Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu, we introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn2+ or by coexpressing the clamped αIIb with a β3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (Mr 7,500) or αIIbβ3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for αIIb extension about the genu in the binding of ligands of 340,000 and 900,000 Mr with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with αIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in β3 that affect the intrinsic affinity of the binding pocket for ligand. Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu, we introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn2+ or by coexpressing the clamped αIIb with a β3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (Mr 7,500) or αIIbβ3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for αIIb extension about the genu in the binding of ligands of 340,000 and 900,000 Mr with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with αIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in β3 that affect the intrinsic affinity of the binding pocket for ligand. IntroductionThe platelet αIIbβ3 receptor plays an important role in both hemostasis and thrombosis (1Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (479) Google Scholar). Ligand binding to αIIbβ3 is controlled by an activation process that affects the conformation of the receptor and ligand binding, in turn, can also affect the conformation of the receptor (2Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (436) Google Scholar). Several different conformations of αIIbβ3 have been identified based on inferences from biochemical analyses (3Parise L.V. Helgerson S.L. Steiner B. Nannizzi L. Phillips D.R. J. Biol. Chem. 1987; 262: 12597-12602Abstract Full Text PDF PubMed Google Scholar), studies employing monoclonal antibodies (4Kouns W.C. Wall C.D. White M.M. Fox C.F. Jennings L.K. J. Biol. Chem. 1990; 265: 20594-20601Abstract Full Text PDF PubMed Google Scholar, 5Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Frelinger 3rd, A.L. Cohen I. Plow E.F. Smith M.A. Roberts J. Lam S.C. Ginsberg M.H. J. Biol. Chem. 1990; 265: 6346-6352Abstract Full Text PDF PubMed Google Scholar, 7Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) and electron microscopy (8Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar, 9Litvinov R.I. Nagaswami C. Vilaire G. Shuman H. Bennett J.S. Weisel J.W. Blood. 2004; 104: 3979-3985Crossref PubMed Scopus (60) Google Scholar, 10Ye F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. J. Cell. Biol. 2010; 188: 157-173Crossref PubMed Scopus (204) Google Scholar), comparison of the crystal structures of the liganded αIIbβ3 headpiece (11Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (664) Google Scholar) and the unliganded complete ectodomain (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), and analysis of the unliganded and liganded ectodomain of the related αVβ3 receptor (13Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1097) Google Scholar, 14Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1382) Google Scholar). Receptor extension about the regions encompassing the thigh, genu, and calf-1 domains of αIIb and the plexin-semaphorin-integrin (PSI), 2The abbreviations used are: PSIplexin-semaphorin-integrinmAbmonoclonal antibodyIEGFintegrin epidermal growth factorDTTdithiothreitolFITCfluorescein isothiocyanateHBMTHEPES-modified Tyrode's bufferNNFInet normalized fluorescence intensityGMFIgeometric mean fluorescence intensity. integrin epidermal growth factor-1 (IEGF-1), and IEGF-2 domains of β3 or comparable regions of other integrin receptors has been proposed to play an important role in receptor activation (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 15Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (257) Google Scholar, 16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 17Luo B.H. Takagi J. Springer T.A. J. Biol. Chem. 2004; 279: 10215-10221Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18Salas A. Shimaoka M. Phan U. Kim M. Springer T.A. J. Biol. Chem. 2006; 281: 10876-10882Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), but there is uncertainty about whether this conformational change occurs prior to or after ligand binding (19Zhu J. Boylan B. Luo B.H. Newman P.J. Springer T.A. J. Biol. Chem. 2007; 282: 11914-11920Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Rocco M. Rosano C. Weisel J.W. Horita D.A. Hantgan R.R. Structure. 2008; 16: 954-964Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 21Arnaout M.A. Goodman S.L. Xiong J.P. Curr. Opin. Cell Biol. 2007; 19: 495-507Crossref PubMed Scopus (312) Google Scholar). Thus, “cross-clasping” the αIIb headpiece β-propeller domain to the β3 IEGF-4 domain in the tail region via a newly engineered disulfide bond prevented the binding of fibrinogen induced by activating mAb in concert with the activating divalent cation Mn2+, and a similar effect was observed with cross-clasped αVβ3 (16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). In both cases the loss of ligand binding could be rescued by reducing the cross-clasped receptors with dithiothreitol (DTT). In contrast, the binding of a fragment of fibronectin (Mr = ∼50 kDa) to the ectodomain of αVβ3 activated by Mn2+ was not associated with receptor extension as judged by electron microscopy (22Adair B.D. Xiong J.P. Maddock C. Goodman S.L. Arnaout M.A. Yeager M. J. Cell Biol. 2005; 168: 1109-1118Crossref PubMed Scopus (153) Google Scholar).To understand better the role of αIIb extension around the αIIb genu region in ligand binding, we selectively clamped the αIIb thigh domain to the αIIb calf-1 domain with a newly engineered disulfide bond created by mutating αIIb Arg597 and Tyr645 to cysteine residues. the of the disulfide bond by and the of both large and ligands to clamped as as both soluble and immobilized also the of αIIbβ3 activation Mn2+ and of and αIIb with a β3 subunit that when with αIIb in fibrinogen binding M. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). Our data support an important role for receptor extension around the genu for the binding of large soluble ligands induced by but not activation In contrast, αIIb extension about the genu had a effect on αIIbβ3-mediated adhesion to immobilized fibrinogen and effect on the binding of soluble data the of αIIb extension around the genu to high affinity ligand studies to the role of αIIb extension about the genu in αIIbβ3 ligand binding. Previous studies the αIIb β-propeller domain to the β3 IEGF-4 domain a role for extension of or both in the binding of fibrinogen to αIIbβ3 when activated by a of Mn2+ and an activating mAb (16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). have studies by the effect of an αIIb clamp to the region to the genu and the binding of both high and low Mr soluble as as the ability of the receptor to support adhesion to immobilized fibrinogen and bind a binding site antibody is on the β3 αIIb clamp was based on the of in the αIIb model we the crystal of the αVβ3 receptor as a studies the of the was in the crystal of the ectodomain of αIIbβ3 (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). for the of the disulfide bond from both data and the of αIIb in both and reducing found that clamped αIIbβ3 has a reduced ability to bind the soluble ligands fibrinogen (Mr = and mAb PAC-1 (Mr = induced by the activating mAb to The could be rescued by the reducing studies have the that on the ability of the mAb to bind to the and clamped the that the in ligand binding was to the clamp the binding of PT25-2 by PT25-2 binding to or clamped αIIbβ3 and binding. was to the receptor and rescued the ability of the clamped receptor to ligand binding in to analysis that reduced the engineered disulfide bond in but not of the clamped αIIbβ3 an for the of ligand binding. the that for and/or affects ligand to the with fibrinogen and the binding of the Mr snake venom an and not receptor was by the receptor extension about the genu is not for binding. The Mr of kistrin with fibrinogen and PAC-1 for the in ability to bind to αIIbβ3 it is that containing the can bind to unactivated platelets J.H. Springer Coller B.S. Blood. PubMed Google activating αIIbβ3 with a mAb not activation by F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. J. Cell. Biol. 2010; 188: 157-173Crossref PubMed Scopus (204) Google Scholar), it a large that we also activated the receptor by with the activating β3 subunit and by with αIIb with the β3 high binding of both fibrinogen and and an activating mAb had little or and PAC-1 both also high binding to the clamped αIIb in with the β3 activating and an activating mAb also did not affect the studies are to the by the but it is in the region of β3 encompassing the and the have been in ligand affinity different in the unliganded and liganded β3 crystal structures (11Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (664) Google Scholar, J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1097) Google Scholar, 14Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1382) Google Scholar). access to play a role in αIIbβ3 ligand binding B.S. J. Cell Biol. PubMed Scopus Google Scholar), it is that the β3 mutation the conformation of the receptor as to access to the ligand-binding or it is that β3 affects the affinity for the ligands by (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) or by facilitating the of the the as to of the to with the ligand that not the (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Our studies of AP5 binding to the and αIIbβ3 receptors data that on Thus, in the of AP5 to αIIbβ3 and clamped αIIbβ3 AP5 the other that the β3 mutation induces conformational in the β3 domain that are to the activation was also to the in binding soluble fibrinogen induced by The by Mn2+ integrin receptors is but studies different regions of αIIb and the region as the process T. M. Y. Y. S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. J. Cell. Biol. 2010; 188: 157-173Crossref PubMed Scopus (204) Google Scholar) that αIIbβ3 did not a change in after activation by Mn2+ increased adhesion of the to immobilized interpreted data as with the for integrin extension for ligand binding, but study had a of of of the of receptors that ligand binding, the of a of receptors that did a conformation in the presence of Mn2+, and the of and in that play an important role in the conformational in αIIbβ3 (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). of the the data from the β3 and Mn2+ activation indicate that it is for high Mr ligands to bind to αIIbβ3 receptors that have about the αIIb as by our ligand is an important of ability to bind to integrin receptors that have not extension about the genu, this an for the by (22Adair B.D. Xiong J.P. Maddock C. Goodman S.L. Arnaout M.A. Yeager M. J. Cell Biol. 2005; 168: 1109-1118Crossref PubMed Scopus (153) Google Scholar) that an fragment of is between kistrin and fibrinogen in can bind to αVβ3 of headpiece Mn2+ was used to the binding of the fibronectin and we found that Mn2+ could the ability of the clamped αIIbβ3 receptor to bind soluble fibrinogen, it is not to the of ligand Mr and Mn2+ to our that Mn2+ activation could soluble ligand binding to clamped Takagi (16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar) found reduced soluble fibrinogen binding to cross-clasped αIIbβ3 in the presence of both Mn2+ and Thus, the induced by from that induced by our our data and of Takagi could be by that PT25-2 induces activation via an effect on αIIb with on the β3 The effect of the β3 and Mn2+ be of the effect of but the Mn2+ effect between regions of αIIb and β3 that are by the introduced by Takagi is αIIbβ3 ligands in that the binding of soluble fibrinogen receptor adhesion to immobilized fibrinogen not B.S. Blood. PubMed Google Scholar, G. L. Blood. PubMed Google Scholar, B. Ruggeri J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, L. Biol. PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar). for this is that αIIbβ3 extension is for the binding of soluble fibrinogen, but not for adhesion to immobilized To this we also the effect of αIIb about the genu on αIIbβ3-mediated adhesion to αIIbβ3 little or in adhesion to fibrinogen immobilized high and a in adhesion to fibrinogen immobilized low Thus, αIIb extension about the genu had of an effect on αIIbβ3 with immobilized fibrinogen on binding of soluble of have been proposed to for the in for αIIbβ3 binding of soluble fibrinogen adhesion to immobilized fibrinogen, conformational in fibrinogen induced by K.A. B. Coller B.S. PubMed Scopus Google Scholar) and the for between receptors and ligand from the of fibrinogen with G. L. Blood. PubMed Google Scholar). a of αIIb the fibrinogen a with ligand but data to be interpreted in of the of fibrinogen on the conformational induced by the ligand G. L. Blood. PubMed Google Scholar, K.A. B. Coller B.S. PubMed Scopus Google Scholar). The study by (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) an of the region of β3 with the to activation of the receptor via receptor extension and β3 Thus, an low affinity with immobilized fibrinogen could affinity by this activation this is a our data indicate that affinity can be extension the found that binding of the head domain to the domain of β3 was to both αIIbβ3 extension and PAC-1 binding F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. J. Cell. Biol. 2010; 188: 157-173Crossref PubMed Scopus (204) Google Scholar), but this not an to enhanced αIIbβ3 affinity via a (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google of ligands to αIIbβ3 is to conformational in the receptor (4Kouns W.C. Wall C.D. White M.M. Fox C.F. Jennings L.K. J. Biol. Chem. 1990; 265: 20594-20601Abstract Full Text PDF PubMed Google Scholar, 5Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Frelinger 3rd, A.L. Cohen I. Plow E.F. Smith M.A. Roberts J. Lam S.C. Ginsberg M.H. J. Biol. Chem. 1990; 265: 6346-6352Abstract Full Text PDF PubMed Google Scholar, 7Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar), resulting in that to and (2Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (436) Google Scholar). receptor extension has been to affect ligand binding, little is about to binding we the effect of αIIb on the ability of the snake venom kistrin we to both and clamped to the AP5 is in the domain of β3 and with ligand binding S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). AP5 binding to αIIbβ3 was low in the of kistrin and increased in the presence of kistrin. AP5 binding to clamped αIIbβ3 was low in the of but the of kistrin was observed with was not to an in the clamped or reduced of receptors with rescued the the αIIbβ3 also decreased AP5 binding. In the crystal of the complete ectodomain of the AP5 is by IEGF-2 in the but it is to be with headpiece extension (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Thus, our data that AP5 to ligand binding extension the genu and the that the also to that the and domains a of in the receptor with a model of the it is also that headpiece extension by to enhanced ligand affinity by reducing the for the of the and is of to our data on the clamped receptor with on the platelets of a with had αIIbβ3 but receptors to as a of a mutation in the β3 domain J. J.P. Ginsberg M.H. Blood. PubMed Google Scholar, I. D. Steiner B. J.P. J.P. PubMed Scopus Google Scholar). The receptors to support platelet adhesion to immobilized fibrinogen but not soluble fibrinogen binding in to The between the data and the data clamped αIIbβ3 are consistent with a model in in by extension the αIIb our data support a role for αIIb extension about the genu in the binding of high but not Mr ligands but not of data are to of activation are by and there is support for αIIb extension binding of high Mr ligands via an effect on access to the ligand-binding site B.S. J. Cell Biol. PubMed Scopus Google Scholar), it that αIIb extension affects the activation and/or affinity of the ligand-binding pocket in to, or is that the loss of the that the β3 and domains in the the β3 subunit to the and the of the the data also a for the in activation of soluble fibrinogen binding adhesion to immobilized our data the that αIIb extension also to binding by facilitating conformational change in IntroductionThe platelet αIIbβ3 receptor plays an important role in both hemostasis and thrombosis (1Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (479) Google Scholar). Ligand binding to αIIbβ3 is controlled by an activation process that affects the conformation of the receptor and ligand binding, in turn, can also affect the conformation of the receptor (2Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (436) Google Scholar). Several different conformations of αIIbβ3 have been identified based on inferences from biochemical analyses (3Parise L.V. Helgerson S.L. Steiner B. Nannizzi L. Phillips D.R. J. Biol. Chem. 1987; 262: 12597-12602Abstract Full Text PDF PubMed Google Scholar), studies employing monoclonal antibodies (4Kouns W.C. Wall C.D. White M.M. Fox C.F. Jennings L.K. J. Biol. Chem. 1990; 265: 20594-20601Abstract Full Text PDF PubMed Google Scholar, 5Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Frelinger 3rd, A.L. Cohen I. Plow E.F. Smith M.A. Roberts J. Lam S.C. Ginsberg M.H. J. Biol. Chem. 1990; 265: 6346-6352Abstract Full Text PDF PubMed Google Scholar, 7Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) and electron microscopy (8Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar, 9Litvinov R.I. Nagaswami C. Vilaire G. Shuman H. Bennett J.S. Weisel J.W. Blood. 2004; 104: 3979-3985Crossref PubMed Scopus (60) Google Scholar, 10Ye F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. J. Cell. Biol. 2010; 188: 157-173Crossref PubMed Scopus (204) Google Scholar), comparison of the crystal structures of the liganded αIIbβ3 headpiece (11Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (664) Google Scholar) and the unliganded complete ectodomain (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), and analysis of the unliganded and liganded ectodomain of the related αVβ3 receptor (13Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1097) Google Scholar, 14Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1382) Google Scholar). Receptor extension about the regions encompassing the thigh, genu, and calf-1 domains of αIIb and the plexin-semaphorin-integrin (PSI), 2The abbreviations used are: PSIplexin-semaphorin-integrinmAbmonoclonal antibodyIEGFintegrin epidermal growth factorDTTdithiothreitolFITCfluorescein isothiocyanateHBMTHEPES-modified Tyrode's bufferNNFInet normalized fluorescence intensityGMFIgeometric mean fluorescence intensity. integrin epidermal growth factor-1 (IEGF-1), and IEGF-2 domains of β3 or comparable regions of other integrin receptors has been proposed to play an important role in receptor activation (12Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 15Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (257) Google Scholar, 16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 17Luo B.H. Takagi J. Springer T.A. J. Biol. Chem. 2004; 279: 10215-10221Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18Salas A. Shimaoka M. Phan U. Kim M. Springer T.A. J. Biol. Chem. 2006; 281: 10876-10882Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), but there is uncertainty about whether this conformational change occurs prior to or after ligand binding (19Zhu J. Boylan B. Luo B.H. Newman P.J. Springer T.A. J. Biol. Chem. 2007; 282: 11914-11920Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Rocco M. Rosano C. Weisel J.W. Horita D.A. Hantgan R.R. Structure. 2008; 16: 954-964Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 21Arnaout M.A. Goodman S.L. Xiong J.P. Curr. Opin. Cell Biol. 2007; 19: 495-507Crossref PubMed Scopus (312) Google Scholar). Thus, “cross-clasping” the αIIb headpiece β-propeller domain to the β3 IEGF-4 domain in the tail region via a newly engineered disulfide bond prevented the binding of fibrinogen induced by activating mAb in concert with the activating divalent cation Mn2+, and a similar effect was observed with cross-clasped αVβ3 (16Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). In both cases the loss of ligand binding could be rescued by reducing the cross-clasped receptors with dithiothreitol (DTT). In contrast, the binding of a fragment of fibronectin (Mr = ∼50 kDa) to the ectodomain of αVβ3 activated by Mn2+ was not associated with receptor extension as judged by electron microscopy (22Adair B.D. Xiong J.P. Maddock C. Goodman S.L. Arnaout M.A. Yeager M. J. Cell Biol. 2005; 168: 1109-1118Crossref PubMed Scopus (153) Google Scholar).To understand better the role of αIIb extension around the αIIb genu region in ligand binding, we selectively clamped the αIIb thigh domain to the αIIb calf-1 domain with a newly engineered disulfide bond created by mutating αIIb Arg597 and Tyr645 to cysteine residues. the of the disulfide bond by and the of both large and ligands to clamped as as both soluble and immobilized also the of αIIbβ3 activation Mn2+ and of and αIIb with a β3 subunit that when with αIIb in fibrinogen binding M. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). Our data support an important role for receptor extension around the genu for the binding of large soluble ligands induced by but not activation In contrast, αIIb extension about the genu had a effect on αIIbβ3-mediated adhesion to immobilized fibrinogen and effect on the binding of soluble data the of αIIb extension around the genu to high affinity ligand binding.