol. Chem. 1998; 273: 23504-23508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Komalavilas P. Shah P.K. Jo H. Lincoln T.M. J. Biol. Chem. 1999; 274: 34301-34309Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). PKG directly phosphorylates and activates Raf-1, the upstream kinase in the ERK pathway (37Hood J. Granger H.J. J. Biol. Chem. 1998; 273: 23504-23508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Our results are consistent with the latter studies. One possible reason for the apparent contradicting results is the difference in the incubation time after the addition of cGMP analogs. Our data indicate that vWF or cGMP analogs induce a rapid increase in ERK2 phosphorylation in platelets that peaks at 30 s (1 min in some donors) and decreases after prolonged incubation (Fig. 6), suggesting that PKG-mediated ERK phosphorylation is a transient event. This is consistent with our observations that cGMP analogs promoted platelet activation only when added simultaneously or immediately after the addition of an agonist such as vWF or thrombin.2 Prolonged preincubation of cGMP analogs with platelets inhibits subsequent platelet response to platelet agonists in a protein kinase A-dependent manner and induces protein kinase A-mediated phosphorylation of vasodilator-stimulated phosphoprotein.2 Thus, it is possible that after a prolonged incubation, cGMP-induced activation of a cAMP-protein kinase A pathway may diminish the function of PKG to induce ERK pathway activation. It is also possible that after the initial activation of MAPK pathway and the integrin, the outside-in signaling of integrin may cause MAPK dephosphorylation, since ligand binding to integrin has been shown to negatively regulate agonist-stimulated ERK activity in platelets (39Nadal F. Levy-Toledano S. Grelac F. Caen J.P. Rosa J.P. Bryckaert M. J. Biol. Chem. 1997; 272: 22381-22384Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).There have been several interesting observations on the relationship between MAPK pathway and integrin signaling in recent years. Although Zhang et al. (40Zhang Z. Vuori K. Wang H. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) reports that Ra-Ras enhanced integrin activity, Hughes et al. (41Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) find that the activity of a constitutively active αIIb/α5β3/β1chimera integrin expressed in CHO cells was enhanced by the expression of a dominant negative mutant of Ha-Ras but was inhibited by the constitutively active Raf mutant or Ha-Ras. The results by Hughes et al. (41Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) apparently contradict our data that dominant negative mutants of Raf and MEK inhibited αIIbβ3 activation induced by GPIb-IX. However, there is a major difference between the experiments of Hughes et al. (41Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) and ours. The integrin mutant used by Hughes et al. (41Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) was a chimera integrin with the cytoplasmic domain of the αIIb replaced by the cytoplasmic domain of α5 (or α subunits other than αIIb) and the cytoplasmic domain of the β3 replaced by the cytoplasmic domain of β1. Thus, in effect, the cytoplasmic regulatory domain of this integrin mutant was not an αIIbβ3 but a β1 integrin or a hybrid between β3 and one of the α subunits that are constitutively active in cells. This may explain why the chimera mutants, unlike wild type integrin αIIbβ3, are constitutively active. In contrast, we used wild type integrin αIIbβ3 coexpressed with platelet GPIb-IX. The wild type αIIbβ3 is normally in resting state but becomes activated only after agonist stimulation. Thus, the results from Hughes et al. (41Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) may reflect a negative regulation of constitutively active integrins (such as β1integrins), but our results reflect stimulatory roles of MAPK pathway in the inside-out signaling of the integrin αIIbβ3. In this respect, it is interesting to note that the effects of integrin outside-in signaling on ERK pathway is also different between different integrins. Although β1 integrin stimulates activation of ERK pathway in several cell types (42Chen Q. Kinch M.S. Lin T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar), ligand binding to αIIbβ3 has been shown to inhibit ERK pathway in platelets (39Nadal F. Levy-Toledano S. Grelac F. Caen J.P. Rosa J.P. Bryckaert M. J. Biol. Chem. 1997; 272: 22381-22384Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). It would be interesting to further investigate the mechanisms of the different interrelationships between MAPK pathway and different members of the integrin family.Although we have identified that the cGMP-PKG-MAPK pathway mediates GPIb-IX-dependent integrin activation signaling, it is important to note that activation of PKG-MAPK pathway by adding cGMP analogs alone in the absence of vWF or thrombin stimulation is not sufficient to activate integrin αIIbβ3.2 Thus, it appears that additional parallel signaling pathways induced by vWF or thrombin is required for integrin activation in addition to the cGMP-PKG-MAPK pathway. In this respect, it has been reported that vWF-induced platelet activation requires activation of the Fc receptor γII (or Fc receptor γ-chain)-protein-tyrosine kinase Syk-signaling pathway (Fig. 7) (43Sullam P.M. Hyun W.C. Szollosi J. Dong J. Foss W.M. Lopez J.A. J. Biol. Chem. 1998; 273: 5331-5336Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 44Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar, 45Wu Y. Suzuki-Inoue K. Satoh K. Asazuma N. Yatomi Y. Berndt M.C. Ozaki Y. Blood. 2001; 97: 3836-3845Crossref PubMed Scopus (105) Google Scholar, 46Canobbio I. Bertoni A. Lova P. Paganini S. Hirsch E. Sinigaglia F. Balduini C. Torti M. J. Biol. Chem. 2001; 276: 26022-26029Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, GPIb-IX is associated with several intracellular signaling proteins including 14-3-3ζ (47Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar, 48Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), phosphatidylinositol 3-kinase (via 14-3-3ζ) (49Munday A.D. Berndt M.C. Mitchell C.A. Blood. 2000; 96: 577-584Crossref PubMed Google Scholar), and calmodulin (50Andrews R.K. Munday A.D. Mitchell C.A. Berndt M.C. Blood. 2001; 98: 681-687Crossref PubMed Scopus (95) Google Scholar). Although the roles of these proteins in GPIb-IX signaling are still unclear, it is possible that these proteins are also involved in GPIb-IX-mediated signaling. Thus, GPIb-IX-mediated integrin activation requires coordination of two or more signaling pathways (Fig. 7), one of which is the cGMP-PKG-MAPK-signaling pathway.The downstream signaling pathway of MAPK-mediated integrin activation is still unknown. Because ERK has been shown to phosphorylate and activate cytoplasmic phospholipase A2 (27Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar), it is a possibility that activation of cytoplasmic phospholipase A2 may be a downstream link between ERK and integrin activation. However, previous studies suggest that the release of arachidonic acid during platelet activation is not affected by MEK inhibitor PD98059 (29Borsch-Haubold A.G. Pasquet S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), and vWF-induced TXA2 production is preceded by integrin activation (35Ruan C.G. Du X.P. Xi X.D. Castaldi P.A. Berndt M.C. Blood. 1987; 69: 570-577Crossref PubMed Google Scholar). We show that vWF-induced integrin activation and low dose-thrombin-induced platelet aggregation are not inhibited by aspirin. These data suggest that ERK-dependent activation of integrin does not require the TXA2 pathway. Although TXA2 is important in the irreversible second wave of platelet aggregation induced by certain agonists, the second wave of platelet aggregation not only requires integrin activation but also requires integrin-dependent outside-in signaling and release of granule contents. It would be interesting to further investigate the downstream mechanism of the MAP kinase-dependent integrin activation pathway. The integrin αIIbβ3 mediates platelet adhesion, spreading, and aggregation and thus plays a critical role in thrombosis and hemostasis (1Ginsberg M.H. Du X. O'Toole T.E. Loftus J.C. Thromb. Haemostasis. 1995; 74: 352-359Crossref PubMed Scopus (65) Google Scholar). In normal circulating platelets, the integrin αIIbβ3 is in a resting state with a low affinity for its ligands such as fibrinogen and von Willebrand factor (vWF).1 At sites of vascular injury, exposure of platelets to soluble agonists (such as thrombin and ADP) or to matrix-bound adhesive proteins (such as collagen and vWF) induces platelet activation. A common consequence of platelet activation is the activation of the ligand binding function of the integrin αIIbβ3 (2Phillips D.R. Charo I.F. Scarborough R.M. 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